U.S. patent application number 11/389343 was filed with the patent office on 2006-10-05 for nucleic acid molecules and other molecules associated with soybean cyst nematode resistance.
Invention is credited to Brian M. Hauge, Laurence David Parnell, Jeremy David Parsons, Ming Li Wang.
Application Number | 20060225150 11/389343 |
Document ID | / |
Family ID | 22637918 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060225150 |
Kind Code |
A1 |
Hauge; Brian M. ; et
al. |
October 5, 2006 |
Nucleic acid molecules and other molecules associated with soybean
cyst nematode resistance
Abstract
The present invention is in the field of soybean genetics. More
specifically, the invention relates to nucleic acid molecules from
regions the soybean genome, which are associated with soybean cyst
nematode resistance. The invention also relates to proteins encoded
by such nucleic acid molecules as well as antibodies capable of
recognizing these proteins. The invention also relates to nucleic
acid markers from regions the soybean genome, which are associated
with soybean cyst nematode resistance. Moreover, the invention
relates to uses of such molecules, including, transforming soybean
cyst nematode resistant soybean with constructs containing nucleic
acid molecules from regions the soybean genome, which are
associated with soybean cyst nematode resistance. Furthermore, the
invention relates to the use of such molecules in a plant breeding
program.
Inventors: |
Hauge; Brian M.; (Beverly,
MA) ; Wang; Ming Li; (Lexington, MA) ;
Parsons; Jeremy David; (Cambridge, GB) ; Parnell;
Laurence David; (Cambridge, MA) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
22637918 |
Appl. No.: |
11/389343 |
Filed: |
March 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09754853 |
Jan 5, 2001 |
|
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11389343 |
Mar 27, 2006 |
|
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60174880 |
Jan 7, 2000 |
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Current U.S.
Class: |
800/279 ;
435/468; 800/312 |
Current CPC
Class: |
C07K 14/415
20130101 |
Class at
Publication: |
800/279 ;
800/312; 435/468 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82; A01H 5/00 20060101
A01H005/00 |
Claims
1-72. (canceled)
73. A method of introgressing an allele into a soybean plant
comprising (A) crossing at least one SCN resistant soybean plant
with at least one SCN sensitive soybean plant in order to form a
segregating population, (B) screening said segregating population
with one or more nucleic acid markers to determine if one or more
soybean plants from said segregating population contains an rhg1
SCN resistant allele, or an Rhg4 SCN resistant allele, wherein said
rhg1 SCN resistant allele is an allele having a deletion of 19
nucleotides corresponding to SEQ ID NO: 2 and encompassing position
48881, and said Rhg4 SCN resistant allele is an allele having one
or more polymorphisms at a position in SEQ ID NO: 4 selected from
the group consisting of 111933, 112065, 112101, and 112461, and (C)
selecting, if present, one or more soybean plants of said
segregating population containing said deletion, or said one or
more polymorphisms located at a position in SEQ ID NO: 4.
74. The method according to claim 73, wherein said one or more
soybean plants of said segregating population have a yellow soybean
seed.
75. A method of introgressing an allele into a soybean plant
comprising (A) crossing at least one SCN resistant soybean plant
with at least one SCN sensitive soybean plant in order to form a
segregating population, (B) screening said segregating population
with one or more nucleic acid markers to determine if one or more
soybean plants from said segregating population contains an rhg1
SCN resistant allele, or an Rhg4 SCN resistant allele, wherein said
rhg1 SCN resistant allele is an allele having one or more first
polymorphisms located at a position in SEQ ID NO: 2 selected from
the group consisting of 45173, 45309, 47057, 47140, 47208, 47571,
47617, 47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279,
48413, 48681, 48881, 49012, and 493161, and said Rhg4 SCN resistant
allele is an allele having one or more second polymorphisms at a
position in SEQ ID NO: 4 selected from the group consisting of
111933, 112065, 112101, and 112461, and (C) selecting, if present,
one or more soybean plants of said segregating population
containing said one or more first polymorphisms, or said one or
more second polymorphisms.
76. The method according to claim 75, wherein said one or more
soybean plants of said segregating population have a yellow soybean
seed.
77. A method of introgressing an allele into a soybean plant
comprising (A) crossing at least one SCN resistant soybean plant
with at least one SCN sensitive soybean plant in order to form a
segregating population, (B) screening said segregating population
with one or more nucleic acid markers to determine if one or more
soybean plants from said segregating population contains an rhg1
SCN resistant allele, or an Rhg4 SCN resistant allele, wherein said
rhg1 SCN resistant allele is an allele having one or more first
polymorphisms in a protein coding region corresponding to
nucleotides 45163 to 45314, 45450 to 45509, 46941 to 48763 or 48975
to 49573 of SEQ ID NO: 2, and said Rhg4 SCN resistant allele is an
allele having one or more second polymorphisms in a protein coding
region corresponding to nucleotides 111805 to 113968 or 114684 to
115204 of SEQ ID NO: 4, and (C) selecting, if present, one or more
soybean plants of said segregating population containing said one
or more first polymorphisms, or said one or more second
polymorphisms.
78. The method according to claim 77, wherein said one or more
soybean plants of said segregating population have a yellow soybean
seed.
79. The method according to claim 77, wherein said one or more
first polymorphisms, or said one or more second polymorphisms, are
single nucleotide polymorphisms.
80. The method according to claim 77, wherein said one or more
first polymorphisms, or said one or more second polymorphisms, are
INDEL or simple sequence repeat (SSR) polymorphisms.
81. The method according to claim 77, wherein said one or more
first polymorphisms are selected from the group consisting of
45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856,
47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, and
49316 of SEQ ID NO: 2.
82. The method according to claim 77, wherein said one or more
second polymorphisms are selected from the group consisting of
111933, 112065, 112101, and 112461 of SEQ ID NO: 4.
83. A method of introgressing an allele into a soybean plant
comprising (A) crossing at least one SCN resistant soybean plant
with at least one SCN sensitive soybean plant in order to form a
segregating population, (B) screening said segregating population
with one or more nucleic acid markers to determine if one or more
soybean plants from said segregating population contains an rhg1
SCN resistant allele or an Rhg4 SCN resistant allele, wherein said
rhg1 SCN resistant allele is an allele having a deletion of 19
nucleotides corresponding to SEQ ID NO: 2 and encompassing position
48881, and said Rhg4 SCN resistant allele is an allele having one
or more polymorphisms within SEQ ID NO: 4, and (C) selecting, if
present, one or more soybean plants of said segregating population
containing said deletion, or said one or more polymorphisms within
SEQ ID NO: 4.
84. The method according to claim 83, wherein said one or more
soybean plants of said segregating population have a yellow soybean
seed.
85. The method according to claim 83, wherein said one or more
polymorphisms within SEQ ID NO: 4 are single nucleotide
polymorphisms.
86. The method according to claim 83, wherein said one or more
polymorphisms within SEQ ID NO: 4 are INDEL or simple sequence
repeat (SSR) polymorphisms.
87. The method according to claim 83, wherein said one or more
polymorphisms within SEQ ID NO: 4 are selected from the group
consisting of 111933, 112065, 112101, and 112461 of SEQ ID NO:
4.
88. A method of introgressing an allele into a soybean plant
comprising (A) crossing at least one SCN resistant soybean plant
with at least one SCN sensitive soybean plant in order to form a
segregating population, (B) screening said segregating population
with one or more nucleic acid markers to determine if one or more
soybean plants from said segregating population contains an rhg1
SCN resistant allele, or an Rhg4 SCN resistant allele, wherein said
rhg1 SCN resistant allele is an allele having one or more first
polymorphisms in a protein coding region corresponding to
nucleotides 45163 to 49573 of SEQ ID NO: 2, and said Rhg4 SCN
resistant allele is an allele having one or more second
polymorphisms in a protein coding region corresponding to
nucleotides 111805 to 115204 of SEQ ID NO: 4, and (C) selecting, if
present, one or more soybean plants of said segregating population
containing said one or more first polymorphisms, or said one or
more second polymorphisms.
89. The method according to claim 88, wherein said one or more
soybean plants of said segregating population have a yellow soybean
seed.
90. The method according to claim 88, wherein said one or more
first polymorphisms, or said one or more second polymorphisms, are
single nucleotide polymorphisms.
91. The method according to claim 88, wherein said one or more
first polymorphisms, or said one or more second polymorphisms, are
INDEL or simple sequence repeat (SSR) polymorphisms.
92. The method according to claim 88, wherein said rhg1 SCN
resistant allele is an allele having one or more first
polymorphisms in a protein coding region corresponding to
nucleotides 45163 to 45314, 45450 to 45509, 46941 to 48763 or 48975
to 49573 of SEQ ID NO: 2.
93. The method according to claim 88, wherein said one or more
first polymorphisms are selected from the group consisting of
45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856,
47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, and
49316 of SEQ ID NO: 2.
94. The method according to claim 88, wherein said rhg1 resistant
allele is an allele having a deletion of 19 nucleotides
corresponding to SEQ ID NO: 2 and encompassing position 48881.
95. The method according to claim 88, wherein said Rhg4 SCN
resistant allele is an allele having one or more second
polymorphisms in a protein coding region corresponding to
nucleotides 111805 to 113968 or 114684 to 115204 of SEQ ID NO:
4.
96. The method according to claim 88, wherein said one or more
second polymorphisms are selected from the group consisting of
111933,.112065, 112101, and 112461 of SEQ ID NO: 4.
97. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 5, 6, 8-23, 28-43, complements thereof, and fragments of
either.
98. A substantially purified first nucleic acid molecule with
nucleic acid sequence which specifically hybridizes to a second
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of a complement of SEQ ID NOs: 5, 6, 8-23,
28-43.
99. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 7, 44-47, and 50-53, complements thereof, and fragments of
either.
100. A substantially purified first nucleic acid molecule with
nucleic acid sequence which specifically hybridizes to a second
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of a complement of SEQ ID NOs: 7, 44-47, and
50-53.
101. A substantially purified protein or fragment thereof
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1097, 1098, and 1100-1115 and fragments
thereof.
102. A substantially purified protein or fragment thereof
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1099, and 1116-1119 and fragments
thereof.
103. A transformed plant having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions in a
plant cell to cause the production of a mRNA molecule; (B) a
structural nucleic acid molecule encoding a protein or fragment
thereof comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1097, 1100, 1098, 1101, 1102-1115; and
(C) a 3' non-translated sequence that functions in the plant cell
to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
104. The transformed plant according to claim 103, wherein said
plant is soybean.
105. The transformed plant according to claim 104, wherein said
plant is soybean is selected from the group consisting of PI548402
(Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4), PI404166
(Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654 (Er-hej-jan),
PI438489 (Chiquita), PI507354 (Tokei 421), PI548655 (Forrest),
PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid, Nathan,
AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501, AG4601,
PION9492, PI88788, Dyer, Custer, Manokin, and Doles.
106. The transformed plant according to claim 103, wherein said
promoter is an rhg1 promoter.
107. The transformed plant according to claim 103, wherein said
promoter is an Rhg4 promoter.
108. A transformed plant having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions in a
plant cell to cause the production of a mRNA molecule; (B) a
structural nucleic acid molecule encoding a protein or fragment
thereof comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1099, 1116-1119; and (C) a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
109. The transformed plant according to claim 108, wherein said
plant is soybean.
110. The transformed plant according to claim 109, wherein said
plant is soybean is selected from the group consisting of PI548402
(Peking), PI437654 (Er-hej-jan), PI438489 (Chiquita), PI507354
(Tokei 421), PI548655 (Forrest), PI548988 (Pickett), PI88788,
PI404198 (Sun Huan Do), PI404166 (Krasnoaarmejkaja), Hartwig,
Manokin, Doles, Dyer, and Custer.
111. The transformed plant according to claim 108, wherein said
promoter is an rgh 1 promoter.
112. The transformed plant according to claim 108, wherein said
promoter is an Rhg4 promoter.
113. A transgenic seed having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions to
cause the production of a mRNA molecule; (B) a structural nucleic
acid molecule encoding a protein or fragment thereof comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1097, 1100, 1098, 1101, 1102-1115; and (C) a 3' non-translated
sequence that functions to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
114. A transgenic seed having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions to
cause the production of a mRNA molecule; (B) a structural nucleic
acid molecule encoding a protein or fragment thereof comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1099, 1116-1119; and (C) a 3' non-translated sequence that
functions to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
115. A transformed plant having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions in a
plant cell to cause the production of a mRNA molecule; (B) a
structural nucleic acid molecule comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 5, 6, 8-23,
28-43, complements thereof, and fragments of either; and (C) a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
116. The transformed plant according to claim 115, wherein said
plant is soybean.
117. The transformed plant according to claim 116, wherein said
plant is soybean is selected from the group consisting of PI548402
(Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4), PI404166
(Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654 (Er-hej-jan),
PI438489 (Chiquita), PI507354 (Tokei 421), PI548655 (Forrest),
PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid, Nathan,
AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501, AG4601,
PION9492, PI88788, Dyer, Custer, Manokin, and Doles.
118. The transformed plant according to claim 115, wherein said
promoter is an rhg1 promoter.
119. The transformed plant according to claim 115, wherein said
promoter is an Rhg4 promoter.
120. A transformed plant having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions in a
plant cell to cause the production of a mRNA molecule; (B) a
structural nucleic acid molecule comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 7, 44-77 and
50-53, complements thereof, and fragments of either; and (C) a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
121. The transformed plant according to claim 120, wherein said
plant is soybean.
122. The transformed plant according to claim 121, wherein said
plant is soybean is selected from the group consisting of PI548402
(Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4), PI404166
(Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654 (Er-hej-jan),
PI438489 (Chiquita), PI507354 (Tokei 421), PI548655 (Forrest),
PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid, Nathan,
AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501, AG4601,
PION9492, PI88788, Dyer, Custer, Manokin, and Doles.
123. The transformed plant according to claim 120, wherein said
promoter is an rhg1 promoter.
124. The transformed plant according to claim 120, wherein said
promoter is an Rhg4 promoter.
125. A transgenic seed having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions to
cause the production of a mRNA molecule; (B) a structural nucleic
acid molecule comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 5, 6, 8-23, 28-43, complements
thereof, and fragments of either; and (C) a 3' non-translated
sequence that functions to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
126. A transgenic seed having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions to
cause the production of a mRNA molecule; (B) a structural nucleic
acid molecule comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 7, 44-77 and 50-53, complements
thereof, and fragments of either; and (C) a 3' non-translated
sequence that functions to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 09/754,853, filed Jan. 5, 2001, which claims the benefit under
35 U.S.C. .sctn.119(e) of U.S. Application No. 60/174,880, filed
Jan. 7, 2000. The disclosures of U.S. application Ser. Nos.
09/754,853 and 60/174,880 are both herein incorporated by reference
in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] A paper copy of the Sequence Listing and a computer readable
form (CRF) of the sequence listing on diskette, containing the file
named 00330V2.TXT, which is 2,521,108 bytes in size (measured in
Windows XP), and which was recorded on Jul. 27, 2001, are herein
incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention is in the field of soybean genetics.
More specifically, the invention relates to nucleic acid molecules
from regions of the soybean genome, which are associated with
soybean cyst nematode (SCN) resistance. The invention also relates
to proteins encoded by such nucleic acid molecules as well as
antibodies capable of recognizing these proteins. The invention
also relates to nucleic acid markers from regions of the soybean
genome, which are associated with SCN resistance. Moreover, the
invention relates to uses of such molecules, including,
transforming SCN sensitive soybean with constructs containing
nucleic acid molecules from regions in the soybean genome, which
are associated with SCN resistance. Furthermore, the invention
relates to the use of such molecules in a plant breeding
program.
BACKGROUND OF THE INVENTION
[0004] The soybean, Glycine max (L.) Merril (Glycine max or
soybean), is one of the major economic crops grown worldwide as a
primary source of vegetable oil and protein (Sinclair and Backman,
Compendium of Soybean Diseases, 3.sup.rd Ed. APS Press, St. Paul,
Minn., p. 106. (1989)). The growing demand for low cholesterol and
high fiber diets has also increased soybean's importance as a
health food.
[0005] Prior to 1940, soybean cultivars were either direct releases
of introductions brought from Asia or pure line selections from
genetically diverse plant introductions. The soybean plant was
primarily used as a hay crop in the early part of the 19th century.
Only a few introductions were large-seeded types useful for feed
grain and oil production. From the mid 1930's through the 1960's,
gains in soybean seed yields were achieved by changing the breeding
method from evaluation and selection of introduced germplasm to
crossing elite by elite lines. The continuous cycle of cross
hybridizing the elite strains selected from the progenies of
previous crosses resulted in the modern day cultivars.
[0006] Over 10,000 soybean strains have now been introduced into
the United States since the early 1900's (Bernard et al., United
States National Germplasm Collections. In: L. D. Hil (ed.), World
Soybean Research, pp. 286-289. Interstate Printers and Publ.,
Danville, Ill. (1976)). A limited number of those introductions
form the genetic base of cultivars developed from the hybridization
and selection programs (Johnson and Bernard, The Soybean, Norman
Ed., Academic Press, N.Y., pp. 1-73 (1963)). For example, in a
survey conducted by Specht and Williams, Genetic Contributions,
Fehr eds. American Soil Association, Wisconsin, pp. 49-73 (1984),
for the 136 cultivars released from 1939 to 1989, only 16 different
introductions were the source of cytoplasm for 121 of that 136.
Certain soybean strains are sensitive to one or more pathogens. One
economically important pathogen is SCN.
[0007] SCN accounts for roughly 40% of the total disease in soybean
and can result in significant yield losses (up to 90%). SCN is the
most destructive pest of soybean to date and accounts for an
estimated yield loss of up to $809 million dollars annually.
Currently, the most cost effective control measures are crop
rotation and the use of host plant resistance. While breeders have
successfully developed SCN resistant soybean lines, breeding is
both difficult and time consuming due to the complex and polygenic
nature of resistance. The resistance is often race specific and
does not provide stability over time due to changing SCN
populations in the field. In addition, many of the resistant
soybean varieties carry a significant yield penalty when grown in
the absence of SCN.
[0008] SCN, Heterodera glycines Ichinohe, was identified on
soybeans in the United States in 1954 at Castle Hayne, N. C.
Winstead, et al., Plant Dis. Rep. 39:9-11 (1955). Since its
discovery the SCN has been recognized as one of the most
destructive pests in soybean. It has been reported in nearly all
states in which soybeans are grown, and it causes major production
problems in several states, being particularly destructive in the
Midwestern states. See generally: Caldwell, et al., Agron. J.
52:635-636 (1960); Rao-Arelli and Anand, Crop. Sci. 28:650-652,
(1988); Baltazar and Mansur, Soybean Genet. Newsl. 19:120-122
(1992); Concibido, et al., Crop. Sci., (1993). For example,
sensitive soybean cultivars had 5.7-35.8% lower seed yields than
did resistant cultivars on SCN race-3 infested sites in Iowa.
(Niblack and Norton, Plant Dis. 76:943-948 (1992)).
[0009] Shortly after the discovery of SCN in the United States,
sources of SCN resistance were identified (Ross and Brim, Plant
Dis. Rep. 41:923-924 (1957)). Some lines such as Peking and Plant
Introduction (PI) P188788, were quickly incorporated into breeding
programs. Peking became widely used as a source of resistance due
to its lack of agronomically undesirable traits, with Pickett as
the first SCN resistant cultivar released (Brim and Ross, Crop Sci.
6:305 (1966)). The recognition that certain SCN resistant
populations could overcome resistant cultivars lead to an extensive
screen for additional sources of SCN resistance. P188788 emerged as
a popular source of race 3 and 4 resistance even though it had a
cyst index greater than 10% (but less than 20%) against race 4, and
Peking and its derivatives emerged as a popular source for races 1
and 3. P1437654 was subsequently identified as having resistance to
all known races and its SCN resistance was backcrossed into
Forrest. Currently there are more than 130 PIs known to have SCN
resistance.
[0010] SCN race 3 is considered to be the prominent race in the
Midwestern soybean producing states. Considerable effort has been
devoted to the genetics and breeding for resistance to race 3.
While both Peking and P188788 are resistant to SCN race 3,
classical genetics studies suggest that they harbor different genes
for race 3 resistance (Rao-Arelli and Anand, Crop Sci. 28:650-652
(1988)). Crosses between P188788(R) and Essex(S) segregate 9(R):
55(S) in the F.sub.2 population and 1(R): 26(Seg): 37(S) families
in the F.sub.3 generation, suggesting that resistance to race 3 in
P188788 is conditioned by one recessive and two dominant genes,
where as Peking and PI90763 resistance is conditioned by one
dominant and two recessive genes. Based on reciprocal crosses,
Peking, Forrest, and P190763 have genes in common for resistance to
SCN race 3 (Rao-Arelli and Anand, Crop Sci., 28:650-652 (1988)). A
cross between Peking and P188788 segregates 13(R):3(S) in the
F.sub.2 generation, indicating a major difference between the
parents for race 3 resistance. Generation mean analysis based on
four crosses between resistant and sensitive genotypes; A20 (R),
Jack (R), Cordell (R) and A2234 (S), suggests that an additive
genetic model is sufficient to explain most of the genetic
variation of race 3 SCN resistance in each cross, while the
analysis of the pooled data indicates the presence of dominant
effects as well (Mansur, Carriquiry and Roa-Arelli, Crop Sci.
33:1249-1253 (1993)). This analysis further indicates that race 3
resistance is probably under the genetic control of three, but not
more than four genes.
[0011] RFLP analysis of segregating populations between resistant
and sensitive lines; PI209332 (R), PI90763 (R), PI88788 (R), Peking
(R) and Evan (S), identified a major SCN resistance QTL (rhg1)
which maps to linkage group G (Concibido et al., Theor Appl. Genet.
93:234-241 (1996)). In this study, rhg1 explains 51.4% of the
phenotypic variation in PI209322, 52.7% of the variation in
PI90763, 40.0% of the variation in PI88788 and 28.1% of the
variation in Peking. This major resistance QTL was assumed be one
and the same in all of the mapping populations employed. However,
as pointed out by the authors, it is possible that the genomic
interval contains distinct but tightly linked QTLs. In a related
study using PI209332 as the source of resistance, Concibido et al.,
Crop Sci. 36:1643-1650 (1996), show that a QTL on linkage group G
(rhg1) is effective against the three SCN races tested, explaining
35% of the phenotypic variation to race 1, 50% of the variation to
race 3, and 54% of the variation to race 6. In addition to the
major QTL on linkage group G. 4 other QTLs mapping to linkage
groups D, J, L and K were identified, with some of the resistance
loci behaving in a race specific manner.
[0012] Concibido et al. (Crop Sci. 37:258-264 (1997)) found
significant association of marker C006V to a major QTL on linkage
group G (rhg1) and resistance to race 1, race 3 and race 6, in
Peking and PI90763 (Evan X Peking, Evan X PI90763) and races 3 and
6 in PI88788 (Evan X PI88788), in agreement with the previous study
based on the P209332 source of resistance (Concibido et al., Crop
Sci. 36:1643-1650 (1996)). The resistance locus near C006V was
effective against all races tested in all of the resistance
sources. While statistically significant against all races, this
locus accounts for different proportions of the total phenotypic
variation with the races tested. For example, in PI90763 the
resistance locus near C006V explains more than three times the
phenotypic variation against race 1 than against race 3. The
variability can be attributed to differences in the genetic
backgrounds, variability among the SCN populations or may be a
reflection of the limited size of the plant populations which were
employed. This study further identified three additional
independent SCN resistance QTLs; one near the RFLP marker A378H
mapping to the opposite end of linkage group G from C006V (rhg1),
one near the marker B032V-1 on linkage group J and a third linked
to A28OHae-1 on linkage group N. Comparisons between the different
SCN races indicated that some of the putative SCN QTLs behave in a
race specific manner.
[0013] PI437654 was identified as having resistance to all known
races. Based on analysis of 328 recombinant inbreed lines (RIL)
derived from a cross between PI437654 and BSR101, Webb reported six
QTLs associated with SCN resistance on linkage groups A2, C1, G, M,
L25 and L26 (U.S. Pat. No. 5,491,081). An allele on linkage group
G, presumed to be rhg1, is involved with certain SCN races tested
(races 1, 2, 3, 5 and 14), and has the largest reported phenotypic
effect on resistance to every race. In contrast, the QTLs on
linkage groups A2, C1, M, L25 and L26 act in a race specific
manner. The QTL on linkage group L25 was reportedly involved with
four of the five races, while the QTLs on linkage groups, A2, C1
and L26 were each involved in resistance to two of the five races
(U.S. Pat. No. 5,491,081). Webb further reports data that the
resistance to any of the five races is likely to result from the
combined effects of the QTL involved in each race (U.S. Pat. No.
5,491,081).
[0014] Qui et al. (Theor Appl Genet 98:356-364 (1999)) screened 200
F2:3 families derived from a cross between Peking and Essex and
identified RFLP markers which are associated with SCN resistance
QTLs on linkage groups B, E, I and H. The three QTLs on linkage
groups B, E and H jointly account for 57.7% of the phenotypic
variation to race 1, the QTLs on linkage groups H and B account for
21.4% of the variation to race 3, while the QTLs on linkage groups
I and E are associated with resistance to race 5 accounting for
14.0% of the phenotypic variation. In contrast to previous mapping
studies which use Peking as the source of resistance, no
significant association was detected to the rhg1 locus on linkage
group G. The authors point out that the marker Bng122, which has
been shown to have significant linkage to rhg1, is not polymorphic
in the population employed (Concibido et al., Crop Sci.
36:1643-1650 (1996)).
[0015] It has been reported that the rhg1 locus on linkage group G
is necessary for the development of resistance to any of the SCN
races. There have been efforts to develop molecular markers to
identify breeding lines harboring the rhg1 SCN resistant allele.
One of the most commonly used markers for marker assisted selection
(MAS) of rhg1 is an SSR locus that co-segregates and maps roughly
0.4 cM from rhg1. This SRR marker, BARC-Satt.sub.--309 is able to
distinguish most, if not all, of the SCN sensitive genotypes from
those harboring rhg1 from important sources of resistance such as
Peking and PI437654. Two simple sequence repeat markers have been
reported that can be used to select for SCN resistance at the rhg1
locus (Concibido et al., Theor Appl Genet 99: 811-818 (1999)).
Satt.sub.--309 was also effective in distinguishing SCN resistant
sources PI88788 and PI209332 in many, but not all, sensitive
genotypes. In particular, Satt.sub.--309 can not be used for MAS in
populations developed from "typical" southern US cultivars (e.g.,
Lee, Bragg and Essex) crossed with resistance sources PI88788 or
PI209332.
[0016] Matson and Williams have reported a dominant SCN resistance
locus, Rhg4, which is tightly linked to the `i` locus on linkage
group A2 (Matson and Williams, Crop Sci. 5:447 (1965)). The QTL
reported by Webb on linkage group A2 maps near the `i` locus and is
considered to be Rhg4 (U.S. Pat. No. 5,491,081). Webb concludes
that only two loci on linkage groups A2 (Rhg4) and G (rhg1) explain
the genetic variation to race 3.
SUMMARY OF THE INVENTION
[0017] The present invention includes and provides a method for the
production of a soybean plant having an rhg1 SCN resistant allele
comprising: (A) crossing a first soybean plant having an rhg1 SCN
resistant allele with a second soybean plant having an rhg1 SCN
sensitive allele to produce a segregating population; (B) screening
the segregating population for a member having an rhg1 SCN
resistant allele with a first nucleic acid molecule capable of
specifically hybridizing to linkage group G, wherein the first
nucleic acid molecule specifically hybridizes to a second nucleic
acid molecule that is linked to the rhg1 SCN resistant allele; and,
(C) selecting the member for further crossing and selection.
[0018] The present invention includes and provides a method of
investigating an rhg1 haplotype of a soybean plant comprising: (A)
isolating nucleic acid molecules from the soybean plant; (B)
determining the nucleic acid sequence of an rhg1 allele or part
thereof; and, (C) comparing the nucleic acid sequence of the rhg1
allele or part thereof to a reference nucleic acid sequence. The
present invention includes and provides a method of introgressing
SCN resistance or partial SCN resistance into a soybean plant
comprising: performing marker assisted selection of the soybean
plant with a nucleic acid marker, wherein the nucleic acid marker
specifically hybridizes with a nucleic acid molecule having a first
nucleic acid sequence that is physically linked to a second nucleic
acid sequence that is located on linkage group G of soybean A3244,
wherein the second nucleic acid sequence is within 500 kb of a
third nucleic acid sequence which is capable of specifically
hybridizing with the nucleic acid sequence of SEQ ID NO: 5, 6,
complements thereof, or fragments thereof having at least 15
nucleotides; and, selecting the soybean plant based on the marker
assisted selection.
[0019] The present invention includes and provides a method for the
production of a soybean plant having an Rhg4 SCN resistant allele
comprising: (A) crossing a first soybean plant having an Rhg4 SCN
resistant allele with a second soybean plant having an Rhg4 SCN
sensitive allele to produce a segregating population; (B) screening
the segregating population for a member having an Rhg4 SCN
resistant allele with a first nucleic acid molecule capable of
specifically hybridizing to linkage group A2, wherein the first
nucleic acid molecule specifically hybridizes to a second nucleic
acid molecule linked to the Rhg4 SCN resistant allele; and, (C)
selecting the member for further crossing and selection.
[0020] The present invention includes and provides a method of
investigating an Rhg4 haplotype of a soybean plant comprising: (A)
isolating nucleic acid molecules from the soybean plant; (B)
determining the nucleic acid sequence of an Rhg4 allele or part
thereof; and (C) comparing the nucleic acid sequence of the Rhg4
allele or part thereof to a reference nucleic acid sequence.
[0021] The present invention includes and provides a method of
introgressing SCN resistance or partial SCN resistance into a
soybean plant comprising: performing marker assisted selection of
the soybean plant with a nucleic acid marker, wherein the nucleic
acid marker specifically hybridizes with a nucleic acid molecule
having a first nucleic acid sequence that is physically linked to a
second nucleic acid sequence that is located on linkage group A2 of
soybean A3244, wherein the second nucleic acid sequence is within
500 kb of a third nucleic acid sequence which specifically
hybridizes with the nucleic acid sequence of SEQ ID NO: 7,
complements thereof, or fragments thereof having at least 15
nucleotides; and, selecting the soybean plant based on the marker
assisted selection.
[0022] The present invention includes and provides a substantially
purified nucleic acid molecule comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 5, 6, 8-23,
28-43, complements thereof, and fragments of either.
[0023] The present invention includes and provides a substantially
purified first nucleic acid molecule with nucleic acid sequence
which specifically hybridizes to a second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NOs: 5, 6, 8-23, 28-43.
[0024] The present invention includes and provides a substantially
purified nucleic acid molecule comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 7, 44-47, and
50-53, complements thereof, and fragments of either.
[0025] The present invention includes and provides a substantially
purified first nucleic acid molecule with nucleic acid sequence
which specifically hybridizes to a second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NOs: 50-53.
[0026] The present invention includes and provides a substantially
purified protein or fragment thereof comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1097,
1098, and 1100-1115 and fragments thereof.
[0027] The present invention includes and provides a substantially
purified protein or fragment thereof comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs 1099, and
1116-1119 and fragments thereof.
[0028] The present invention includes and provides a transformed
plant having a nucleic acid molecule which comprises: (A) an
exogenous promoter region which functions in a plant cell to cause
the production of a mRNA molecule; (B) a structural nucleic acid
molecule encoding a protein or fragment thereof comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1097, 1100, 1098, 1101, 1102-1115; and (C) a 3' non-translated
sequence that functions in the plant cell to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA molecule.
[0029] The present invention includes and provides a transformed
plant having a nucleic acid molecule which comprises: (A) an
exogenous promoter region which functions in a plant cell to cause
the production of a mRNA molecule; (B) a structural nucleic acid
molecule encoding a protein or fragment thereof comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1099, 1116-1119; and (C) a 3' non-translated sequence that
functions in the plant cell to cause termination of transcription
and addition of polyadenylated ribonucleotides to a 3' end of the
mRNA molecule.
[0030] The present invention includes and provides a transgenic
seed having a nucleic acid molecule which comprises: (A) an
exogenous promoter region which functions to cause the production
of a mRNA molecule; (B) a structural nucleic acid molecule encoding
a protein or fragment thereof comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1097, 1100, 1098,
1101, 1102-1115; and (C) a 3' non-translated sequence that
functions to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
[0031] The present invention includes and provides a transgenic
seed having a nucleic acid molecule which comprises: (A) an
exogenous promoter region which functions to cause the production
of a mRNA molecule; (B) a structural nucleic acid molecule encoding
a protein or fragment thereof comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1099, 1116-1119;
and (C) a 3' non-translated sequence that functions to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
DESCRIPTION OF THE FIGURES
[0032] FIG. 1 is an amino acid sequence alignment of the leucine
rich repeat domain of rhg1.
[0033] FIG. 2 is an amino acid sequence alignment of the leucine
rich repeat domain of Rhg4.
DESCRIPTION OF THE SEQUENCE LISTINGS
[0034] The following sequence listings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these sequences in
combination with the detailed description presented herein.
[0035] SEQ ID NOs: 1-7 and 1097-1099 all refer to sequences from
the line A3244.
[0036] SEQ ID NO: 1 is sequence ID 515002_region_G2 from line
A3244, and is adjacent to the contig containing rhg1.
[0037] SEQ ID NO: 2 is sequence ID 240017_region_G3 from line
A3244, and contains the rhg1, v.1 four exon gene at coding
coordinates 45163-45314, 45450-45509, 46941-48763, 48975-49573. The
amino acid translation for SEQ ID NO: 2 is SEQ ID NO: 1097.
[0038] SEQ ID NO: 3 is sequence ID 240017_region_G3 from line
A3244, and contains the rhg1, v.2 two exon gene at coding
coordinates 46798-48763 and 48975-49573. The amino acid translation
for SEQ ID NO: 3 is SEQ ID NO: 1098.
[0039] SEQ ID NO: 4 is sequence ID 318013_region_A3 from line
A3244, contains the Rhg4 gene at coding coordinates 111805-113968
and 114684-115204, and has an amino acid translation of SEQ ID NO:
1099.
[0040] SEQ ID NO: 5 is sequence ID 240017_region_G3.sub.--8_mRNA,
and comprises the two rhg1, v.2 exons from the coding sequence
portion of SEQ ID NO: 3.
[0041] SEQ ID NO: 6 is sequence ID 240017_region_G3.sub.--8_cds,
and comprises the four rhg1, v.1 exons from the coding sequence
portion of SEQ ID NO: 2.
[0042] SEQ ID NO: 7 is sequence ID 318013_region_A3.sub.--17_cds,
and comprises the Rhg4 coding sequence portion from SEQ ID NO:
4.
[0043] SEQ ID NOs: 8-43 and 1100-1115 all refer to rhg1
sequences.
[0044] SEQ ID NO: 8 is sequence ID rhg1_A3244_amplicon from line
A3244, contains four rhg1, v.1 exons at coding coordinates 113-264,
400-459, 1891-3713, and 3925-4523, and has an amino acid
translation of SEQ ID NO: 1100 and 1097.
[0045] SEQ ID NO: 9 is sequence ID rhg1_A3244_amplicon, contains
two rhg1, v.2 exons at coding coordinates 1748-3713 and 3925-4523
and has an amino acid translation of SEQ ID NO: 1101 and 1098.
[0046] SEQ ID NO: 10 is sequence ID rhg1_peking_amplicon from the
line peking, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1888-3710, and 3903-4501, and has an amino acid
translation of SEQ ID NO: 1102.
[0047] SEQ ID NO: 11 is sequence ID rhg1_peking_amplicon, contains
two rhg1, v.2 exons at coding coordinates 1745-3710 and 3903-4501,
and has an amino acid translation of SEQ ID NO: 1103.
[0048] SEQ ID NO: 12 is sequence ID rhg1_toyosuzu_amplicon from the
line toyosuzu, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1890-3712, and 3924-4522, and has an amino acid
translation of SEQ ID NO: 1104.
[0049] SEQ ID NO: 13 is sequence ID rhg1_toyosuzu_amplicon,
contains two rhg1, v.2 exons at coding coordinates 1747-3712 and
3924-4522, and has an amino acid translation of SEQ ID NO:
1105.
[0050] SEQ ID NO: 14 is sequence ID rhg1_will_amplicon from the
line will, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1891-3713, and 3925-4523, and has an amino acid
translation of SEQ ID NO: 1106.
[0051] SEQ ID NO: 15 is sequence ID rhg1_will_amplicon, contains
two rhg1, v.2 exons at coding coordinates 1748-3713 and 3925-4523,
and has an amino acid translation of SEQ ID NO: 1107.
[0052] SEQ ID NO: 16 is sequence ID rhg_a2704_amplicon from the
line A2704, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1891-3713, and 3925-4523, and has an amino acid
translation of SEQ ID NO: 1108.
[0053] SEQ ID NO: 17 is sequence ID rhg1_a2704_amplicon, contains
two rhg1, v.2 exons at coding coordinates 1748-3713 and 3925-4523,
and has an amino acid translation of SEQ ID NO: 1109.
[0054] SEQ ID NO: 18 is sequence ID rhg1_noir_amplicon from the
line noir, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1876-3698, and 3910-4508, and has an amino acid
translation of SEQ ID NO: 1110.
[0055] SEQ ID NO: 19 is sequence ID rhg1_noir_amplicon, contains
two rhg1, v.2 exons at coding coordinates 1733-3698 and 3910-4508,
and has an amino acid translation of SEQ ID NO: 1111.
[0056] SEQ ID NO: 20 is sequence ID rhg1-lee-amplicon from the line
lee, contains four rhg1, v.1 exons at coding coordinates 113-264,
400-459, 1876-3698, and 3910-4508, and has an amino acid
translation of SEQ ID NO: 1112.
[0057] SEQ ID NO: 21 is sequence ID rhg1_lee_amplicon, contains two
rhg1, v.2 exons at coding coordinates 1733-3698 and 3910-4508, and
has an amino acid translation of SEQ ID NO: 1113.
[0058] SEQ ID NO: 22 is sequence ID rhg_pi200499_amplicon from the
line P1200499, contains four rhg1, v.1 exons at coding coordinates
113-264, 400-459, 1876-3698, and 3910-4508, and has an amino acid
translation of SEQ ID NO: 1114.
[0059] SEQ ID NO: 23 is sequence ID rhg1_pi200499_amplicon,
contains two rhg1, v.2 exons at coding coordinates 1733-3698 and
3910-4508, and has an amino acid translation of SEQ ID NO:
1115.
[0060] SEQ ID NO: 24 is sequence ID
240017_region_G3_forward.sub.--1, is a primer that hybridizes to
coordinates 45051-45077 on contig 240017_region_G3 before the start
codon, and can be used with SEQ ID NO: 25.
[0061] SEQ ID NO: 25 is sequence ID
240017_region_G3_reverse.sub.--1, is a primer that hybridizes to
coordinates 47942-47918 on contig 240017_region_G3, and can be used
with SEQ ID NO: 24.
[0062] SEQ ID NO: 26 is sequence ID
240017_region_G3_forward.sub.--2, is a primer that hybridizes to
coordinates 47808-47831 on contig 240017_region_G3, and can be used
with SEQ ID NO: 27.
[0063] SEQ ID NO: 27 is sequence ID
240017_region_G3_reverse.sub.--2, is a primer that hybridizes to
coordinates 49553-49531 of contig 240017-region-G3 prior to the
stop codon, and can be used with SEQ ID NO: 26.
[0064] Primers given by SEQ ID NOs: 24-27 are used to create the
amplicons of SEQ ID NOs: 8-23. The final 22 bases are added to the
actual amplicons in order to simulate the rest of the gene to the
stop codon, in order to allow complete translation.
[0065] SEQ ID NO: 28 is sequence ID rhg1_A3244_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 8.
[0066] SEQ ID NO: 29 is sequence ID rhg1_peking_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 10.
[0067] SEQ ID NO: 30 is sequence ID rhg1_toyosuzu_amplicon_cds,
which is the coding sequence portion of SEQ ID NO: 12.
[0068] SEQ ID NO: 31 is sequence ID rhg1_will_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 14.
[0069] SEQ ID NO: 32 is sequence ID rhg1_a2704_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 16.
[0070] SEQ ID NO: 33 is sequence ID rhg1_noir_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 18.
[0071] SEQ ID NO: 34 is sequence ID rhg1_lee_amplicon_cds, which is
the coding sequence portion of SEQ ID NO: 20.
[0072] SEQ ID NO: 35 is sequence ID rhg1_pi200499_amplicon_cds,
which is the coding sequence portion of SEQ ID NO: 22.
[0073] SEQ ID NO: 36 is sequence ID
rhg1_A3244_amplicon_cds.sub.--2, which is the coding sequence
portion of SEQ ID NO: 9.
[0074] SEQ ID NO: 37 is sequence ID
rhg1_peking_amplicon_cds.sub.--2, which is the coding sequence
portion of SEQ ID NO: 11.
[0075] SEQ ID NO: 38 is sequence ID
rhg1_toyosuzu_amplicon_cds.sub.--2, which is the coding sequence
portion of SEQ ID NO: 13.
[0076] SEQ ID NO: 39 is sequence ID rhg1_will_amplicon_cds.sub.--2,
which is the coding sequence portion of SEQ ID NO: 15.
[0077] SEQ ID NO: 40 is sequence ID
rhg1_a2704_amplicon_cds.sub.--2, which is the coding sequence
portion of SEQ ID NO: 17.
[0078] SEQ ID NO: 41 is sequence ID rhg1_noir_amplicon_cds.sub.--2,
which is the coding sequence portion of SEQ ID NO: 19.
[0079] SEQ ID NO: 42 is sequence ID rhg1_lee_amplicon_cds.sub.--2,
which is the coding sequence portion of SEQ ID NO: 21.
[0080] SEQ ID NO: 43 is sequence ID
rhg1_pi200499_amplicon_cds.sub.--2, which is the coding sequence
portion of SEQ ID NO: 23.
[0081] SEQ ID NOs: 44-53 and 1116-1119 all refer to Rhg4
sequences.
[0082] SEQ ID NO: 44 is sequence ID rhg4_a3244_amplicon from the
line A3244, contains Rhg4 at coding coordinates 79-2242 and
2958-3478, is made using SEQ ID NOs: 48 and 49, and has an amino
acid translation of SEQ ID NO: 1116 and 1099.
[0083] SEQ ID NO: 45 is sequence ID rhg4_Minsoy_amplicon from the
line Minsoy, contains Rhg4 at coding coordinates 79-2242 and
2958-3478, is made using SEQ ID NOs: 48 and 49, and has an amino
acid translation of SEQ ID NO: 1117.
[0084] SEQ ID NO: 46 is sequence ID rhg4_Jack_amplicon from the
line Jack, contains Rhg4 at coding coordinates 79-2242 and
2958-3478, is made using SEQ ID NO: 48 and 49, and has an amino
acid translation of SEQ ID NO: 1118.
[0085] SEQ ID NO: 47 is sequence ID rhg4_peking_amplicon from the
line Peking, contains Rhg4 at coding coordinates 79-2242 and
2958-3478, is made using SEQ ID NOs: 48 and 49, and has an amino
acid translation of SEQ ID NO: 1119.
[0086] SEQ ID NO: 48 is sequence ID 318013_region_A3_forward,
hybridizes to coordinates 111727-111756 of contig 318013_region_A3,
and is a primer used with SEQ ID NO: 49 to create Rhg4
amplicons.
[0087] SEQ ID NO: 49 is sequence ID 318013_region_A3_reverse,
hybridizes to coordinates 115206-115177 of contig 318013_region_A3,
and is a primer used with SEQ ID NO: 48 to create Rhg4
amplicons.
[0088] SEQ ID NO: 50 is sequence ID rhg4_A3244_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 44.
[0089] SEQ ID NO: 51 is sequence ID rhg4_Minsoy_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 45.
[0090] SEQ ID NO: 52 is sequence ID rhg4_Jack_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 46.
[0091] SEQ ID NO: 53 is sequence ID rhg4_peking_amplicon_cds, which
is the coding sequence portion of SEQ ID NO: 47.
[0092] SEQ ID NO: 1120 is sequence ID consensusLRR, which is a
consensus sequence for the LRR repeats shown in FIGS. 1 and 2.
[0093] SEQ ID NO: 1121 is sequence ID rhg1LRR, which is the amino
acid sequence of the LRR domain shown in FIG. 1.
[0094] SEQ ID NO: 1122 is sequence ID Rhg4LRR, which is the amino
acid sequence of the LRR domain shown in FIG. 2.
[0095] SEQ ID NO: 1123 is sequence ID
240017_region_G3_forward.sub.--1_b, which is an alternate primer
that hybridizes to coordinates 45046-45072 on contig
240017_region_G3 before the start codon, and which can be used with
SEQ ID NO: 25.
[0096] Table 1 below provides further information on the sequences
described herein.
[0097] In table 1, for all rows, "Seq Num" refers to the
corresponding SEQ ID NO in the sequence listing.
[0098] For rows with SEQ ID NOs: 1-53 and 1120-1123 "Seq ID" refers
to the name of the SEQ ID NO given in the "Seq Num" column.
[0099] For rows with SEQ ID NOs: 2-4, 8-23, and 44-47 "Coding
Sequence" refers to the coordinates of the coding portion of the
SEQ ID NO given in the "Seq Num" column, and "AA" refers to the SEQ
ID NO that is the amino acid translation of the SEQ ID NO given in
the "Seq Num" column.
[0100] For rows with SEQ ID NOs: 24-27 and 1123, "Primer location
on 240017_region_G3" refers to the coordinates of the
240017_region_G3 contig to which the SEQ ID NO given in the "Seq
Num" column hybridizes.
[0101] For rows with SEQ ID NOs: 48 and 49, "Primer location on
318013_region_A3" refers to the coordinates of the 318013_region_A3
contig to which the SEQ ID NO given in the "Seq Num" column
hybridizes.
[0102] For rows with SEQ ID NOs: 54-400, "Seq ID" refers to the
names of amplicon sequences. Within the Seq ID is the "_" (double
length underscore) symbol. The name before this symbol refers to
the name of the contig in which the amplicon is found, and the
numbers after this symbol refer to the nucleotide location of the
SSR on the contig.
[0103] For rows with SEQ ID NOs: 401-1096, "Seq ID" refers to the
names of primer sequences used in PCR to generate the amplicon
sequences in table 1. For these rows, the "Seq ID" name contains
the same name as the amplicon that is generated by the pair of
primers of which the SEQ ID NO referred to in the first column is a
member. The "Seq ID" name also contains either "Forward" or
"Reverse," which indicates the orientation of the primer. For these
sequences, "location of primer on contig start" and "location of
primer on contig end" refer, respectively, to the first and last
base number of the contig on which the primer aligns.
TABLE-US-00001 TABLE 1 Seq Num Seq ID 1 515O02_region_G2 Coding
Sequence AA No. 2 240O17_region_G3 45163-45314, 45450-45509,
46941-48763, 1097 48975-49573 3 240O17_region_G3 46798-48763,
48975-49573 1098 4 318O13_region_A3 111805-113968, 114684-115204
1099 5 240O17_region_G3_8_mRNA 6 240O17_region_G3_8_cds 7
318O13_region_A3_17_cds Coding Sequence AA No. 8
rhg1_A3244_amplicon 113-264, 400-459, 1891-3713, 3925-4523 1100 9
rhg1_A3244_amplicon 1748-3713, 3925-4523 1101 10
rhg1_peking_amplicon 113-264, 400-459, 1888-3710, 3903-4501 1102 11
rhg1_peking_amplicon 1745-3710, 3903-4501 1103 12
rhg1_toyosuzu_amplicon 113-264, 400-459, 1890-3712, 3924-4522 1104
13 rhg1_toyosuzu_amplicon 1747-3712, 3924-4522 1105 14
rhg1_will_amplicon 113-264, 400-459, 1891-3713, 3925-4523 1106 15
rhg1_will_amplicon 1748-3713, 3925-4523 1107 16 rhg1_a2704_amplicon
113-264, 400-459, 1891-3713, 3925-4523 1108 17 rhg1_a2704_amplicon
1748-3713, 3925-4523 1109 18 rhg1_noir_amplicon 113-264, 400-459,
1876-3698, 3910-4508 1110 19 rhg1_noir_amplicon 1733-3698,
3910-4508 1111 20 rhg1_lee_amplicon 113-264, 400-459, 1876-3698,
3910-4508 1112 21 rhg1_lee_amplicon 1733-3698, 3910-4508 1113 22
rhg1_pi200499_amplicon 113-264, 400-459, 1876-3698, 3910-4508 1114
23 rhg1_pi200499_amplicon 1733-3698, 3910-4508 1115 Primer location
on 240O17_region_G3 24 240O17_region_G3_forward_1 45051-45077 25
240O17_region_G3_reverse_1 47942-47918 26
240O17_region_G3_forward_2 47808-47831 27
240O17_region_G3_reverse_2 49553-49531 28 rhg1_A3244_amplicon_cds
29 rhg1_peking_amplicon_cds 30 rhg1_toyosuzu_amplicon_cds 31
rhg1_will_amplicon_cds 32 rhg1_a2704_amplicon_cds 33
rhg1_noir_amplicon_cds 34 rhg1_lee_amplicon_cds 35
rhg1_pi200499_amplicon_cds 36 rhg1_A3244_amplicon_cds_2 37
rhg1_peking_amplicon_cds_2 38 rhg1_toyosuzu_amplicon_cds_2 39
rhg1_will_amplicon_cds_2 40 rhg1_a2704_amplicon_cds_2 41
rhg1_noir_amplicon_cds_2 42 rhg1_lee_amplicon_cds_2 43
rhg1_pi200499_amplicon_cds_2 Coding Sequence AA No. 44
rhg4_a3244_amplicon 79-2242, 2958-3478 1116 45 rhg4_Minsoy_amplicon
79-2242, 2958-3478 1117 46 rhg4_Jack_amplicon 79-2242, 2958-3478
1118 47 rhg4_peking_amplicon 79-2242, 2958-3478 1119 Primer
location on 318O13_region_A3 48 318O13_region_A3_forward
111727-111756 49 318O13_region_A3_reverse 115206-115177 50
rhg4_A3244_amplicon_cds 51 rhg4_Minsoy_amplicon_cds 52
rhg4_Jack_amplicon_cds 53 rhg4_peking_amplicon_cds 54
240O17_region_G3_289711_11 55 240O17_region_G3_236585_14 56
240O17_region_G3_168772_13 57 240O17_region_G3_332420_21 58
240O17_region_G3_228126_18 59 240O17_region_G3_139723_11 60
240O17_region_G3_280585_14 61 240O17_region_G3_70509_14 62
240O17_region_G3_50537_17 63 240O17_region_G3_231556_17 64
240O17_region_G3_117057_11 65 240O17_region_G3_23092_13 66
240O17_region_G3_297741_14 67 240O17_region_G3_206502_14 68
240O17_region_G3_221223_13 69 240O17_region_G3_169084_14 70
240O17_region_G3_94891_14 71 240O17_region_G3_281852_61 72
240O17_region_G3_46583_12 73 240O17_region_G3_306835_13 74
240O17_region_G3_85471_12 75 240O17_region_G3_257208_12 76
240O17_region_G3_150390_17 77 240O17_region_G3_34697_75 78
240O17_region_G3_150374_13 79 240O17_region_G3_40513_22 80
240O17_region_G3_268602_14 81 240O17_region_G3_25357_13 82
240O17_region_G3_137548_13 83 240O17_region_G3_139131_13 84
240O17_region_G3_203855_12 85 240O17_region_G3_199049_15 86
240O17_region_G3_320907_12 87 240O17_region_G3_16407_17 88
240O17_region_G3_206516_17 89 240O17_region_G3_264495_13 90
240O17_region_G3_156785_13 91 240O17_region_G3_187129_12 92
240O17_region_G3_214106_13 93 240O17_region_G3_149013_12 94
240O17_region_G3_326352_16 95 240O17_region_G3_278962_12 96
240O17_region_G3_256930_13 97 240O17_region_G3_29646_14 98
240O17_region_G3_29618_13 99 240O17_region_G3_108561_14 100
240O17_region_G3_143975_14 101 240O17_region_G3_108431_20 102
240O17_region_G3_281764_11 103 240O17_region_G3_130058_15 104
240O17_region_G3_310590_52 105 240O17_region_G3_313405_14 106
240O17_region_G3_302190_13 107 240O17_region_G3_225343_17 108
240O17_region_G3_208823_14 109 240O17_region_G3_74285_11 110
240O17_region_G3_109052_16 111 240O17_region_G3_6395_12 112
240O17_region_G3_244905_16 113 240O17_region_G3_244956_13 114
240O17_region_G3_117220_13 115 240O17_region_G3_134707_14 116
240O17_region_G3_35078_13 117 240O17_region_G3_210506_16 118
240O17_region_G3_116961_26 119 240O17_region_G3_51073_13 120
240O17_region_G3_55291_15 121 240O17_region_G3_229651_18 122
240O17_region_G3_303308_19 123 240O17_region_G3_168373_20 124
240O17_region_G3_253333_17 125 240O17_region_G3_5791_13 126
240O17_region_G3_206841_19 127 240O17_region_G3_202827_12 128
240O17_region_G3_322656_13 129 240O17_region_G3_111841_14 130
240O17_region_G3_192719_13 131 240O17_region_G3_195630_17 132
240O17_region_G3_69999_13 133 240O17_region_G3_11176_13 134
240O17_region_G3_228643_13 135 240O17_region_G3_88478_19 136
240O17_region_G3_108950_13 137 240O17_region_G3_121054_14 138
240O17_region_G3_188337_14 139 240O17_region_G3_255944_21 140
240O17_region_G3_219518_14 141 240O17_region_G3_235601_15 142
240O17_region_G3_301529_13 143 240O17_region_G3_94795_14 144
240O17_region_G3_46703_23 145 240O17_region_G3_59616_14 146
240O17_region_G3_296933_15 147 240O17_region_G3_192428_17 148
240O17_region_G3_191490_14 149 240O17_region_G3_201115_11 150
240O17_region_G3_72882_15 151 240O17_region_G3_69514_13 152
240O17_region_G3_37699_47 153 240O17_region_G3_11301_29 154
240O17_region_G3_141875_12 155 240O17_region_G3_98090_18 156
240O17_region_G3_43298_35 157 240O17_region_G3_262094_11 158
240O17_region_G3_262079_15 159 240O17_region_G3_59090_12 160
240O17_region_G3_245723_13 161 240O17_region_G3_194628_54 162
240O17_region_G3_4566_16 163 240O17_region_G3_96209_14 164
240O17_region_G3_248715_17 165 240O17_region_G3_71410_40 166
240O17_region_G3_226519_13 167 240O17_region_G3_11282_19 168
240O17_region_G3_170504_12 169 240O17_region_G3_40864_14 170
240O17_region_G3_13529_14 171 240O17_region_G3_22858_14 172
240O17_region_G3_309211_13 173 240O17_region_G3_55568_26 174
240O17_region_G3_73238_16 175 240O17_region_G3_52488_19 176
318O13_region_A3_471518_14 177 318O13_region_A3_231599_23 178
318O13_region_A3_375912_13 179 318O13_region_A3_180013_12 180
318O13_region_A3_171606_14 181 318O13_region_A3_416256_13 182
318O13_region_A3_231395_15 183 318O13_region_A3_5502_47 184
318O13_region_A3_93061_14 185 318O13_region_A3_111684_19 186
318O13_region_A3_69328_14 187 318O13_region_A3_36529_17 188
318O13_region_A3_139128_12 189 318O13_region_A3_495674_13 190
318O13_region_A3_187577_13 191 318O13_region_A3_453036_14 192
318O13_region_A3_374041_13 193 318O13_region_A3_3412_11 194
318O13_region_A3_276495_28 195 318O13_region_A3_151839_17 196
318O13_region_A3_292912_12 197 318O13_region_A3_104560_12 198
318O13_region_A3_65193_11 199 318O13_region_A3_110573_70 200
318O13_region_A3_65117_12 201 318O13_region_A3_490837_16 202
318O13_region_A3_107448_11 203 318O13_region_A3_331_23 204
318O13_region_A3_193470_13 205 318O13_region_A3_183305_14 206
318O13_region_A3_55050_14 207 318O13_region_A3_224693_21 208
318O13_region_A3_207216_12 209 318O13_region_A3_4654_22 210
318O13_region_A3_408959_13 211 318O13_region_A3_132288_22 212
318O13_region_A3_292822_20 213 318O13_region_A3_311076_12 214
318O13_region_A3_509623_13 215 318O13_region_A3_190404_14 216
318O13_region_A3_164916_15 217 318O13_region_A3_21028_13 218
318O13_region_A3_208012_17 219 318O13_region_A3_484089_14 220
318O13_region_A3_332780_17 221 318O13_region_A3_480137_37 222
318O13_region_A3_441056_14 223 318O13_region_A3_77486_11 224
318O13_region_A3_272468_11 225 318O13_region_A3_425319_17 226
318O13_region_A3_413879_31 227 318O13_region_A3_80477_64 228
318O13_region_A3_277272_50 229 318O13_region_A3_509642_13
230 318O13_region_A3_321771_14 231 318O13_region_A3_26788_12 232
318O13_region_A3_262706_16 233 318O13_region_A3_243928_16 234
318O13_region_A3_23246_14 235 318O13_region_A3_165406_12 236
318O13_region_A3_486294_14 237 318O13_region_A3_46754_12 238
318O13_region_A3_381116_15 239 318O13_region_A3_350369_11 240
318O13_region_A3_138841_13 241 318O13_region_A3_12158_14 242
318O13_region_A3_315368_13 243 318O13_region_A3_307549_13 244
318O13_region_A3_159857_14 245 318O13_region_A3_140551_15 246
318O13_region_A3_279869_11 247 318O13_region_A3_78292_35 248
318O13_region_A3_185019_12 249 318O13_region_A3_409164_13 250
318O13_region_A3_75392_14 251 318O13_region_A3_231320_12 252
318O13_region_A3_381102_14 253 318O13_region_A3_491826_15 254
318O13_region_A3_56365_21 255 318O13_region_A3_372628_15 256
318O13_region_A3_302609_11 257 318O13_region_A3_341804_11 258
318O13_region_A3_217037_11 259 318O13_region_A3_264929_68 260
318O13_region_A3_55499_12 261 318O13_region_A3_295634_14 262
318O13_region_A3_269358_15 263 318O13_region_A3_457009_24 264
318O13_region_A3_176598_14 265 318O13_region_A3_278266_12 266
318O13_region_A3_391810_12 267 318O13_region_A3_269485_15 268
318O13_region_A3_359247_17 269 318O13_region_A3_315094_13 270
318O13_region_A3_307823_13 271 318O13_region_A3_248588_15 272
318O13_region_A3_252426_85 273 318O13_region_A3_513314_16 274
318O13_region_A3_68183_14 275 318O13_region_A3_471191_13 276
318O13_region_A3_163547_18 277 318O13_region_A3_417867_15 278
318O13_region_A3_332465_14 279 318O13_region_A3_207697_14 280
318O13_region_A3_277229_43 281 318O13_region_A3_36366_11 282
318O13_region_A3_91970_12 283 318O13_region_A3_211533_11 284
318O13_region_A3_336301_11 285 318O13_region_A3_441603_14 286
318O13_region_A3_468354_15 287 318O13_region_A3_188983_18 288
318O13_region_A3_115502_17 289 318O13_region_A3_163006_13 290
318O13_region_A3_119283_14 291 318O13_region_A3_491126_11 292
318O13_region_A3_99512_21 293 318O13_region_A3_280291_17 294
318O13_region_A3_138443_19 295 318O13_region_A3_115973_14 296
318O13_region_A3_329977_14 297 318O13_region_A3_205203_14 298
318O13_region_A3_153114_12 299 318O13_region_A3_34581_13 300
318O13_region_A3_292577_19 301 318O13_region_A3_445391_20 302
318O13_region_A3_350540_17 303 318O13_region_A3_453879_15 304
318O13_region_A3_201246_13 305 318O13_region_A3_326020_13 306
318O13_region_A3_503801_14 307 318O13_region_A3_302400_52 308
318O13_region_A3_448857_15 309 318O13_region_A3_48364_14 310
318O13_region_A3_251804_48 311 318O13_region_A3_382583_13 312
318O13_region_A3_124737_14 313 318O13_region_A3_124766_13 314
318O13_region_A3_461351_16 315 318O13_region_A3_64953_19 316
318O13_region_A3_366586_13 317 318O13_region_A3_46190_15 318
318O13_region_A3_81016_11 319 318O13_region_A3_134426_14 320
318O13_region_A3_292724_14 321 318O13_region_A3_187096_17 322
318O13_region_A3_381693_13 323 318O13_region_A3_361286_33 324
318O13_region_A3_482668_14 325 318O13_region_A3_128002_12 326
318O13_region_A3_499270_14 327 318O13_region_A3_231650_12 328
318O13_region_A3_199851_13 329 318O13_region_A3_324629_13 330
318O13_region_A3_374190_19 331 318O13_region_A3_460603_13 332
318O13_region_A3_108681_14 333 318O13_region_A3_459791_47 334
318O13_region_A3_4257_20 335 318O13_region_A3_238810_14 336
318O13_region_A3_245817_14 337 318O13_region_A3_245956_14 338
318O13_region_A3_74148_14 339 318O13_region_A3_74089_15 340
318O13_region_A3_241686_12 341 318O13_region_A3_47476_12 342
318O13_region_A3_164550_12 343 318O13_region_A3_101255_15 344
515O02_region_G2_16189_11 345 515O02_region_G2_71925_13 346
515O02_region_G2_4707_12 347 515O02_region_G2_118904_18 348
515O02_region_G2_13655_17 349 515O02_region_G2_53900_13 350
515O02_region_G2_8079_14 351 515O02_region_G2_9969_28 352
515O02_region_G2_72308_77 353 515O02_region_G2_99475_19 354
515O02_region_G2_118615_18 355 515O02_region_G2_119001_46 356
515O02_region_G2_118958_43 357 515O02_region_G2_17197_13 358
515O02_region_G2_105163_29 359 515O02_region_G2_111335_13 360
515O02_region_G2_106396_13 361 515O02_region_G2_59229_17 362
515O02_region_G2_73795_20 363 515O02_region_G2_85664_20 364
515O02_region_G2_36921_17 365 515O02_region_G2_124150_19 366
515O02_region_G2_5089_14 367 515O02_region_G2_58221_15 368
515O02_region_G2_96139_14 369 515O02_region_G2_70595_13 370
515O02_region_G2_4340_15 371 515O02_region_G2_90417_11 372
515O02_region_G2_49711_17 373 515O02_region_G2_63053_13 374
515O02_region_G2_63076_14 375 515O02_region_G2_44442_12 376
515O02_region_G2_44422_19 377 515O02_region_G2_44158_19 378
515O02_region_G2_44141_17 379 515O02_region_G2_90762_17 380
515O02_region_G2_106241_14 381 515O02_region_G2_109676_12 382
515O02_region_G2_86242_14 383 515O02_region_G2_83109_12 384
515O02_region_G2_10461_15 385 515O02_region_G2_67608_15 386
515O02_region_G2_63275_46 387 515O02_region_G2_62405_14 388
515O02_region_G2_33563_12 389 515O02_region_G2_33146_14 390
515O02_region_G2_102179_29 391 515O02_region_G2_2646_15 392
515O02_region_G2_76652_24 393 515O02_region_G2_66280_14 394
515O02_region_G2_54768_13 395 515O02_region_G2_62580_14 396
515O02_region_G2_34598_55 397 515O02_region_G2_77680_13 398
515O02_region_G2_77693_12 399 515O02_region_G2_97392_14 400
515O02_region_G2_97359_15 location of primer location of primer on
contig start on contig end 401
240O17_region_G3_289711_11_Forward_Primer 289637 289661 402
240O17_region_G3_289711_11_Reverse_Primer 289756 289732 403
240O17_region_G3_236585_14_Forward_Primer 236511 236535 404
240O17_region_G3_236585_14_Reverse_Primer 236638 236614 405
240O17_region_G3_168772_13_Forward_Primer 168683 168707 406
240O17_region_G3_168772_13_Reverse_Primer 168811 168786 407
240O17_region_G3_332420_21_Forward_Primer 332375 332399 408
240O17_region_G3_332420_21_Reverse_Primer 332505 332481 409
240O17_region_G3_228126_18_Forward_Primer 228048 228072 410
240O17_region_G3_228126_18_Reverse_Primer 228182 228158 411
240O17_region_G3_139723_11_Forward_Primer 139666 139690 412
240O17_region_G3_139723_11_Reverse_Primer 139802 139778 413
240O17_region_G3_280585_14_Forward_Primer 280524 280550 414
240O17_region_G3_280585_14_Reverse_Primer 280661 280637 415
240O17_region_G3_70509_14_Forward_Primer 70478 70502 416
240O17_region_G3_70509_14_Reverse_Primer 70616 70592 417
240O17_region_G3_50537_17_Forward_Primer 50455 50479 418
240O17_region_G3_50537_17_Reverse_Primer 50593 50569 419
240O17_region_G3_231556_17_Forward_Primer 231468 231492 420
240O17_region_G3_231556_17_Reverse_Primer 231606 231582 421
240O17_region_G3_117057_11_Forward_Primer 117029 117053 422
240O17_region_G3_117057_11_Reverse_Primer 117169 117145 423
240O17_region_G3_23092_13_Forward_Primer 23010 23034 424
240O17_region_G3_23092_13_Reverse_Primer 23151 23127 425
240O17_region_G3_297741_14_Forward_Primer 297680 297704 426
240O17_region_G3_297741_14_Reverse_Primer 297823 297799 427
240O17_region_G3_206502_14_Forward_Primer 206456 206480 428
240O17_region_G3_206502_14_Reverse_Primer 206600 206581 429
240O17_region_G3_221223_13_Forward_Primer 221134 221158 430
240O17_region_G3_221223_13_Reverse_Primer 221278 221254 431
240O17_region_G3_169084_14_Forward_Primer 169051 169075 432
240O17_region_G3_169084_14_Reverse_Primer 169196 169173 433
240O17_region_G3_94891_14_Forward_Primer 94784 94808 434
240O17_region_G3_94891_14_Reverse_Primer 94929 94905 435
240O17_region_G3_7439_12_Forward_Primer 7397 7421 436
240O17_region_G3_7439_12_Reverse_Primer 7542 7518 437
240O17_region_G3_281852_61_Forward_Primer 281797 281821 438
240O17_region_G3_281852_61_Reverse_Primer 281943 281919 439
240O17_region_G3_46583_12_Forward_Primer 46554 46578 440
240O17_region_G3_46583_12_Reverse_Primer 46700 46676 441
240O17_region_G3_306835_13_Forward_Primer 306727 306751 442
240O17_region_G3_306835_13_Reverse_Primer 306874 306849 443
240O17_region_G3_85471_12_Forward_Primer 85359 85383 444
240O17_region_G3_85471_12_Reverse_Primer 85507 85483 445
240O17_region_G3_257208_12_Forward_Primer 257129 257153 446
240O17_region_G3_257208_12_Reverse_Primer 257278 257254 447
240O17_region_G3_150390_17_Forward_Primer 150327 150351 448
240O17_region_G3_150390_17_Reverse_Primer 150476 150452 449
240O17_region_G3_34697_75_Forward_Primer 34662 34685 450
240O17_region_G3_34697_75_Reverse_Primer 34811 34787 451
240O17_region_G3_150374_13_Forward_Primer 150327 150351 452
240O17_region_G3_150374_13_Reverse_Primer 150476 150452 453
240O17_region_G3_40513_22_Forward_Primer 40422 40446 454
240O17_region_G3_40513_22_Reverse_Primer 40572 40548 455
240O17_region_G3_268602_14_Forward_Primer 268555 268579 456
240O17_region_G3_268602_14_Reverse_Primer 268705 268681 457
240O17_region_G3_25357_13_Forward_Primer 25271 25295 458
240O17_region_G3_25357_13_Reverse_Primer 25422 25402 459
240O17_region_G3_137548_13_Forward_Primer 139088 139111 459
240O17_region_G3_137548_13_Forward_Primer 137505 137528 460
240O17_region_G3_137548_13_Reverse_Primer 139239 139215 460
240O17_region_G3_137548_13_Reverse Primer 137656 137632 461
240O17_region_G3_139131_13_Forward_Primer 139088 139111 462
240O17_region_G3_139131_13_Reverse_Primer 139239 139215 463
240O17_region_G3_203855_12_Forward_Primer 203749 203773 464
240O17_region_G3_203855_12_Reverse_Primer 203901 203877 465
240O17_region_G3_199049_15_Forward_Primer 199008 199033 466
240O17_region_G3_199049_15_Reverse_Primer 199160 199136 467
240O17_region_G3_320907_12_Forward_Primer 320885 320906 468
240O17_region_G3_320907_12_Reverse_Primer 321038 321015 469
240O17_region_G3_16407_17_Forward_Primer 16330 16354 470
240O17_region_G3_16407_17_Reverse_Primer 16483 16459 471
240O17_region_G3_206516_17_Forward_Primer 206482 206506 472
240O17_region_G3_206516_17_Reverse_Primer 206635 206616 473
240O17_region_G3_264495_13_Forward_Primer 264423 264447 474
240O17_region_G3_264495_13_Reverse_Primer 264577 264553 475
240O17_region_G3_156785_13_Forward_Primer 156713 156737 476
240O17_region_G3_156785_13_Reverse_Primer 156868 156844
477 240O17_region_G3_187129_12_Forward_Primer 187068 187092 478
240O17_region_G3_187129_12_Reverse_Primer 187223 187199 479
240O17_region_G3_214106_13_Forward_Primer 214042 214067 480
240O17_region_G3_214106_13_Reverse_Primer 214197 214173 481
240O17_region_G3_149013_12_Forward_Primer 148898 148922 482
240O17_region_G3_149013_12_Reverse_Primer 149053 149027 483
240O17_region_G3_326352_16_Forward_Primer 326311 326335 484
240O17_region_G3_326352_16_Reverse_Primer 326467 326443 485
240O17_region_G3_278962_12_Forward_Primer 278933 278957 486
240O17_region_G3_278962_12_Reverse_Primer 279089 279065 487
240O17_region_G3_256930_13_Forward_Primer 256850 256874 488
240O17_region_G3_256930_13_Reverse_Primer 257006 256982 489
240O17_region_G3_29646_14_Forward_Primer 29589 29613 490
240O17_region_G3_29646_14_Reverse_Primer 29746 29721 491
240O17_region_G3_29618_13_Forward_Primer 29589 29613 492
40O17_region_G3_29618_13_Reverse_Primer 29746 29721 493
240O17_region_G3_108561_14_Forward_Primer 108518 108542 494
240O17_region_G3_108561_14_Reverse_Primer 108675 108651 495
240O17_region_G3_143975_14_Forward_Primer 143939 143964 496
240O17_region_G3_143975_14_Reverse_Primer 144096 144072 497
240O17_region_G3_108431_20_Forward_Primer 108362 108386 498
240O17_region_G3_108431_20_Reverse_Primer 108520 108497 499
240O17_region_G3_281764_11_Forward_Primer 281645 281669 500
240O17_region_G3_281764_11_Reverse_Primer 281803 281779 501
240O17_region_G3_130058_15_Forward_Primer 129994 130018 502
240O17_region_G3_130058_15_Reverse_Primer 130153 130129 503
240O17_region_G3_310590_52_Forward_Primer 310533 310557 504
240O17_region_G3_310590_52_Reverse_Primer 310692 310668 505
240O17_region_G3_313405_14_Forward_Primer 313345 313369 506
240O17_region_G3_313405_14_Reverse_Primer 313505 313481 507
240O17_region_G3_302190_13_Forward_Primer 302093 302119 508
240O17_region_G3_302190_13_Reverse_Primer 302253 302229 509
240O17_region_G3_225343_17_Forward_Primer 225315 225338 510
240O17_region_G3_225343_17_Reverse_Primer 225475 225451 511
240O17_region_G3_208823_14_Forward_Primer 208760 208784 512
240O17_region_G3_208823_14_Reverse_Primer 208921 208897 513
240O17_region_G3_74285_11_Forward_Primer 74220 74244 514
240O17_region_G3_74285_11_Reverse_Primer 74382 74358 515
240O17_region_G3_109052_16_Forward_Primer 108999 109023 516
240O17_region_G3_109052 16_Reverse_Primer 109161 109137 517
240O17_region_G3_6395_12_Forward_Primer 6285 6309 518
240O17_region_G3_6395_12_Reverse_Primer 6447 6423 519
240O17_region_G3_244905_16_Forward_Primer 244865 244890 520
240O17_region_G3_244905_16_Reverse_Primer 245028 245004 521
240O17_region_G3_244956_13_Forward_Primer 244865 244890 522
240O17_region_G3_244956_13_Reverse_Primer 245028 245004 523
240O17_region_G3_117220_13_Forward_Primer 117175 117199 524
240O17_region_G3_117220_13_Reverse_Primer 117339 117315 525
240O17_region_G3_134707_14_Forward_Primer 134584 134608 526
240O17_region_G3_134707_14_Reverse_Primer 134749 134725 527
240O17_region_G3_35078_13_Forward_Primer 34990 35013 528
240O17_region_G3_35078_13_Reverse_Primer 35157 35133 529
240O17_region_G3_210506_16_Forward_Primer 210477 210501 530
240O17_region_G3_210506_16_Reverse_Primer 210644 210620 531
240O17_region_G3_116961_26_Forward_Primer 116885 116909 532
240O17_region_G3_116961_26_Reverse_Primer 117053 117029 533
240O17_region_G3_51073_13_Forward_Primer 50979 51003 534
240O17_region_G3_51073_13_Reverse_Primer 51147 51123 535
240O17_region_G3_55291_15_Forward_Primer 55164 55188 536
240O17_region_G3_55291_15_Reverse_Primer 55333 55309 537
240O17_region_G3_229651_18_Forward_Primer 229615 229639 538
240O17_region_G3_229651_18_Reverse_Primer 229784 229760 539
240O17_region_G3_303308_19_Forward_Primer 303284 303307 540
240O17_region_G3_303308_19_Reverse_Primer 303454 303429 541
240O17_region_G3_168373_20_Forward_Primer 168262 168286 542
240O17_region_G3_168373_20_Reverse_Primer 168432 168408 543
240O17_region_G3_253333_17_Forward_Primer 253257 253281 544
240O17_region_G3_253333_17_Reverse_Primer 253428 253404 545
240O17_region_G3_5791_13_Forward_Primer 5766 5790 546
240O17_region_G3_5791_13_Reverse_Primer 5937 5912 547
240O17_region_G3_206841_19_Forward_Primer 206821 206840 548
240O17_region_G3_206841_19_Reverse_Primer 206993 206969 549
240O17_region_G3_202827_12_Forward_Primer 202782 202806 550
240O17_region_G3_202827_12_Reverse_Primer 202956 202932 551
240O17_region_G3_322656_13_Forward_Primer 322572 322598 552
240O17_region_G3_322656_13_Reverse_Primer 322748 322724 553
240O17_region_G3_111841_14_Forward_Primer 111709 111733 554
240O17_region_G3_111841_14_Reverse_Primer 111886 111861 555
240O17_region_G3_192719_13_Forward_Primer 192589 192613 556
240O17_region_G3_192719_13_Reverse_Primer 192767 192743 557
240O17_region_G3_195630_17_Forward_Primer 195490 195514 558
240O17_region_G3_195630_17_Reverse_Primer 195672 195648 559
240O17_region_G3_69999_13_Forward_Primer 69858 69881 560
240O17_region_G3_69999_13_Reverse_Primer 70040 70016 561
240O17_region_G3_11176_13_Forward_Primer 11060 11084 562
240O17_region_G3_11176_13_Reverse_Primer 11243 11219 563
240O17_region_G3_228643_13_Forward_Primer 228529 228553 564
240O17_region_G3_228643_13_Reverse_Primer 228713 228689 565
240O17_region_G3_88478_19_Forward_Primer 88378 88402 566
240O17_region_G3_88478_19_Reverse_Primer 88562 88538 567
240O17_region_G3_108950_13_Forward_Primer 108838 108858 568
240O17_region_G3_108950_13_Reverse_Primer 109023 108998 569
240O17_region_G3_121054_14_Forward_Primer 120911 120935 570
240O17_region_G3_121054_14_Reverse_Primer 121096 121072 571
240O17_region_G3_188337_14_Forward_Primer 188204 188228 572
240O17_region_G3_188337_14_Reverse_Primer 191544 191520 572
240O17_region_G3_188337_14_Reverse_Primer 188391 188367 573
240O17_region_G3_255944_21_Forward_Primer 255879 255903 574
240O17_region_G3_255944_21_Reverse_Primer 256068 256044 575
240O17_region_G3_219518_14_Forward_Primer 219420 219444 576
240O17_region_G3_219518_14_Reverse_Primer 219609 219585 577
240O17_region_G3_235601_15_Forward_Primer 235483 235507 578
240O17_region_G3_235601_15_Reverse_Primer 235673 235649 579
240O17_region_G3_301529_13_Forward_Primer 301498 301522 580
240O17_region_G3_301529_13_Reverse_Primer 301689 301665 581
240O17_region_G3_94795_14_Forward_Primer 94735 94756 582
240O17_region_G3_94795_14_Reverse_Primer 94929 94905 583
240O17_region_G3_46703_23_Forward_Primer 46676 46700 584
240O17_region_G3_46703_23_Reverse_Primer 46870 46846 585
240O17_region_G3_59616_14_Forward_Primer 59539 59563 586
240O17_region_G3_59616_14_Reverse_Primer 59738 59714 587
240O17_region_G3_296933_15_Forward_Primer 296908 296932 588
240O17_region_G3_296933_15_Reverse_Primer 297113 297089 589
240O17_region_G3_192428_17_Forward_Primer 192402 192426 590
240O17_region_G3_192428_17_Reverse_Primer 192613 192589 591
240O17_region_G3_191490_14_Forward_Primer 191332 191356 592
240O17_region_G3_191490_14_Reverse_Primer 191544 191520 593
240O17_region_G3_201115_11_Forward_Primer 200994 201018 594
240O17_region_G3_201115_11_Reverse_Primer 201214 201189 595
240O17_region_G3_72882_15_Forward_Primer 72848 72874 596
240O17_region_G3_72882_15_Reverse_Primer 73068 73042 597
240O17_region_G3_69514_13_Forward_Primer 69411 69437 598
240O17_region_G3_69514_13_Reverse_Primer 69632 69608 599
240O17_region_G3_37699_47_Forward_Primer 37601 37625 600
240O17_region_G3_37699_47_Reverse_Primer 37827 37802 601
240O17_region_G3_11301_29_Forward_Primer 11274 11300 602
240O17_region_G3_11301_29_Reverse_Primer 11501 11477 603
240O17_region_G3_141875_12_Forward_Primer 141729 141750 604
240O17_region_G3_141875_12_Reverse_Primer 141964 141939 605
240O17_region_G3_98090_18_Forward_Primer 98037 98062 606
240O17_region_G3_98090_18_Reverse_Primer 98274 98250 607
240O17_region_G3_43298_35_Forward_Primer 43144 43168 608
240O17_region_G3_43298_35_Reverse_Primer 43387 43363 609
240O17_region_G3_262094_11_Forward_Primer 261989 262014 610
240O17_region_G3_262094_11_Reverse_Primer 262236 262211 611
240O17_region_G3_262079_15_Forward_Primer 261989 262014 612
240O17_region_G3_262079_15_Reverse_Primer 262236 262211 613
240O17_region_G3_59090_12_Forward_Primer 58986 59012 614
240O17_region_G3_59090_12_Reverse_Primer 59248 59224 615
240O17_region_G3_245723_13_Forward_Primer 245502 245526 616
240O17_region_G3_245723_13_Reverse_Primer 245766 245742 617
240O17_region_G3_194628_54_Forward_Primer 194581 194607 618
240O17_region_G3_194628_54_Reverse_Primer 194846 194822 619
240O17_region_G3_4566_16_Forward_Primer 4455 4479 620
240O17_region_G3_4566_16_Reverse_Primer 4722 4696 621
240O17_region_G3_96209_14_Forward_Primer 96119 96143 622
240O17_region_G3_96209_14_Reverse_Primer 96392 96368 623
240O17_region_G3_248715_17_Forward_Primer 248633 248657 624
240O17_region_G3_248715_17_Reverse_Primer 248906 248882 625
240O17_region_G3_71410_40_Forward_Primer 71357 71379 626
240O17_region_G3_71410_40_Reverse_Primer 71636 71611 627
240O17_region_G3_226519_13_Forward_Primer 226315 226339 628
240O17_region_G3_226519_13_Reverse_Primer 226598 226574 629
240O17_region_G3_11282_19_Forward_Primer 11217 11242 630
240O17_region_G3_11282_19_Reverse_Primer 11501 11477 631
240O17_region_G3_170504_12_Forward_Primer 170409 170433 632
240O17_region_G3_170504_12_Reverse_Primer 170694 170671 633
240O17_region_G3_40864_14_Forward_Primer 40652 40678 634
240O17_region_G3_40864_14_Reverse_Primer 40938 40912 635
240O17_region_G3_13529_14_Forward_Primer 13332 13356 636
240O17_region_G3_13529_14_Reverse_Primer 13622 13598 637
240O17_region_G3_22858_14_Forward_Primer 22675 22699 638
240O17_region_G3_22858_14_Reverse_Primer 22966 22942 639
240O17_region_G3_309211_13_Forward_Primer 309092 309118 640
240O17_region_G3_309211_13_Reverse_Primer 309383 309358 641
240O17_region_G3_55568_26_Forward_Primer 55375 55399 642
240O17_region_G3_55568_26_Reverse_Primer 55667 55642 643
240O17_region_G3_73238_16_Forward_Primer 73043 73069 644
240O17_region_G3_73238_16_Reverse_Primer 73342 73318 645
240O17_region_G3_352488_19_Forward_Primer 52413 52437 646
240O17_region_G3_52488_19_Reverse_Primer 52712 52688 647
318O13_region_A3_471518_14_Forward_Primer_Seq 471464 471488 648
318O13_region_A3_471518_14_Reverse_Primer_Seq 471567 471541 649
318O13_region_A3_231599_23_Forward_Primer_Seq 231568 231592 650
318O13_region_A3_231599_23_Reverse_Primer_Seq 231672 231651 651
318O13_region_A3_375912_13_Forward_Primer_Seq 375845 375865 652
318O13_region_A3_375912_13_Reverse_Primer_Seq 375954 375932 653
318O13_region_A3_180013_12_Forward_Primer_Seq 179951 179974 654
318O13_region_A3_180013_12_Reverse_Primer_Seq 180060 180038 655
318O13_region_A3_171606_14_Forward_Primer_Seq 171545 171569 656
318O13_region_A3_171606_14_Reverse_Primer_Seq 171657 171633 657
318O13_region_A3_416256_13_Forward_Primer_Seq 416180 416203 658
318O13_region_A3_416256_13_Reverse_Primer_Seq 416293 416269 659
318O13_region_A3_231395_15_Forward_Primer_Seq 231339 231363 660
318O13_region_A3_231395_15_Reverse_Primer_Seq 231461 231438 661
318O13_region_A3_5502_47_Forward_Primer_Seq 5461 5485 662
318O13_region_A3_5502_47_Reverse_Primer_Seq 5585 5561 663
318O13_region_A3_93061_14_Forward_Primer_Seq 92988 93012 664
318O13_region_A3_93061_14_Reverse_Primer_Seq 93112 93090 665
318O13_region_A3_111684_19_Forward_Primer_Seq 111646 111670 666
318O13_region_A3_111684_19_Reverse_Primer_Seq 111772 111748 667
318O13_region_A3_69328_14_Forward_Primer_Seq 69246 69269 668
318O13_region_A3_69328_14_Reverse_Primer_Seq 69373 69349 669
318O13_region_A3_36529_17_Forward_Primer_Seq 36488 36512 670
318O13_region_A3_36529_17_Reverse_Primer_Seq 36617 36593 671
318O13_region_A3_139128_12_Forward_Primer_Seq 139043 139067 672
318O13_region_A3_139128_12_Reverse_Primer_Seq 139174 139150 673
318O13_region_A3_495674_13_Forward_Primer_Seq 495592 495616 674
318O13_region_A3_495674_13_Reverse_Primer_Seq 495723 495699 675
318O13_region_A3_187577_13_Forward_Primer_Seq 187482 187506 676
318O13_region_A3_187577_13_Reverse_Primer_Seq 187613 187590 677
318O13_region_A3_453036_14_Forward_Primer_Seq 452999 453023 678
318O13_region_A3_453036_14_Reverse_Primer_Seq 453132 453108 679
318O13_region_A3_374041_13_Forward_Primer_Seq 373964 373988 680
318O13_region_A3_374041_13_Reverse_Primer_Seq 374097 374073 681
318O13_region_A3_3412_11_Forward_Primer_Seq 3319 3341 682
318O13_region_A3_3412_11_Reverse_Primer_Seq 3454 3430 683
318O13_region_A3_276495_28_Forward_Primer_Seq 276462 276485 684
318O13_region_A3_276495_28_Reverse_Primer_Seq 276598 276574 685
318O13_region_A3_151839_17_Forward_Primer_Seq 151744 151768 686
318O13_region_A3_151839_17_Reverse_Primer_Seq 151882 151858 687
318O13_region_A3_292912_12_Forward_Primer_Seq 292875 292899 688
318O13_region_A3_292912_12_Reverse_Primer_Seq 293014 292990 689
318O13_region_A3_104560_12_Forward_Primer_Seq 104464 104488 690
318O13_region_A3_104560_12_Reverse_Primer_Seq 104604 104580 691
318O13_region_A3_65193_11_Forward_Primer_Seq 65155 65179 692
318O13_region_A3_65193_11_Reverse_Primer_Seq 65295 65271 693
318O13_region_A3_110573_70_Forward_Primer_Seq 110533 110559 694
318O13_region_A3_110573_70_Reverse_Primer_Seq 110674 110648 695
318O13_region_A3_65117_12_Forward_Primer_Seq 65034 65058 696
318O13_region_A3_65117_12_Reverse_Primer_Seq 65177 65153 697
318O13_region_A3_490837_16_Forward_Primer_Seq 490762 490786 698
318O13_region_A3_490837_16_Reverse_Primer_Seq 490905 490881 699
318O13_region_A3_107448_11_Forward_Primer_Seq 107385 107411 700
318O13_region_A3_107448_11_Reverse_Primer_Seq 107529 107505 701
318O13_region_A3_331_23_Forward_Primer_Seq 276 301 702
318O13_region_A3_331_23_Reverse_Primer_Seq 421 397 703
318O13_region_A3_193470_13_Forward_Primer_Seq 193444 193468 704
318O13_region_A3_193470_13_Reverse_Primer_Seq 193589 193565 705
318O13_region_A3_183305_14_Forward_Primer_Seq 183239 183263 706
318O13_region_A3_183305_14_Reverse_Primer_Seq 183384 183360 707
318O13_region_A3_55050_14_Forward_Primer_Seq 54998 55022 708
318O13_region_A3_55050_14_Reverse_Primer_Seq 55144 55120 709
318O13_region_A3_224693_21_Forward_Primer_Seq 224656 224682 710
318O13_region_A3_224693_21_Reverse_Primer_Seq 224803 224779 711
318O13_region_A3_207216_12_Forward_Primer_Seq 207152 207176 712
318O13_region_A3_207216_12_Reverse_Primer_Seq 207299 207276 713
318O13_region_A3_4654_22_Forward_Primer_Seq 4612 4636 714
318O13_region_A3_4654_22_Reverse_Primer_Seq 4760 4736 715
318O13_region_A3_408959_13_Forward_Primer_Seq 408918 408942 716
318O13_region_A3_408959_13_Reverse_Primer_Seq 409066 409042 717
318O13_region_A3_132288_22_Forward_Primer_Seq 132192 132216 718
318O13_region_A3_132288_22_Reverse_Primer_Seq 132340 132316 719
318O13_region_A3_292822_20_Forward_Primer_Seq 292747 292771 720
318O13_region_A3_292822_20_Reverse_Primer_Seq 292895 292871 721
318O13_region_A3_311076_12_Forward_Primer_Seq 311027 311051 722
318O13_region_A3_311076_12_Reverse_Primer_Seq 311175 311152 723
318O13_region_A3_509623_13_Forward_Primer_Seq 509584 509608 724
318O13_region_A3_509623_13_Reverse_Primer_Seq 509732 509708 725
318O13_region_A3_190404_14_Forward_Primer_Seq 190358 190382 726
318O13_region_A3_190404_14_Reverse_Primer_Seq 190506 190482
727 318O13_region_A3_164916_15_Forward_Primer_Seq 164808 164832 728
318O13_region_A3_164916_15_Reverse_Primer_Seq 164957 164933 729
318O13_region_A3_21028_13_Forward_Primer_Seq 21001 21026 730
318O13_region_A3_21028_13_Reverse_Primer_Seq 21150 21126 731
318O13_region_A3_208012_17_Forward_Primer_Seq 207955 207979 732
318O13_region_A3_208012_17_Reverse_Primer_Seq 208104 208085 733
318O13_region_A3_484089_14_Forward_Primer_Seq 484036 484060 734
318O13_region_A3_484089_14_Reverse_Primer_Seq 484185 484161 735
318O13_region_A3_332780_17_Forward_Primer_Seq 332723 332747 736
318O13_region_A3_332780_17_Reverse_Primer_Seq 332872 332853 737
318O13_region_A3_480137_37_Forward_Primer_Seq 480059 480084 738
318O13_region_A3_480137_37_Reverse_Primer_Seq 480208 480182 739
318O13_region_A3_441056_14_Forward_Primer_Seq 441011 441035 740
318O13_region_A3_441056_14_Reverse_Primer_Seq 441161 441138 741
318O13_region_A3_77486_11_Forward_Primer_Seq 77447 77471 742
318O13_region_A3_77486_11_Reverse_Primer_Seq 77597 77573 743
318O13_region_A3_272468_11_Forward_Primer_Seq 272423 272447 744
318O13_region_A3_272468_11_Reverse_Primer_Seq 272573 272549 745
318O13_region_A3_425319_17_Forward_Primer_Seq 425233 425257 746
318O13_region_A3_425319_17_Reverse_Primer_Seq 425383 425359 747
318O13_region_A3_413879_31_Forward_Primer_Seq 413835 413859 748
318O13_region_A3_413879_31_Reverse_Primer_Seq 413985 413961 749
318O13_region_A3_80477_64_Forward_Primer_Seq 80440 80464 750
318O13_region_A3_80477_64_Reverse_Prime_Seq 80591 80567 751
318O13_region_A3_277272_50_Forward_Primer_Seq 277213 277237 752
318O13_region_A3_277272_50_Reverse_Primer_Seq 277364 277340 753
318O13_region_A3_509642_13_Forward_Primer_Seq 509604 509628 754
318O13_region_A3_509642_13_Reverse_Primer_Seq 509755 509731 755
318O13_region_A3_321771_14_Forward_Primer_Seq 321663 321687 756
318O13_region_A3_321771_14_Reverse_Primer_Seq 321815 321791 757
318O13_region_A3_26788_12_Forward_Primer_Seq 26734 26758 758
318O13_region_A3_26788_12_Reverse_Primer_Seq 26886 26862 759
318O13_region_A3_262706_16_Forward_Primer_Seq 262649 262673 760
318O13_region_A3_262706_16_Reverse_Primer_Seq 262802 262778 761
318O13_region_A3_243928_16_Forward_Primer_Seq 243891 243915 762
318O13_region_A3_243928_16_Reverse_Primer_Seq 244044 244020 763
318O13_region_A3_23246_148_Forward_Primer_Seq 23215 23239 764
318O13_region_A3_23246_148_Reverse_Primer_Seq 23368 23344 765
318O13_region_A3_165406_12_Forward_Primer_Seq 165367 165391 766
318O13_region_A3_165406_12_Reverse_Primer_Seq 165521 165497 767
318O13_region_A3_486294_14_Forward_Primer_Seq 486208 486232 768
318O13_region_A3_486294_14_Reverse_Primer_Seq 486362 486338 769
318O13_region_A3_46754_12_Forward_Primer_Seq 46661 46685 770
318O13_region_A3_46754_12_Reverse_Primer_Seq 46816 46792 771
318O13_region_A3_381116_15_Forward_Primer_Seq 381080 381104 772
318O13_region_A3_381116_15_Reverse_Primer_Seq 381235 381211 773
318O13_region_A3_350369_11_Forward_Primer_Seq 350295 350319 774
318O13_region_A3_350369_11_Reverse_Primer_Seq 350450 350426 775
318O13_region_A3_138841_13_Forward_Primer_Seq 138795 138819 776
318O13_region_A3_138841_13_Reverse_Primer_Seq 138950 138926 777
318O13_region_A3_12158_142_Forward_Primer_Seq 12117 12141 778
318O13_region_A3_12158_142_Reverse_Primer_Seq 12272 12248 779
318O13_region_A3_315368_13_Forward_Primer_Seq 315310 315334 780
318O13_region_A3_315368_13_Reverse_Primer_Seq 315465 315441 781
318O13_region_A3_307549_13_Forward_Primer_Seq 307464 307488 782
318O13_region_A3_307549_13_Reverse_Primer_Seq 307619 307595 783
318O13_region_A3_159857_14_Forward_Primer_Seq 159772 159796 784
318O13_region_A3_159857_14_Reverse_Primer_Seq 159928 159904 785
318O13_region_A3_140551_15_Forward_Primer_Seq 140454 140478 786
318O13_region_A3_140551_15_Reverse_Primer_Seq 140610 140586 787
318O13_region_A3_279869_11_Forward_Primer_Seq 279797 279821 788
318O13_region_A3_279869_11_Reverse_Primer_Seq 279953 279929 789
318O13_region_A3_78292_35_Forward_Primer_Seq 78265 78291 790
318O13_region_A3_78292_35_Reverse_Primer_Seq 78422 78397 791
318O13_region_A3_185019_12_Forward_Primer_Seq 184953 184977 792
318O13_region_A3_185019_12_Reverse_Primer_Seq 185111 185087 793
318O13_region_A3_409164_13_Forward_Primer_Seq 409082 409106 794
318O13_region_A3_409164_13_Reverse_Primer_Seq 409240 409219 795
318O13_region_A3_75392_14_Forward_Primer_Seq 75287 75311 796
318O13_region_A3_75392_14_Reverse_Primer_Seq 75445 75421 797
318O13_region_A3_231320_12_Forward_Primer_Seq 231269 231293 798
318O13_region_A3_231320_12_Reverse_Primer_Seq 231429 231405 799
318O13_region_A3_381102_14_Forward_Primer_Seq 381041 381064 800
318O13_region_A3_381102_14_Reverse_Primer_Seq 381201 381176 801
318O13_region_A3_491826_15_Forward_Primer_Seq 491753 491777 802
318O13_region_A3_491826_15_Reverse_Primer_Seq 491914 491891 803
318O13_region_A3_56365_21_Forward_Primer_Seq 56336 56360 804
318O13_region_A3_56365_21_Reverse_Primer_Seq 56497 56473 805
318O13_region_A3_372628_15_Forward_Primer_Seq 372554 372578 806
318O13_region_A3_372628_15_Reverse_Primer_Seq 372715 372691 807
318O13_region_A3_217037_11_Forward_Primer_Seq 216919 216943 808
318O13_region_A3_217037_11_Reverse_Primer_Seq 217081 217057 809
318O13_region_A3_302609_11_Forward_Primer_Seq 302575 302599 810
318O13_region_A3_302609_11_Reverse_Primer_Seq 302737 302713 811
318O13_region_A3_341804_11_Forward_Primer_Seq 341686 341710 812
318O13_region_A3_341804_11_Reverse_Primer_Seq 341848 341824 807
318O13_region_A3_217037_11_Forward_Primer_Seq 216919 216943 808
318O13_region_A3_217037_11_Reverse_Primer_Seq 217081 217057 813
318O13_region_A3_264929_68_Forward_Primer_Seq 264862 264886 814
318O13_region_A3_264929_68_Reverse_Primer_Seq 265024 265000 815
318O13_region_A3_55499_12_Forward_Primer_Seq 55400 55424 816
318O13_region_A3_55499_12_Reverse_Primer_Seq 55563 55539 817
318O13_region_A3_295634_14_Forward_Primer_Seq 295538 295562 818
318O13_region_A3_295634_14_Reverse_Primer_Seq 295702 295677 819
318O13_region_A3_269358_15_Forward_Primer_Seq 269242 269266 820
318O13_region_A3_269358_15_Reverse_Primer_Seq 269406 269382 821
318O13_region_A3_457009_24_Forward_Primer_Seq 456924 456948 822
318O13_region_A3_457009_24_Reverse_Primer_Seq 457088 457064 823
318O13_region_A3_176598_14_Forward_Primer_Seq 176554 176578 824
318O13_region_A3_176598_14_Reverse_Primer_Seq 176718 176694 825
318O13_region_A3_278266_12_Forward_Primer_Seq 278210 278234 826
318O13_region_A3_278266_12_Reverse_Primer_Seq 278376 278350 827
318O13_region_A3_391810_12_Forward_Primer_Seq 391683 391707 828
318O13_region_A3_391810_12_Reverse_Primer_Seq 391851 391826 829
318O13_region_A3_269485_15_Forward_Primer_Seq 269383 269407 830
318O13_region_A3_269485_15_Reverse_Primer_Seq 269551 269527 831
318O13_region_A3_359247_17_Forward_Primer_Seq 359218 359243 832
318O13_region_A3_359247_17_Reverse_Primer_Seq 359386 359362 833
318O13_region_A3_315094_13_Forward_Primer_Seq 314976 315002 834
318O13_region_A3_315094_13_Reverse_Primer_Seq 315145 315120 835
318O13_region_A3_307823_13_Forward_Primer_Seq 307784 307809 836
318O13_region_A3_307823_13_Reverse_Primer_Seq 307953 307927 837
318O13_region_A3_248588_15_Forward_Primer_Seq 248540 248564 838
318O13_region_A3_248588_15_Reverse_Primer_Seq 248709 248684 839
318O13_region_A3_252426_85_Forward_Primer_Seq 252398 252423 840
318O13_region_A3_252426_85_Reverse_Primer_Seq 252568 252543 841
318O13_region_A3_513314_16_Forward_Primer_Seq 513209 513233 842
318O13_region_A3_513314_16_Reverse_Primer_Seq 513379 513355 843
318O13_region_A3_68183_14_Forward_Primer_Seq 68108 68132 844
318O13_region_A3_68183_14_Reverse_Primer_Seq 68279 68255 845
318O13_region_A3_471191_13_Forward_Primer_Seq 471059 471083 846
318O13_region_A3_471191_13_Reverse_Primer_Seq 471231 471206 847
318O13_region_A3_163547_18_Forward_Primer_Seq 163459 163483 848
318O13_region_A3_163547_18_Reverse_Primer_Seq 163632 163608 849
318O13_region_A3_417867_15_Forward_Primer_Seq 417839 417863 850
318O13_region_A3_417867_15_Reverse_Primer_Seq 418014 417990 851
318O13_region_A3_332465_14_Forward_Primer_Seq 332346 332370 852
318O13_region_A3_332465_14_Reverse_Primer_Seq 332523 332499 853
318O13_region_A3_207697_14_Forward_Primer_Seq 207578 207602 854
318O13_region_A3_207697_14_Reverse_Primer_Seq 207755 207731 855
318O13_region_A3_277229_43_Forward_Primer_Seq 277186 277210 856
318O13_region_A3_277229_43_Reverse_Primer_Seq 277364 277340 857
318O13_region_A3_36366_11_Forward_Primer_Seq 36323 36345 858
318O13_region_A3_36366_11_Reverse_Primer_Seq 36501 36477 859
318O13_region_A3_91970_12_Forward_Primer_Seq 91938 91962 860
318O13_region_A3_91970_12_Reverse_Primer_Seq 92116 92091 861
318O13_region_A3_211533_11_Forward_Primer_Seq 211406 211430 862
318O13_region_A3_211533_11_Reverse_Primer_Seq 211585 211561 863
318O13_region_A3_336301_11_Forward_Primer_Seq 336174 336198 864
318O13_region_A3_336301_11_Reverse_Primer_Seq 336353 336329 865
318O13_region_A3_441603_14_Forward_Primer_Seq 441539 441563 866
318O13_region_A3_441603_14_Reverse_Primer_Seq 441718 441694 867
318O13_region_A3_468354_15_Forward_Primer_Seq 468263 468287 868
318O13_region_A3_468354_15_Reverse_Primer_Seq 468442 468418 869
318O13_region_A3_188983_18_Forward_Primer_Seq 188855 188879 870
318O13_region_A3_188983_18_Reverse_Primer_Seq 189035 189009 871
318O13_region_A3_115502_17_Forward_Primer_Seq 115469 115493 872
318O13_region_A3_115502_17_Reverse_Primer_Seq 115649 115625 873
318O13_region_A3_163006_13_Forward_Primer_Seq 162972 162996 874
318O13_region_A3_163006_13_Reverse_Primer_Seq 163153 163129 875
318O13_region_A3_119283_14_Forward_Primer_Seq 119199 119224 876
318O13_region_A3_119283_14_Reverse_Primer_Seq 119381 119357 877
318O13_region_A3_491126_11_Forward_Primer_Seq 491062 491086 878
318O13_region_A3_491126_11_Reverse_Primer_Seq 491244 491220 879
318O13_region_A3_99512_21_Forward_Primer_Seq 99398 99422 880
318O13_region_A3_99512_21_Reverse_Primer_Seq 99581 99557 881
318O13_region_A3_280291_17_Forward_Primer_Seq 280201 280226 882
318O13_region_A3_280291_17_Reverse_Primer_Seq 280385 280361 883
318O13_region_A3_138443_19_Forward_Primer_Seq 138304 138329 884
318O13_region_A3_138443_19_Reverse_Primer_Seq 138488 138465 885
318O13_region_A3_115973_14_Forward_Primer_Seq 115832 115856 886
318O13_region_A3_115973_14_Reverse_Primer_Seq 116016 115992 887
318O13_region_A3_329977_14_Forward_Primer_Seq 329864 329889 888
318O13_region_A3_329977_14_Reverse_Primer_Seq 330050 330026 889
318O13_region_A3_205203_14_Forward_Primer_Seq 205090 205115 890
318O13_region_A3_205203_14_Reverse_Primer_Seq 205276 205252 891
318O13_region_A3_153114_12_Forward_Primer_Seq 152969 152993 892
318O13_region_A3_153114_12_Reverse_Primer_Seq 153156 153132 893
318O13_region_A3_34581_13_Forward_Primer_Seq 34523 34547 894
318O13_region_A3_34581_13_Reverse_Primer_Seq 34712 34688 895
318O13_region_A3_292577_19_Forward_Primer_Seq 292549 292573 896
318O13_region_A3_292577_19_Reverse_Primer_Seq 292739 292715 897
318O13_region_A3_445391_20_Forward_Primer_Seq 445356 445382 898
318O13_region_A3_445391_20_Reverse_Primer_Seq 445547 445523 899
318O13_region_A3_350540_17_Forward_Primer_Seq 350421 350445 900
318O13_region_A3_350540_17_Reverse_Primer_Seq 350612 350588 901
318O13_region_A3_453879_15_Forward_Primer_Seq 453725 453750 902
318O13_region_A3_453879_15_Reverse_Primer_Seq 453918 453894 903
318O13_region_A3_201246_13_Forward_Primer_Seq 201128 201153 904
318O13_region_A3_201246_13_Reverse_Primer_Seq 201321 201297 905
318O13_region_A3_326020_13_Forward_Primer_Seq 325902 325927 906
318O13_region_A3_326020_13_Reverse_Primer_Seq 326095 326071 907
318O13_region_A3_503801_14_Forward_Primer_Seq 503656 503680 908
318O13_region_A3_503801_14_Reverse_Primer_Seq 503849 503823 909
318O13_region_A3_302400_52_Forward_Primer_Seq 302283 302307 910
318O13_region_A3_302400_52_Reverse_Primer_Seq 302481 302456 911
318O13_region_A3_448857_15_Forward_Primer_Seq 448748 448772 912
318O13_region_A3_448857_15_Reverse_Primer_Seq 448947 448924 913
318O13_region_A3_48364_14_Forward_Primer_Seq 48232 48256 914
318O13_region_A3_48364_14_Reverse_Primer_Seq 48435 48412 915
318O13_region_A3_251804_48_Forward_Primer_Seq 251738 251762 916
318O13_region_A3_251804_48_Reverse_Primer_Seq 251942 251918 917
318O13_region_A3_382583_13_Forward_Primer_Seq 382549 382574 918
318O13_region_A3_382583_13_Reverse_Primer_Seq 382753 382728 919
318O13_region_A3_124737_14_Forward_Primer_Seq 124641 124665 920
318O13_region_A3_124737_14_Reverse_Primer_Seq 124846 124822 921
318O13_region_A3_124766_13_Forward_Primer_Seq 124641 124665 922
318O13_region_A3_124766_13_Reverse_Primer_Seq 124846 124822 923
318O13_region_A3_461351_16_Forward_Primer_Seq 461218 461242 924
318O13_region_A3_461351_16_Reverse_Primer_Seq 461426 461402 925
318O13_region_A3_64953_19_Forward_Primer_Seq 64798 64823 926
318O13_region_A3_64953_19_Reverse_Primer_Seq 65011 64987 927
318O13_region_A3_366586_13_Forward_Primer_Seq 366508 366532 928
318O13_region_A3_366586_13_Reverse_Primer_Seq 366722 366698 929
318O13_region_A3_46190_15_Forward_Primer_Seq 46012 46037 930
318O13_region_A3_46190_15_Reverse_Primer_Seq 46228 46205 931
318O13_region_A3_81016_11_Forward_Primer_Seq 80927 80952 932
318O13_region_A3_81016_11_Reverse_Primer_Seq 81146 81122 933
318O13_region_A3_134426_14_Forward_Primer_Seq 134253 134277 934
318O13_region_A3_134426_14_Reverse_Primer_Seq 134474 134449 935
318O13_region_A3_292724_14_Forward_Primer_Seq 292549 292573 936
318O13_region_A3_292724_14_Reverse_Primer_Seq 292771 292747 937
318O13_region_A3_187096_17_Forward_Primer_Seq 187058 187082 938
318O13_region_A3_187096_17_Reverse_Primer_Seq 187282 187257 939
318O13_region_A3_381693_13_Forward_Primer_Seq 381658 381683 940
318O13_region_A3_381693_13_Reverse_Primer_Seq 381885 381863 941
318O13_region_A3_361286_33_Forward_Primer_Seq 361173 361197 942
318O13_region_A3_361286_33_Reverse_Primer_Seq 361401 361376 943
318O13_region_A3_482668_14_Forward_Primer_Seq 482592 482616 944
318O13_region_A3_482668_14_Reverse_Primer_Seq 482821 482796 945
318O13_region_A3_128002_12_Forward_Primer_Seq 127882 127906 946
318O13_region_A3_128002_12_Reverse_Primer_Seq 128112 128087 947
318O13_region_A3_499270_14_Forward_Primer_Seq 499184 499208 948
318O13_region_A3_499270_14_Reverse_Primer_Seq 499422 499398 949
318O13_region_A3_231650_12_Forward_Primer_Seq 231568 231592 950
318O13_region_A3_231650_12_Reverse_Primer_Seq 231809 231788 951
318O13_region_A3_199851_13_Forward_Primer_Seq 199762 199786 952
318O13_region_A3_199851_13_Reverse_Primer_Seq 200012 199988 953
318O13_region_A3_324629_13_Forward_Primer_Seq 324540 324564 954
318O13_region_A3_324629_13_Reverse_Primer_Seq 324790 324766 955
318O13_region_A3_374190_19_Forward_Primer_Seq 374129 374152 956
318O13_region_A3_374190_19_Reverse_Primer_Seq 374394 374370 957
318O13_region_A3_460603_13_Forward_Primer_Seq 460450 460474 958
318O13_region_A3_460603_13_Reverse_Primer_Seq 460715 460691 959
318O13_region_A3_108681_14_Forward_Primer_Seq 108524 108548 960
318O13_region_A3_108681_14_Reverse_Primer_Seq 108791 108768 961
318O13_region_A3_459791_47_Forward_Primer_Seq 459639 459663 962
318O13_region_A3_459791_47_Reverse_Primer_Seq 459907 459883 963
318O13_region_A3_4257_20_Forward_Primer_Seq 4172 4196 964
318O13_region_A3_4257_20_Reverse_Primer_Seq 4450 4425 965
318O13_region_A3_238810_14_Forward_Primer_Seq 238563 238589 966
318O13_region_A3_238810_14_Reverse_Primer_Seq 238850 238826 967
318O13_region_A3_245817_14_Forward_Primer_Seq 245713 245738 968
318O13_region_A3_245817_14_Reverse_Primer_Seq 246001 245977 969
318O13_region_A3_245956_14_Forward_Primer_Seq 245713 245738 970
318O13_region_A3_245956_14_Reverse_Primer_Seq 246001 245977 971
318O13_region_A3_74148_14_Forward_Primer_Seq 74050 74075 972
318O13_region_A3_74148_14_Reverse_Primer_Seq 74338 74314 973
318O13_region_A3_74089_15_Forward_Primer_Seq 74050 74075 974
318O13_region_A3_74089_15_Reverse_Primer_Seq 74338 74314 975
318O13_region_A3_241686_12_Forward_Primer_Seq 241470 241494
976 318O13_region_A3_241686_12_Reverse_Primer_Seq 241765 241741 977
318O13_region_A3_47476_12_Forward_Primer_Seq 47280 47304 978
318O13_region_A3_47476_127_Reverse_Primer_Seq 47577 47554 979
318O13_region_A3_164550_12_Forward_Primer_Seq 164323 164347 980
318O13_region_A3_164550_12_Reverse_Primer_Seq 164621 164598 981
318O13_region_A3_101255_15_Forward_Primer_Seq 101119 101144 982
318O13_region_A3_101255_15_Reverse_Primer_Seq 101418 101392 983
515O02_region_G2_16189_11_Forward_Primer 16144 16168 984
515O02_region_G2_16189_11_Reverse_Primer 16244 16220 985
515O02_region_G2_71925_13_Forward_Primer 71880 71905 986
515O02_region_G2_71925_13_Reverse_Primer 71987 71963 987
515O02_region_G2_4707_12_Forward_Primer 4660 4684 988
515O02_region_G2_4707_12_Reverse_Primer 4769 4743 989
515O02_region_G2_118904_18_Forward_Primer 118847 118871 990
515O02_region_G2_118904_18_Reverse_Primer 118957 118932 991
515O02_region_G2_13655_17_Forward_Primer 13567 13592 992
515O02_region_G2_13655_17_Reverse_Primer 13698 13673 993
515O02_region_G2_53900_13_Forward_Primer 53843 53867 994
515O02_region_G2_53900_13_Reverse_Primer 53985 53961 995
515O02_region_G2_8079_14_Forward_Primer 8023 8047 996
515O02_region_G2_8079_14_Reverse_Primer 8167 8143 997
515O02_region_G2_9969_28_Forward_Primer 9917 9941 998
515O02_region_G2_9969_28_Reverse_Primer 10062 10038 999
515O02_region_G2_72308_77_Forward_Primer 72272 72298 1000
515O02_region_G2_72308_77_Reverse_Primer 10062 10038 1001
515O02_region_G2_99475_19_Forward_Primer 99408 99433 1002
515O02_region_G2_99475_19_Reverse_Primer 99554 99530 1003
515O02_region_G2_118615_18_Forward_Primer 118512 118535 1004
515O02_region_G2_118615_18_Reverse_Primer 118658 118634 1005
515O02_region_G2_119001_46_Forward_Primer 118931 118956 1006
515O02_region_G2_119001_46_Reverse_Primer 119079 119055 1007
515O02_region_G2_118958_43_Forward_Primer 118931 118956 1008
515O02_region_G2_118958_43_Reverse_Primer 119079 119055 1009
515O02_region_G2_17197_13_Forward_Primer 17128 17152 1010
515O02_region_G2_17197_13_Reverse_Primer 17276 17252 1011
515O02_region_G2_105163_29_Forward_Primer 105068 105092 1012
515O02_region_G2_105163_29_Reverse_Primer 105217 105192 1013
515O02_region_G2_111335_13_Forward_Primer 111308 111332 1014
515O02_region_G2_111335_13_Reverse_Primer 111458 111434 1015
515O02_region_G2_106396_13_Forward_Primer 106318 106342 1016
515O02_region_G2_106396_13_Reverse_Primer 106469 106445 1017
515O02_region_G2_59229_17_Forward_Primer 59203 59227 1018
515O02_region_G2_59229_17_Reverse_Primer 59354 59330 1019
515O02_region_G2_73795_20_Forward_Primer 73769 73793 1020
515O02_region_G2_73795_20_Reverse_Primer 73921 73896 1021
515O02_region_G2_85664_20_Forward_Primer 85586 85611 1022
515O02_region_G2_85664_20_Reverse_Primer 85738 85714 1023
515O02_region_G2_36921_17_Forward_Primer 36830 36854 1024
515O02_region_G2_36921_17_Reverse_Primer 36983 36959 1025
515O02_region_G2_124150_19_Forward_Primer 124073 124096 1026
515O02_region_G2_124150_19_Reverse_Primer 124227 124203 1027
515O02_region_G2_5089_14_Forward_Primer 4999 5024 1028
515O02_region_G2_5089_14_Reverse_Primer 5156 5132 1029
515O02_region_G2_58221_15_Forward_Primer 58197 58220 1030
515O02_region_G2_58221_15_Reverse_Primer 58354 58330 1031
515O02_region_G2_96139_14_Forward_Primer 96022 96046 1032
515O02_region_G2_96139_14_Reverse_Primer 96182 96158 1033
515O02_region_G2_70595_13_Forward_Primer 70472 70496 1034
515O02_region_G2_70595_13_Reverse_Primer 70634 70608 1035
515O02_region_G2_4340_15_Forward_Primer 4312 4337 1036
515O02_region_G2_4340_15_Reverse_Primer 4477 4454 1037
515O02_region_G2_90417_11_Forward_Primer 90335 90359 1038
515O02_region_G2_90417_11_Reverse_Primer 90503 90479 1039
515O02_region_G2_49711_17_Forward_Primer 49652 49676 1040
515O02_region_G2_49711_17_Reverse_Primer 49820 49796 1041
515O02_region_G2_63053_13_Forward_Primer 63005 63029 1042
515O02_region_G2_63053_13_Reverse_Primer 63173 63148 1043
515O02_region_G2_63076_14_Forward_Primer 63005 63029 1044
515O02_region_G2_63076_14_Reverse_Primer 63173 63148 1045
515O02_region_G2_44442_12_Forward_Primer 44335 44359 1046
515O02_region_G2_44442_12_Reverse_Primer 44505 44481 1047
515O02_region_G2_44422_19_Forward_Primer 44335 44359 1048
515O02_region_G2_44422_19_Reverse_Primer 44505 44481 1049
515O02_region_G2_44158_19_Forward_Primer 44075 44100 1050
515O02_region_G2_44158_19_Reverse_Primer 44252 44227 1051
515O02_region_G2_44141_17_Forward_Primer 44075 44100 1052
515O02_region_G2_44141_17_Reverse_Primer 44252 44227 1053
515O02_region_G2_90762_17_Forward_Primer 90637 90663 1054
515O02_region_G2_90762_17_Reverse_Primer 90814 90790 1055
515O02_region_G2_106241_14_Forward_Primer 106160 106184 1056
515O02_region_G2_106241_14_Reverse_Primer 106341 106317 1057
515O02_region_G2_109676_12_Forward_Primer 109609 109634 1058
515O02_region_G3_109676_12_Reverse_Primer 109793 109768 1059
515O02_region_G2_86242_14_Forward_Primer 86134 86158 1060
515O02_region_G2_86242_14_Reverse_Primer 86318 86293 1061
515O02_region_G2_83109_12_Forward_Primer 83017 83041 1062
515O02_region_G2_83109_12_Reverse_Primer 83202 83178 1063
515O02_region_G2_10461_15_Forward_Primer 10418 10442 1064
515O02_region_G2_10461_15_Reverse_Primer 10609 10585 1065
515O02_region_G2_67608_15_Forward_Primer 67552 67577 1066
515O02_region_G2_67608_15_Reverse_Primer 67745 67721 1067
515O02_region_G2_63275_46_Forward_Primer 63148 63173 1068
515O02_region_G2_63275_46_Reverse_Primer 63347 63323 1069
515O02_region_G2_62405_14_Forward_Primer 62374 62399 1070
515O02_region_G2_62405_14_Reverse_Primer 62576 62552 1071
515O02_region_G2_33563_12_Forward_Primer 33460 33484 1072
515O02_region_G2_33563_12_Reverse_Primer 33670 33646 1073
515O02_region_G2_33146_14_Forward_Primer 32949 32973 1074
515O02_region_G2_33146_14_Reverse_Primer 33191 33167 1075
515O02_region_G2_102179_29_Forward_Primer 102102 102126 1076
515O02_region_G2_102179_29_Reverse_Primer 102352 102327 1077
515O02_region_G2_2646_15_Forward_Primer 2553 2577 1078
515O02_region_G2_2646_15_Reverse_Primer 2809 2784 1079
515O02_region_G2_76652_24_Forward_Primer 76567 76591 1080
515O02_region_G2_76652_24_Reverse_Primer 76835 76812 1081
515O02_region_G2_66280_14_Forward_Primer 66052 66077 1082
515O02_region_G2_66280_14_Reverse_Primer 66334 66309 1083
515O02_region_G2_54768_13_Forward_Primer 54640 54666 1084
515O02_region_G2_54768_13_Reverse_Primer 54923 54899 1085
515O02_region_G2_62580_14_Forward_Primer 62552 62576 1086
515O02_region_G2_62580_14_Reverse_Primer 62840 62816 1087
515O02_region_G2_34598_55_Forward_Primer 34473 34497 1088
515O02_region_G2_34598_55_Reverse_Primer 34765 34739 1089
515O02_region_G2_77680_13_Forward_Primer 77444 77470 1090
515O02_region_G2_77680_13_Reverse_Primer 77741 77716 1091
515O02_region_G2_77693_12_Forward_Primer 77444 77470 1092
515O02_region_G2_77693_12_Reverse_Primer 77741 77716 1093
515O02_region_G2_97392_14_Forward_Primer 97255 97280 1094
515O02_region_G2_97392_14_Reverse_Primer 97554 97530 1095
515O02_region_G2_97359_15_Forward_Primer 97255 97280 1096
515O02_region_G2_97359_15_Reverse_Primer 97554 97530 1120
consensusLRR 1121 rhg1LRR 1122 Rhg4LRR Primer location on
240O17_region_G3 1123 240O17_region_G3_forward_1_b 45046-45072
DETAILED DESCRIPTION OF THE INVENTION
[0104] A) rhg1
[0105] The present invention provides a method for the production
of a soybean plant having an rhg1 SCN resistant allele comprising:
(A) crossing a first soybean plant having an rhg1 SCN rersistant
allele with a second soybean plant having an rhg1 SCN sensitive
allele to produce a segregating population; (B) screening the
segregating population for a member having an rhg1 SCN resistant
allele with a first nucleic acid molecule capable of specifically
hybridizing to linkage group G, wherein the first nucleic acid
molecule specifically hybridizes to a second nucleic acid molecule
that is linked to the rhg1 SCN resistant allele; and, (C) selecting
the member for further crossing and selection.
[0106] rhg1 is located on linkage group G (Concibido et al., Crop
Sci. 36:1643-1650 (1996)). SCN resistant alleles of rhg1 provide
partial resistance to SCN races 1, 2, 3, 5, 6, and 14 (Concibido et
al. (Crop Sci. 37:258-264 (1997)). Also, Webb (U.S. Pat. No.
5,491,081) reports that a QTL on linkage group G (rhg1) provides
partial resistance to SCN races 1, 2, 3, 5, and 14. rhg1 and Rhg4
provide complete or nearly complete resistance to SCN race 3 (U.S.
Pat. No. 5,491,081). While initially thought to be a recessive
gene, rhg1 classification as a recessive gene has been
questioned.
[0107] Using bioinformatic approaches, the rhg1 coding region is
predicted to contain either four exons (rhg1, v.1)(coding
coordinates 45163-45314, 45450-45509, 46941-48763, and 48975-49573
of SEQ ID NO: 2) or two exons (rhg1, v.2) (coding coordinates
46798-48763 and 48975-49573 of SEQ ID NO: 3). rhg1, v.1 encodes an
877 amino acid polypeptide. rhg1, v.2 encodes an 854 amino acid
length polypeptide. rhg1 codes for a Xa21-like receptor kinase (SEQ
ID NOs: 1097, 1098, and 1100-1115) (Song, et al., Science 270,
1804-1806 (1995)). rhg1 has an extracellular leucine rich repeat
(LRR) domain (rhg1, v.1, SEQ ID NO: 1097, residues 164-457; rhg1,
v.2, SEQ ID NO: 1098, residues 141-434), a transmembrane domain
(rhg1, v.1, SEQ ID NO: 1097, residues 508-530; rhg1, v.2, SEQ ID
NO: 1098, residues 33-51, and 485-507) and serine/threonine protein
kinase (STK) domain (rhg1, v.1, SEQ ID NO: 1097, residues 578-869;
rhg1, v.2, SEQ ID NO: 1098, residues 555-846). In a preferred
embodiment, the LRR domain has multiple LRR repeats. In a more
preferred embodiment, the LRR domain has 12 LRR H repeats.
[0108] To identify proteins similar to the proteins encoded by rhg1
candidates, database searches are performed using the predicted
peptide sequences. The rhg1 candidate shows similarity to CAA18124,
which is the Arabidopsis putative receptor kinase (58.4% similarity
and 35.8% identity, (CLUSTALW (default parameters), Thompson et
al., Nucleic Acids Res. 22:4673-4680 (1994)), GCG package, Genetics
Computer Group, Madison, Wis.), and the apple leucine-rich
receptor-like protein kinase (g3641252) (53.2% similarity and 31.5%
identity, (CLUSTALW (default parameters))), which has both LLR and
STK domains, showing conservation in both the LLR and STK domains.
The predicted LRR extracellular domain shows similarity to the
tomato resistance genes Cf-2.1 (Lycopersicon pimpinellifolium)
(66.9% similarity and 45.4% identity (CLUSTALW (default
parameters))) and Cf-2.2 (Lycopersicon pimpinellifolium) (66.9%
similarity and 45.4% identity (CLUSTALW (default parameters))).
[0109] FIG. 1 is an alignment of the LRR domain of the rhg1 gene. A
consensus sequence for the LRR is shown as the top row of the
alignment. Each row of amino acids represents an LRR domain. The
boxed region indicates the putative .beta.-turn/.beta.-sheet
structural motif postulated to be involved in ligand binding (Jones
and Jones, Adv. Bot. Res. Incorp. Adv. Plant Path. 24;89-167
(1997)). The hydrophobic leucine residues are thought to project
into the core of the protein while the flanking amino acids are
thought to be solvent exposed where they may interact with the
ligand (Kobe and Deisenhofer, Nature 374; 183-186 (1995)).
Non-conservative changes in this region are thought to affect
folding. An "x" represents an arbitrary amino acid while an "a"
represents a hydrophobic residue (leucine, isoleucine, methionine,
valine, or phenylalanine). Amino acid substitutions between
resistant and sensitive phenotypes are bordered by a double line.
The amino acid substitution within the 302-325 region is a
histidine/asparagine substitution, and the amino acid substitution
within the 422-445 region is a phenylalanine/serine
substitution.
[0110] As used herein, a naturally occurring rhg1 allele is any
allele that encodes for a protein having an extracellular LRR, a
transmembrane domain, and STK domain where the naturally occurring
allele is present on linkage group G and where certain rhg1
alleles, but not all rhg1 alleles, are capable of providing or
contributing to resistance or partial resistance to a race of SCN.
It is understood that such an allele can, using for example methods
disclosed herein, be manipulated so that the nucleic acid molecule
encoding the protein is no longer present on linkage group G. It is
also understood that such an allele can, using for example methods
disclosed herein, be manipulated so that the nucleic acid molecule
sequence is altered.
[0111] As used herein, an rhg1 SCN resistant allele is any rhg1
allele where that allele alone or in combination with other SCN
resistant alleles present in the plant, such as an Rhg4 SCN
resistant allele, provides resistance to a race of SCN, and that
resistance is due, at least in part, to the genetic contribution of
the rhg1 allele.
[0112] SCN resistance or partial resistance is determined by a
comparison of the plant in question with a known SCN sensitive
host, Lee 74, according to the method set forth in Schmitt, J.
Nematol. 20:392-395 (1988). As used herein, resistance to a
particular race of SCN is defined as having less than 10% of cyst
development relative to the SCN sensitive host Lee 74. Moreover, as
used herein, partial resistance to a particular race of SCN is
defined as having more than 10% but less than 75% of cyst
development relative to the SCN sensitive host Lee 74.
[0113] Any soybean plant having an rhg1 SCN resistant allele can be
used in conjunction with the present invention. Soybeans with known
rhg1 SCN resistant alleles can be used. Such soybeans include but
are not limited to PI548402 (Peking), PI200499, A2869, Jack, A2069,
PI209332 (No:4), PI404166 (Krasnoaarmejkaja), PI404198 (Sun huan
do), PI437654 (Er-hej-jan), PI438489 (Chiquita), PI507354 (Tokei
421), PI548655 (Forrest), P1548988 (Pickett), PI84751, PI437654,
PI40792, Pyramid, Nathan, AG2201, A3469, AG3901, A3904, AG4301,
AG4401, AG4501, AG4601, PION9492, P188788, Dyer, Custer, Manokin,
and Doles. In a preferred aspect, the soybean plant having an rhg1
SCN resistant allele is an rhg1 haplotype 2 allele. Examples of
soybeans with an rhg1 haplotype 2 allele are PI548402 (Peking),
PI404166 (Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654
(Er-hejjan), PI438489 (Chiquita), PI507354 (Tokei 421), PI548655
(Forrest), PI548988 (Pickett), PI84751, PI437654, and PI40792. In
addition, using the methods or agents of the present invention,
soybeans and wild relative of soybean such as Glycine soja can be
screened for the presence of rhg1 SCN resistant alleles.
[0114] Any soybean plant having an rhg1 SCN sensitive allele can be
used in conjunction with the present invention. Such soybeans
include A3244, A2833, AG3001, Williams, Will, A2704, Noir, DK23-51,
Lee 74, Essex, Minsoy, A1923, and Hutcheson. In a preferred aspect,
the soybean plant ing an rhg1 SCN sensitive allele is an rhg1 A3244
allele. In addition, using the methods or nts of the present
invention, soybeans and wild relatives of soybean such as Glycine
soja can be eened for the presence of rhg1 SCN sensitive
alleles.
[0115] Table 2, below, is a table showing single nucleotide
polymorphisms (SNPs) and rtions/deletions (INDEL) sites for eight
haplotype sequences of rhg1. TABLE-US-00002 TABLE 2 Identification
Base number of contig 240O17_region_G3 of reference line A3244 Hap
PI# Line Ph 45173 45309 45400 45416 45439 45611 45916 45958 1 --
A3244 S G G A T A A A A 2 PI548402 Peking R G A C C T A G A 3
PI423871 Toyosuzu -- G A A T A A G A 4 PI518672 Will S G G A T A A
A A 5 -- A2704 S G G A T A A A A 6 PI290136 Noir S A A A C T G A T
7 PI548658 Lee 74 S A A A C T G A T 8 PI200499 -- R G A A C A A A A
N/A PI548667 Essex S A A A C T G A T N/A PI548389 Minsoy S G G A T
A A A A N/A PI360843 Oshima. -- -- -- -- -- -- -- -- -- N/A --
A2869 R -- -- -- -- -- -- -- -- N/A PI540556 Jack R -- -- -- -- --
-- -- -- N/A -- A2069 R -- -- -- -- -- -- -- -- N/A PI209332 No. 4
R -- -- -- -- -- -- -- -- Base number of contig Identification
240O17_region_G3 of reference line A3244 Hap PI# Line Ph 46049
46113 46227 46703 47057 47140 47208 1 -- A3244 S C A d1 0 T C C 2
PI548402 Peking R T G 0 d2 C C C 3 PI423871 Toyosuzu -- T G 0 0 T C
C 4 PI518672 Will S C A d1 0 T A T 5 -- A2704 S C A d1 0 T A T 6
PI290136 Noir S T A 0 d14 T C C 7 PI548658 Lee 74 S T A 0 d14 T C C
8 PI200499 -- R T A 0 d14 T C C N/A PI548667 Essex S T A 0 d14 T C
C N/A PI548389 Minsoy S C A d1 0 T A T N/A PI360843 Oshima. -- --
-- -- 0 T A T N/A -- A2869 R -- -- 0 d14 T C C N/A PI540556 Jack R
-- -- -- -- -- -- -- N/A -- A2069 R -- -- -- -- -- -- -- N/A
PI209332 No. 4 R -- -- -- -- -- -- -- Identification Base number of
contig 240O17_region_G3 of reference line A3244 Hap PI# Line Ph
47571 47617 47796 47856 47937 48012 48060 48073 1 -- A3244 S G C A
T T T C C 2 PI548402 Peking R G C C C C T C C 3 PI423871 Toyosuzu
-- G C C C C T C C 4 PI518672 Will S G C A T T T C C 5 -- A2704 S G
C A T T C T T 6 PI290136 Noir S A A C C C C T T 7 PI548658 Lee 74 S
G A C C C C T T 8 PI200499 -- R G A C C C C T T N/A PI548667 Essex
S G A C C C C T T N/A PI548389 Minsoy S G C A T T C/T C/T C/T N/A
PI360843 Oshima. -- G C A T/C T/C T C C N/A -- A2869 R G A C C C C
T T N/A PI540556 Jack R -- -- C C C C T T N/A -- A2069 R -- -- C
T/C T/C C T T N/A PI209332 No. 4 R -- -- C C C C T T Base number of
contig Identification 240O17_region_G3 of reference line A3244 Hap
PI# Line Ph 48135 48279 48413 48681 48881 49012 49316 1 -- A3244 S
A C G A 0 A T 2 PI548402 Peking R G C G G d19 G T 3 PI423871
Toyosuzu -- G C G A 0 A T 4 PI518672 Will S A C G A 0 A T 5 --
A2704 S G T C -- 0 G C 6 PI290136 Noir S G T C G 0 G C 7 PI548658
Lee 74 S G T C G 0 G C 8 PI200499 -- R G T C G 0 G C N/A PI548667
Essex S G T C A/G 0 G C N/A PI548389 Minsoy S A C G A 0 A T N/A
PI360843 Oshima. -- A/G C G A 0 A T N/A -- A2869 R G T C G 0 G C
N/A PI540556 Jack R G T C G 0 G C N/A -- A2069 R A T C G 0 G C N/A
PI209332 No. 4 R A/G T C G 0 G C
[0116] In Table 2, discrete haplotypes are designated 1 through 8.
N/A refers to a haplotype that is not characterized. The Plant
Introduction classification number is indicated in the "PI#"
column. A dash indicates that no PI number is known or assigned for
the line under investigation. The line from which the sequences are
derived is indicated in the "line" column, with a dash indicating
an unknown or unnamed line. The "Ph." (phenotype) column of table 2
indicates whether a given line has been reported as resistant (R)
to at least one race of SCN or sensitive (S).
[0117] The nucleotide bases located at each of 30 positions in each
of the haplotype sequences is shown in the columns labeled "Base
number of contig 240017_region_G3 of reference line A3244.'' The
base number at the top of each column corresponds to the base
number in contig 240O17_region_G3 of reference line A3224 (SEQ ID
NOs: 2 and 3). The letters G, A, C, and T correspond to the bases
guanine, adenine, cytosine, and thymine. Two bases separated by a
slash (A/G, C/T, or T/C) indicate uncertainty at the specified
position of the haplotype sequence. A "d" followed by a number
indicates a deletion of a the length specified. That is, dl is a
one base deletion, d2 is a two base deletion, d14 is a fourteen
base deletion, and d19 is a nineteen base deletion. A zero (0)
indicates no deletion. A dash indicates that the identity of the
base is undetermined.
[0118] Examination of table 2 reveals that the amino acid
substitutions in the rhg1 coding region are common to the resistant
lines PI467312 (Cha-mo-shi-dou), PI88788 and the southern
susceptible lines Essex, Hutchenson, Noir and A1923. As used
herein, a "southern" cultivar is any cultivar from maturity groups
VI, VII, VIII, IX, or X, and a "northern" cultivar is any cultivar
from maturity groups 000, 00, 0, I, II, III, IV, or V. This data is
consistent with the mapping experiments of Qui et al. (Theor Appl
Genet 98:356-364 (1999)). Based on analysis of 200 F.sub.2:.sub.3
families derived from a cross between Peking and Essex, the authors
failed to detect any significant association with SCN resistance to
races 1, 2, and 3, and the rhg1 locus on linkage group G. The
authors point out that one of the markers, Bng122, which has been
shown to have significant linkage to rhg1 (Concibido et al., Crop
Sci. 36:1643-1650 (1996)), is not polymorphic in the population
employed. It is also possible that the susceptible southern lines
contain rhg1 and the susceptible phenotype reflects the polygenic
nature of SCN resistance. In a study to uncover QTLs for sudden
death syndrome (SDS) in soybean, two SCN resistant alleles
originating from the susceptible parent Essex have been described
(Hnetkovsky et al., Crop Sci. 36:393-400). TABLE-US-00003 TABLE 3a
Haplotype 2 Lines PI# Line Ph. PI548402 Peking R PI404166
Krasnoaarmejkaja R PI404198 (Sun huan do) R PI437654 Er-hej-jan R
PI438489 (Chiquita) R PI507354 Tokei 421 R PI548655 Forrest R
PI548988 Pickett R PI84751 -- R PI437654 -- R PI40792 -- --
[0119] TABLE-US-00004 TABLE 3b Haplotype 4 Lines PI# Line Ph. --
Will S PI467312 Cha-mo-shi-dou R PI88788 -- R
[0120] TABLE-US-00005 TABLE 3c Haplotype 6 Lines PI# Line Ph. --
Noir S -- A1923 S -- Hutcheson S
[0121] In Tables 3a, 3b, and 3c, Plant Introduction classification
number is indicated in the "PI#" column. A dash indicates that no
PI number is known or assigned for the line in question. The line
from which the sequences are derived is indicated in the "line"
column, with a dash indicating an unknown or unnamed line. The
"Ph." column indicates whether a given line has been reported as
resistant (R) to at least one race of SCN or sensitive (S), with a
dash indicating that the phenotype is unknown.
[0122] In a preferred aspect, the source of either an rhg1 SCN
sensitive allele or an rhg1 SCN resistant allele, or more
preferably both, is an elite plant. An "elite line" is any line
that has resulted from breeding and selection for superior
agronomic performance. Examples of elite lines are lines that are
commercially available to farmers or soybean breeders such as
HARTZT.TM. variety H4994, HARTZ.TM. variety H5218, HARTZ.TM.
variety H5350, HARTZ.TM. variety H5545, HARTZ.TM. variety H5050,
HARTZ.TM. variety H5454, HARTZ.TM. variety H5233, HARTZ.TM. variety
H5488, HARTZ.TM. variety HLA572, HARTZ.TM. variety H6200, HARTZ.TM.
variety H6104, HARTZ.TM. variety H6255, HARTZ.TM. variety H6586,
HARTZ.TM. variety H6191, HARTZ.TM. variety H7440, HARTZ.TM. variety
H4452 Roundup Ready.TM., HARTZ.TM. variety H4994 Roundup Ready.TM.,
HARTZ.TM. variety H4988 Roundup Ready.TM., HARTZ.TM. variety H5000
Roundup Ready.TM., HARTZ.TM. variety H5147 Roundup Ready.TM.,
HARTZ.TM. variety H5247 Roundup Ready.TM., HARTZ.TM. variety H5350
Roundup Ready.TM., HARTZ.TM. variety H5545 Roundup Ready, HARTZ.TM.
variety H5855 Roundup Ready.TM., HARTZ.TM. variety H5088 Roundup
Ready.TM., HARTZ.TM. variety H5164 Roundup Ready.TM., HARTZ.TM.
variety H5361 Roundup Ready.TM., HARTZ.TM. variety H5566 Roundup
Ready.TM., HARTZ.TM. variety H5318 Roundup Ready.TM., HARTZ.TM.
variety H5868 Roundup Ready.TM., HARTZ.TM. variety HM59 Roundup
Ready.TM., HARTZ.TM. variety H6013 Roundup Ready.TM., HARTZ.TM.
variety H6285 Roundup Ready.TM., HARTZ.TM. variety H6454 Roundup
Ready.TM., HARTZ.TM. variety H6686 Roundup Ready.TM., HARTZ.TM.
variety H7152 Roundup Ready.TM., HARTZ.TM. variety H7550 Roundup
Ready.TM., HARTZ.TM. variety H8001 Roundup Ready (HARTZ SEED,
Stuttgart, Ark., U.S.A.); A0868, AGO901, A1553, A1900, AG1901,
A1923, A2069, AG2101, AG2201, A2247, AG2301, A2304, A2396, AG2401,
AG2501, A2506, A2553, AG2701, AG2702, A2704, A2833, A2869, AG2901,
AG2902, AG3001, AG3002, A3204, A3237, A3244, AG3301, AG3302, A3404,
A3469, AG3502, A3559, AG3601, AG3701, AG3704, AG3750, A3834,
AG3901, A3904, A4045 AG4301, A4341, AG4401, AG4501, AG4601, AG4602,
A4604, AG4702, AG4901, A4922, AG5401, A5547, AG5602, A5704, AG5801,
AG5901, A5944, A5959, AG6101, QR4459 and QP4544 (Asgrow Seeds, Des
Moines, Iowa, U.S.A.); DeKalb variety CX445 (DeKalb, Ill.). An
elite plant is any plant from an elite line.
[0123] B) Rhg4
[0124] The present invention provides a method for the production
of a soybean plant having an Rhg4 SCN resistant allele comprising:
(A) crossing a first soybean plant having an Rhg4 SCN resistant
allele with a second soybean plant having an Rhg4 SCN sensitive
allele to produce a segregating population; (B) screening the
segregating population for a member having an Rhg4 SCN resistant
allele with a first nucleic acid molecule capable of specifically
hybridizing to linkage group A2, wherein the first nucleic acid
molecule specifically hybridizes to a second nucleic acid molecule
linked to the Rhg4 SCN resistant allele; and, (C) selecting the
member for further crossing and selection.
[0125] Rhg4 is located on linkage group A2 (Matson and Williams,
Crop Sci. 5:447 (1965)). SCN resistant alleles of Rhg4 provide
partial resistance to SCN races 1 and 3 (U.S. Pat. No. 5,491,081).
Together, rhg1 and Rhg4 provide complete or nearly complete
resistance to SCN race 3. The dominant gene, Rhg4, was found to be
closely linked to the seed coat color locus (i) (Matson and
Williams, Crop Sci. 5:447 (1965)). The i locus in Peking was also
reported to be linked with a recessive gene for resistance to SCN
(Sugiyama and Katsumi, Jpn. J. Breed. 16:83-86 (1966)). It is
possible that Rhg4 and the recessive gene linked to the i locus are
one and the same, which would call into question the classification
of Rhg4 as a dominant gene.
[0126] Using bioinformatic approaches the Rhg4 coding region is
predicted to contain 2 exons (coding coordinates 111805-113968 and
114684-115204 of SEQ ID NO: 4). Rhg4 encodes an 894 amino acid
polypeptide. Rhg4 codes for a Xa21-like receptor kinase (SEQ ID
NOs: 1099 and 1116-1119) (Song et al., Science 270, 1804-1806,
(1995)). Rhg4 has an extracellular LRR domain (Rhg4, SEQ ID NO:
1099, residues 34-44), a transmembrane domain (Rhg4 SEQ ID NO:
1099, residues 449-471), and STK domain (Rhg4, SEQ ID NO: 1099,
residues 531-830). In a preferred embodiment, the LRR domain has
multiple LRR repeats. In a more preferred embodiment, the LRR
domain has 12 LRR repeats.
[0127] To identify proteins similar to the Rhg4 candidate, database
searches are performed using the predicted peptide sequences. The
Rhg4 candidate shows similarity to TMK (Y07748)(73.0% similarity
and 54.8% identity (CLUSTALW (default parameters))) and TMK1
PRECURSOR (70.6% similarity and 55.1% identity (CLUSTALW (default
parameters))), which are rice and Arabidopsis receptor kinases,
respectively. The predicted LRR extracellular domain reveals
similarity to TMK (Y07748)(70. I% similarity and 46.6% identity
(CLUSTALW (default parameters))), TMK1 PRECURSOR (g1707642) (65.8%
similarity and 48.8% identity (CLUSTALW (default parameters))), and
F21J9.1 (g2213607) (65.5% similarity and 45.6% identity (CLUSTALW
(default parameters))).
[0128] FIG. 2 is an alignment of the LRR domain of the Rhg4 gene. A
consensus sequence is shown as the top row. Each row of amino acids
represents an LRR domain. The boxed region indicates the putative
.beta.-turn/.beta.-sheet structural motif postulated to be involved
in ligand binding (Jones and Jones, Adv. Bot. Res. Incorp. Adv.
Plant Path. 24;89-167 (1997)). The hydrophobic leucine residues are
thought to project into the core of the protein while the flanking
amino acids are thought to be solvent exposed where they may
interact with the ligand (Kobe and Deisenhofer, Nature 374; 183-186
(1995)). An "x" represents an arbitrary amino acid while an "a"
represents a hydrophobic residue (leucine, isoleucine, methionine,
valine, or phenylalanine). Amino acid substitutions between
resistant and sensitive phenotypes are bordered by a double line.
The amino acid substitution within the 35-57 region is a
histidine/glutamine substitution, and the amino acid substitution
within the 81-104 region is a leucine/phenylalanine
substitution.
[0129] As used herein, a naturally-occurring Rhg4 allele is any
allele that encodes for a protein having an extracellular LRR
domain, a transmembrane domain, and STK domain where the naturally
occurring allele is present on linkage group A2 and where certain
Rgh4 alleles, but not all Rgh4 alleles, are capable of providing or
contributing to resistance or partial resistance to a race of SCN.
It is understood that such an allele can, using, for example
methods disclosed herein, be manipulated so that the nucleic acid
molecule encoding the protein is no longer present on linkage group
A2. It is also understood that such an allele can, using, for
example methods disclosed herein, be manipulated so that the
nucleic acid molecule sequence is altered.
[0130] As used herein, an Rhg4 SCN resistant allele is any Rhg4
allele where that allele alone or in combination with other SCN
resistant alleles present in the plant, such as an rhg1 SCN
resistant allele, provides resistance to a race of SCN, and that
resistance is due, at least in part, to the genetic contribution of
the Rhg4 allele.
[0131] Any soybean plant having an Rhg4 SCN resistant allele can be
used in conjunction with the present invention. Soybeans with known
Rhg4 SCN resistant alleles can be used. Such soybeans include, but
are not limited to, PI548402 (Peking), PI437654 (Er-hej-jan),
PI438489 (Chiquita), PI507354 (Tokei 421), PI548655 (Forrest),
PI548988 (Pickett), PI88788, PI404198 (Sun Huan Do), PI404166
(Krasnoaarmejkaja), Hartwig, Manokin, Doles, Dyer, and Custer. In a
preferred aspect, the soybean plant having an Rhg4 SCN resistant
allele is an Rhg4 haplotype 3 allele in a plant having either an
rhg1 haplotype 2 or rhg1 haplotype 4 allele. Examples of soybeans
with an Rhg4 haplotype 3 allele are PI548402 (Peking), PI88788,
PI404198 (Sun huan do), PI438489 (Chiquita), PI437654 (Er-hej-jan),
PI404166 (Krasnoaarmejkaja), PI548655 (Forrest), PI548988
(Pickett), and PI507354 (Tokei 421). In addition, using the methods
or agents of the present invention, soybeans and wild relatives of
soybeans such as Glycine soja can be screened for the presence of
Rhg4 SCN resistant alleles.
[0132] Table 4 below is a table showing single nucleotide
polymorphisms (SNPs) for three haplolotype sequences of Rhg4.
TABLE-US-00006 TABLE 4 Identification Base number of contig
318O13_region_A3 Markers Hap PI number Line Ph Coat 111933 112065
112101 112461 114066 scn279 scnb267 scn273 1 -- A2069 R yellow T A
T A T 2 2 2 1 -- A2869 R yellow T A T A T 2 2 2 1 -- A3244 S yellow
T A T A T 2 2 2 1 PI87631 Kindaizu R yellow T A T A T 2 2 2 1
PI548389 Minsoy S yellow T A T A T 2 2 2 1 PI518664 Hutcheson S
yellow T A T A T 2 2 2 1 PI548658 Lee 74 S yellow T A T A T -- 2 2
2 PI540556 Jack R yellow G A T A T 2 2 1 2 PI360843 Oshimashirome R
yellow G A T A T -- -- -- 2 PI423871 Toyosuzu R yellow G A T A T --
-- -- 3 PI548402 Peking R black G C C T G 1 1 1 3 PI88788 -- R
black G C C T G 1 1 1 3 PI404198 B (Sun huan do) R black G C C T G
1 1 1 3 PI438489 B (Chiquita) R black G C C T G 1 1 1 3 PI437654
Er-hej-jan R black G C C T G 2 1 1 3 PI404166 Krasnoaarmejkaja R
black G C C T G 1 1 -- 3 PI290136 Noir S black G C C T G 1 1 1 3
PI548655 Forrest R yellow G C C T G 1 1 1 3 PI548988 Pickett R
yellow G C C T G 1 1 1 3 PI507354 Tokei 421 R yellow G C C T G 1 1
1 N/A PI467312 Cha-mo-shi-dou R GnBr G C C T -- 1 1 1 N/A PI209332
No. 4 R black T A T -- -- 2 2 2 N/A PI518672 Will S yellow T A T --
T 2 2 2 N/A PI548667 Essex S yellow T A T -- T 2 2 2
[0133] In Table 4, discrete haplotypes are designated 1 through 3.
N/A refers to a haplotype that is not characterized. In Table 4,
the Plant Introduction classification number is indicated in the
"PI#" column. A dash indicates that no PI number is known or
assigned for the line under investigation. The line from which the
sequences are derived is indicated in the "line" column, with a
dash indicating an unknown or unnamed line. The "Ph." column of
Table 4 indicates whether a given line has been reported to be
resistant (R) to at least one race of SCN, or sensitive (S). The
"coat" column shows the phenotypic coat color of a seed as either
yellow, black, green/brown (GnBr), or unknown/unassigned (dash). At
the I locus, black seeded varieties harbor the i allele for black
or imperfect black seed coat. In a preferred embodiment, the seed
has a yellow coat.
[0134] The nucleotide base located at each of 5 positions in each
of the haplotype sequences is shown in the columns labeled "Base
number of contig 318013_region_A3." The base number at the top of
each column correspond to the base number in the contig
318013_region_A3 of reference line A3244 (SEQ ID NO: 4). The
letters G, A, C, and T correspond to the bases guanine, adenine,
cytosine, and thymine. A dash indicates that the identity of the
base is unknown.
[0135] Three different simple sequence repeat (SSR) or
microsatellite markers that occur within the sequences, scn279 (SEQ
ID NO: 292), scn267 (SEQ ID NO: 282), and scn273 (SEQ ID NO: 294),
are listed under "markers." The allele of each marker occurring in
a haplotype is indicated by a 1 or a 2, with a dash indicating that
the information is not determined.
[0136] Any soybean plant having an Rhg4 SCN sensitive allele can be
used in conjunction with the present invention. Such soybeans
include A3244, Will, Noir, Lee 74, Essex, Minsoy, A2704, A2833,
AG3001, Williams, DK23-51, and Hutcheson. In a preferred aspect,
the soybean plant having an Rhg4 SCN sensitive allele is an Rhg4
A3244 allele. In addition, using the methods or agents of the
present invention, soybeans and wild relative of soybean such as
Glycine soja can be screened for 15 the presence of Rhg4 SCN
sensitive alleles.
[0137] In a preferred aspect, the source of either an Rhg4 SCN
sensitive allele or an Rhg4 SCN resistant allele, or more
preferably both, is an elite plant.
[0138] In table 5, below, rhg1 and Rhg4 haplotypes for various
cultivars are compared. TABLE-US-00007 TABLE 5 Identification
Haplotype PI# Line Coat Ph. rhg4 rhg1 -- A3244 yellow S 1 1
PI548402 Peking black R 3 2 PI404198 B (Sun huan do) black R 3 2
PI438489 B (Chiquita) black R 3 2 PI437654 Er-hej-jan black R 3 2
PI404166 Krasnoaarmejkaja black R 3 2 PI548655 Forrest yellow R 3 2
PI548988 Pickett yellow R 3 2 PI507354 Tokei 421 yellow R 3 2
PI88788 -- black R 3 4 PI467312 Cha-mo-shi-dou GnBr R N/A 4 -- Noir
black S 3 6 -- Jack yellow R 2 N/A PI360843 Oshimashirome yellow R
2 N/A PI423871 Toyosuzu yellow R 2 3 PI209332 No. 4 black R N/A N/A
PI87631 Kindaizu yellow R 1 -- -- Minsoy yellow S 1 N/A -- Will
yellow S N/A 4 -- Hutcheson yellow S 1 6 -- Lee 74 yellow S N/A 7
-- Essex yellow S N/A N/A -- A2069 yellow R 1 N/A -- A2869 yellow R
1 N/A
[0139] In Table 5, haplotypes, as used in Tables 2 through 4, are
listed for each line. N/A refers to a haplotype that is not
characterized. The Plant Introduction classification number is
indicated in the "PI#" column. A dash indicates that no PI number
is known or assigned for the line under investigation. The line
from which the sequences are derived is indicated in the "line"
column, with a dash indicating an unknown or unnamed line. The
"Ph." column of table 5 indicates whether a given line has been
reported to be resistant (R) to at least one race of SCN, or
sensitive (S). The "coat" column shows the phenotypic coat color of
a seed as either yellow, black, green/brown (GnBr), or
unknown/unassigned (dash). At the I locus, black seeded varieties
harbor the i allele for black or imperfect black seed coat. In a
preferred embodiment, the seed has a yellow coat.
[0140] Screening for rhg1 and Rhg4 Alleles
[0141] Any appropriate method can be used to screen for a plant
having an rhg1 SCN resistant allele. Any appropriate method can be
used to screen for a plant having an Rhg4 SCN resistant allele. In
a preferred aspect of the present invention, a nucleic acid marker
of the present invention can be used (see section entitled
"Screening for rhg1 and Rhg4 alleles" and subsection (ii) of the
section entitled "Agents").
[0142] Additional markers, such as SSRs, AFLP markers, RFLP
markers, RAPD markers, phenotypic markers, SNPs, isozyme markers,
microarray transcription profiles that are genetically linked to or
correlated with alleles of a QTL of the present invention can be
utilized (Walton, Seed World 22-29 (July, 1993); Burow and Blake,
Molecular Dissection of Complex Traits, 13-29, Eds. Paterson, CRC
Press, New York (1988)). Methods to isolate such markers are known
in the art. For example, locus-specific SSRs can be obtained by
screening a genomic library for SSRs, sequencing of "positive"
clones, designing primers which flank the repeats, and amplifying
genomic DNA with these primers. The size of the resulting
amplification products can vary by integral numbers of the basic
repeat unit. To detect a polymorphism, PCR products can be
radiolabeled, separated on denaturing polyacrylamide gels, and
detected by autoradiography. Fragments with size differences >4
bp can also be resolved on agarose gels, thus avoiding
radioactivity.
[0143] Other SSR markers may be utilized. Amplification of simple
tandem repeats, mainly of the [CA].sub.n type were reported by Litt
and Luty, Amer. J Human Genet. 44:397-401 (1989); Smeets et al.,
Human Genet. 83:245-251 (1989); Tautz, Nucleic Acids Res. 1
7:6463-6472 (1989); Weber and May, Am. J. Hum. Genet. 44:388-396
(1989). Weber, Genomics 7:524-530 (1990), reported that the level
of polymorphism detected by PCR-amplified [CA].sub.n type SSRs
depends on the number of the "perfect" (i.e., uninterrupted),
tandemly repeated motifs. Below a certain threshold (i.e., 12
CA-repeats), the SSRs were reported to be primarily monomorphic.
Above this threshold, however, the probability of polymorphism
increases with SSR length. Consequently, long, perfect arrays of
SSRs are preferred for the generation of markers, i.e., for the
design and synthesis of flanking primers.
[0144] Suitable primers can be deduced from DNA databases (e.g.,
Akkaya et al., Genetics. 132:1131-1139 (1992)). Alternatively,
size-selected genomic libraries (200 to 500 bp) can be constructed
by, for example, using the following steps: (1) isolation of
genomic DNA; (2) digestion with one or more 4 base-specific
restriction enzymes; (3) size-selection of restriction fragments by
agarose gel electrophoresis, excision and purification of the
desire size fraction; (4) ligation of the DNA into a suitable
vector and transformation into a suitable E. coli strain; (5)
screening for the presence of SSRs by colony or plaque
hybridization with a labeled probe; (6) isolation of positive
clones and sequencing of the inserts; and (7) design of suitable
primers flanking the SSR.
[0145] Establishing libraries with small, size-selected inserts can
be advantageous for SSR isolation for two reasons: (1) long SSRs
are often unstable in E. coli, and (2) positive clones can be
sequenced without subdloning. A number of approaches have been
reported for the enrichment of SSRs in genomic libraries. Such
enrichment procedures are particularly useful if libraries are
screened with comparatively rare tri- and tetranucleotide repeat
motifs. One such approach has been described by Ostrander et al.,
Proc. Natl. Acad. Sci. (U.S.A.). 89:3419-3423 (1992), who reported
the generation of a small-insert phagemid library in an E. coli
strain deficient in UTPase (d8t) and uracil-N-glycosylase (ung)
genes. In the absence of UTPase and uracil-N-glycosylase, dUTP can
compete with dTTP for the incorporation into DNA. Single-stranded
phagemid DNA isolated from such a library can be primed with
[CA].sub.n and [TG].sub.n primers for second strand synthesis, and
the products used to transform a wild-type E. coli strain. Since
under these conditions there will be selection against
single-stranded, uracil-containing DNA molecules, the resulting
library will consist of primer-extended, double-stranded products
and an about 50-fold enrichment in CA-repeats.
[0146] Other reported enrichment strategies rely on hybridization
selection of simple sequence repeats prior to cloning (Karagyozov
et al., Nucleic Acids Res. 21:3911-3912 (1993); Armour et al., Hum.
Mol. Gen. 3:599-605 (1994); Kijas et al., Genome 38:349-355 (1994);
Kandpal et al., Proc. Natl. Acad. Sci. (U.S.A.) 91:88-92 (1994);
Edwards et al., Am. J. Hum. Genet. 49:746-756 (1991)).
Hybridization selection, can for example, involve the following
steps: (1) genomic DNA is fragmented, either by sonication, or by
digestion with a restriction enzyme; (2) genomic DNA fragments are
ligated to adapters that allow a "whole genome PCR" at this or a
later stage of the procedure; (3) genomic DNA fragments are
amplified, denatured and hybridized with single-stranded SSR
sequences bound to a nylon membrane; (4) after washing off unbound
DNA, hybridizing fragments enriched for SSRs are eluted from the
membrane by boiling or alkali treatment, reamplified using
adapter-complementary primers, and digested with a restriction
enzyme to remove the adapters; and (5) DNA fragments are ligated
into a suitable 15 vector and transformed into a suitable E. coli
strain. SSRs can be found in up to 50-70% of the clones obtained
from these procedures (Armour et al., Hum. Mol. Gen. 3:599-605
(1994); Edwards et al., Am. J. Hum. Genet. 49:746-756 (1991)).
[0147] An alternative hybridization selection strategy was reported
by Kijas et al., Genome 38:599-605 (1994), which replaced the nylon
membrane with biotinylated, SSR-complementary oligonucleotides
attached to streptavidin-coated magnetic particles. SSR-containing
DNA fragments are selectively bound to the magnetic beads,
reamplified, restriction-digested and cloned.
[0148] It is further understood that other additional markers on
linkage group G or A2 may be utilized (Morgante et al., Genome
37:763-769 (1994)). As used herein, reference to the linkage group
of G or A2 refers to the linkage group that corresponds to linkage
groups U5 and U3, respectively from the genetic map of Glycine max
(Mansur et al., Crop Sci. 36: 1327-1336 (1996), and linkage groups
G and A2, respectively, of Glycine max x. Glycine soja (Shoemaker
et al., Genetics 144: 329-336 (1996)) that is present in Glycine
soja (Soybase, an Agricultural Research Service, United States
Department of Agriculture (http-129.186.26.940 and
USDA-Agricultural Research Service: http-www.ars.usda.gov/)).
[0149] PCR-amplified SSRs can be used, because they are
locus-specific, codominant, occur in large numbers and allow the
unambiguous identification of alleles. Standard PCR-amplified SSR
protocols use radioisotopes and denaturing polyacrylamide gels to
detect amplified SSRs. In many situations, however, allele sizes
are sufficiently different to be resolved on high percentage
agarose gels in combination with ethidium bromide staining (Bell
and Ecker, Genomics 19:137-144 (1994); Becker and Heun, Genome
38:991-998 (1995); Huttel, Ph.D. Thesis, University of Frankfurt,
Germany (1996)). High resolution without applying radioactivity is
also provided by nondenaturing polyacrylamide gels in combination
with either ethidium bromide (Scrimshaw, Biotechniques 13:2189
(1992)) or silver straining (Klinkicht and Tautz, Molecular Ecology
1: 133-134 (1992); Neilan et al., Biotechniques 17:708-712 (1994)).
An alternative of PCR-amplified SSRs typing involves the use of
fluorescent primers in combination with a semi-automated DNA
sequencer (Schwengel et al., Genomics 22:46-54 (1994)). Fluorescent
PCR products can be detected by real-time laser scanning during gel
electrophoresis. An advantage of this technology is that different
amplification reactions as well as a size marker (each labeled with
a different fluorophore) can be combined into one lane during
electrophoresis. Multiplex analysis of up to 24 different SSR loci
per lane has been reported (Schwengel et al, Genomics 22:46-54
(1994)).
[0150] The detection of polymorphic sites in a sample of DNA may be
facilitated through the use of nucleic acid amplification methods.
Such methods specifically increase the concentration of
polynucleotides that span the polymorphic site, or include that
site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis
or other means.
[0151] The most preferred method of achieving such amplification
employs the polymerase chain reaction ("PCR") (Mullis et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al.,
European Patent Appln. 50,424; European Patent Appln. 84,796,
European Patent Application 258,017, European Patent Appln.
237,362; Mullis, European Patent Appln. 201,184; Mullis et al.,
U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki
et al., U.S. Pat. No. 4,683,194), using primer pairs that are
capable of hybridizing to the proximal sequences that define a
polymorphism in its double-stranded form.
[0152] In lieu of PCR, alternative methods, such as the "Ligase
Chain Reaction" ("LCR") may be used (Barany, Proc. Natl. Acad. Sci.
(U.S.A.) 88:189-193 (1991)). LCR uses two pairs of oligonucleotide
probes to exponentially amplify a specific target. The sequences of
each pair of oligonucleotides is selected to permit the pair to
hybridize to abutting sequences of the same strand of the target.
Such hybridization forms a substrate for a template-dependent
ligase. As with PCR, the resulting products thus serve as a
template in subsequent cycles and an exponential amplification of
the desired sequence is obtained.
[0153] LCR can be performed with oligonucleotides having the
proximal and distal sequences of the same strand of a polymorphic
site. In one embodiment, either oligonucleotide will be HE designed
to include the actual polymorphic site of the polymorphism. In such
an embodiment, the reaction conditions are selected such that the
oligonucleotides can be ligated together only if the target
molecule either contains or lacks the specific nucleotide that is
complementary to the polymorphic site present on the
oligonucleotide. Alternatively, the oligonucleotides may be
selected such that they do not include the polymorphic site (see,
Segev, PCT Application WO 90/01069).
[0154] The "Oligonucleotide Ligation Assay" ("OLA") may
alternatively be employed (Landegren et al., Science 241:1077-1080
(1988)). The OLA protocol uses two oligonucleotides that are
designed to be capable of hybridizing to abutting sequences of a
single strand of a target. OLA, like LCR, is particularly suited
for the detection of point mutations. Unlike LCR, however, OLA
results in "linear" rather than exponential amplification of the
target sequence.
[0155] Nickerson et al. have described a nucleic acid detection
assay that combines attributes of PCR and OLA (Nickerson et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990)). In this
method, PCR is used to achieve the exponential amplification of
target DNA, which is then detected using OLA. In addition to
requiring multiple, and separate, processing steps, one problem
associated with such combinations is that they inherit all of the
problems associated with PCR and OLA.
[0156] Schemes based on ligation of two (or more) oligonucleotides
in the presence of a nucleic acid having the sequence of the
resulting "di-oligonucleotide," thereby amplifying the
di-oligonucleotide, are also known (Wu et al., Genomics 4:560-569
(1989)), and may be readily adapted to the purposes of the present
invention.
[0157] Other known nucleic acid amplification procedures, such as
allele-specific oligomers, branched DNA technology,
transcription-based amplification systems, or isothermal
amplification methods may also be used to amplify and analyze such
polymorphisms (Malek et al., U.S. Pat. No. 5,130,238; Davey et al.,
European Patent Application 329,822; Schuster et al., U.S. Pat. No.
5,169,766; Miller et al., PCT Patent Application WO 89/06700; Kwoh,
et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173-1177 (1989);
Gingeras et al., PCT Patent Application WO 88/10315; Walker et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)).
[0158] Polymorphisms can also be identified by Single Strand
Conformation Polymorphism (SSCP) analysis. SSCP is a method capable
of identifying most sequence variations in a single strand of DNA,
typically between 150 and 250 nucleotides in length (Elles, Methods
in Molecular Medicine: Molecular Diagnosis of Genetic Diseases,
Humana Press (1996); Orita et al., Genomics 5: 874-879 (1989)).
Under denaturing conditions a single strand of DNA will adopt a
conformation that is uniquely dependent on its sequence
conformation. This conformation usually will be different, even if
only a single base is changed. Most conformations have been
reported to alter the physical configuration or size sufficiently
to be detectable by electrdphoresis. A number of protocols have
been described for SSCP including, but not limited to, Lee et al.,
Anal. Biochem. 205: 289-293 (1992); Suzuki et al., Anal. Biochem.
192: 82-84 (1991); Lo et al., Nucleic Acids Research 20: 1005-1009
(1992); Sarkar et al., Genomics 13:441-443 (1992). It is understood
that one or more of the nucleic acids of the present invention can
be utilized as markers or probes to detect polymorphisms by SSCP
analysis.
[0159] Polymorphisms may also be found using random amplified
polymorphic DNA (RAPD) (Williams et al., Nuc. Acids Res. 18:
6531-6535 (1990)) and cleaveable amplified polymorphic sequences
(CAPS) (Lyamichev et al., Science 260: 778-783 (1993)). It is
understood that one or more of the nucleic acid molecules of the
present invention can be utilized as markers or probes to detect
polymorphisms by RAPD or CAPS analysis.
[0160] The identification of a polymorphism can be determined in a
variety of ways. By correlating the presence or absence of it in a
plant with the presence or absence of a phenotype, it is possible
to predict the phenotype of that plant. If a polymorphism creates
or destroys a restriction endonuclease cleavage site, or if it
results in the loss or insertion of DNA (e.g., a variable
nucleotide tandem repeat (VNTR) polymorphism), it will alter the
size or profile of the DNA fragments that are generated by
digestion with that restriction endonuclease. As such, individuals
that possess a variant sequence can be distinguished from those
having the original sequence by restriction fragment analysis.
Polymorphisms that can be identified in this manner are termed
"restriction fragment length polymorphisms" ("RFLPs"). RFLPs have
been widely used in human and plant genetic analyses (Glassberg, UK
Patent Application 2135774; Skolnick et al., Cytogen. Cell Genet.
32:58-67 (1982); Botstein et al., Ann. J. Hum. Genet. 32:314-331
(1980); Fischer et al. (PCT Application WO90/13668); Uhlen, PCT
Application WO90/11369)).
[0161] A central attribute of "single nucleotide polymorphisms," or
"SNPs" is that the site of the polymorphism is at a single
nucleotide. SNPs have certain reported advantages over RFLPs and
VNTRs. First, SNPs are more stable than other classes of
polymorphisms. Their spontaneous mutation rate is approximately
10.sup.-9 (Komberg, DNA Replication, W.H. Freeman & Co., San
Francisco, 1980), approximately 1,000 times less frequent than
VNTRs (U.S. Pat. No. 5,679,524). Second, SNPs occur at greater
frequency, and with greater uniformity than RFLPs and VNTRs. As
SNPs result from sequence variation, new polymorphisms can be
identified by sequencing random genomic or cDNA molecules. SNPs can
also result from deletions, point mutations and insertions. Any
single base alteration, whatever the cause, can be an SNP. The
greater frequency of SNPs means that they can be more readily
identified than the other classes of polymorphisms.
[0162] SNPs and insertion/deletions can be detected by methods, by
any of a variety of methods including those disclosed in U.S. Pat.
Nos. 5,210,015; 5,876,930 and 6,030,787 in which an oligonucleotide
probe having reporter and quencher molecules is hybridized to a
target polynucleotide. The probe is degraded by 5'.fwdarw.3'
activity of a nucleic acid polymerase. A useful assay is available
from AB Biosystems (850 Lincoln Centre Drive, Foster City, Calif.)
as the Taqman.RTM. assay.
[0163] Specific nucleotide variations such as SNPs and
insertion/deletions can also be detected by labeled base extension
methods as disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;
5,595,890; 5,762,876; and 5,945,283. These methods are based on
primer extension and incorporation of detectable nucleoside
triphosphates. The primer is designed to anneal to the sequence
immediately adjacent to the variable nucleotide which can be can be
detected after incorporation of as few as one labeled nucleoside
triphosphate. U.S. Pat. No. 5,468,613 discloses allele specific
oligonucleotide hybridizations where single or multiple nucleotide
variations in nucleic acid sequence can be detected in nucleic
acids by a process in which the sequence containing the nucleotide
variation is amplified, spotted on a membrane and treated with a
labeled sequence-specific oligonucleotide probe.
[0164] Such methods also include the direct or indirect sequencing
of the site, the use of restriction enzymes where the respective
alleles of the site create or destroy a restriction site, the use
of allele-specific hybridization probes, the use of antibodies that
are specific for the proteins encoded by the different alleles of
the polymorphism or by other biochemical interpretation. SNPs can
be sequenced by a number of methods. Two basic methods may be used
for DNA sequencing, the chain termination method of Sanger et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 74: 5463-5467 (1977), and the
chemical degradation method of Maxam and Gilbert, Proc. Nat. Acad.
Sci. (U.S.A) 74: 560-564 (1977). Automation and advances in
technology such as the replacement of radioisotopes with
fluorescence-based sequencing have reduced the effort required to
sequence DNA (Craxton, Methods, 2: 20-26 (1991); Ju et al., Proc.
Natl. Acad. Sci. (U.S.A.) 92: 4347-4351 (1995); Tabor and
Richardson, Proc. Natl. Acad. Sci. (U.S.A.) 92: 6339-6343 (1995)).
Automated sequencers are available from, for example, Pharmacia
Biotech, Inc., Piscataway, N.J. (Pharmacia ALF), LI-COR, Inc.,
Lincoln, Nebr. (LI-COR 4,000) and Millipore, Bedford, Mass.
(Millipore BaseStation).
[0165] In addition, advances in capillary gel electrophoresis have
also reduced the effort required to sequence DNA and such advances
provide a rapid high resolution approach for sequencing DNA samples
(Swerdlow and Gesteland, Nucleic Acids Res. 18:1415-1419 (1990);
Smith, Nature 349:812-813 (1991); Luckey et al., Methods Enzymol.
218:154-172 (1993); Lu et al., J. Chromatog. A. 680:497-501 (1994);
Carson et al., Anal. Chem. 65:3219-3226 (1993); Huang et al., Anal.
Chem. 64:2149-2154 (1992); Kheterpal et al., Electrophoresis
17:1852-1859 (1996); Quesada and Zhang, Electrophoresis
17:1841-1851 (1996); Baba, Yakugaku Zasshi 117:265-281 (1997),
Marino, Appl. Theor. Electrophor. 5:1-5 (1995)).
[0166] The genetic linkage of marker molecules can be established
by a gene mapping model such as, without limitation, the flanking
marker model reported by Lander and Botstein, Genetics, 121:185-199
(1989), and the interval mapping, based on maximum likelihood
methods described by Lander and Botstein, Genetics, 121:185-199
(1989), and implemented in the software package MAPMAKER/QTL
(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits
Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,
Massachusetts, (1990). Additional software includes Qgene, Version
2.23 (1996), Department of Plant Breeding and Biometry, 266 Emerson
Hall, Cornell University, Ithaca, N.Y.). Use of Qgene software is a
particularly preferred approach.
[0167] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A logio of an odds ratio (LOD) is then
calculated as: LOD=log.sub.10 (MLE for the presence of a QTL(MLE
given no linked QTL).
[0168] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics, 121:185-199 (1989), and
further described by Arus and Moreno-Gonzdlez, Plant Breeding,
Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.
314-331 (1993).
[0169] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use of non-parametric methods (Kruglyak and Lander,
Genetics, 139:1421-1428 (1995)). Multiple regression methods or
models can be also be used, in which the trait is regressed on a
large number of markers (Jansen, Biometrics in Plant Breed, van
Oijen, Jansen (eds.) Proceedings of the Ninth Meeting of the
Eucarpia Section Biometrics in Plant Breeding, The Netherlands, pp.
116-124 (1994); Weber and Wricke, Advances in Plant Breeding,
Blackwell, Berlin, 16 (1994)). Procedures combining interval
mapping with regression analysis, whereby the phenotype is
regressed onto a single putative QTL at a given marker interval,
and at the same time onto a number of markers that serve as
`cofactors,` have been reported by Jansen and Stam, Genetics,
136:1447-1455 (1994) and Zeng, Genetics, 136:1457-1468 (1994).
Generally, the use of cofactors reduces the bias and sampling error
of the estimated QTL positions (Utz and Melchinger, Biometrics in
Plant Breeding, van Oijen, Jansen (eds.) Proceedings of the Ninth
Meeting of the Eucarpia Section Biometrics in Plant Breeding, The
Netherlands, pp. 195-204 (1994), thereby improving the precision
and efficiency of QTL mapping (Zeng, Genetics, 136:1457-1468
(1994)). These models can be extended to multi-environment
experiments to analyze genotype-environment interactions (Jansen et
al., Theo. Appl. Genet. 91:33-37 (1995)).
[0170] Selection of an appropriate mapping or segregation
populations is important to map construction. The choice of
appropriate mapping population depends on the type of marker
systems employed (Tanksley et al., Molecular mapping plant
chromosomes. Chromosome structure and function: Impact of new
concepts J. P. Gustafson and R. Appels (eds.), Plenum Press, New
York, pp. 157-173 (1988)). Consideration must be given to the
source of parents (adapted vs. exotic) used in the mapping
population. Chromosome pairing and recombination rates can be
severely disturbed (suppressed) in wide crosses (adapted x exotic)
and generally yield greatly reduced linkage distances. Wide crosses
will usually provide segregating populations with a relatively
large array of polymorphisms when compared to progeny in a narrow
cross (adapted x adapted).
[0171] As used herein, the progeny include not only, without
limitation, the products of any cross (be it a backcross or
otherwise) between two plants, but all progeny whose pedigree
traces back to the original cross. Specifically, without
limitation, such progeny include plants that have 12.5% or less
genetic material derived from one of the two originally crossed
plants. As used herein, a second plant is derived from a first
plant if the second plant's pedigree includes the first plant.
[0172] An F.sub.2 population is the first generation of selfing
after the hybrid seed is produced. Usually a single F.sub.1 plant
is selfed to generate a population segregating for all the genes in
Mendelian (1:2:1) fashion. Maximum genetic information is obtained
from a completely classified F.sub.2 population using a codorninant
marker system (Mather, Measurement of Linkage in Heredity: Methuen
and Co., (1938)). In the case of dominant markers, progeny tests
(e.g., F.sub.3, BCF.sub.2) are required to identify the
heterozygotes, thus making it equivalent to a completely classified
F.sub.2 population. However, this procedure is often prohibitive
because of the cost and time involved in progeny testing. Progeny
testing of F.sub.2 individuals is often used in map construction
where phenotypes do not consistently reflect genotype (e.g.,
disease resistance) or where trait expression is controlled by a
QTL. Segregation data from progeny test populations (e.g., F.sub.3
or BCF.sub.2) can be used in map construction. Marker-assisted
selection can then be applied to cross progeny based on
marker-trait map associations (F.sub.2, F.sub.3), where linkage
groups have not been completely disassociated by recombination
events (i.e., maximum disequilibrium).
[0173] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter et al., Proc. Natl. Acad. Sci.
(U.S.A.) 89:1477-1481 (1992)). However, as the distance between
markers becomes larger (i.e., loci become more independent), the
information in RIL populations decreases dramatically when compared
to codominant markers.
[0174] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992)). Information
obtained from backcross populations using either codominant or
dominant markers is less than that obtained from F.sub.2
populations because one, rather than two, recombinant gametes are
sampled per plant. Backcross populations, however, are more
informative (at low marker saturation) when compared to RELs as the
distance between linked loci increases in RIL populations (i.e.,
about 0.15% recombination). Increased recombination can be
beneficial for resolution of tight linkages, but may be undesirable
in the construction of maps with low marker saturation.
[0175] Near-isogenic lines (NIL) created by many backcrosses to
produce an array of individuals that are nearly identical in
genetic composition except for the trait or genomic region under
interrogation can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci are expected to map to
a selected region.
[0176] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore, et al., Proc. Natl. Acad. Sci. (U.S.A.)
88:9828-9832 (1991)). In BSA, two bulked DNA samples are drawn from
a segregating population originating from a single cross. These
bulks contain individuals that are identical for a particular trait
(resistant or sensitive to particular disease) or genomic region
but arbitrary at unlinked regions (i.e., heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
[0177] Plants generated using a method of the present invention can
be part of or generated from a breeding program. The choice of
breeding method depends on the mode of plant reproduction, the
heritability of the trait(s) being improved, and the type of
cultivar used commercially (e.g., F.sub.1 hybrid cultivar, pureline
cultivar, etc). Selected, non-limiting approaches, for breeding the
plants of the present invention are set forth below. A breeding
program can be enhanced using marker assisted selection of the
progeny of any cross. It is further understood that any commercial
and non-commercial cultivars can be utilized in a breeding program.
Factors such as, for example, emergence vigor, vegetative vigor,
stress tolerance, disease resistance, branching, flowering, seed
set, seed size, seed density, standability, and threshability etc.
will generally dictate the choice.
[0178] For highly heritable traits, a choice of superior individual
plants evaluated at a single location will be effective, whereas
for traits with low heritability, selection should be based on mean
values obtained from replicated evaluations of families of related
plants. Popular selection methods commonly include pedigree
selection, modified pedigree selection, mass selection, and
recurrent selection. In a preferred embodiment a backcross or
recurrent breeding program is undertaken.
[0179] The complexity of inheritance influences choice of the
breeding method. Backcross breeding can be used to transfer one or
a few favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes. The use of recurrent selection in self-pollinating
crops depends on the ease of pollination, the frequency of
successful hybrids from each pollination, and the number of hybrid
offspring from each successful cross.
[0180] Breeding lines can be tested and compared to appropriate
standards in environments representative of the commercial target
area(s) for two or more generations. The best lines are candidates
for new commercial cultivars; those still deficient in traits may
be used as parents to produce new populations for further
selection.
[0181] One method of identifying a superior plant is to observe its
performance relative to other experimental plants and to a widely
grown standard cultivar. If a single observation is inconclusive,
replicated observations can provide a better estimate of its
genetic worth. A breeder can select and cross two or more parental
lines, followed by repeated selfing and selection, producing many
new genetic combinations.
[0182] The development of new soybean cultivars requires the
development and selection of soybean varieties, the crossing of
these varieties and selection of superior hybrid crosses. The
hybrid seed can be produced by manual crosses between selected
male-fertile parents or by using male sterility systems. Hybrids
are selected for certain single gene traits such as pod color,
flower color, seed yield, pubescence color or herbicide resistance
which indicate that the seed is truly a hybrid. Additional data on
parental lines, as well as the phenotype of the hybrid, influence
the breeder's decision whether to continue with the specific hybrid
cross.
[0183] Pedigree breeding and recurrent selection breeding methods
can be used to develop cultivars from breeding populations.
Breeding programs combine desirable traits from two or more
cultivars or various broad-based sources into breeding pools from
which cultivars are developed by selfing and selection of desired
phenotypes. New cultivars can be evaluated to determine which have
commercial potential.
[0184] Pedigree breeding is used commonly for the improvement of
self-pollinating crops. Two parents who possess favorable,
complementary traits are crossed to produce an F.sub.1. An F.sub.2
population is produced by selfing one or several F.sub.1's.
Selection of the best individuals in the best families is
performed. Replicated testing of families can begin in the F.sub.4
generation to improve the effectiveness of selection for traits
with low heritability. At an advanced stage of inbreeding (i.e.,
F.sub.6 and F.sub.7), the best lines or mixtures of phenotypically
similar lines are tested for potential release as new
cultivars.
[0185] Backcross breeding has been used to transfer genes for a
simply inherited, highly heritable trait into a desirable
homozygous cultivar or inbred line, which is the recurrent parent.
The source of the trait to be transferred is called the donor
parent. The resulting plant is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent. After the initial cross,
individuals possessing the phenotype of the donor parent are
selected and repeatedly crossed (backcrossed) to the recurrent
parent. The resulting parent is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent.
[0186] The single-seed descent procedure in the strict sense refers
to planting a segregating population, harvesting a sample of one
seed per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F.sub.2
to the desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2 individuals. The
number of plants in a population declines each generation due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
[0187] In a multiple-seed procedure, soybean breeders commonly
harvest one or more pods from each plant in a population and thresh
them together to form a bulk. Part of the bulk is used to plant the
next generation and part is put in reserve. The procedure has been
referred to as modified single-seed descent or the pod-bulk
technique.
[0188] The multiple-seed procedure has been used to save labor at
harvest. It is considerably faster to thresh pods with a machine
than to remove one seed from each by hand for the single-seed
procedure. The multiple-seed procedure also makes it possible to
plant the same number of seeds of a population each generation of
inbreeding.
[0189] Descriptions of other breeding methods that are commonly
used for different traits and crops can be found in one of several
reference books (e.g., Fehr, Principles of Cultivar Development
Vol. 1, pp. 2-3 (1987)).
[0190] In a preferred aspect of the present invention the source of
the rhg1 SCN resistant allele for use in a breeding program is
derived from a plant selected from the group consisting of PI548402
(Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4), PI404166
(Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654 (Er-hej-jan),
PI438489 (Chiquita), PI507354 (Tokei 421), PI548655 (Forrest),
PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid, Nathan,
AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501, AG4601,
PION9492, PI88788, Dyer, Custer, Manokin, Doles, and SCN resistant
progeny thereof (USDA, Soybean Germplasm Collection, University of
Illinois, Ill.). In a more preferred aspect, the source of the rhg1
SCN resistant allele for use in a breeding program is derived from
a plant selected from the group consisting of PI548402 (Peking),
PI404166 (Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654
(Er-hej-jan), PI438489 (Chiquita), PI507354 (Tokei 421), PI548655
(Forrest), PI548988 (Pickett), PI84751, PI437654, PI40792, and SCN
resistant progeny thereof.
[0191] In a preferred aspect of the present invention the source of
the rhg1 SCN sensitive allele for use in a breeding program is
derived from a plant selected from the group consisting of A3244,
A2833, AG3001, Williams, Will, A2704, Noir, DK23-5 1, Lee 74,
Essex, Minsoy, A1923, Hutcheson, and SCN sensitive progeny thereof.
In a more preferred aspect, the source of the rhg1 SCN sensitive
allele for use in a breeding program is derived from an A3244
plant, and SCN sensitive progeny thereof.
[0192] In a preferred aspect of the present invention the source of
the Rhg4 SCN resistant allele for use in a breeding program is
derived from a plant selected from the group consisting of PI548402
(Peking), PI437654 (Er-hejjan), PI438489 (Chiquita), PI507354
(Tokei 421), PI548655 (Forrest), PI548988 (Pickett), PI88788,
PI404198 (Sun Huan Do), PI404166 (Krasnoaarmejkaja), Hartwig,
Manokin, Doles, Dyer, Custer, and SCN resistant progeny thereof. In
a more preferred aspect, the source of the Rhg4 SCN resistant
allele for use in a breeding program is derived from a plant
selected from the group consisting of PI548402 (Peking), PI88788,
PI404198 (Sun huan do), PI438489 (Chiquita), PI437654 (Er-hej-jan),
PI404166 (Krasnoaarmejkaja), PI548655 (Forrest), PI548988
(Pickett), PI507354 (Tokei 421), and SCN resistant progeny
thereof.
[0193] In a preferred aspect of the present invention the source of
the Rhg4 SCN sensitive allele for use in a breeding program is
derived from a plant selected from the group consisting of A3244,
Will, Noir, Lee 74, Essex, Minsoy, A2704, A2833, AG3001, Williams,
DK23-51, and Hutcheson, and SCN sensitive progeny thereof. In a
more preferred aspect, the source of the Rhg4 SCN sensitive allele
for use in a breeding program is derived from an A3244 plant, and
SCN sensitive progeny thereof.
[0194] As used herein linkage of a nucleic acid sequence with
another nucleic acid sequence may be genetic or physical. In a
preferred embodiment, a nucleic acid marker is genetically linked
to either rhg1 or Rhg4, where the marker nucleic acid molecule
exhibits a LOD score of greater than 2.0, as judged by interval
mapping, for SCN resistance or partial resistance, preferably where
the marker nucleic acid molecule exhibits a LOD score of greater
than 3.0, as judged by interval mapping, for SCN resistance or
partial resistance, more preferably where the marker nucleic acid
molecule exhibits a LOD score of greater than 3.5, as judged by
interval mapping, for SCN resistance or partial resistance and even
more preferably where the marker nucleic acid molecule exhibits a
LOD score of about 4.0, as judged by interval mapping, for SCN
resistance or partial resistance based on maximum likelihood
methods described by Lander and Botstein, Genetics, 121:185-199
(1989), and implemented in the software package MAPMAKER/QTL
(default parameters)(Lincoln and Lander, Mapping Genes Controlling
Quantitative Traits Using MAPMAKER/QTL, Whitehead Institute for
Biomedical Research, Massachusetts, (1990)).
[0195] In another embodiment the nucleic acid molecule may be
physically linked to either rhg1 or Rhg4. In a preferred
embodiment, the nucleic acid marker specifically hybridizes to a
nucleic acid molecule having a sequence that is present on linkage
group G within 500 kb or 100 kb, more preferably within 50 kb, even
more preferably within 25 kb of an rhg1 allele, where the rgh1
allele is preferably a sensitive allele, and more preferably a
sensitive allele from A3244. In a preferred embodiment the nucleic
acid marker is capable of specifically hybridizing to a nucleic
acid molecule having a sequence that is present on linkage group A2
within 500 kb or 100 kb, more preferably within 50 kb, even more
preferably within 25 kb of an Rhg4 allele, where the Rgh4 allele is
preferably a sensitive allele, and more preferably a sensitive
allele from A3244.
[0196] The present invention provides a method of investigating an
rhg1 haplotype of a soybean plant comprising: (A) isolating nucleic
acid molecules from the soybean plant; (B) determining the nucleic
acid sequence of an rhg1 allele or part thereof; and, (C) comparing
the nucleic acid sequence of the rhg1 allele or part thereof to a
reference nucleic acid sequence.
[0197] As used herein, the term "investigating" refers to any
method capable of detecting a feature, such as a polymorphism or
haplotype. Nucleic acid molecules only need to be isolated from a
soybean plant to the degree of purity necessary for the task
required or to a greater purity if desired. A person of ordinary
skill in the art has available techniques to isolate nucleic acid
molecules from plants to a sufficient purity, for example without
limitation, to sequence the desired region of the nucleic acid
molecule or to carry out a marker assay.
[0198] The determination of an rhg1 or Rhg4 allele or part thereof
may be carried out using any technique. Illustration of such
techniques include techniques that provide the nucleic acid
sequence for an rhg1 or rhg4 allele or part thereof include
amplification of a desired allele or part thereof (see, for
example, the Examples and SEQ ID NOs: 8-53). In a preferred
embodiment, the nucleic acid sequence determined is that of an exon
of an rhg1 allele, more preferably exon 1 or exon 3 of an rhg1
allele, or of an LRR domain. In another preferred embodiment, a
single nucleotide is determined. In another preferred embodiment,
the nucleic acid sequence determined is that of an LRR domain.
[0199] A comparison of a sequence with a reference sequence can be
carried out with any appropriate sequence comparison method.
[0200] As used herein, a reference sequence is any rhg1 allele
sequence or consensus sequence. A reference sequence may be a
nucleic acid sequence or an amino acid sequence. In a preferred
embodiment, the reference sequence is any SCN resistant rhg1 allele
sequence. In a further preferred embodiment, the rhg1 reference
sequence is selected from the group consisting of SEQ ID NOs: 2, 3,
5, 6, 8-23, 28-43, 1097, 1098, and 1100-1115.
[0201] The present invention provides a method of investigating an
Rhg4 haplotype of a soybean plant comprising: (A) isolating nucleic
acid molecules from the soybean plant; (B) determining the nucleic
acid sequence of an Rhg4 allele or part thereof; and (C) comparing
the nucleic acid sequence of the Rhg4 allele or part thereof to a
reference nucleic acid sequence.
[0202] As used herein, a reference sequence is any Rhg4 allele
sequence or consensus sequence. A reference sequence ma be a
nucleic acid sequence or an amino acid sequence. In a preferred
embodiment, the reference sequence is any SCN resistant Rhg4 allele
sequence. In a further preferred embodiment, the Rhg4 reference
sequence is selected from the group consisting of SEQ ID NOs: 4, 7,
44-47, 50-53, 1099, and 1116-1119.
[0203] The present invention provides a method of introgressing SCN
resistance or partial SCN resistance into a soybean plant
comprising: performing marker assisted selection of the soybean
plant with a nucleic acid marker, wherein the nucleic acid marker
specifically hybridizes with a nucleic acid molecule having a first
nucleic acid sequence that is physically linked to a second nucleic
acid sequence that is located on linkage group G of soybean A3244,
wherein the second nucleic acid sequence is within 500 kb of a
third nucleic acid sequence which is capable of specifically
hybridizing with the nucleic acid sequence of SEQ ID NO: 5, 6,
complements thereof, or fragments thereof; and, selecting the
soybean plant based on the marker assisted selection.
[0204] The present invention provides a method of introgressing SCN
resistance or partial SCN resistance into a soybean plant
comprising: performing marker assisted selection of the soybean
plant with a nucleic acid marker, wherein the nucleic acid marker
specifically hybridizes with a nucleic acid molecule having a first
nucleic acid sequence that is physically linked to a second nucleic
acid sequence that is located on linkage group A2 of soybean A3244,
wherein the second nucleic acid sequence is within 500 kb of a
third nucleic acid sequence which is capable of specifically
hybridizing with the nucleic acid sequence of SEQ ID NO: 7,
complements thereor, or fragments thereof; and, selecting the
soybean plant based on the marker assisted selection. Marker
assisted introgression of traits into plants has been reported.
Marker assisted introgression involves the transfer of a chromosome
region defined by one or more markers from one germplasm to a
second germplasm. In a preferred embodiment the introgression is
carried out by backcrossing with an rhg1 or Rhg4 SCN resistant
soybean recurrent parent.
[0205] In light of the current disclosure, plant introductions and
germplasm can be screened with a marker nucleic acid molecule of
the present invention to screen for alleles of rhg1 or Rhg4 using
one or more of techniques disclosed herein or known in the art.
[0206] The present invention also provides for parts of the plants
produced by a method of the present invention. Plant parts, without
limitation, include seed, endosperm, ovule and pollen. In a
particularly preferred embodiment of the present invention, the
plant part is a seed.
[0207] Plants or parts thereof produced by a method of the present
invention may be grown in culture and regenerated. Methods for the
regeneration of soybean plants from various tissue types and
methods for the tissue culture of soybean are known in the art
(See, for example, Widholm et al., In Vitro Selection and
Culture-induced Variation in Soybean, In Soybean: Genetics,
Molecular Biology and Biotechnology, Eds. Verma and Shoemaker, CAB
International, Wallingford, Oxon, England (1996)). Regeneration
techniques for plants such as soybean can use as the starting
material a variety of tissue or cell types. With soybean in
particular, regeneration processes have been developed that begin
with certain differentiated tissue types such as meristems, Cartha
et al., Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections,
Cameya et al., Plant Science Letters 21: 289-294 (1981), and stem
node segments, Saka et al., Plant Science Letters, 19: 193-201
(1980); Cheng et al., Plant Science Letters, 19: 91-99 (1980).
Regeneration of whole sexually mature soybean plants from somatic
embryos generated from explants of immature soybean embryos has
been reported (Ranch et al., In Vitro Cellular & Developmental
Biology 21: 653-658 (1985). Regeneration of mature soybean plants
from tissue culture by organogenesis and embryogenesis has also
been reported (Barwale et al., Planta 167: 473-481 (1986); Wright
et al., Plant Cell Reports 5: 150-154 (1986)).
Agents
[0208] One skilled in the art can refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Current Protocols in
Molecular Biology Ausubel, et al., eds., John Wiley & Sons,
N.Y. (1989), and supplements through September (1998), Molecular
Cloning, A Laboratory Manual, Sambrook et al., 2.sup.nd Ed., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Genome
Analysis: A Laboratory Manual 1: Analyzing DNA, Birren et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1997); Genome
Analysis: A Laboratory Manual 2: Detecting Genes, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1998); Genome
Analysis: A Laboratory Manual 3: Cloning Systems, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); Genome
Analysis: A Laboratory Manual 4: Mapping Genomes, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); Plant
Molecular Biology: A Laboratory Manual, Clark, Springer-Verlag,
Berlin, (1997), Methods in Plant Molecular Biology, Maliga et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1995). These
texts can, of course, also be referred to in making or using an
aspect of the invention. It is understood that any of the agents of
the invention can be substantially purified and/or be biologically
active and/or recombinant. (a) Nucleic Acid Molecules
[0209] Nucleic acid molecules of the present invention include,
without limitation, nucleic acid molecules having a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1-1096
and complements thereof. A subset of the nucleic acid molecules of
the present invention includes nucleic acid molecules that encode a
protein or fragment thereof. Another subset of the nucleic acid
molecules of the present invention are cDNA molecules. Another
subset of the nucleic acid molecules of the present invention
includes nucleic acid molecules that are marker molecules. A
further subset of the nucleic acid molecules of the present
invention are those nucleic acid molecules having promoter
sequences.
[0210] Fragment nucleic acid molecules may comprise significant
portion(s) of, or indeed most of, these nucleic acid molecules. In
preferred embodiments, the fragments may comprise smaller
polynucleotides, e.g., oligonucleotides having from about 20 to
about 250 nucleotide residues and more preferably, about 20 to
about 100 nucleotide residues, or about 40 to about 60 nucleotide
residues. In another preferred embodiment, fragment molecules may
be at least 15 nucleotides, at least 30 nucleotides, at least 50
nucleotides, or at least 100 nucleotides.
[0211] The term "substantially purified," as used herein, refers to
a molecule separated from substantially all other molecules
normally associated with it in its native state. More preferably a
substantially purified molecule is the predominant species present
in a preparation. A substantially purified molecule may be greater
than 60% free, preferably 75% free, more preferably 90% free, and
most preferably 95% free from the other molecules (exclusive of
solvent) present in the natural mixture. The term "substantially
purified" is not intended to encompass molecules present in their
native state.
[0212] The agents of the present invention will preferably be
"biologically active" with respect to either a structural
attribute, such as the capacity of a nucleic acid to hybridize to
another nucleic acid molecule, or the ability of a protein to be
bound by an antibody (or to compete with another molecule for such
binding). Alternatively, such an attribute may be catalytic and
thus involve the capacity of the agent to mediate a chemical
reaction or response.
[0213] The agents of the present invention may also be recombinant.
As used herein, the term recombinant describes (a) nucleic acid
molecules that are constructed or modified outside of cells and
that can replicate or function in a living cell, (b) molecules that
result from the transcription, replication or translation of
recombinant nucleic acid molecules, or (c) organisms that contain
recombinant nucleic acid molecules or are modified using
recombinant nucleic acid molecules.
[0214] It is understood that the agents of the present invention
may be labeled with reagents that facilitate detection of the
agent, e.g., fluorescent labels, (Prober et al., Science
238:336-340 (1987); Albarella et al., EP 144914), chemical labels,
(Sheldon et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S.
Pat. No. 4,563,417), and modified bases, (Miyoshi et al., EP
119448) including nucleotides with radioactive elements, e.g.,
.sup.32P, .sup.33P, 35S or .sup.125I, such as .sup.32P dCTP.
[0215] It is further understood, that the present invention
provides recombinant bacterial, animal, fungal and plant cells and
viral constructs comprising the agents of the present
invention.
[0216] Nucleic acid molecules or fragments thereof of the present
invention are capable of specifically hybridizing to other nucleic
acid molecules under certain circumstances. As used herein, two
nucleic acid molecules are said to be capable of specifically
hybridizing to one another if the two molecules are capable of
forming an anti-parallel, double-stranded nucleic acid structure. A
nucleic acid molecule is said to be the "complement" of another
nucleic acid molecule if they exhibit "complete complementarity,"
i.e., each nucleotide in one sequence is complementary to its base
pairing partner nucleotide in another sequence. Two molecules are
said to be "minimally complementary" if they can hybridize to one
another with sufficient stability to permit them to remain annealed
to one another under at least conventional "low-stringency"
conditions. Similarly, the molecules are said to be "complementary"
if they can hybridize to one another with sufficient stability to
permit them to remain annealed to one another under conventional
"high-stringency" conditions. Nucleic acid molecules which
hybridize to other nucleic acid molecules, e.g., at least under low
stringency conditions are said to be "hybridizable cognates" of the
other nucleic acid molecules. Conventional stringency conditions
are described by Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(1989) and by Haymes et al., Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, D.C. (1985). Departures
from complete complementarity are therefore permissible, as long as
such departures do not completely preclude the capacity of the
molecules to form a double-stranded structure. Thus, in order for a
nucleic acid molecule to serve as a primer or probe it need only be
sufficiently complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0217] Appropriate stringency conditions which promote DNA
hybridization, for example, 6.0.times.sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed.
[0218] In a preferred embodiment, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 1096 or
complements thereof under moderately stringent conditions, for
example at about 2.0.times.SSC and about65.degree. C.
[0219] In a particularly preferred embodiment, a nucleic acid of
the present invention will include those nucleic acid molecules
that specifically hybridize to one or more of the nucleic acid
molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 1096 or
complements thereof under high stringency conditions such as
0.2.times.SSC and about 65.degree. C.
[0220] In one aspect of the present invention, the nucleic acid
molecules of the present invention comprise one or more of the
nucleic acid sequences set forth in SEQ ID NO: 1 through SEQ ID NO:
1096 or complements thereof or fragments of either. In another
aspect of the present invention, one or more of the nucleic acid
molecules of the present invention share at least 60% sequence
identity with one or more of the nucleic acid sequences set forth
in SEQ ID NO: 1 through SEQ ID NO: 1096 or complements thereof or
fragments of either. In a further aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
share at least 70% or more, e.g., at least 80%, sequence identity
with one or more of the nucleic acid sequences set forth in SEQ ID
NO: 1 through SEQ ID NO: 1096 or complements thereof or fragments
of either. In a more preferred aspect of the present invention, one
or more of the nucleic acid molecules of the present invention
share at least 90% or more, e.g., at least 95% and up to 100%
sequence identity with one or more of the nucleic acid sequences
set forth in SEQ ID NO: 1 through SEQ ID NO: 1096 complements
thereof or fragments of either.
[0221] As used herein "sequence identity" refers to the extent to
which two optimally aligned polynucleotide or peptide sequences are
invariant throughout a window of alignment of components, e.g.,
nucleotides or amino acids. An "identity fraction" for aligned
segments of a test sequence and a reference sequence is the number
of identical components which are shared by the two aligned
sequences divided by the total number of components in reference
sequence segment, i.e., the entire reference sequence or a smaller
defined part of the reference sequence. "Percent identity" is the
identity fraction times 100.
[0222] Useful methods for determining sequence identity are
disclosed in Guide to Huge Computers, Martin J. Bishop, ed.,
Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D.,
SIAM J Applied Math (1988) 48:1073. More particularly, preferred
computer programs for determining sequence identity include the
Basic Local Alignment Search Tool (BLAST) programs which are
publicly available from National Center Biotechnology Information
(NCBI) at the National Library of Medicine, National Institute of
Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al.,
NCBI, NLM, NIH; Altschul et al., J. Mol. Biol. 215:403-410 (1990);
version 2.0 or higher of BLAST programs allows the introduction of
gaps (deletions and insertions) into alignments; BLASTX can be used
to determine sequence identity between a polynucleotide sequence
query and a protein sequence database; and, BLASTN can be used to
determine sequence identity between between sequences.
[0223] For purposes of this invention "percent identity" shall be
determined using BLASTX version 2.0.14 (default parameters), BLASTN
version 2.0.14, or BLASTP 2.0.14.
[0224] A particularly preferred group of nucleic acid sequences are
those present in the soybean insert of the clones set forth in
table 6 below. TABLE-US-00008 TABLE 6 Names of Clones Containing
the Specified Gene Line Rhg4 rhg1/frag 1 rhg1/frag 2 Forrest
Forrest 1 Forrest 7 Forrest13 Peking Peking 1 Peking 7 Peking 13
Pickett Pickett 1 Pickett 7 Pickett 13 PI84751 PI 84751.1 PI
84751.7 PI 84751.13 PI87631 PI 87631.1 PI 87631.7 PI 87631.13
PI87631-1 PI 87631-1.1 PI 87631-1.13 PI88788R PI88788R.1 PI
88788R.7 PI 88788R.13 PI89772 PI 89772.13 PI90763 PI 90763.7 PI
90763.13 PI200499 PI 200499.1 PI 200499.7 PI 200499.13 PI209332 PI
209332.1 PI 209332.13 PI404166 PI 404166.1 PI 404166.7 PI 404166.13
PI404198A PI 404198A.7 PI 404198A.13 PI404198B PI 404198B.1 PI
404198B.7 PI404198B.13 PI437654 PI 437654.1 PI 437654.7 PI
437654.13 PI438489B PI 438489.1 PI 438489.7 PI 438489B.13 PI467312
PI 467312.1 PI 467312.7 PI 467312.13 PI507354 PI 507354.1 PI
507354.7 PI 507354.13 PI423871 PI 423871.1 PI 423871.7 PI 423871.13
PI407922 PI 407922.7 PI 407922.13 PI360843 PI 360843.1 PI 360843.7
PI 360843.13 A2869 A2869.1 A2869.7 A2869.13 A2069 A2069.1 A2069.13
Jack JACK1 JACK13 Will WILL1 WILL.7 WILL13 Minsoy Minsoy1 Minsoy.7
MINSOY13 Noir Noir1 Noir.7 NOIR13 Hutcheson Hutcheson1 Hutcheson.7
Hutcheson.13 A1923 A1923.1 A1923.7 A1923.13 A2704 A2704.7 A2704.13
Essex Essex1 Essex.7 ESSEX13 A3244 A3244.1 A3244.7 A3244.13 Lee74
Lee74.1 Lee74.7 Lee74.13 PI437654 R107C17.7 R107C17.13
[0225] Table 5 shows clones comprising rhg1 and Rhg4 sequences. The
"Lines" column indicates the cultivar from which the sequence in
the clone is derived. The Rhg4, rhg1/frag1, and rhg1/frag2 columns
show the clones derived from the lines that have the Rhg4, rhg1
fragment 1, or rhg1 fragment 2, respectively. Rhg4 is amplified
with SEQ ID NOs: 48 and 49, which produces a 3.5 kb product. rhg1
fragment 1 is amplified with SEQ ID NOs: 24 and 25, which produces
a 2.9 kb product, and rhg1 fragment 2 is amplified with SEQ ID NOs:
26 and 27, which produces a 1.75 kb product. All fragments are
subcloned into a pCR4-TOPO vector. (i) Nucleic Acid Molecules
Encoding Proteins or Fragments Thereof
A) rhg1
[0226] The present invention includes nucleic acid molecules that
code for an rhg1 protein or fragment thereof. Examples of such
nucleic acid molecules include those that code for the proteins set
forth in SEQ ID NOs: 1097, 1100, 1098, 1101, and 1102-1115.
Examples of illustrative fragment molecules include, without
limitation, an extracellular LRR domain (rhg1, v.1, SEQ ID NO:
1097, residues 164-457; rhg1, v.2, SEQ ID NO: 1098, residues
141-434), a transmembrane domain (rhg1, v.1, SEQ ID NO: 1097,
residues 508-530; rhg1, v.2, SEQ ID NO: 1098, residues 33-51 and
507), and an STK domain (rhg1, v.1, SEQ ID NO: 1097, residues
578-869; rhg1, v.2, SEQ ID NO: 1098, residues 555-846). Examples of
illustrative nucleic acid molecules include SEQ ID NOs: 5, 6, 8-23,
and 28-43.
B) Rhg4
[0227] The present invention includes nucleic acid molecules that
code for an Rhg4 protein or fragment thereof. Examples of such
nucleic acid molecules include those that code for the proteins set
forth in SEQ ID NOs: 1099 and 1116-1119. Examples of illustrative
fragment molecules include, without limitation, an extracellular
LRR domain (SEQ ID NO: 1099, residues 34-44), a transmembrane
domain (SEQ ID NO: 1099, residues 449-471), and an STK domain (SEQ
ID NO: 1099, residues 531-830). Examples of illustrative nucleic
acid molecules include SEQ ID NOs: 7, 44-47, and 50-53.
C) Rhg1 and Rhg4
[0228] In another further aspect of the present invention, nucleic
acid molecules of the present invention can comprise sequences
which differ from those encoding a protein or fragment thereof in
SEQ ID NO: 1097 through SEQ ID NO: 1119 due to fact that the
different nucleic acid sequence encodes a protein having one or
more conservative amino acid changes. It is understood that codons
capable of coding for such conservative amino acid substitutions
are known in the art.
[0229] It is well known in the art that one or more amino acids in
a native sequence can be substituted with another amino acid(s),
the charge and polarity of which are similar to that of the native
amino acid, i.e., a conservative amino acid substitution. Conserved
substitutions for an amino acid within the native polypeptide
sequence can be selected from other members of the class to which
the naturally occurring amino acid belongs. Amino acids can be
divided into the following four groups: (1) acidic amino acids, (2)
basic amino acids, (3) neutral polar amino acids, and (4) neutral
nonpolar amino acids. Representative amino acids within these
various groups include, but are not limited to: (1) acidic
(negatively charged) amino acids such as aspartic acid and glutamic
acid; (2) basic (positively charged) amino acids such as arginine,
histidine, and lysine; (3) neutral polar amino acids such as
glycine, serine, threonine, cysteine, cystine, tyrosine,
asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic)
amino acids such as alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine.
[0230] Conservative amino acid changes within the native
polypeptides sequence can be made by substituting one amino acid
within one of these groups with another amino acid within the same
group. Biologically functional equivalents of the proteins or
fragments thereof of the present invention can have ten or fewer
conservative amino acid changes, more preferably seven or fewer
conservative amino acid changes, and most preferably five or fewer
conservative amino acid changes. The encoding nucleotide sequence
will thus have corresponding base substitutions, permitting it to
encode biologically functional equivalent forms of the proteins or
fragments of the present invention.
[0231] It is understood that certain amino acids may be substituted
for other amino acids in a protein structure without appreciable
loss of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Because it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence and, of course, its underlying DNA
coding sequence and, nevertheless, obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the peptide sequences of the proteins or
fragments of the present invention, or corresponding DNA sequences
that encode said peptides, without appreciable loss of their
biological utility or activity. It is understood that codons
capable of coding for such amino acid changes are known in the
art.
[0232] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, J. Mol. Biol.
157, 105-132 (1982). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction of
the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like. In making such
changes, the substitution of amino acids whose hydropathic indices
are within .+-.2 is preferred, those which are within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0233] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, states that the greatest
local average hydrophilicity of a protein, as govern by the
hydrophilicity of its adjacent amino acids, correlates with a
biological property of the protein. In a further aspect of the
present invention, one or more of the nucleic acid molecules of the
present invention differ in nucleic acid sequence from those
encoding a peptide set forth in SEQ ID NO: 1097 through SEQ ID NO:
1119 or fragment thereof due to the fact that one or more codons
encoding an amino acid has been substituted for a codon that
encodes a nonessential substitution of the amino acid originally
encoded.
[0234] Agents of the invention include nucleic acid molecules that
encode at least about a contiguous 10 amino acid region of a
protein of the present invention, more preferably at least about a
contiguous 11 to 14 or larger amino acid region of a protein of the
present invention. It is understood that the present invention
includes nucleic acid molecules that specifically hybridize or
exhibit a particular identity to the nucleic acid molecules
described in (i). See (a) above.
[0235] (ii) Nucleic Acid Molecule Markers and Collections of Such
Molecules
[0236] One aspect of the present invention concerns nucleic acid
molecules of the present invention that can act as markers. As used
herein, a "marker" is an indicator for the presence of at least one
phenotype or polymorphism, such as single nucleotide polymorphisms
(SNPs), cleaveable amplified polymorphic sequences (CAPS),
amplified fragment length polymorphisms (AFLPs), restriction
fragment length polymorphisms (RFLPs), simple sequence repeats
(SSRs), or random amplified polymorphic DNA (RAPDs). A "nucleic
acid marker" as used herein means a nucleic acid molecule that is
capable of being a marker for detecting a polymorphism or
phenotype.
[0237] In one embodiment of the present invention, the nucleic acid
marker specifically hybridizes to a nucleic acid molecule having a
nucleic acid sequence selected from the group SEQ NOs: 1-1096 and
complements thereof. In a preferred embodiment, the nucleic acid
marker is capable of detecting an rhg1 SNP or INDEL set forth in
table 2. In a preferred embodiment, the nucleic acid marker is
capable of detecting an Rgh4 SNP or INDEL set forth in table 4. In
another preferred embodiment the nucleic acid marker is a nucleic
acid molecule capable of acting as a PCR primer to amplify an rhg1
or Rhg4 coding region. Examples of such primers include, without
limitation, nucleic acid molecules having a nucleic acid sequence
set forth in SEQ ID NO: 401-1096 and complements thereof. Such
primers can be used in pairs to amplify a region (amplicons, e.g.,
without limitation, SEQ ID NOs: 54-400) that can be further
investigated using techniques known in the art such as nucleic acid
sequencing. Preferred pairs are those with identical "Seq ID" (see
Description of the Sequence Listing) except for the fact that one
"Seq ID" recites forward primer and one recites reverse primer.
[0238] In another embodiment of the present invention, the nucleic
acid marker specifically hybridizes to a nucleic acid molecule
having a sequence that is present on linkage group G within 500 kb
or 100 kb, more preferably within 50 kb, even more preferably
within 25 kb of an rhg1 allele, where the Rgh4 allele is preferably
a sensitive allele, and more preferably a sensitive allele from
A3244. In a preferred embodiment the nucleic acid marker
specifically hybridizes to M5 a nucleic acid molecule having a
sequence that is present on linkage group A2 within 500 kb or 100
kb, more preferably within 50 kb, even more preferably within 25 kb
of an Rhg4 allele, where the Rgh4 allele is preferably a sensitive
allele, and more preferably a sensitive allele from A3244.
[0239] As used herein, a "collection of nucleic acid molecules" is
a population of nucleic acid molecules where at least two,
preferably all, of the nucleic acid molecules differ, at least in
part, in their nucleic acid sequence. It is understood, that as
used herein, an individual species within a collection of nucleic
acid molecules may be physically separate or alternatively not
physically separate from one or more other species within the
collection of nucleic acid molecules. An example of a situation
where individual species may be physically separate but considered
a collection of nucleic acid molecules is where more than two
species are present in a single location such as an array.
[0240] As used herein, where a collection of nucleic acid molecules
is a marker for a particular attribute, the level, pattern,
occurrence and/or absence of the nucleic acid molecules associated
with the attribute are not required to be the same between species
of the collection. For example, the increase in the level of a
species when in combination with the decrease in a second species
could be diagnostic for a particular attribute. In a preferred
embodiment of the present invention, the level, pattern, occurrence
and/or absence of a nucleic acid molecule and/or collection of
nucleic acid molecules of the present invention is a marker for SCN
resistance.
[0241] In one embodiment, the marker is any nucleic acid molecule
that specifically hybridizes to any nucleic acid sequence set forth
herein. In another embodiment, the marker is a marker capable of
distinguishing among the haplotypes of either rhg1 or Rhg4. In yet
another embodiment, more than one marker is used to simultaneously
distinguish more than one haplotype. In a preferred embodiment,
two, three, four, six, eight, twenty five or fifty or more nucleic
acid markers are used simultaneously. In another embodiment, one or
more markers that are capable of distinguishing among the
haplotypes of rhg1 and one or more markers that are capable of
distinguishing among the haplotypes of Rhg4 are used together.
[0242] (iii) Nucleic Acid Molecules Having Promoter Sequences and
Other Regulatory Sequences
[0243] The present invention includes nucleic acid molecules that
are an rhg1 or Rhg4 promoter or fragment thereof. Examples of such
nucleic acid molecules include those set forth in SEQ ID NO: 2,
upstream of coordinate 45163 and SEQ ID NO: 3, upstream of
coordinate 46798. As used herein a promoter is a nucleic acid
sequence that when joined with a coding region is capable of
expressing the protein or fragment thereof so encoded. In a
preferred embodiment the promoter sequence corresponds to between
500 nucleotides and 5,000 nucleotides or between 300 nucleotides
and 700 nucleotides of the nucleic acid sequence set forth in SEQ
ID NO: 2 between coordinates 45163 and 40163, or SEQ ID NO:3
between coordinates 46798 and 41798, or the nucleic acid sequence
set forth in SEQ ID NO: 4 between coordinates 111805 and 106805
Preferred partial promoter regions include the TATA box region,
e.g. at coordinates 44234 through 44246 of SEQ ID NO: 2 and at
coordinates 107826 through 107835 of SEQ ID NO: 4, and CAAT box
region, e.g. at coordinates 106243 through 106259 of SEQ ID NO:
4.
[0244] Other regulatory sequences include introns or 3'
untranslated regions (3'UTRs) associated with rhg1 and Rhg4. In a
preferred embodiment, an intron is selected from a nucleic acid
comprising a sequence selected from SEQ ID NO: 2 (rhg1 v.1 at
coordinates 45315-45449, 45510-46940, and 48764-48974), SEQ ID NO:
3 (rhg1 v.2 at coordinates 48764-48974) and SEQ ID NO: 4 (Rhg4 at
coordinates 113969-114683). In another preferred embodiment, a
3'UTR is located within 5,000 nucleotides, more preferable within
1000 nucleotides in the 3' direction of the last coding nucleotide
of either rhg1 or Rhg4 (SEQ ID NO: 2, rhg1 v.1, coordinate 49573,
SEQ ID NO: 3, rhg1, v.2, coordinate 49573, SEQ ID NO: 4, Rhg4,
coordinate 115204).
[0245] It is understood that the present invention includes nucleic
acid molecules that specifically hybridize or exhibit a particular
identity to the nucleic acid molecules described in (iii). See (a)
above.
[0246] (b) Protein and Peptide Molecules
[0247] A class of agents comprises one or more of the protein or
peptide molecules encoded by SEQ ID NO: 1097 through SEQ ID NO:
1119 or one or more of the protein or fragment thereof or peptide
molecules encoded by other nucleic acid agents of the present
invention. As used herein, the term "protein molecule" and "peptide
molecule" mean any protein or protein fragment or peptide or
polypeptide molecule that comprises ten or more amino acids,
preferably at least 1I1 or 12 or more, more preferably at least 13
or 14 amino acids. It is well know in the art that proteins may
undergo modification, including post-translational modifications,
such as, but not limited to, disulfide bond formation,
glycosylation, phosphorylation, or oligomerization. Thus, as used
herein, the terms "protein molecule" and "peptide molecule" include
molecules that are modified by any biological or non-biological
process. The terms "amino acid" and "amino acids" refer to all
naturally occurring L-amino acids. This definition is meant to
include norleucine, omithine, homocysteine, and homoserine.
[0248] One or more of the protein or peptide molecules may be
produced via chemical synthesis, or more preferably, by expression
in a suitable bacterial or eukaryotic host. Suitable methods for
expression are described by Sambrook, et al., (In: Molecular
Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1989), or similar texts.
[0249] Another class of agents comprise protein or peptide
molecules encoded by SEQ ID NO: 1097 through SEQ ID NO: 1119 or
complements thereof or, fragments or fusions thereof in which
non-essential, or not relevant, amino acid residues have been
added, replaced, or deleted. An example of such a homolog is a
protein homolog of each soybean species, including but not limited
to alfalfa, barley, Brassica, broccoli, cabbage, citrus, garlic,
oat, oilseed rape, onion, canola, flax, pea, peanut, pepper,
potato, rye, soybean, strawberry, sugarcane, sugarbeet, soybean,
maize, rice, cotton, sorghum, Arabidopsis, wheat, pine, fir,
eucalyptus, apple, lettuce, peas, lentils, grape, banana, tea, turf
grasses, etc. Particularly preferred non-soybean plants to utilize
for the isolation of homologs would include alfalfa, barley, oat,
oilseed rape, canola, ornamentals, sugarcane, sugarbeet, soybean,
maize, rice, cotton, sorghum, Arabidopsis, wheat, potato, and turf
grasses. Such a homolog can be obtained by any of a variety of
methods. Most preferably, as indicated above, one or more of the
disclosed sequences (SEQ ID NO: 1 through SEQ ID NO: 1096 or
complements thereof) will be used to define a pair of primers that
may be used to isolate the protein homolog-encoding nucleic acid
molecules from any desired species. Such molecules can be expressed
to yield protein homologs by recombinant means.
[0250] (c) Plant Constructs and Plant Transformants
[0251] One or more of the nucleic acid molecules of the invention
may be used in plant transformation or transfection. Exogenous
genetic material may be transferred into a plant cell and the plant
cell regenerated into a whole, fertile or sterile plant. Exogenous
genetic material is any genetic material, whether naturally
occurring or otherwise, from any source that is capable of being
inserted into any organism. In a preferred embodiment the exogenous
genetic material includes a nucleic acid molecule of the present
invention, preferably a nucleic acid molecule having at least 20
nucleotides of a sequence selected from the group consisting of SEQ
ID NO: 1 through SEQ ID NO: 1096 and complements thereof. In a
preferred embodiment, the nucleic acid molecule codes for a protein
or fragment thereof described in Section (i). In another preferred
embodiment, the nucleic acid molecule is a promoter or fragment
thereof described in Section (iii).
[0252] Such genetic material may be transferred into either
monocotyledons and dicotyledons including, but not limited to
tomato, eggplant, maize, soybean, Arabidopsis, phaseolus, peanut,
alfalfa, wheat, rice, oat, sorghum, rye, tritordeum, millet,
fescue, perennial ryegrass, sugarcane, cranberry, papaya, banana,
banana, muskmelon, apple, cucumber, dendrobium, gladiolus,
chrysanthemum, liliacea, cotton, eucalyptus, sunflower, canola,
turfgrass, sugarbeet, coffee and dioscorea (Christou, In: Particle
Bombardment for Genetic Engineering of Plants, Biotechnology
Intelligence Unit. Academic Press, San Diego, Calif. (1996).
[0253] In a preferred embodiment, the genetic material is
transferred to a soybean. Preferred soybeans to transfer an rhg1
SCN resistance allele are selected from the group consisting of
PI548402 (Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4),
PI404166 (Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654
(Er-hej-jan), PI438489 (Chiquita), PI507354 (rokei 421), PI548655
(Forrest), PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid,
Nathan, AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501,
AG4601, PION9492, PI88788, Dyer, Custer, Manokin, and Doles.
[0254] Preferred soybeans to transfer an Rhg4 SCN resistance allele
are selected from the group consisting of PI548402 (Peking),
PI437654 (Er-hej-jan), PI438489 (Chiquita), PI507354 (Tokei 421),
PI548655 (Forrest), PI548988 (Pickett), PI88788, PI404198 (Sun Huan
Do), PI404166 (Krasnoaarmejkaja), Hartwig, Manokin, Doles, Dyer,
and Custer.
[0255] Transfer of a nucleic acid that encodes for a protein can
result in overexpression of that protein in a transformed cell or
transgenic plant. One or more of the proteins or fragments thereof
encoded by nucleic acid molecules of the invention may be
overexpressed in a transformed cell or transformed plant. Such
overexpression may be the result of transient or stable transfer of
the exogenous genetic material. Such overexpression can also result
in SCN resistance to one or more races of SCN.
[0256] Exogenous genetic material may be transferred into a host
cell by the use of a DNA vector or construct designed for such a
purpose. Design of such a vector is generally within the skill of
the art (See, Plant Molecular Biology: A Laboratory Manual, Clark
(ed.), Springier, New York (1997).
[0257] A construct or vector may include a plant promoter to
express the protein or protein fragment of choice. A number of
promoters, which are active in plant cells, have been described in
the literature. These include the nopaline synthase (NOS) promoter
(Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987),
the octopine synthase (OCS) promoter (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), and
the CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985), the
figwort mosaic virus 35S-promoter, the light-inducible promoter
from the small subunit of ribulose-1,5-bis-phosphate carboxylase
(ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad.
Sci. (U.S.A.) 84:6624-6628 (1987), the sucrose synthase promoter
(Yang et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990),
the R gene complex promoter (Chandler et al., The Plant Cell 1:
1175-1183 (1989), and the chlorophyll a/b binding protein gene
promoter, etc. These promoters have been used to create DNA
constructs that have been expressed in plants; see, e.g., PCT
publication WO 84/02913. The CaMV 35S promoters are preferred for
use in plants. Promoters known or found to cause transcription of
DNA in plant cells can be used in the invention.
[0258] For the purpose of expression in source tissues of the
plant, such as the leaf, seed, root or stem, it is preferred that
the promoters utilized have relatively high expression in these
specific tissues. Tissue-specific expression of a protein of the
present invention is a particularly preferred embodiment. For this
purpose, one may choose from a number of promoters for genes with
tissue- or cell-specific or -enhanced expression. Examples of such
promoters reported in the literature include the chloroplast
glutamine synthetase GS2 promoter from pea (Edwards et al., Proc.
Natl. Acad. Sci. (U.S.A.) 87:3459-3463 (1990), the chloroplast
fructose-1,6-biphosphatase (FBPase) promoter from wheat (Lloyd et
al., Mol. Gen. Genet. 225:209-216 (1991), the nuclear
photosynthetic ST-LS1 promoter from potato (Stockhaus et al., EMBO
J. 8:2445-2451 (1989), the STK (PAL) promoter and the glucoamylase
(CHS) promoter from Arabidopsis thaliana. Also reported to be
active in photosynthetically active tissues are the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoter from eastern
larch (Larix laricina), the promoter for the cab gene, cab6, from
pine (Yamamoto et al., Plant Cell Physiol. 35:773-778 (1994), the
promoter for the Cab-1 gene from wheat (Fejes et al., Plant Mol.
Biol. 15:921-932 (1990), the promoter for the CAB-1 gene from
spinach (Lubberstedt et al., Plant Physiol. 104:997-1006 (1994),
the promoter for the cab I R gene from rice (Luan et al., Plant
Cell. 4:971-981 (1992), the pyruvate, orthophosphate dikinase
(PPDK) promoter from maize (Matsuoka et al., Proc. Natl. Acad. Sci.
(U.S.A.) 90: 9586-9590 (1993), the promoter for the tobacco Lhcb1*2
gene (Cerdan et al., Plant Mol. Biol. 33:245-255 (1997), the
Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truemit et
al., Planta. 196:564-570 (1995), and the promoter for the thylakoid
membrane proteins from spinach (psaD, psaF, psaE, PC, FNR, atpC,
atpD, cab, rbcS). Other promoters for the chlorophyll a/b-binding
proteins may also be utilized in the invention, such as the
promoters for LhcB gene and PsbP gene from white mustard (Sinapis
alba; Kretsch et al., Plant Mol. Biol. 28:219-229 (1995)).
[0259] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of maize, wheat, rice and barley, it is preferred that the
promoters utilized in the invention have relatively high expression
in these specific tissues. A number of promoters for genes with
tuber-specific or -enhanced expression are known, including the
class I patatin promoter (Bevan et al., EMBO J. 8:1899-1906 (1986);
Jefferson et al., Plant Mol. Biol. 14:995-1006 (1990)), the
promoter for the potato tuber ADPGPP genes, both the large and
small subunits, the sucrose synthase promoter (Salanoubat and
Belliard, Gene 60:47-56 (1987), Salanoubat and Belliard, Gene
84:181-185 (1989)), the promoter for the major tuber proteins
including the 22 kd protein complexes and proteinase inhibitors
(Hannapel, Plant Physiol. 101:703-704 (1993)), the promoter for the
granule bound starch synthase gene (GBSS) (Visser et al., Plant
Mol. Biol. 1 7:691-699 (1991)), and other class I and II patatins
promoters (Koster-Topfer et al., Mol Gen Genet. 219:390-396 (1989);
Mignery et al., Gene. 62:27-44 (1988)).
[0260] Other promoters can also be used to express a protein or
fragment thereof in specific tissues, such as seeds or fruits. The
promoter for .beta.-conglycinin (Chen et al., Dev. Genet. 10:
112-122 (1989)) or other seed-specific promoters such as the napin
and phaseolin promoters, can be used. The zeins are a group of
storage proteins found in maize endosperm. Genomic clones for zein
genes have been isolated (Pedersen et al., Cell 29:1015-1026
(1982)) and the promoters from these clones, including the 15 kD,
16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used. Other
promoters known to function, for example, in maize include the
promoters for the following genes: waxy, Brittle, Shrunken 2,
Branching enzymes I and II, starch synthases, debranching enzymes,
oleosins, glutelins and sucrose synthases. A particularly preferred
promoter for maize endosperm expression is the promoter for the
glutelin gene from rice, more particularly the Osgt-1 promoter
(Zheng et al., Mol. Cell Biol. 13:5829-5842 (1993)). Examples of
promoters suitable for expression in wheat include those promoters
for the ADPglucose pyrosynthase (ADPGPP) subunits, the granule
bound and other starch synthase, the branching and debranching
enzymes, the embryogenesis-abundant proteins, the gliadins and the
glutenins. Examples of such promoters in rice include those
promoters for the ADPGPP subunits, the granule bound and other
starch synthase, the branching enzymes, the debranching enzymes,
sucrose synthases and the glutelins. A particularly preferred
promoter is the promoter for rice glutelin, Osgt-1. Examples of
such promoters for barley include those for the ADPGPP subunits,
the granule bound and other starch synthase, the branching enzymes,
the debranching enzymes, sucrose synthases, the hordeins, the
embryo globulins and the aleurone specific proteins.
[0261] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994)). Expression in root tissue
could also be accomplished by utilizing the root specific
subdomains of the CaMV35S promoter that have been identified (Lam
et al., Proc. Nati. Acad. Sci. (USA.) 86:7890-7894 (1989)). Other
root cell specific promoters include those reported by Conkling et
al. (Conkling et al., Plant Physiol. 93:1203-1211 (1990)).
[0262] Additional promoters that may be utilized are described, for
example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147;
5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435;
and 4,633,436. In addition, a tissue specific enhancer may be used
(From et al., The Plant Cell 1:977-984 (1989)).
[0263] Preferred promoters are those set forth in Section (a)(iii)
of Agents.
[0264] Constructs or vectors may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. A number of
such sequences have been isolated, including the Tr7 3' sequence
and the NOS 3' sequence (Ingelbrecht et al., The Plant Cell
1:671-680 (1989); Bevan et al., Nucleic Acids Res. 11:369-385
(1983)).
[0265] A vector or construct may also include regulatory elements.
Examples of such include the Adh intron 1 (Callis et al., Genes and
Develop. 1:1183-1200 (1987)), the sucrose synthase intron (Vasil et
al., Plant Physiol. 91:1575-1579 (1989)) and the TMV omega element
(Gallie et al., The Plant Cell 1:301-311 (1989)). These and other
regulatory elements may be included when appropriate.
[0266] A vector or construct may also include a selectable marker.
Selectable markers may also be used to select for plants or plant
cells that contain the exogenous genetic material. Examples of such
include, but are not limited to: a neomycin phosphotransferase gene
(U.S. Pat. No. 5,034,322), which codes for kanamycin resistance and
can be selected for using kanamycin, G418, etc.; a bar gene which
codes for bialaphos resistance; genes which encode glyphosate
resistance (U.S. Pat. Nos. 4,940,835; 5,188,642; 4,971,908;
5,627,061); a nitrilase gene which confers resistance to bromoxynil
(Stalker et al., J. Biol. Chem. 263:6310-6314 (1988)); a mutant
acetolactate synthase gene (ALS) which confers imidazolinone or
sulphonylurea resistance (European Patent Application 154,204 (Sep.
11, 1985)); and a methotrexate resistant DHFR gene (Thillet et al.,
J. Biol. Chem. 263:12500-12508 (1988)).
[0267] A vector or construct may also include DNA sequence which
encodes a transit peptide. Incorporation of a suitable chloroplast
transit peptide may also be employed (European Patent Application
Publication Number 0218571). Translational enhancers may also be
incorporated as part of the vector DNA. DNA constructs could
contain one or more 5' non-translated leader sequences which may
serve to enhance expression of the gene products from the resulting
mRNA transcripts. Such sequences may be derived from the promoter
selected to express the gene or can be specifically modified to
increase translation of the mRNA. Such regions may also be obtained
from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence. For a review of optimizing expression of
transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405
(1996).
[0268] A vector or construct may also include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include: a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405
(1987); Jefferson et al., EMBO J. 6:3901-3907 (1987)); an R-locus
gene, which encodes a product that regulates the production of
anthocyanin pigments (red color) in plant tissues (Dellaporta et
al., Stadler Symposium 11:263-282 (1988)); a .beta.-lactamase gene
(Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741
(1978)), a gene which encodes an enzyme for which various
chromogenic substrates are known (e.g., PADAC, a chromogenic
cephalosporin); a luciferase gene (Ow et al., Science 234:856-859
(1986)); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci.
(U.S.A) 80:1101-1105 (1983)) which encodes a catechol dioxygenase
that can convert chromogenic catechols; an .alpha.-amylase gene
(Ikatu et al., Bio/Technol. 8:241-242 (1990)); a tyrosinase gene
(Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which
encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin; an a-galactosidase,
which will turn a chromogenic a-galactose substrate.
[0269] Included within the terms "selectable or screenable marker
genes" are also genes which encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes which can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins which are detectable, (e.g., by ELISA), small
active enzymes which are detectable in extracellular solution
(e.g., .alpha.-amylase, .beta.-lactamase, phosphinothricin
transferase), or proteins which are inserted or trapped in the cell
wall (such as proteins which include a leader sequence such as that
found in the expression unit of extension or tobacco PR-S). Other
possible selectable and/or screenable marker genes will be apparent
to those of skill in the art.
[0270] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method by which nucleic acid molecules may be
introduced into a cell, such as by Agrobacterium infection or
direct delivery of nucleic acid molecules such as, for example, by
PEG-mediated transformation, by electroporation or by acceleration
of DNA coated particles, etc (Potrykus, Ann. Rev. Plant Physiol.
Plant Mol. Biol. 42:205-225 (1991); Vasil, Plant Mol. Biol.
25:925-937 (1994)). For example, electroporation has been used to
transform maize protoplasts (Fromm et al., Nature 312:791-793
(1986)).
[0271] Other vector systems suitable for introducing transforming
DNA into a host plant cell include but are not limited to binary
artificial chromosome (BIBAC) vectors (Hamilton et al., Gene
200:107-116 (1997)); and transfection with RNA viral vectors
(Della-Cioppa et al., Ann. N.Y. Acad. Sci. (1996), 792 (Engineering
Plants for Commercial Products and Applications), 57-61).
Additional vector systems also include plant selectable YAC vectors
such as those described in Mullen et al., Molecular Breeding
4:449-457 (1988)).
[0272] Technology for introduction of DNA into cells is well known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, Virology 54:536-539 (1973)); (2) physical methods
such as microinjection (Capecchi, Cell 22:479-488 (1980)),
electroporation (Wong and Neumann, Biochem. Biophys. Res. Commun.
107:584-587 (1982); Fromm et al., Proc. Natl. Acad. Sci. (USA.)
82:5824-5828 (1985); U.S. Pat. No. 5,384,253); and the gene gun
(Johnston and Tang, Methods Cell Biol. 43:353-365 (1994)); (3)
viral vectors (Clapp, Clin. Perinatol. 20:155-168 (1993); Lu et
al., J. Exp. Med 178:2089-2096 (1993); Eglitis and Anderson,
Biotechniques 6:608-614 (1988)); and (4) receptor-mediated
mechanisms (Curiel et al., Hum. Gen. Ther. 3:147-154 (1992), Wagner
et al., Proc. Natl. Acad. Sci. (USA) 89:6099-6103 (1992)).
[0273] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules to plant cells
is microprojectile bombardment. This method has been reviewed by
Yang and Christou (eds.), Particle Bombardment Technologyfor Gene
Transfer, Oxford Press, Oxford, England (1994)). Non-biological
particles (microprojectiles) that may be coated with nucleic acids
and delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum and the
like.
[0274] A particular advantage of microprojectile bombardment, in
addition to it being an effective means of reproducibly
transforming monocots, is that neither the isolation of protoplasts
(Cristou et al., Plant Physiol. 87:671-674 (1988)) nor the
susceptibility of Agrobacterium infection are required. An
illustrative embodiment of a method for delivering DNA into maize
cells by acceleration is a biolistics a-particle delivery system,
which can be used to propel particles coated with DNA through a
screen, such as a stainless steel or Nytex screen, onto a filter
surface covered with corn cells cultured in suspension. Gordon-Kamm
et al., describes the basic procedure for coating tungsten
particles with DNA (Gordon-Kamm et al., Plant Cell 2:603-618
(1990)). The screen disperses the tungsten nucleic acid particles
so that they are not delivered to the recipient cells in large
aggregates. A particle delivery system suitable for use with the
invention is the helium acceleration PDS-1000/He gun is available
from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.)(Sanford et
al., Technique 3:3-16 (1991)).
[0275] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0276] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain up to
1000 or more foci of cells transiently expressing a screenable or
selectable marker gene. The number of cells in a focus which
express the exogenous gene product 48 hours post-bombardment often
range from one to ten and average one to three.
[0277] In bombardment transformation, one may optimize the
pre-bombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment and also the nature of the transforming
DNA, such as linearized DNA or intact supercoiled plasmids. It is
believed that pre-bombardment manipulations are especially
important for successful transformation of immature embryos.
[0278] In another alternative embodiment, plastids can be stably
transformed. Methods disclosed for plastid transformation in higher
plants include the particle gun delivery of DNA containing a
selectable marker and targeting of the DNA to the plastid genome
through homologous recombination (Svab et al., Proc. Natl. Acad.
Sci. (U.S.A.) 87:8526-8530 (1990); Svab and Maliga, Proc. Natl.
Acad. Sci. (U.S.A.) 90:913-917 (1993); Staub and Maliga, EMBO J.
12:601-606 (1993); U.S. Pat. Nos. 5,451,513 and 5,545,818).
[0279] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small-scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions which
influence the physiological state of the recipient cells and which
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0280] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated E15s plant integrating vectors to introduce
DNA into plant cells is well known in the art. See, for example the
methods described by Fraley et al., Bio/Technology 3:629-635 (1985)
and Rogers et al., Methods Enzymol. 153:253-277 (1987). Further,
the integration of the T-DNA is a relatively precise process
resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet. 205:34 (1986)).
[0281] Modem Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New
York, pp. 179-203 (1985)). Moreover, technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Methods Enzymol.
153:253-277 (1987)). In addition, Agrobacterium containing both
armed and disarmed Ti genes can be used for the transformations. In
those plant strains where Agrobacterium-mediated transformation is
efficient, it is the method of choice because of the facile and
defined nature of the gene transfer.
[0282] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. More preferred is a transgenic plant that is homozygous
for the added structural gene; i.e., a transgenic plant that
contains two added genes, one gene at the same locus on each
chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants
produced for the gene of interest.
[0283] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating, exogenous genes. Selfing of appropriate
progeny can produce plants that are homozygous for both added,
exogenous genes that encode a polypeptide of interest. Backcrossing
to a parental plant and out-crossing with a non-transgenic plant
are also contemplated, as is vegetative propagation.
[0284] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation and combinations of these
treatments (See, for example, Potrykus et al., Mol. Gen. Genet.
205:193-200 (1986); Lorz et al., Mol. Gen. Genet. 199:178 (1985);
Fromm et al., Nature 319:791 (1986); Uchimiya et al., Mol. Gen.
Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457
(1988)).
[0285] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al., Plant
Tissue Culture Letters 2:74 (1985); Toriyama et al., Theor Appl.
Genet. 205:34 (1986); Yamada et al., Plant Cell Rep. 4:85 (1986);
Abdullah et al., Biotechnology 4:1087 (1986)).
[0286] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, Biotechnology 6:397 (1988)). In addition,
"particle gun" or high-velocity microprojectile technology can be
utilized (Vasil et al., Bio/Technology 10:667 (1992)).
[0287] Using the latter technology, DNA is carried through the cell
wall and into the cytoplasm on the surface of small metal particles
as described (Klein et al., Nature 328:70 (1987); Klein et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al.,
Bio/Technology 6:923 (1988)). The metal particles penetrate through
several layers of cells and thus allow the transformation of cells
within tissue explants.
[0288] The regeneration, development and cultivation of plants from
single plant protoplast transformants or from various transformed
explants are well known in the art (Weissbach and Weissbach, In:
Methods for Plant Molecular Biology, Academic Press, San Diego,
Calif., (1988)). This regeneration and growth process typically
includes the steps of selection of transformed cells, culturing
those individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0289] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a protein of interest is well
known in the art. Preferably, the regenerated plants are
self-pollinated to provide homozygous transgenic plants. Otherwise,
pollen obtained from the regenerated plants is crossed to
seed-grown plants of agronomically important lines. Conversely,
pollen from plants of these important lines is used to pollinate
regenerated plants. A transgenic plant of the invention containing
a desired polypeptide is cultivated using methods well known to one
skilled in the art.
[0290] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0291] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens and obtaining transgenic plants have been
published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. Nos.
5,159,135; 5,518,908); soybean (U.S. Pat. No. 5,569,834; U.S. Pat.
No. 5,416,011; McCabe et al., Biotechnology 6-923 (1988); Christou
et al., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Pat. No.
5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657
(1996), McKently et al., Plant Cell Rep. 14:699-703 (1995));
papaya; and pea (Grant et al., Plant Cell Rep. 15:254-258
(1995)).
[0292] Transformation of monocotyledons using electroporation,
particle bombardment and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354
(1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994));
maize (Rhodes et al., Science 240:204 (1988); Gordon-Kamm et al.,
Plant Cell 2:603-618 (1990); Fromm et al., Bio/Technology 8:833
(1990); Koziel et al., Bio/Technology 11:194 (1993); Armstrong et
al., Crop Science 35:550-557 (1995)); oat (Somers et al.,
Bio/Technology 10:1589 (1992)); orchard grass (Horn et al, Plant
Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor Appl. Genet.
205:34 (1986); Part et al., Plant Mol. Biol. 32:1135-1148 (1996);
Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang
and Wu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell
Rep. 7:379 (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992);
Christou et al., Bio/Technology 9:957 (1991)); rye (De la Pena et
al., Nature 325:274 (1987)); sugarcane (Bower and Birch, Plant J.
2:409 (1992)); tall fescue (Wang et al., Bio/Technology 10:691
(1992)) and wheat (Vasil et al., Bio/Technology 10:667 (1992); U.S.
Pat. No. 5,631,152).
[0293] Assays for gene expression based on the transient expression
of cloned nucleic acid constructs have been developed by
introducing the nucleic acid molecules into plant cells by
polyethylene glycol treatment, electroporation, or particle
bombardment (Marcotte et al., Nature 335:454-457 (1988); Marcotte
et al., Plant Cell 1:523-532 (1989); McCarty et al., Cell
66:895-905 (1991); Hattori et al., Genes Dev. 6:609-618 (1992);
Goffet al, EMBO J. 9:2517-2522 (1990)). Transient expression
systems may be used to functionally dissect gene constructs (see
generally, Mailga et al., Methods in Plant Molecular Biology, Cold
Spring Harbor Press (1995)).
[0294] Any of the nucleic acid molecules of the invention may be
introduced into a plant cell in a permanent or transient manner in
combination with other genetic elements such as vectors, promoters,
enhancers, etc. Further, any of the nucleic acid molecules of the
invention may be introduced into a plant cell in a manner that
allows for overexpression of the protein or fragment thereof
encoded by the nucleic acid molecule.
[0295] Cosuppression is the reduction in expression levels, usually
at the level of RNA, of a particular endogenous gene or gene family
by the expression of a homologous sense construct that is capable
of transcribing mRNA of the same strandedness as the transcript of
the endogenous gene (Napoli et al., Plant Cell 2:279-289 (1990);
van der Krol et al., Plant Cell 2:291-299 (1990)). Cosuppression
may result from stable transformation with a single copy nucleic
acid molecule that is homologous to a nucleic acid sequence found
within the cell (Prolls and Meyer, Plant J. 2:465-475 (1992)) or
with multiple copies of a nucleic acid molecule that is homologous
to a nucleic acid sequence found within the cell (Mittlesten et
al., Mol. Gen. Genet. 244:325-330 (1994)). Genes, even though
different, linked to homologous promoters may result in the
cosuppression of the linked genes (Vaucheret, C. R. Acad. Sci. III
316:1471-1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)
91:3490-3496 (1994)); van Blokland et al., Plant J. 6:861-877
(1994); Jorgensen, Trends Biotechnol. 8:340-344 (1990); Meins and
Kunz, In: Gene Inactivation and Homologous Recombination in Plants,
Paszkowski (ed.), pp. 335-348, Kluwer Academic, Netherlands
(1994)).
[0296] It is understood that one or more of the nucleic acids of
the invention may be introduced into a plant cell and transcribed
using an appropriate promoter with such transcription resulting in
the cosuppression of an endogenous protein.
[0297] Antisense approaches are a way of preventing or reducing
gene function by targeting the genetic material (U.S. Pat. Nos.
4,801,540 and 5,107,065 Mol et al., FEBS Lett. 268:427-430 (1990)).
The objective of the antisense approach is to use a sequence
complementary to the target gene to block its expression and create
a mutant cell line or organism in which the level of a single
chosen protein is selectively reduced or abolished. Antisense
techniques have several advantages over other `reverse genetic`
approaches. The site of inactivation and its developmental effect
can be manipulated by the choice of promoter for antisense genes or
by the timing of external application or microinjection. Antisense
can manipulate its specificity by selecting either unique regions
of the target gene or regions where it shares homology to other
related genes (Hiatt et al., In: Genetic Engineering, Setlow (ed.),
Vol. 11, New York: Plenum 49-63 (1989)).
[0298] The principle of regulation by antisense RNA is that RNA
that is complementary to the target mRNA is introduced into cells,
resulting in specific RNA:RNA duplexes being formed by base pairing
between the antisense substrate and the target mRNA (Green et al.,
Annu. Rev. Biochem. 55:569-597 (1986)). Under one embodiment, the
process involves the introduction and expression of an antisense
gene sequence. Such a sequence is one in which part or all of the
normal gene sequences are placed under a promoter in inverted
orientation so that the `wrong` or complementary strand is
transcribed into a noncoding antisense RNA that hybridizes with the
target mRNA and interferes with its expression (Takayama and
Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990)). An
antisense vector is constructed by standard procedures and
introduced into cells by transformation, transfection,
electroporation, microinjection, infection, etc. The type of
transformation and choice of vector will determine whether
expression is transient or stable. The promoter used for the
antisense gene may influence the level, timing, tissue,
specificity, or inducibility of the antisense inhibition.
[0299] It is understood that the activity of a protein in a plant
cell may be reduced or depressed by growing a transformed plant
cell containing a nucleic acid molecule whose non-transcribed
strand encodes a protein or fragment thereof.
[0300] Post transcriptional gene silencing (PTGS) can result in
virus immunity or gene silencing in plants. PTGS is induced by
dsRNA and is mediated by an RNA-dependent RNA polymerase, present
in the cytoplasm, that requires a dsRNA template. The dsRNA is
formed by hybridization of complementary transgene mRNAs or
complementary regions of the same transcript. Duplex formation can
be accomplished by using transcripts from one sense gene and one
antisense gene co-located in the plant genome, a single transcript
that has self-complementarity, or sense and antisense transcripts
from genes brought together by crossing. The dsRNA-dependent RNA
polymerase makes a complementary strand from the transgene mRNA and
RNAse molecules attach to this complementary strand (cRNA). These
cRNA-RNAse molecules hybridize to the endogene mnRNA and cleave the
single-stranded RNA adjacent to the hybrid. The cleaved
single-stranded RNAs are further degraded by other host RNAses
because one will lack a capped 5' end and the other will lack a
poly(A) tail (Waterhouse et al., PNAS 95: 13959-13964 (1998)).
[0301] It is understood that one or more of the nucleic acids of
the invention may be introduced into a plant cell and transcribed
using an appropriate promoter with such transcription resulting in
the postranscriptional gene silencing of an endogenous
transcript.
[0302] Antibodies have been expressed in plants (Hiatt et al.,
Nature 342:76-78 (1989); Conrad and Fielder, Plant Mol. Biol.
26:1023-1030 (1994)). Cytoplasmic expression of a scFv
(single-chain Fv antibodies) has been reported to delay infection
by artichoke mottled crinkle virus. Transgenic plants that express
antibodies directed against endogenous proteins may exhibit a
physiological effect (Philips et al., EMBO J. 16:4489-4496 (1997);
Marion-Poll, Trends in Plant Science 2:447-448 (1997)). For
example, expressed anti-abscissic antibodies have been reported to
result in a general perturbation of seed development (Philips et
al., EMBO J. 16: 4489-4496 (1997)).
[0303] Antibodies that are catalytic may also be expressed in
plants (abzymes). The principle behind abzymes is that since
antibodies may be raised against many molecules, this recognition
ability can be directed toward generating antibodies that bind
transition states to force a chemical reaction forward (Persidas,
Nature Biotechnology 15:1313-1315 (1997); Baca et al., Ann. Rev.
Biophys. Biomol. Struct. 26:461-493 (1997)). The catalytic
abilities of abzymes may be enhanced by site directed mutagenesis.
Examples of abzymes are, for example, set forth in U.S. Pat. No.
5,658,753; U.S. Pat. No. 5,632,990; U.S. Pat. No. 5,631,137; U.S.
Pat. No. 5,602,015; U.S. Pat. No. 5,559,538; U.S. Pat. No.
5,576,174; U.S. Pat. No. 5,500,358; U.S. Patent 5,318,897; U.S.
Pat. No. 5,298,409; U.S. Pat. No. 5,258,289 and U.S. Pat. No.
5,194,585.
[0304] It is understood that any of the antibodies of the invention
may be expressed in plants and that such expression can result in a
physiological effect. It is also understood that any of the
expressed antibodies may be catalytic.
[0305] (d) Antibodies
[0306] One aspect of the present invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the present invention and their homologues, fusions or
fragments. Such antibodies may be used to quantitatively or
qualitatively detect the protein or peptide molecules of the
present invention. As used herein, an antibody or peptide is said
to "specifically bind" to a protein or peptide molecule of the
present invention if such binding is not competitively inhibited by
the presence of non-related molecules.
[0307] Nucleic acid molecules that encode all or part of the
protein of the present invention can be expressed, via recombinant
means, to yield protein or peptides that can in turn be used to
elicit antibodies that are capable of binding the expressed protein
or peptide. Such antibodies may be used in immunoassays for that
protein. Such protein-encoding molecules, or their fragments may be
a "fusion" molecule (i.e., a part of a larger nucleic acid
molecule) such that, upon expression, a fusion protein is produced.
It is understood that any of the nucleic acid molecules of the
present invention may be expressed, via recombinant means, to yield
proteins or peptides encoded by these nucleic acid molecules.
[0308] The antibodies that specifically bind proteins and protein
fragments of the present invention may be polyclonal or monoclonal
and may comprise intact immunoglobulins, or antigen binding
portions of immunoglobulins fragments (such as (F(ab'),
F(ab').sub.2), or single-chain immunoglobulins producible, for
example, via recombinant means. It is understood that practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of antibodies (see, for example, Harlow
and Lane, In: Antibodies: A Laboratory Manual, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1988)).
[0309] Murine monoclonal antibodies are particularly preferred.
BALB/c mice are preferred for this purpose, however, equivalent
strains may also be used. The animals are preferably immunized with
approximately 25 .mu.g of purified protein (or fragment thereof)
that has been emulsified in a suitable adjuvant (such as TiterMax
adjuvant (Vaxcel, Norcross, Ga.)). Immunization is preferably
conducted at two intramuscular sites, one intraperitoneal site and
one subcutaneous site at the base of the tail. An additional i.v.
injection of approximately 25 .mu.g of antigen is preferably given
in normal saline three weeks later. After approximately 11 days
following the second injection, the mice may be bled and the blood
screened for the presence of anti-protein or peptide antibodies.
Preferably, a direct binding Enzyme-Linked Immunoassay (ELISA) is
employed for this purpose.
[0310] More preferably, the mouse having the highest antibody titer
is given a third i.v. injection of approximately 25 .mu.g of the
same protein or fragment. The splenic leukocytes from this animal
may be recovered 3 days later and then permitted to fuse, most
preferably, using polyethylene glycol, with cells of a suitable
myeloma cell line (such as, for example, the P3X63Ag8.653 myeloma
cell line). Hybridoma cells are selected by culturing the cells
under "HAT" (hypoxanthine-aminopterin-thy-mine) selection for about
one week. The resulting clones may then be screened for their
capacity to produce monoclonal antibodies ("mAbs"), preferably by
direct ELISA.
[0311] In one embodiment, anti-protein or peptide monoclonal
antibodies are isolated using a fusion of a protein or peptide of
the present invention, or conjugate of a protein or peptide of the
present invention, as immunogens. Thus, for example, a group of
mice can be immunized using a fusion protein emulsified in Freund's
complete adjuvant (e.g., approximately 50 .mu.g of antigen per
immunization). At three week intervals, an identical amount of
antigen is emulsified in Freund's incomplete adjuvant and used to
immunize the animals. Ten days following the third immunization,
serum samples are taken and evaluated for the presence of antibody.
If antibody titers are too low, a fourth booster can be employed.
Polysera capable of binding the protein or peptide can also be
obtained using this method.
[0312] In a preferred procedure for obtaining monoclonal
antibodies, the spleens of the above-described immunized mice are
removed, disrupted and immune splenocytes are isolated over a
ficoll gradient. The isolated splenocytes are fused, using
polyethylene glycol with BALB/c-derived HGPRT (hypoxanthine guanine
phosphoribosyl transferase) deficient P3x63xAg8.653 plasmacytoma
cells. The fused cells are plated into 96 well microtiter plates
and screened for hybridoma fusion cells by their capacity to grow
in culture medium supplemented with hypothanthine, aminopterin and
thymidine for approximately 2-3 weeks.
[0313] Hybridoma cells that arise from such incubation are
preferably screened for their capacity to produce an immunoglobulin
that binds to a protein of interest. An indirect ELISA may be used
for this purpose. In brief, the supernatants of hybridomas are
incubated in microtiter wells that contain immobilized protein.
After washing, the titer of bound immunoglobulin can be determined
using, for example, a goat anti-mouse antibody conjugated to
horseradish peroxidase. After additional washing, the amount of
immobilized enzyme is determined (for example through the use of a
chromogenic substrate). Such screening is performed as quickly as
possible after the identification of the hybridoma in order to
ensure that a desired clone is not overgrown by non-secreting
neighbor cells. Desirably, the fusion plates are screened several
times since the rates of hybridoma growth vary. In a preferred
sub-embodiment, a different antigenic form may be used to screen
the hybridoma. Thus, for example, the splenocytes may be immunized
with one immunogen, but the resulting hybridomas can be screened
using a different immunogen. It is understood that any of the
protein or peptide molecules of the present invention may be used
to raise antibodies.
[0314] Such antibody molecules or their fragments may be used for
diagnostic purposes. Where the antibodies are intended for
diagnostic purposes, it may be desirable to derivatize them, for
example with a ligand group (such as biotin) or a detectable marker
group (such as a fluorescent group, a radioisotope or an
enzyme).
[0315] The ability to produce antibodies that bind the protein or
peptide molecules of the present invention permits the
identification of mimetic compounds of those molecules. A "mimetic
compound" is a compound that is not that compound, or a fragment of
that compound, but which nonetheless exhibits an ability to
specifically bind to antibodies directed against that compound.
[0316] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
[0317] In this example, DNA is extracted from soybean plants,
amplified, and mapped.
[0318] A single trifoliate leaf is collected from the newest growth
of four week old soybean plants. Leaf tissue from the leaf is
placed on ice and stored at -80.degree. C. The frozen tissue is
lyophilized, and approximately 0.01 grams of the tissue is used for
DNA extraction. The 0.01 grams of leaf tissue is ground to powder
in 1.4 ml tubes. 600 microliters (.mu.l) of DNA extraction buffer
consisting of 0.5M NaCl, 0.1M Tris-(hydroxymethyl) aminomethane pH
8.0, 0.05 M ethylenediaminetetra-acetic acid (EDTA), 10.0 g
L.sup.-1 sodium dodecyl sulfate (SDS), and 2 g L.sup.-1
phenantroline (dissolved in 0.01 L ethanol) is heated to 65.degree.
C. (with 0.77 g L.sup.-1 dithiothreitol added immediately before
use) is added to each tube, and each tube is mixed thoroughly. The
samples are placed in a 65.degree. C. water bath for 15 minutes and
shaken by hand after 10 minutes. The samples are taken out of the
water bath and cooled to room temperature, and then 200 .mu.l of 5
M KOAc is added to each tube. The samples are inverted and placed
at 4.degree. C. for 20 minutes. Samples are then centrifuged for 12
minutes at 6200.times.g and the supernatant (about 600 .mu.l) is
transferred to new tubes. DNA is precipitated with 330 .mu.l of
cold isopropanol and placed at -20.degree. C. for 1 hr. The DNA is
pelleted by centrifuging at 6200.times. g for 10 minutes and washed
with 70% EtOH. The DNA is pelleted by centrifugation at 6200.times.
g for 10 minutes and dried using a Speed-Vac. The DNA is dissolved
in 100 .mu.l of TEO.sub.0.1 (0.01 M Tris-HCl pH 8.0, 0.0001 M
EDTA). The extraction will generally yield 500 ng DNA
.mu.l.sup.-1.
[0319] A polymerase chain reaction (PCR) is conducted with 5 to 10
ng genomic DNA in 10 .mu.l volumes of 10 mM Tris-HCl (pH 8.3), 50
mM KCl, 0.001% gelatin, 1.5 mM MgCl.sub.2, 0.1 mM of each dNTP, 150
nM of each primer, 0.01 mM Cresol Red, 2% sucrose and 0.32 units of
AmpliTaq DNA Polymerase (Perkin Elmer Instruments Inc., USA). For
thermocycling, the Gene Amp PCR System 9700 (Perkin Elmer
Instruments Inc., USA) is used with one step of 94.degree. C. for 3
minutes, then 32 cycles of 94.degree. C., 47.degree. C., and
72.degree. C. steps of 25 sec each and one final step of 72.degree.
C. for 3 minutes. The PCR products are run on a 6% polyacrylamide
gel (30 cm.times.8 cm.times.1 mm) in 1.times. TAE (40 mM Tris-HCl,
pH 8.3, 1 mM EDTA) at 180 v for 45 minutes. The gels are stained
using SYBR Gold (Molecular Probes, Eugene, Oreg.) according to the
manufacturer's instructions.
[0320] SSR primer screening for polymorphism is performed using
PIC, HS-1, Will and PI507354 genotypes. SSRs that are polymorphic
and easy to score (i.e., clear banding pattern and good separation
between alleles) are mapped using the HS-1 X PIC (F2) and/or Will x
PI507354 (RIL) mapping populations. At least one SSR per BAC
sequence is mapped. DNA markers that exhibited codominant banding
patterns are scored as homozygous for one or the other parent or as
heterozygous, exhibiting both parental alleles. Marker scores are
checked for segregation distortion using the chi-squared test for
goodness of fit to expected ratios. Linkage relationships are
determined using Mapmaker Version 3.0b with a LOD of 3.0 (Whitehead
Institute, Cambridge, Mass.).
EXAMPLE 2
[0321] DNA fragments containing candidates for genes rhg1 and Rhg4
from susceptible and resistant soybean lines are subdloned into a
TA cloning plasmid (TOPO TA Cloning Kit, Version E, Invitrogen
Corporation, 1600 Faraday Avenue, Carlsbad, Calif.).
[0322] Genomic DNA from 24 susceptible and 9 resistant lines is
isolated using standard techniques. Approximately 500 nanograms
(ng) of DNA is used for PCR amplification. Resistant BAC DNA is
isolated by using AUTOGEN (AutoGen Corp., 35 Loring Drive
Framingham, Mass.). PCR amplification is then performed using
0.1-0.2 ng of resistant BAC DNA. The primers that are used to
amplify candidate rhg1 genes PCR are as follows:
[0323] Fragment 1(2,892 bp) primer (SEQ ID NO: 25), GCA ATA CTT GAA
GGA ATA TGT CCA C; primer (SEQ ID NO: 24), beginning at start
codon, ATG GAT GGT AAA AAT TCA AAA CTA AAC; modified reverse primer
1 (SEQ ID NO: 1123), beginning 5 bp before start codon; GTT GTA TGG
ATG GTA AAA ATT CAA AAC. Fragment 11 (1,746 bp) reverse primer 2
(SEQ ID NO: 27), ending at 13 bp after stop codon, GAC TGG CTG TGA
CTG ATC TCT CT; primer 2 (SEQ ID NO: 26), CTC ACT TAC ACT GCT GAA
TGC AGA.
[0324] The primers for Rgh4 PCR are as follows:
[0325] Forward primer (SEQ ID NO: 48), ATG TCT CTC CCC AAA ACC CTA
CTT TCT CTC; reverse primer (SEQ ID NO: 49), ending at 2 bp after
stop codon, GGT TAA CGG CAA TCC ATT GAA TCA AAG GAG.
[0326] PCR amplification is performed in an MJ Research PTC DNA
Engine TM System, Model PTC-225 (MJ Research Inc, 590 Lincoln
Street Waltham, Mass.). PCR is performed using the following
components: 1 .mu.g DNA, 5 .mu.l 10.times. buffer, 1 .mu.l primer
1, 1 .mu.l primer 2, 1 .mu.l 10 mM dNTP, 1.5 .mu.l 50 mM
MgCl.sub.2, 0.2 .mu.l Taq. (Platinum), 39.3 .mu.l H.sub.2O. The PCR
program used is as follows: 95.degree. C. for 10 minutes (step 1),
95.degree. C. for 30 seconds (step 2), 70.degree. C. for 30
seconds/-1.degree. C. per cycle/72.degree. C. for 3 minutes (step
3), repeat steps two through three 9 times (step 4), 95.degree. C.
for 30 seconds (step 5), 60.degree. C. for 30 seconds (step 6),
72.degree. C. for 3 minutes (step 7), repeat steps five through
seven 34 times (step 8), 4.degree. C. forever (step 9), end.
[0327] PCR products are separated on 1% agarose gel by
electrophoresis. A single DNA band is excised from gel. Gel
extraction is done using CLONTECH NucleoSpin Extraction Kit
(Clonetech Laboratories Inc., 1020 East Meadow Circle, Palo Alto,
Calif.). 2 .mu.l of purified DNA is loaded on 1% agarose gel to
check concentration. 40-100 ng of DNA is used for subcloning.
[0328] A TOPO cloning reaction is done according to the following:
4 .mu.l of fresh PCR product, 1 .mu.l Clontech Salt Solution, and 1
.mu.l TOPO vector. The solution is mixed gently, incubated for 10
minutes at room temperature, and then placed on ice.
[0329] A one shot chemical transformation is performed as follows.
2 .mu.l of the TOPO Cloning reaction is added to a vial of TOP 10
One Shot Chemically Competent E. coli and mixed gently. The mixture
is then Incubated on ice for 30 minutes. The cells are then
heat-shocked for 30 seconds at 42.degree. C., and immediately
transferred to ice. 250 .mu.l of SOC medium is then added, and the
mixture is incubated at 37.degree. C. for 1 hour. 80 .mu.l is then
spread onto a selective plate, and 170 .mu.l is spread onto another
plate. The plates are incubated at 37.degree. C. for 18-20 hours.
The selective plates are LB agar plates with 100 .mu.g/ml
ampicillin, 40 .mu.g/ml IPTG, and 40 .mu.g/ml X-GAL.
[0330] After incubation, 8-10 white or light blue colonies are
selected. The positive colonies are inoculated into LB medium
containing 50 .mu.g/ml ampicillin and incubated at 37.degree. C.
overnight. Sterilized glycerol is added to make 15% glycerol stock,
which can be stored at -80.degree. C.
[0331] Sanger sequencing reactions are performed on subclones using
BigDye Terminators (Applied Biosystems, 850 Lincoln Centre Drive,
Foster City, Calif.) and then analyzed on ABI 377/ABI 3700
automated sequencing machines (Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, Calif.). The sequences are evaluated for
quality and error probability using the program, PHRED (Ewing and
Green, Genome Res., 8:186-194 (1998), Ewing et al., Genome Res.,
8:175-185, (1998)), assembled using thephrap assembler and viewed
using consed (Gordon et al., Genome Res., 8:195-202). An rhg1
candidate gene is found in BAC 240017, and is about 4.5 kb in size.
An Rhg4 candidate was found in BAC 318013, and is about 3.5 kb in
size.
EXAMPLE 3
[0332] The physical mapping of a QTL (quantitative trait locus) is
described in this example. Mapping is initiated with linkage
analysis of SSR (simple sequence repeats) markers. Markers that are
shown to be linked to the QTL of interest are used to PCR screen
the soy BAC library and identify candidate BACs. Confirmed BACs are
subcloned and sequenced, BAC-end sequenced, and fingerprinted. New
markers are designed from good BAC-end sequences and used to screen
the library, by either PCR or hybridization to high density grid
filters, in order to extend the contigs. A BAC-end sequence and
fingerprint database of soy BACs is used in conjunction with the
above methods to help build and extend contigs. Sequenced BACs are
aligned, and overlapping BACs are placed into contigs. These
contigs, which contain unique sequences, are put into an ACEDB
database, and predicted genes are annotated by hand using various
programs. Candidates genes (for the gene of interest) are subdloned
from genomic DNA of different lines by PCR using primers from
outside the predicted coding regions. These subclones are sequenced
and screened for SNPs (single nucleotide polymorphisms) and INDELs
(insertions/deletions), and different haplotypes of the lines with
and without the desired phenotype are examined for correlations
between the haplotype and phenotype.
[0333] A single trifoliate leaf is collected from the newest growth
of four week old soybean plants. The leaf tissue is placed on ice
and stored at -80.degree. C. The frozen tissue is lyophilized and
approximately 0.01 grams of tissue is used for DNA extraction. The
leaf tissue is ground to powder in 1.4 ml tubes and 600 .mu.l of
DNA extraction buffer [0.5M NaCl, 0.1 M Tris-(hydroxymethyl)
aminomethane pH 8.0, 0.05 M ethylenediaminetetra-acetic acid
(EDTA), 10.0 g L.sup.-1 sodium dodecyl sulfate (SDS), 2 g L.sup.-1
phenantroline (dissolved in 0.01 L ethanol)] heated to 65.degree.
C. (with 0.77 g L.sup.-1 dithiothreitol added immediately before
use) is added to each tube and mixed thoroughly. The samples are
placed in a 65.degree. C. water bath for 15 minutes and shaken by
hand after 10 min. The samples are taken out of the water bath,
cooled to room temperature, and 200 .mu.l of 5 M KOAc is added to
each tube. The samples are inverted and placed at 4.degree. C. for
20 min. Samples are then centrifuged for 12 minutes at 6200.times.
g and the supernatant (about 600 pI) is transferred to new tubes.
DNA is precipitated with 330 .mu.l of cold isopropanol and placed
at -20.degree. C. for 1 hr. The DNA is pelleted by centrifuging at
6200.times. g for 10 minutes and is washed with 70% EtOH. The DNA
is pelleted by centrifugation at 6200.times. g for 10 minutes and
dried using a Speed-Vac. The DNA is dissolved in 100 .mu.l of
TE.sub.0.1 (0.01 M Tris-HCl pH 8.0, 0.0001 M EDTA). The extraction
yields 500 ng DNA .mu.l.sup.-1.
[0334] The polymerase chain reaction (PCR) is conducted with 5 to
10 ng genomic DNA in 10 .mu.l volumes of 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 0.001% gelatin, 1.5 mM MgCl.sub.2, 0.1 mM of each dNTP,
150 nM of each primer, 0.01 mM Cresol Red, 2% sucrose and 0.32
units of AmpliTaq DNA Polymerase (Perkin Elmer Instruments Inc.,
USA, 761 Main Avenue, Norwalk, Conn.). For thermocycling, the Gene
Amp PCR System 9700 (Perkin Elmer Instruments Inc., USA, 761 Main
Avenue, Norwalk, Conn.) is used with one step of 94.degree. C. for
3 min, then 32 cycles of 94.degree. C., 47.degree. C. and
72.degree. C. steps of 25 sec each and one final step of 72.degree.
C. for 3 min. The PCR products are run on a 6% polyacrylamide gel
(30 cm.times.8 cm.times.1 mm) in 1.times. TAE (40 MM Tris-dHC, pH
8.3, 1 mM EDTA) at 180 v for 45 min. The gels are stained using
SYBR Gold (Molecular Probes, Eugene, Oreg.) per manufacturers
instructions.
[0335] SSR primer screening for polymorphisms is performed using
PIC, HS-1, Will and PI507354 genotypes. SSRs that are polymorphic
and easy to score (i.e., Clear banding pattern and good separation
between alleles) are mapped using the HS-1 x PIC (F2) and/or Will x
PI507354 (RIL) mapping populations. At least one SSR per BAC
sequence is mapped. DNA markers that exhibited codominant banding
patterns are scored as homozygous for one or the other parent or as
heterozygous, exhibiting both parental alleles. Marker scores are
checked for segregation distortion using the chi-squared test for
goodness of fit to expected ratios. Linkage relationships were
determined using Mapmaker Version 3.0b with a LOD of 3.0 (Whitehead
Institute for Biomedical Research, Cambridge Mass.).
[0336] Thirty-two BAC DNA superpools (10 genomic equivalents)
extracted from either 4608 clones (48 96-well microtiter plates)
are used as templates for the first round of PCR screening.
Following identification of the positive superpools, the second
screening is performed against 4-D BAC DNA pools. Each clone of the
superpool is addressed 4-dimentionally (7.times.7.times.12.times.8)
and pooled in each dimension. Each set of 48 plates is divided into
6 sets of 7 plates and one set of 6 plates, and partitioned in two
ways. The first partition is in numerical order, plates 1-7, 8-14,
. . . 43-48 representing 7 group or stack pools. The second
partition is according to plate position within each of the
respective stacks, plates [1, 8, 15, 22, 29, 36], [2, 9, 16, 23,
30, 37, 43] etc., representing 7 plate pools. Each well of the
96-well plates contains 12 columns and 8 rows. Clones from row 1
are pooled from all 48 plates to generate the row 1 pool. Clones of
rows 2, 3, 4 . . . 8, and columns 1, 2, 3 . . . 12 are pooled to
generate 8 row pools and 12 column pools respectively.
[0337] For each superpool, BAC DNA is extracted from a total of 34
subpools (7+7+8+12). Positive clones are identified by TaqMan/PCR
screening of the 34 subpools if one positive clone is present. If
more than one positive clone is present in a superpool, a third
round of screening with N4 PCR reactions is performed.
[0338] Addresses of candidate BACs are identified, and the
candidates are streaked out for single colony isolation and grown
overnight at 37.degree. C. A single, isolated colony is picked and
streaked out and grown overnight at 37.degree. C. PCR is repeated
for the marker of interest (using the program designed for the
relevant marker) using a smear of cells from the plate streaked
from a single colony. The PCR product is run on a 2% agarose gel
and purified using the Clonetech NucleoSpin Gel Extraction Kit
(according to the manufacturer's instructions, Clonetech
Laboratories Inc., 1020 East Meadow Circle, Palo Alto, Calif.) and
10-50 ng of the purified DNA are added to 10 pmol of each primer
(forward and reverse), in a total volume of 6 .mu.l of ddH2O and 2
pl of BigDye Terminators (Applied Biosystems, 850 Lincoln Centre
Drive, Foster City, Calif.). The cycling conditions are: 96.degree.
C. for 1 minute (step 1), 96.degree. C. for 10 seconds (step 2),
50.degree. C. for 5 seconds (step 3), 60.degree. C. for 4 minutes
(step 4), steps 2-4 are repeated for 24 cycles (step 5), and hold
at 4.degree. C.
[0339] The generated sequence is compared to the consensus sequence
using DNA comparison software. Confirmed clones are subcloned,
sequenced, BAC-end sequenced, and Fingerprinted.
[0340] BAC-end sequencing is done using 3.2 pmol of SP6 and T7
primers (separately), approximately 600 ng-1 ug of BAC DNA (Autogen
prepped, AutoGen Corp., 35 Loring Drive Framingham, Mass.)
reaction, resuspended in 6 .mu.l of ddH20, and 4 .mu.l of BigDye
Terminators (Applied Biosystems 850 Lincoln Centre Drive, Foster
City, Calif.) to give a total reaction volume of 10 ul. The cycling
conditions are: 96.degree. C. for 2 minutes (step 1), 96.degree. C.
for 15 seconds (step 2), 50.degree. C. for 15 seconds (step 3),
60.degree. C. for 4 minutes (step 4), steps 2-4 are repeated for
50-60 cycles (step 5), 72.degree. C. for 2 minutes (step 6), hold
at 4.degree. C. or 10.degree. C. (step 7).
[0341] The reactions are ethanol precipitated and loaded on
capillary sequencers. The newly generated BAC-end sequence is
trimmed from the vector sequence, and entered into a database
containing approximately 400,000 BAC-end sequences. Each BAC is
blasted against the database to search for BAC-end matches
extension of the contigs. New markers are designed from good
BAC-end sequences, and these are then used to rescreen the library
in order to build up contigs across the region of interest.
Screening can be done in either of two ways: as above (PCR
strategy), or by hybridization of high-density grid filters from
Research Genetics (Research Genetics, 2130 Memorial Parkway,
Huntsville, Ala.).
[0342] The probes used for hybridization are derived from clones or
genomic DNA by PCR amplification using the vector or gene-specific
primers, with the appropriate cycling conditions. PCR products are
run on a 1% agarose gel containing ethidium bromide (0.2 ug/ml) in
IX TAE buffer at 100 volt for 1-2 hrs. Isolated DNA fragments are
excised and gel-purified using the Clonetech NucleoSpin gel
extraction kit (Clonetech Laboratories Inc., 1020 East Meadow
Circle, Palo Alto, Calif.), before labeling. In order to check the
size of the fragments and concentration, 2 .mu.l of eluted DNA plus
loading buffer are loaded on a 1% agarose gel along with DNA
markers of known concentration and size. All the probes used to
screen the library are tested individually for repetitiveness, with
a smaller filter spotted with random clones from the library along
with some positive control clones according to the protocol
described below.
[0343] The A3244 soy library generated by a an EcoRI digest is
spotted on 3 high density grid filters from Research Genetics
(Research Genetics, 2130 Memorial Parkway, Huntsville, Ala.). Each
filter has six fields, twelve 384 well plates are spotted in each
field in duplicate, with a total of 27,648 clones spotted on each
filter. The plates are spotted in a 5.times.5 grid (12 clones per
5.times.5 grid) pattern within each field. Each clone is spotted in
duplicate with a specific orientation within the 5.times.5 grid,
which, together with the field position, gives information about
its address. In a first round hybridization procedure, multiple
probes are labeled separately and then pooled together to hybridize
to BAC filters. Positive BACs identified in this procedure are
deconvoluted by rehybridization with the individual probes.
[0344] A hybridization oven is set at 65.degree. C., and Church
Buffer (0.5 M Sodium Phosphate, pH 7.0, 7% SDS, 1% bovine serum
albumin, 1 mM EDTA, 100 .mu.g/ml salmon sperm DNA) is prewarmed to
65.degree. C. Membranes are washed in 500 ml of 0.1.times.SSC, 0.1%
SDS in a large container at room temperature for 5 minutes with
gentle shaking (50 rpm) on a rotary shaker. The membranes are
rinsed with 500 ml of 0.1.times.SSC (no SDS) for 1 minute. The wash
solution is poured off, and 500 ml of 6.times.SSC (no SDS) is added
to equilibrate the membranes. Three filters are placed in a tube.
The filters are separated from each other and the sides of the tube
by a layer of mesh. Each tube is filled with 6.times.SSC and shaken
gently with the tube vertical to help eliminate bubbles between the
filters and tube wall. The 6.times.SSC solution is poured off, and
25 ml of pre-warmed Church buffer is added. The bottles are rotated
in a hybridization oven at 60 rpm and 65.degree. C. for 30 minutes
or longer.
[0345] Probes are labeled using 1 .mu.l of 40-50 uCi/.mu.l
[.alpha..sup.32P dCTP], 50 ng of purified DNA in 49 .mu.l of ddH2O,
and Read-To-Go Labeling Beads from Amersham Pharmacia according to
the manufacturers instructions (Amersham Pharmacia, Uppsala,
Sweden). The probes are purified using the Bio-Spin Column P30 from
BioRad according to manufacturers instructions (Bio-Rad
Laboratories, 3316 Spring Garden Street, Philadelphia, Pa.). To 1
.mu.l of the column-purified probe is added to a minipoly-Q vial
(liquid scintillation vial) for each probe. 5 ml of scintillation
liquid is added to each vial, and radiation activity for each vial
is measured using a liquid scintillation counter.
[0346] After the probes are purified and counted for radioactivity,
10-20 probes and one control probe (from 50 .mu.l reaction) are
pooled with 107 cpm/probe each, into one 1.5 ml eppendorf tube. The
pooled probes are denatured at 99.degree. C. in a sand heating
block for 10 minutes. The tubes are cooled on ice or ice water
about 2 minutes, and then spun down at 14,000 rpm for 30 seconds in
microcentrifuge. The tubes are pre-hybridized in 25ml of Church
buffer for at least 30 minutes, which is then poured off. 40 ml of
fresh hybridization solution (pre-warmed Church buffer) is added.
The pooled-probe solution is added to the hybridization tube. The
tube is rotated in the hybridization oven at 60 rpm, 65.degree. C.
overnight.
[0347] The probe solution is poured off, 30 ml of pre-warmed
(65.degree. C.) 1.times.SSC, 0.1% SDS washing solution is added to
the hybridization tube, the hybridization tube is rotated in the
hybridization oven (at 65.degree. C.) for 15 minutes, and the
process is repeat two times. At the last wash, the tube is rotated
180.degree. and at the same speed for 15 minutes at 65.degree. C.
The washing solution is poured off, and 2.times.SSC (no SDS) is
added.
[0348] Excess liquid is removed from each filter by placing the
filter on a piece of 3MM paper. The washed filter is placed on
developed film with the DNA-side up (the side BACs were spotted
on), covered with Saran wrap, and squeezed to force out liquid and
bubbles. The Saran wrap is folded to the other side of the film,
fixed it with tape, and then dried Kimwipes. The wrapped filters
are placed into a film cassette with the DNA-side up (the side BACs
were spotted on), which is placed on BioMax MS film (Biomax
Technologies Inc., Vancouver, BC, Canada) in a darkroom, and
exposed overnight at room temperature without an intensifying
screen. Film is developed with a film developer in the dark room
the next day, and each film is labeled with filter number, probe
used for hybridization, exposure time, and date.
[0349] Starting from Field 3, a 384-well grid is put on the field
with the Al position of the grid on the upper right, and the grid
is aligned to the image. The row and column position for each
positive clone on the BAC recording spreadsheet is determined and
recorded. The pattern of the hybridization signal is matched to
known patterns. There are 6 plate reference numbers for each of
twelve patterns, which are arranged in the same manner as the 6
fields. Based on the signal pattern and field number, a plate
reference number is determined for each positive clone. The grid is
moved to the next field and the process is repeated. The original
plate number (P) is determined using the following formula:
P=(N-1).times.72+R, where N is the filter number on which the
identified clone is present and R is the plate reference number
previously determined. The complete address of the identified clone
is given by the original plate number plus its position on the
plate determined previously. BACs' addresses are identified and
converted to "imp" files according to a Q-bot file format.
[0350] 24 working plates are loaded into a Q-bot (Genetix,
Queensway, New Milton, Hampshire, United Kingdom) 6-high hotel and
media-filled 96-well plates are placed on the deck. The Q-bot is
run following the standard manual using the program called
"Rearraying98" with the settings given in Appendix III of the
accompanying manual: BAC-Picking. Plates containing picked clones
are placed in a shaker incubator and grown overnight at 37.degree.
C. at 200 rpm.
[0351] 35 .mu.l DNA solution are transferred from 96-well plates
into a 384-well plate using a Platemate such that 4 96-well plates
of DNA are combined into one 384-well plate. The 384-pin head
(puck) is washed in 10% SDS solution for 5 minutes, ultrasonicated
in a water bath for 3 minutes, washed with 70% ethanol for 1 min.,
and air dried for 3 minutes. The 384-well DNA source plates and
membranes are arranged on the deck according to the instruction
from the manual and the spotted grid design chosen for the
membrane. Spotting pattern are designed so that there is one
control probe at each of the 4 corners of the membrane. An
asymmetric pattern is used to orient filters. The control probe
concentration is about 5 ng/ul. Zeus is run according to
instructions. If the DNA concentration is lower than 5 ng/ul, the
Zeus is run a second time to double the amount of spotted DNA on
the membrane. One of the empty spots is spot dyed, if available,
using one 384-well dye plate. If an empty spot is not available, it
is printed on one of the DNA spots. This spot marks the position
for cutting filters into small membranes (9.times.12 cm). Membranes
are interleaved between 3M papers and left to air-dry. Each corner
of each membrane is marked with a permanent marker and numbered.
Filters are denatured on the surface of 3M paper soaked with
denaturalization solution for 4 minutes, and then neutralized on
the surface of 3 M paper soaked with neutralization solution for 5
minutes. The filters are washed with 2.times.SSC for 5 minutes and
then air dried. The filters are then baked at 80.degree. C. for 1
hr. and cut into individual small membranes (9.times.12 cm)
according to the marked corner.
[0352] To confirm and deconvolute, hybridizations are done as
before, but with newly generated filters, and each probe is done
separately with a single filter using the smaller tube. 15 ml of
Church buffer is used for the hybridization.
[0353] Fingerprints are generated by digesting the BAC DNA with
Hind III for 3 hours at 37.degree. C. and running the reaction on a
0.8% gel at 200V for 19 hours. The gels are stained with SybrGreen,
while shaking at room temperature for 45 minutes, and scanned with
a Flourimager. The bands are sized using Frag software and the
fingerprints are assembled into contigs within FPC. Every time new
clones are added the contigs are rebuilt using a tolerance of 10
and a cutoff of 10.sup.-9.
[0354] Subclones are generated and Sanger sequencing reactions were
performed on randomly chosen subclones using BigDye Terminators
(Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif.)
then analyzed on ABI 377/ABI 3700 automated sequencing machines
(Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif.
7-8 fold sequence coverage is thereby generated across the BAC. The
sequences are evaluated for quality and error probability using the
program, phred, assembled using the phrap assembler, and viewed
using consed, as in example 2. For Bermuda standard BACs, all
contigs are ordered and oriented and all gaps are closed using a
directed primer walking strategy. A final quality value of phred40
(1 base error in 10,000 bases) with no gap regions, double coverage
or two chemistries across single stranded areas is achieved.
[0355] The sequence contigs are put into an ACEDB database along
with soy EST and plant EST matches, along with Blastx, Thlastx, and
Plant Blastn hits. Genemark.hmm is used to predict possible genes,
and GeneFinder is used to predict splicing sites, ORFs, potential
coding regions, as well as start and stop codons. The contigs are
then annotated by hand and predicted genes accepted, edited, and
modified based on the characteristics present in the sequence and
matches to protein, nucleotide, and EST databases.
[0356] The high-density BAC library membranes used for
hybridization are made by Research Genetics (Research Genetics,
2130 Memorial Parkway, Huntsville, Ala., using a modified Q-bot
(Genetix, Queensway, New Milton, Hampshire, United Kingdom),
384-well plates containing BACs are spotted onto 22 cm.times.22 cm
Hybond N+ membranes (Amersham Pharmacia, Uppsala, Sweden). Bacteria
from 72 plates are spotted twice onto one membrane, giving 55,296
colonies in total, or 27,648 unique clones per membrane. The plates
are spotted into six "fields" per membrane, with each field having
12 plates spotted in duplicate. This spotting format results in six
fields with 384 grids in each field. Each grid is a 5.times.5
matrix containing 12 unique clones in duplicate, with the center
position left empty. The two positions occupied by each clone in
duplicate are designed to give a unique pattern that indicates the
plate location of each clone. After spotting, the bacteria on the
membrane are incubated for 8 hours on LB-agar plates containing
12.5 ug/ml chloramphenicol. The membranes are then denatured,
neutralized, washed in a standard procedure, UV-light crosslinked,
and air-dried. The membranes can be stored and shipped at room
temperature.
[0357] Every reference, patent, or other published work cited above
is herein incorporated by reference in its entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060225150A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060225150A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References