U.S. patent application number 16/062597 was filed with the patent office on 2018-12-27 for compositions and methods for manipulating the development of plants.
This patent application is currently assigned to The New Zealand Institute for Plant and Food Research Limited. The applicant listed for this patent is The New Zealand Institute for Plant and Food Research Limited. Invention is credited to Toshi Marie FOSTER.
Application Number | 20180371481 16/062597 |
Document ID | / |
Family ID | 59055889 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371481 |
Kind Code |
A1 |
FOSTER; Toshi Marie |
December 27, 2018 |
Compositions and Methods for Manipulating the Development of
Plants
Abstract
The invention provides a methods and materials for producing and
selecting plants with at least one dwarfing-associated phenotype.
The methods and materials relate to altering the expression, or
activity, of an ARF3 poypeptide in the plant, and selecting plants
with altered the expression, or activity, of an ARF3 poypeptide.
The invention also provides plants produced or selected by the
methods. The methods also involve crossing plants of the invention
with other plants to produce further plants with at least one
dwarfing-associated phenotype.
Inventors: |
FOSTER; Toshi Marie;
(Ashhurst, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The New Zealand Institute for Plant and Food Research
Limited |
Auckland |
|
NZ |
|
|
Assignee: |
The New Zealand Institute for Plant
and Food Research Limited
Auckland
NZ
|
Family ID: |
59055889 |
Appl. No.: |
16/062597 |
Filed: |
December 14, 2016 |
PCT Filed: |
December 14, 2016 |
PCT NO: |
PCT/IB2016/057631 |
371 Date: |
June 14, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8261 20130101;
A01H 5/08 20130101; C12N 15/67 20130101; C12N 15/827 20130101; C12Q
1/6895 20130101; C12N 15/8241 20130101; C12N 15/8294 20130101; A01H
6/7418 20180501; C12Q 1/68 20130101; C07K 14/415 20130101; C12N
15/8223 20130101; Y02A 40/146 20180101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2015 |
NZ |
714988 |
Claims
1-47. (canceled)
48. A method for producing a plant with at least one
dwarfing-associated phenotype the method comprising altering the
expression, or activity, of an ARF3 poypeptide in the plant,
wherein the dwarfing-associated phenotype is selected from: i) one
of the following phenotypes in the plant: a) altered auxin
transport, b) slower auxin transport, c) reduced apical dominance,
d) an altered xylem/phloem ratio, e) an increased number of phloem
elements, f) smaller phloem elements, g) thicker bark, h) a bushier
habit, i) reduced root mass, and ii) competence to induce one of
the following phenotypes in a scion grafted on to the plant: j)
reduced vigour, k) less vegetative growth, l) earlier termination
of shoot growth, m) earlier competence to flower, n) precocity, o)
earlier phase change, p) smaller canopy, q) reduced stem
circumference, r) reduced branch diameter, s) fewer sylleptic
branches, t) shorter sylleptic branches, u) more axillary flowers,
v) an earlier teminating primary axis, w) earlier teminating
secondary axes, and x) shorter intenode length y) reduced scion
mass.
49. The method of claim 48 comprising increasing the expression of
the ARF3 poypeptide in the plant.
50. The method of claim 48 comprising transforming the plant to
express the ARF3 poypeptide in the plant.
51. The method of claim 50 comprising transforming the plant with
polynucleotide encoding the ARF3 polypeptide.
52. The method of claim 51 wherein polynucleotide is operably
linked to a heterologous promoter.
53. The method of claim 48 comprising modifying the sequence of an
endogenous polynucleotide encoding the ARF3 polypeptide in the
plant.
54. The method of claim 53 wherein modifying the endogenous
polynucleotide alters the activity of the ARF3 polypeptide in the
plant to induce the dwarfing-associated phenotype.
55. The method of claim 48 wherein the dwarfing-associated
phenotype in the plant is at least one of reduced apical dominance,
a bushier habit, an altered xylem/phloem ratio, an increased number
of phloem elements and reduced root mass.
56. The method of claim 48 wherein the dwarfing-associated
phenotype is the competence to induce at least one of: reduced
vigour, less vegetative growth, earlier termination of shoot
growth, a smaller canopy, reduced stem circumference, and reduced
scion mass, in a scion grafted on to the plant.
57. The method of claim 48 wherein the method includes the step of
grafting a scion on to a plant produced by the method.
58. A method for producing a plant with at least one
dwarfing-associated phenotype selected from: j) reduced vigour, k)
less vegetative growth l) earlier termination of shoot growth m)
earlier competence to flower n) precocity o) earlier phase change
p) smaller canopy, q) reduced stem circumference r) reduced branch
diameter s) fewer sylleptic branches t) shorter sylleptic branches
u) more axillary flowers v) an earlier teminating primary axis, w)
earlier teminating secondary axes, x) shorter intenode length y)
reduced scion mass the method comprising the steps: A. providing a
plant with altered the expression or activity of a ARF3 poypeptide
produced by the method of claim 48, B. grafting a scion onto the
plant in A wherein at least one of j) to y) is exhibited in the
scion grafted on to the plant in A.
59. The method of claim 58 wherein the phenotype exhibited in the
scion is at least one of: reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, reduced stem
circumference, and reduced scion mass.
60. The method of claim 48 in which the ARF3 polypeptide has a
sequence with at least 70% identity to any one of SEQ ID NO:1 to
11, 28 and 29.
61. The method of claim 60 in which the ARF3 polypeptide has a
sequence with at least 70% identity to SEQ ID NO:1 or 28
(MdARF3).
62. The method of claim 60 in which the ARF3 polypeptide comprises
a Leucine residue at the position corresponding amino acid residue
72 in SEQ ID NO:1 or 28 (MdARF3).
63. The method of claim 60 in which the ARF3 polypeptide comprises
the sequence of SEQ ID NO:2 or 29 (M9 MdARF3)
64. The method of claim 48 in which the alteration results in
expression of an ARF3 polypeptide with a Leucine residue at a
position corresponding the amino acid residue 72 in SEQ ID NO:1 or
28 (MdARF3).
65. A construct, cell or plant comprising a polynucleotide encoding
an ARF3 polypeptide, or a fragment or variant thereof with ARF3
activity, comprising a Leucine residue at a position corresponding
the amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).
66. The construct, cell or plant of claim 65 wherein the ARF3
polypeptide comprises at least 70% identity to SEQ ID NO:2 or 29
(MdARF3).
67. The construct, cell or plant of claim 65 wherein the
polypeptide comprises the sequence of SEQ ID NO:2 or 29 (M9
MdARF3).
68. The construct, cell or plant of claim 65 wherein the
polynucleotide has at least 70% identity to at least one of SEQ ID
NO:14 and 15.
69. An isolated polynucleotide comprising the sequence with at
least 70% identity to at least one of SEQ ID NO:14 or 15, or a
fragment thereof, encoding a polypeptide with ARF3 activity.
70. An isolated ARF3 polypeptide encoded by the polynucleotide of
claim 65, or a fragment thereof with ARF3 activity, comprising a
Leucine residue at a position corresponding the amino acid residue
72 in SEQ ID NO:1 or 28.
71. A construct comprising the polynucleotide of claim 65 operably
linked to a heterologous promoter.
72. A construct comprising the polynucleotide of claim 69 operably
linked to a heterologous promoter.
73. A cell plant, plant part, propagule or progeny comprising the
polynucleotide of claim 65.
74. A cell plant, plant part, propagule or progeny comprising the
polynucleotide of claim 69.
75. A method for identifying a plant with a genotype indicative of
at least one dwarfing-associated phenotype, the method comprising
testing a plant for at least one of: a) altered expression of at
least one ARF3 polypeptide, b) altered expression of at least one
ARF3 polynucleotide, c) presence of a marker associated with
altered expression of at least one ARF3 polypeptide, d) presence of
a marker associated with altered expression of at least one ARF3
polynucleotide, e) presence of a marker associated with altered
activity of at least one ARF3 polypeptide, wherein presence of any
of A) to E) indicates that the plant has at least one
dwarfing--associated phenotype, and wherein the dwarfing-associated
phenotype is selected from: i) one of the following phenotypes in
the plant: a) altered auxin transport, b) slower auxin transport,
c) reduced apical dominance, d) an altered xylem/phloem ratio, e)
an increased number of phloem elements, f) smaller phloem elements,
g) thicker bark, h) a bushier habit, i) reduced root mass, and ii)
competence to induce one of the following phenotypes in a scion
grafted on to the plant: j) reduced vigour, k) less vegetative
growth, l) earlier termination of shoot growth, m) earlier
competence to flower, n) precocity, o) earlier phase change, p)
smaller canopy, q) reduced stem circumference, r) reduced branch
diameter, s) fewer sylleptic branches, t) shorter sylleptic
branches, u) more axillary flowers, v) an earlier teminating
primary axis, w) earlier teminating secondary axes, and x) shorter
intenode length y) reduced scion mass.
76. The method of claim 75 in which the marker associated with
altered activity of at least one ARF3 polypeptide is presence of a
Leucine residue at a position corresponding the amino acid residue
72 in SEQ ID NO:1 or 28 (MdARF3).
77. The method of claim 76 in which the method involves detection
of a polynucleotide encoding the Leucine residue at a position
corresponding the amino acid residue 72 in SEQ ID NO:1 or 28
(MdARF3).
78. The method of claim 75 including an additional step of at least
one of: a) cultivating the identified plant, and b) breeding from
the identified plant.
79. A method for producing a plant with at least one
dwarfing-associated phenotype, the method comprising crossing a
plant produced by a method of claim 48 with another plant, wherein
the off-spring produced by the crossing is a plant with at least
one dwarfing-associated phenotype.
80. A method of producing a plant with at least one
dwarfing-associated phenotype selected from: j) reduced vigour, k)
less vegetative growth, l) earlier termination of shoot growth, m)
earlier competence to flower, n) precocity, o) earlier phase
change, p) smaller canopy, q) reduced stem circumference, r)
reduced branch diameter, s) fewer sylleptic branches, t) shorter
sylleptic branches, u) more axillary flowers, v) an earlier
teminating primary axis, w) earlier teminating secondary axes, x)
shorter intenode length, y) reduced scion mass, the method
comprising grafting a scion onto a plant produced by a method of
claim 48.
81. The method of claim 80 in which the at least one dwarfing
associated phenotype is exhibited in the grafted scion.
82. The method of claim 81 in which the grafted scion exhibits at
least one of reduced vigour, less vegetative growth, earlier
termination of shoot growth, a smaller canopy, reduced stem
circumference and reduced scion mass.
83. A plant that has been altered from the wild type to include a
Leucine residue at the position corresponding to amino acid residue
72 in SEQ ID NO: 1 or 28.
84. The plant of claim 83 that has at least one dwarfing associated
phenotype selected from: i) one of the following phenotypes in the
plant: a) altered auxin transport, b) slower auxin transport, c)
reduced apical dominance, d) an altered xylem/phloem ratio, e) an
increased number of phloem elements, f) smaller phloem elements, g)
thicker bark, h) a bushier habit, i) reduced root mass, and ii)
competence to induce one of the following phenotypes in a scion
grafted on to the plant: j) reduced vigour, k) less vegetative
growth, l) earlier termination of shoot growth, m) earlier
competence to flower, n) precocity, o) earlier phase change, p)
smaller canopy, q) reduced stem circumference, r) reduced branch
diameter, s) fewer sylleptic branches, t) shorter sylleptic
branches, u) more axillary flowers, v) an earlier teminating
primary axis, w) earlier teminating secondary axes, and x) shorter
intenode length y) reduced scion mass.
Description
TECHNICAL FIELD
[0001] The invention relates to compositions and methods for the
manipulation of plant development.
BACKGROUND
[0002] Dwarfing rootstocks have revolutionized the production of
some tree and vine crops, by permitting high-density plantings that
increase fruit yield in the early years of orchard establishment
(Ferree and Carlson 1987; Webster and Wertheim 2003; Gregory and
George 2011). The widespread use of dwarfing rootstocks has led to
a steady increase in the efficiency of apple production over the
past century (Hirst and Ferree 1995; Webster 1995).
[0003] `Malling9` (`M9`) is the most frequently used apple dwarfing
rootstock in both commercial and home orchards (Webster 1995).
`M9`, originally called `Jaune de Metz`, was discovered as single
seedling in the 1800s and was clonally propagated as a rootstock
because of its effects on both precocity and vigour control of the
grafted scion (Carriere 1897). At the beginning of the 20.sup.th
century, all the apple rootstocks grown in Western Europe were
collected at the East Mailing Research Station (UK) and classified
according to their effect on the grafted scion (Hatton 1917). Many
of the apple rootstock varieties bred worldwide have parentage
derived from this `Mailing` series, particularly `M9` (Manhart
1995; Webster and Wertheim 2003). Progeny of `M9` segregate for
rootstock-induced dwarfing, indicating that this trait is
determined by one or more genetic factors.
[0004] Dwarfing is a complex phenomenon, with some
dwarfing-associated phenotypes being exhibited in the root stock
plant, and other dwarfing-associated phenotypes being exhibited in
scions grafted onto the root stock plants.
[0005] Phenotypes reported in M9 root stock plants include: altered
xylem/phloem ratio, more phloem elements, smaller phloem elements,
thicker bark, altered auxin transport, slower auxin transport, and
reduced apical dominance. Grown as an ungrafted plant, M9 is also
bushier than other types of non-grafted apples.
[0006] Based on the altered xylem/phloem phenotypes, researchers
have suggested that dwarfing roots tocks function by altering the
transport of water, nutrients or hormones. A number of studies have
measured hormone concentration and/or movement in dwarfing
rootstocks; auxin in particular seems to play a major role in
rootstock induced dwarfing (Hooijdonk, Woolley et al. 2011).
Soumelidou was the first to demonstrate that `M9` apple stems
transport auxin at a slower rate than non-dwarfing stems
(Soumelidou K 1994). More recently, it has been shown that treating
apple trees with NPA, a polar auxin transport inhibitor,
phenocopies the effect of a dwarfing rootstock (van Hooijdonk
2010).
[0007] Despite M9 rootstocks being so widely used and the subjects
of numerous studies, the underlying mechanism by which dwarfing
rootstocks control both scion vigour and flowering remains
unresolved.
[0008] In woody perennials where a dwarfing or vigour-reducing
rootstock exists, the overall effect on the grafted scion is
characterised by less vegetative growth, earlier termination of
shoot growth, earlier competency to flower than non-grafted trees
or trees on vigorous rootstocks (also called precocity), earlier
phase change (a term which is related to earlier flowering, but
also encompasses other traits, such as thorns, leaf shape, etc), a
smaller canopy, reduced stem circumference (or TCA, Trunk
Cross-sectional Area), weaker shoot system, reduced branch
diameter.
[0009] The first detectable effects on apple scions grafted onto M9
rootstock are fewer and shorter sylleptic branches (axillary
meristems that grow out in the same season they were initiated),
more axillary flowers (these do not appear until the spring of year
two, but are formed in summer of year 1), and a tendency for both
the primary axis and secondary axes to terminate earlier
(Seleznyova, Thorp et al. 2003; Seleznyova, Tustin et al. 2008; van
Hooijdonk, Woolley et al. 2010; van Hooijdonk, Woolley et al.
2011).
[0010] An increased proportion of axillary floral buds along the
primary axis can have a profound impact on the subsequent growth of
the scion. In a floral bud, the sympodial "bourse" shoot that
develops from an axillary meristem is much less vigorous than the
monopodial shoot that continues growth from the apex of a
vegetative bud. Bourse shoots do not begin extension until anthesis
of the flowers and are developmentally delayed relative to
monopodial shoots, which begin growth immediately after budbreak.
The effects of increased flowering and reduced sylleptic shoot
number and length in year one became amplified in successive growth
seasons, and within three years, scions grafted on dwarf or
semi-dwarf rootstocks exhibited a distinctly reduced canopy size
and branching density.
[0011] Quantitantive trait loci (QTL) associated with dwarfing have
been identified in apple dwarf rootstock. For example, Pilcher et
al (2008) generated a segregating rootstock population derived from
a cross of `M9` and the vigorous rootstock `Robusta 5` (`R5`). The
progeny were all grafted with `Braeburn` scions and the scions were
phenotyped over seven years. Using a bulked segregant analysis
(comparing pooled rootstock DNAs from dwarfed and vigorous trees)
of a the rootstock population, the authors identified a major
dwarfing locus (Dw1) derived from `M9` and located at the top of
linkage group (LG) 5 (Pilcher, Celton et al. 2008) (FIG. 1a). Some
of the vigorous individuals in this population carried Dw1,
suggesting there were one or more additional rootstock loci that
influence dwarfing of the scion. Using an enlarged population from
the same cross, a genetic map was constructed which enabled a
multi-trait quantitative trait locus (QTL) analysis of
rootstock-induced dwarfing (Celton, Tustin et al. 2009).
[0012] More recently Fazio et al characterised two dwarfing loci
Dw1 and Dw2 and reported that the strongest degree of dwarfing was
conferred by rootstock with both Dw1 and Dw2 whereas either Dw1 or
Dw2 alone affected dwarfing (Celton et al 2009). The authors also
reported the Dw1 QTL to be located between the marking Hi22f12 and
Hi04a08 defining an interval of 2.46 Mb.
[0013] The introduction of dwarfing into new apple cultivars is
only currently achievable, through the laborious and slow
procedures of breeding. Breeding of any fruit is also of course
limited by the compatability of breeding species.
[0014] It would be beneficial to have tools or methods to introduce
dwarfing, or dwarfing-associated phenotypes into new species where
dwarfing technology is not yet available. Furthermore, even in
species where dwarfing technology is available, it would also be
advantageous to be able to more efficiently introduce dwarfing into
certain cultivars, or root stock cultivars, that are well adapted
to their local environment.
[0015] It is an object of the invention to provide materials and
methods for producing dwarfing and/or at least one
dwarfing-associated phenotype in plant, and/or at least to provide
the public with a useful choice.
SUMMARY OF THE INVENTION
[0016] Method
[0017] In the first aspect the invention provides a method for
producing a plant with at least one dwarfing-associated phenotype
the method comprising altering the expression, or activity, of an
ARF3 poypeptide in the plant.
[0018] In one embodiment the the method comprises increasing the
expression of the ARF3 poypeptide in the plant.
[0019] In a further embodiment the method comprises transforming
the plant to express the ARF3 poypeptide in the plant.
[0020] In a further embodiment the method comprises transforming
the plant with polynucleotide encoding the ARF3 polypeptide.
[0021] In a further embodiment the polynucleotide is operably
linked to a heterologous promoter.
[0022] In a further embodiment the method comprises modifying the
sequence of an endogenous polynucleotide encoding the ARF3
polypeptide in the plant.
[0023] In one embodiment, modifying the endogenous polynucleotide
alters the activity of the ARF3 poypeptide in the plant to induce
the dwarfing-associated phenotype.
[0024] In one embodiment the dwarfing-associated phenotype is
selected from:
[0025] a) altered auxin transport, [0026] b) slower auxin
transport, [0027] c) reduced apical dominance, [0028] d) an altered
xylem/phloem ratio, [0029] e) an increased number of phloem
elements, [0030] f) smaller phloem elements, [0031] g) thicker
bark, [0032] h) a bushier habit, [0033] i) reduced root mass,
[0034] j) reduced vigour, [0035] k) less vegetative growth, [0036]
l) earlier termination of shoot growth, [0037] m) earlier
competence to flower, [0038] n) precocity, [0039] o) earlier phase
change, [0040] p) smaller canopy, [0041] q) reduced stem
circumference, [0042] r) reduced branch diameter, [0043] s) fewer
sylleptic branches, [0044] t) shorter sylleptic branches, [0045] u)
more axillary flowers, [0046] v) an earlier teminating primary
axis, [0047] w) earlier teminating secondary axes, [0048] x)
shorter intenode length, and [0049] y) reduced scion mass.
[0050] In one embodiment the dwarfing-associated phenotype is
selected from a) to i). In a further embodiment the
dwarfing-associated phenotype is selected from a) to h). In one
embodiment a plant with at least one of these phenotypes is
suitable for use as a rootstock plant. In a further embodiment the
dwarfing-associated phenotype in this plant is at least one of
reduced apical dominance, a bushier habit, an altered xylem/phloem
ratio, an increased number of phloem elements, and reduced root
mass.
[0051] In a further embodiment the dwarfing-associated phenotype is
the competence to induce at least one of a) to y) in a scion
grafted on to the plant. In a further embodiment the
dwarfing-associated phenotype is the competence to induce at least
one of a) to h) and j) to x) in a scion grafted on to the
plant.
[0052] In a preferred embodiment the dwarfing-associated phenotype
is the competence to induce at least one of j) to y) in a scion
grafted on to the plant.
[0053] In a further embodiment the dwarfing-associated phenotype is
the competence to induce at least one of: reduced vigour, less
vegetative growth, earlier termination of shoot growth, a smaller
canopy, reduced stem circumference, and reduced scion mass in a
scion grafted on to the plant.
[0054] In a further embodiment the method includeds the step of
grafting a scion on to a plant produced by the method.
[0055] In a further embodiment the dwarfing-associated phenotype is
the competence to induce at least one of: reduced vigour, less
vegetative growth, earlier termination of shoot growth, a smaller
canopy, and reduced stem circumference, in a scion grafted on to
the plant.
[0056] In a further embodiment the method includeds the step of
grafting a scion on to a plant produced by the method.
[0057] In one embodiment the dwarfing-associated phenoytype is
exhibited in a scion grafted onto the plant.
[0058] In one embodiment the dwarfing-associated phenoytype
exhibited in the scion is at least one of j) to y). In one
embodiment the dwarfing-associated phenoytype exhibited in the
scion is at least one of j) to x).
[0059] In a further embodiment the dwarfing-associated phenoytype
exhibited in the scion is at least one of: reduced vigour, less
vegetative growth, earlier termination of shoot growth, a smaller
canopy, reduced stem circumference, and reduced scion mass, in a
scion grafted on to the plant.
[0060] In a further embodiment the dwarfing-associated phenoytype
exhibited in the scion is at least one of: reduced vigour, less
vegetative growth, earlier termination of shoot growth, a smaller
canopy, and reduced stem circumference, in a scion grafted on to
the plant.
[0061] In a further embodiment the invention provides a method of
producing a plant with at least one dwarfing-associated phenotype
selected from:
[0062] a) altered auxin transport, [0063] b) slower auxin
transport, [0064] c) reduced apical dominance, [0065] d) an altered
xylem/phloem ratio, [0066] e) an increased number of phloem
elements, [0067] f) smaller phloem elements, [0068] g) thicker
bark, [0069] h) a bushier habit, [0070] i) reduced root mass,
[0071] j) reduced vigour, [0072] k) less vegetative growth, [0073]
l) earlier termination of shoot growth, [0074] m) earlier
competence to flower, [0075] n) precocity, [0076] o) earlier phase
change, [0077] p) smaller canopy, [0078] q) reduced stem
circumference, [0079] r) reduced branch diameter, [0080] s) fewer
sylleptic branches, [0081] t) shorter sylleptic branches, [0082] u)
more axillary flowers, [0083] v) an earlier teminating primary
axis, [0084] w) earlier teminating secondary axes, [0085] x)
shorter intenode length, and [0086] y) reduced scion mass, the
method comprising grafting a scion onto a plant produced by a
method of the invention.
[0087] In this embodiment the at least one dwarfing-associated
phenotype is preferably exhibited in the grafted scion. In this
embodiment the grafted scion exhibits at least one of j) to y). In
a further the grafted scion exhibits at least one of j) to x).
[0088] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, reduced stem
circumference, and reduce scion mass, in a scion grafted on to the
plant.
[0089] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, and reduced
stem circumference, in a scion grafted on to the plant.
[0090] In a further embodiment the invention provides a method for
producing a plant with at least one dwarfing-associated phenotype
selected from: [0091] a) altered auxin transport, [0092] b) slower
auxin transport, [0093] c) reduced apical dominance, [0094] d) an
altered xylem/phloem ratio, [0095] e) an increased number of phloem
elements, [0096] f) smaller phloem elements, [0097] g) thicker
bark, [0098] h) a bushier habit, [0099] i) reduced root mass,
[0100] j) reduced vigour, [0101] k) less vegetative growth, [0102]
l) earlier termination of shoot growth, [0103] m) earlier
competence to flower, [0104] n) precocity, [0105] o) earlier phase
change, [0106] p) smaller canopy, [0107] q) reduced stem
circumference, [0108] r) reduced branch diameter, [0109] s) fewer
sylleptic branches, [0110] t) shorter sylleptic branches, [0111] u)
more axillary flowers, [0112] v) an earlier teminating primary
axis, [0113] w) earlier teminating secondary axes, [0114] x)
shorter intenode length, [0115] y) reduced scion mass, the method
comprising the steps: [0116] A. providing a plant with altered the
expression or activity of a ARF3 poypeptide, [0117] B. grafting a
scion onto the plant in A wherein at least one of j) to y) is
exhibited in the scion grafted on to the plant in A.
[0118] In a further embodiment at least one of j) to x) is
exhibited in the scion grafted on to the plant in A.
[0119] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, reduced stem
circumference, and reduced scion mass, in a scion grafted on to the
plant.
[0120] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, and reduced
stem circumference, in a scion grafted on to the plant.
[0121] In one embodiment the plant in A has increased expression of
the ARF3 poypeptide.
[0122] In a further embodiment the plant in A has been transformed
to express the ARF3 poypeptide.
[0123] In a further embodiment the plant in A is transgenic for a
polynucleotide encoding the ARF3 polypeptide.
[0124] In a further embodiment the polynucleotide is operably
linked to a heterologous promoter.
[0125] In a further embodiment the plant in A comprises a
modification in an endogenous polynucleotide encoding the ARF3
polypeptide in the plant.
[0126] In a further embodiment the modification alters the activity
of the ARF3 poypeptide in the plant to induce the
dwarfing-associated phenotype.
[0127] ARF3 Polypeptide/Polynucleotides Used in the Methods of the
Invention
[0128] In one embodiment of the methods above the ARF3 polypeptide
has a sequence with at least 70% identity to any one of SEQ ID NO:1
to 11, 28 and 29.
[0129] In a further embodiment the polypeptide has a sequence with
at least 70% identity to SEQ ID NO:1 (MdARF3).
[0130] In a further embodiment the polypeptide has a sequence with
at least 70% identity to SEQ ID NO:28 (MdARF3).
[0131] In most known ARF3 polypeptide sequences either a Serine or
Proline residue is found at the position corresponding amino acid
residue 72 in SEQ ID NO:1 or 28 (MdARF3) as shown in FIG. 8.
[0132] In a further embodiment the polypeptide comprises a
hydrophobic amino acid residue at the position corresponding amino
acid residue 72 in SEQ ID NO:28 (MdARF3).
[0133] In a further embodiment the polypeptide comprises a Leucine
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO:28 (MdARF3).
[0134] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:2 (M9 MdARF3).
[0135] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:29 (M9 MdARF3).
[0136] In one embodiment the ARF3 polynucleotide is a
polynucleotide that encodes and ARF3 polypeptide.
[0137] Modification of an Endogenous Polynucleotide
[0138] In one embodiment the modification results in expression of
an ARF3 polypeptide with a hydrophobic amino acid residue at a
position corresponding the amino acid residue 72 in SEQ ID NO:1 or
28 (MdARF3).
[0139] In a preferred embodiment the hydrophobic amino acid is a
Leucine residue.
[0140] In one embodiment the modification results in a codon
encoding the Leucine residue.
[0141] In one embodiment the codon is found at a position
corresponding to nucleotides 214 to 216 in the ARF3 polynucleotide
of SEQ ID NO:12.
[0142] In one embodiment the codon is selected from: TTA, TTG, CU,
CTC, CTA and CTG.
[0143] In a preferred embodiment the codon is TTG.
[0144] Thus in a preferred embodiment, the modification results in
a T nucleotide at a position corresponding to nucleotide 215 in the
ARF3 polynucleotide of SEQ ID NO:12.
[0145] Polynucleotide Encoding a M9 Type ARF3 Polypeptide
[0146] In a further aspect, the invention provides an isolated
polynucleotide encoding an ARF3 polypeptide comprising a
hydrophobic amino acid residue at a position corresponding the
amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).
[0147] In a further embodiment the invention provides a
polynucleotide encoding a variant of fragment of the ARF3
polypeptide.
[0148] In one embodiment, the hydrophobic amino acid residue is a
Leucine residue.
[0149] Thus, in one embodiment, the invention provides an isolated
polynucleotide encoding an ARF3 polypeptide comprising a Leucine
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO:1 or 28 (MdARF3).
[0150] In a further embodiment the ARF3 polypeptide comprising
comprises at least 70% identity to SEQ ID NO:2 or 29 (MdARF3).
[0151] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:2 or 29 (M9 MdARF3).
[0152] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:29 (M9 MdARF3).
[0153] In one embodiment the polynucleotide has at least 70%
identity to at least one of SEQ ID NO:14 and 15.
[0154] In a further embodiment the polynucleotide has at least 70%
identity to SEQ ID NO:14.
[0155] In a further embodiment the polynucleotide has at least 70%
identity to SEQ ID NO:15.
[0156] In a further embodiment the polynucleotide comprises the
sequence of SEQ ID NO:14 or 15.
[0157] In a further embodiment the polynucleotide comprises the
sequence of SEQ ID NO:14.
[0158] In a further embodiment the polynucleotide comprises the
sequence of SEQ ID NO:15.
[0159] Preferably the fragment of the ARF3 polypeptide comprises at
least 50 contiguous amino acids, more preferably at least 100
contiguous amino acids, more preferably at least 150 contiguous
amino acids, more preferably at least 200 contiguous amino acids,
more preferably at least 250 contiguous amino acids, more
preferably at least 300 contiguous amino acids, more preferably at
least 350 contiguous amino acids, more preferably at least 400
contiguous amino acids, more preferably at least 450 contiguous
amino acids of the polypeptide of the invention.
[0160] Preferably the fragment comprises the hydrophobic amino acid
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO:1 or 28 (MdARF3).
[0161] Preferably the fragment comprises the hydrophobic amino acid
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO:28 (MdARF3).
[0162] Preferably the hydrophobic amino acid residue is a Leucine
residue.
[0163] Polynucleotide
[0164] In a further aspect the invention provides an isolated
polynucleotide comprising the sequence of SEQ ID NO:14 or 15.
[0165] In one embodiment the polynucleotide comprising the sequence
of SEQ ID NO:14.
[0166] In one embodiment the polynucleotide comprising the sequence
of SEQ ID NO:15.
[0167] In a further embodiment the invention provides a variant or
fragment of the polynucleotide.
[0168] Polypeptide
[0169] In a further aspect, the invention provides an isolated ARF3
polypeptide comprising a hydrophobic amino acid residue at a
position corresponding the amino acid residue 72 in SEQ ID NO:1 or
28 (MdARF3).
[0170] In a further embodiment the ARF3 polypeptide comprises a
hydrophobic amino acid residue at a position corresponding the
amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).
[0171] In a further embodiment the invention provides a variant of
fragment of the ARF3 polypeptide.
[0172] In one embodiment, the hydrophobic amino acid residue is a
Leucine residue.
[0173] Thus, in one embodiment, the invention provides an isolated
ARF3 polypeptide comprising a Leucine residue at a position
corresponding the amino acid residue 72 in SEQ ID NO:1 or 28
(MdARF3).
[0174] In a further embodiment the ARF3 polypeptide comprising
comprises at least 70% identity to SEQ ID NO:2 or 29 (M9
MdARF3).
[0175] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:2 (M9 MdARF3).
[0176] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:29 (M9 MdARF3).
[0177] Polypeptide Fragment
[0178] Preferably the fragment comprises at least 50 contiguous
amino acids, more preferably at least 100 contiguous amino acids,
more preferably at least 150 contiguous amino acids, more
preferably at least 200 contiguous amino acids, more preferably at
least 250 contiguous amino acids, more preferably at least 300
contiguous amino acids, more preferably at least 350 contiguous
amino acids, more preferably at least 400 contiguous amino acids,
more preferably at least 450 contiguous amino acids of the
polypeptide of the invention.
[0179] Preferably the fragment comprises the hydrophobic amino acid
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO:1 or 28 (MdARF3).
[0180] Preferably the fragment comprises the hydrophobic amino acid
residue at a position corresponding the amino acid residue 72 in
SEQ ID NO: 28 (MdARF3).
[0181] Preferably the hydrophobic amino acid residue is a Leucine
residue.
[0182] Polynucleotide Fragment/Primers and Probes
[0183] Preferably the polynucleotide fragment comprises at least 5
contiguous nucleotides, more preferably at least 10 contiguous
nucleotides, more preferably at least 15 contiguous nucleotides,
more preferably at least 20 contiguous nucleotides, more preferably
at least 21 contiguous nucleotides, more preferably at least 30
contiguous nucleotides, more preferably at least 50 contiguous
nucleotides, more preferably at least 100 contiguous nucleotides,
more preferably at least 150 contiguous nucleotides, more
preferably at least 200 contiguous nucleotides, more preferably at
least 250 contiguous nucleotides, more preferably at least 300
contiguous nucleotides, more preferably at least 350 contiguous
nucleotides, more preferably at least 400 contiguous nucleotides,
more preferably at least 450 contiguous nucleotides of the
polynucleotide of the invention.
[0184] In a preferred embodiment, the fragment of the
polynucleotide of the invention, encodes a polypeptide fragment of
the invention.
[0185] In one embodiment the invention provides a primer consisting
of a polynucleotide fragment of the invention.
[0186] In a further embodiment the invention provides a probe
consisting of a polynucleotide fragment of the invention.
[0187] Construct
[0188] In a further embodiment the invention provides a construct
comprising a polynucleotide of the invention.
[0189] In one embodiment the construct comprises the polynucleotide
sequence operably linked to a heterologous promoter.
[0190] Cells
[0191] In a further embodiment the invention provides a cell
comprising a polynucleotide of the invention.
[0192] Preferably the cell is transgenic for the polynucleotide.
Preferably the transgenic cell, is transformed to comprise the
polynucleotide of the invention. Alternatively, a predecessor of
the cell has been transformed to comprise the polynucleotide, and
the cell is an off-spring of the predecessor cell and has inherited
the polynucleotide that was transformed into the predecessor
cell.
[0193] In a further embodiment the invention provides a cell
comprising a genetic construct of the invention.
[0194] In a preferred embodiment the cell expresses the
polynucleotide of the invention.
[0195] In a preferred embodiment the cell expresses the polypeptide
of the invention.
[0196] In a preferred embodiment the cell is transformed or
genetically modified to expresses the polynucleotide or polypeptide
of the invention.
[0197] In one embodiment the cell is a plant cell.
[0198] Plant
[0199] In a further embodiment the invention provides a plant
comprising a polynucleotide of the invention.
[0200] Preferably the plant is transgenic for the polynucleotide.
Preferably the transgenic plant is transformed to comprise the
polynucleotide of the invention. Alternatively, a predecessor of
the plant has been transformed to comprise the polynucleotide, and
the plant is an off-spring of the predecessor plant and has
inheritied the polynucleotide that was transformed into the
predecessor plant.
[0201] In a further embodiment the invention provides a plant
comprising a genetic construct of the invention.
[0202] In a preferred embodiment the plant expresses the
polynucleotide of the invention.
[0203] In a preferred embodiment the plant expresses the
polypeptide of the invention.
[0204] In a preferred embodiment the plant is transformed or
genetically modified to expresses the polynucleotide or polypeptide
of the invention.
[0205] In one embodiment the plant comprises a plant cell of the
invention.
[0206] In a further embodiment the plant has a dwarfing-associated
phenotype as described above.
[0207] Plant Parts
[0208] In a further embodiment the invention provides a part,
propagule or progeny of a plant of the invention.
[0209] Preferably the part, propagule or progeny is transgenic for
the polynucleotide. Preferably the transgenic part, propagule or
progeny is transformed to comprise the polynucleotide of the
invention. Alternatively, a predecessor of the plant (that provided
the part, propagule or progeny) has been transformed to comprise
the polynucleotide, and the part, propagule or progeny provided by
an off-spring of the predecessor plant and has inherited the
polynucleotide that was transformed into the predecessor plant.
[0210] In a further embodiment the invention provides a part,
propagule or progeny comprising a genetic construct of the
invention.
[0211] In a preferred embodiment the part, propagule or progeny
expresses the polynucleotide of the invention.
[0212] In a preferred embodiment the part, propagule or progeny
expresses the polypeptide of the invention.
[0213] In a preferred embodiment the part, propagule or progeny is
transformed or genetically modified to expresses the polynucleotide
or polypeptide of the invention.
[0214] In one embodiment the part, propagule or progeny comprises a
plant cell of the invention.
[0215] In one embodiment the plant cell, part, propagule or progeny
can be rejgenrated into a plant with a dwarfing-associated
phenotype as described above.
[0216] Marker Assisted Selection
[0217] In a further aspect the invention provides a method for
identifying a plant with a genotype indicative of at least one
dwarfing-associated phenotype, the method comprising testing a
plant for at least one of: [0218] a) altered expression of at least
one ARF3 polypeptide, [0219] b) altered expression of at least one
ARF3 polynucleotide, [0220] c) presence of a marker associated with
altered expression of at least one ARF3 polypeptide, [0221] d)
presence of a marker associated with altered expression of at least
one ARF3 polynucleotide, [0222] e) presence of a marker associated
with altered activity of at least one ARF3 polypeptide,
[0223] In one embodiment presence of any of a) to e) indicates that
the plant has at least one dwarfing-associated phenotype.
[0224] In one embodiment dwarfing-associated phenotype is selected
from those described above.
[0225] In one embodiment the altered expression is increased
expression.
[0226] In one embodiment the marker associated with altered
activity of at least one ARF3 polypeptide is presence of a
hydrophobic amino acid residue at a position corresponding the
amino acid residue 72 in SEQ ID NO:1 (MdARF3).
[0227] In one embodiment, the hydrophobic amino acid residue is a
Leucine residue.
[0228] Thus, in one embodiment, the invention the method involves
identifying presence of a Leucine residue at a position
corresponding the amino acid residue 72 in SEQ ID NO:1
(MdARF3).
[0229] In a further embodiment the ARF3 polypeptide comprising
comprises at least 70% identity to SEQ ID NO:2 (MdARF3).
[0230] In a further embodiment the polypeptide comprises the
sequence of SEQ ID NO:2 (M9 MdARF3).
[0231] Alternatively, the method involves detection of a
polynucleotide encoding the Leucine residue at a position
corresponding the amino acid residue 72 in SEQ ID NO:1
(MdARF3).
[0232] In a further embodiment the method provides the additional
step of cultivating the identified plant.
[0233] In a further embodiment the method provides the additional
step of breeding from the identified plant.
[0234] Methods for Breeding Plants with at Least One Dwarfing
Associated Phenotype
[0235] In a further aspect the invention provides a method for
producing a plant with at least one dwarfing-associated phenotype,
the method comprising crossing one of: [0236] a) a plant of the
invention, [0237] b) a plant produced by a method of the invention,
and [0238] c) a plant selected by a method of the invention, with
another plant, wherein the off-spring produced by the crossing is a
plant with at least one dwarfing-associated phenotype.
[0239] In one embodiment dwarfing-associated phenotype is selected
from those described above.
[0240] Method Using Plant of the Invention
[0241] In a further embodiment the invention provides a method of
producing a plant with at least one dwarfing-associated phenotype
selected from: [0242] a) altered auxin transport, [0243] b) slower
auxin transport, [0244] c) reduced apical dominance, [0245] d) an
altered xylem/phloem ratio, [0246] e) an increased number of phloem
elements, [0247] f) smaller phloem elements, [0248] g) thicker
bark, [0249] h) a bushier habit, [0250] i) reduced root mass,
[0251] j) reduced vigour, [0252] k) less vegetative growth, [0253]
l) earlier termination of shoot growth, [0254] m) earlier
competence to flower, [0255] n) precocity, [0256] o) earlier phase
change, [0257] p) smaller canopy, [0258] q) reduced stem
circumference, [0259] r) reduced branch diameter, [0260] s) fewer
sylleptic branches, [0261] t) shorter sylleptic branches, [0262] u)
more axillary flowers, [0263] v) an earlier teminating primary
axis, [0264] w) earlier teminating secondary axes, [0265] x)
shorter intenode length, [0266] y) reduced scion mass, the method
comprising grafting a scion onto a plant of the invention, a plant
produced by a method of the invention, or a plant selected by a
method of the invention.
[0267] In one embodiment the dwarfing-associated phenotype is at
least one of a) to h) and j) to x).
[0268] In this embodiment the at least one dwarfing associated
phenotype is preferably exhibited in the grafted scion.
[0269] In this embodiment the grafted scion preferably exhibits at
least one of j) to y). Alternatively, the grafted scion preferably
exhibits at least one of j) to x).
[0270] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, reduced stem
circumference, and reduced scion mass in a scion grafted on to the
plant.
[0271] In a further embodiment the grafted scion preferably
exhibits at least one of reduced vigour, less vegetative growth,
earlier termination of shoot growth, a smaller canopy, and reduced
stem circumference, in a scion grafted on to the plant.
DETAILED DESCRIPTION OF THE INVENTION
[0272] The present invention provides methods and materials useful
for producing or selecting plants with at least one dwarfing
associated phenotype.
[0273] The dwarfing-associated phenotype may be exhibited in the
plant produced or selected, or may be exhibited in scions grafted
onto the plants used as root stock, as indicated in Table 1
below.
TABLE-US-00001 TABLE 1 Dwarfing-associated phenotypes
Dwarfing-associated phenotypes found in scions grafted onto found
in dwarfing rootstock plants dwarfing rootstock plants bushier
reduced vigour altered auxin transport less vegetative growth
altered xylem/phloem ratio earlier termination of shoot growth more
phloem elements earlier competency to flower smaller phloem
elements precocity thicker bark earlier phase change slower auxin
transport smaller canopy reduced apical dominance reduced stem
circumference reduced root mass reduced branch diameter fewer
sylleptic branches shorter sylleptic branches more axillary flowers
earlier terminating primary axis earlier terminating secondary axes
reduced branching density reduced internode length reduced scion
mass
[0274] The dwarfing-associated phenotype may be selected from:
[0275] a) altered auxin transport, [0276] b) slower auxin
transport, [0277] c) reduced apical dominance, [0278] d) an altered
xylem/phloem ratio, [0279] e) an increased number of phloem
elements, [0280] f) smaller phloem elements, [0281] g) thicker
bark, [0282] h) a bushier habit, [0283] i) reduced root mass,
[0284] j) reduced vigour, [0285] k) less vegetative growth, [0286]
l) earlier termination of shoot growth, [0287] m) earlier
competence to flower, [0288] n) precocity, [0289] o) earlier phase
change, [0290] p) smaller canopy, [0291] q) reduced stem
circumference, [0292] r) reduced branch diameter, [0293] s) fewer
sylleptic branches, [0294] t) shorter sylleptic branches, [0295] u)
more axillary flowers, [0296] v) an earlier teminating primary
axis, [0297] w) earlier teminating secondary axes, [0298] x)
shorter intenode length, and [0299] y) reduced scion mass.
[0300] In one embodiment the plant exhibits at least 2, more
preferably at least 3, more preferably at least 4, more preferably
at least 5, more preferably at least 6, more preferably at least 7,
more preferably at least 8, more preferably at least 9, more
preferably at least 10, more preferably at least 11, more
preferably at least 12, more preferably at least 13, more
preferably at least 14, more preferably at least 15, more
preferably at least 16, more preferably at least 17, more
preferably at least 18, more preferably at least 19, more
preferably at least 20, more preferably at least 21, more
preferably at least 22, more preferably all 23 of dwarfing
associated phenotypes a) to w).
[0301] In a further embodiment the plant exhibits at least one of
dwarfing associated phenotypes selected from a) to i). In one
embodiment the plant exhibits at least 2, more preferably at least
3, more preferably at least 4, more preferably at least 5, more
preferably at least 6, more preferably at least 7, more preferably
at least 8, of dwarfing associated phenotypes a) to i). In one
embodiment this a plant is suitable for use as a root stock.
[0302] The dwarfing-associated phenotype may also be the capacity
to induce at least one of a) to y) in a scion grafted onto the
plant. In a further embodiment the dwarfing-associated phenotype
may also be the capacity to induce at least one of a) to y) in a
scion grafted onto the plant.
[0303] The dwarfing-associated phenotypes are relative terms. In
one embodiment the dwarfing associated phenotype is relative to
that of a control plant.
[0304] The control plant may be any plant of the same type that is
not transformed with the polynucleotide, or construct, of the
invention of the invention, or used in a method of the invention.
The control plant may also be transformed with an "empty" vector,
wherein the empty vector does not include an insert sequence
corresponding to a polynucleotide of the invention or used in a
method of the invention.
[0305] For the selection methods the control plant may be a
non-selected plant.
[0306] The phrases "altered auxin transport" and "slower auxin
transport" means that auxin transport in the plant of the
invention, or in a method of the invention, is altered or slower
relative to that in a contol plant. Auxin transport may be measured
by methods known to those skilled in the art and explified for
example in (Ulmasov, Murfett et al. 1997; Ljung, Hull et al.
2005)
[0307] The phrase "apical dominance" is the phenomenon whereby the
primary shoot axis suppresses outgrowth of axillary brances. Apical
dominance may be assessed by methods known to those skilled in the
art for example (Napoli, Beveridge et al. 1999; Shimizu-Sato and
Mori 2001; Sussex and Kerk 2001; Bennett, Sieberer et al. 2006)
[0308] The phrases "an altered xylem/phloem ratio", "an increased
number of phloem elements" and "smaller phloem elements" are known
to those skilled in the art, and may be assessed microscopically,
as described in the present Examples section (Ruzin 1999).
[0309] The phrase "thicker bark" is intended to take the standard
meaning, known to those skilled in the art. Thickness of bark can
be assessed by taking transverse sections, using hisological stains
such as safranin/fast green to distinguish xylem from phleom and
observing under a microscope (Ruzin 1999).
[0310] Bushiness of habit is a term well understood and easily
assessed visually by those skilled in the art.
[0311] The phrase "reduced vigour" means a reduction in the number
of metamers intintiated by extension growth units, resulting in
fewer branches, shorter branches and shorter main axis (Costes and
Guedon 2002; Seleznyova, Thorp et al. 2003).
[0312] The pharase "metamer" means the repeating unit of leaf,
axillary meristem, node, and internode (Steeves and Sussex
1989).
[0313] The phrase "extension growth unit" means a vegetative shoot
with internode expansion (Seleznyova, Thorp et al. 2003).
[0314] The phrase "less vegetative growth" means a higher
proportion of floral buds relative to vegetative shoots.
[0315] The phrase "earlier termination of shoot growth" means a
vegetative extension shoot that stops initiating new metamers
earlier in the season, resulting in a shorter shoot (Bohlenius,
Huang et al. 2006; Hsu, Adams et al. 2011).
[0316] The phrase "earlier competence to flower" means the ability
of the plant to respond to flowering cues and begin floral
development (Hsu, Liu et al. 2006).
[0317] The phrase "precocity" means a reduced period in which a
plant is unable to begin floral development (Imamura, Nakatsuka et
al. 2011).
[0318] The phrase "earlier phase change" means the same as
"precocious", a plant that is able to respond to floral cues and
begin floral development before others of the same age (Huijser and
Schmid 2011; Willmann and Poethig 2011).
[0319] The phrase "smaller canopy" is a phrase well understood and
easily assessed by those skilled in the art.
[0320] The phrase "stem circumference" can be easily assessed by
those skilled in the art. Measurement of stem circumference can be
replaced by measurement of "Trunk Cross-sectional Area" (TCA). TCA
of a grafted scion is generally measured 20 cm above the graft
union for grafted trees. For non-tree plants the primary stem is
measured in place of the trunk.
[0321] "Branch diameter" is a term well understood and easily
assessed by those skilled in the art.
[0322] The term "sylleptic branches" means a vegetative bud that
grows out without a dormancy period, i.e. in the same season it was
initiated (Costes and Guedon 1997).
[0323] Number and length of sylleptic branches can be easily
assessed by those skilled in the art.
[0324] The term "axillary flowers" means flowers that are flowers
that form directly from an axillary meristem, as opposed to a
"fruiting spur" (Fulford 1966).
[0325] The term "fruiting spur" means a very short shoot with very
condensed internodes that terminates in a bud containing several
leaves and an inflorescence" (Fulford 1966).
[0326] The phrase "an earlier teminating primary axis means a tree
with a shorter primary axis, comprised of fewer nodes.
[0327] The phrase "earlier teminating secondary axes" means shorter
branches comprised of fewer nodes.
[0328] The term "internode" is intended to take its standard
meaning. Internode length can be easily assessed by those skilled
in the art (Steeves and Sussex 1989).
[0329] Cells
[0330] In one embodiment the cell is a prokaryotic cell.
[0331] In a further embodiment the cell is a eukaryotic cell.
[0332] In one embodiment the cell is selected from a bacterial
cell, a yeast cell, a fungal cell, an insect cell, algal cell, and
a plant cell. In one embodiment the cell is a bacterial cell. In a
further embodiment the cell is a yeast cell. In one embodiment the
yeast cell is a S. ceriviseae cell. In further embodiment the cell
is a fungal cell. In further embodiment the cell is an insect cell.
In further embodiment the cell is an algal cell.
[0333] In a preferred embodiment the cell is a plant cell.
[0334] Plants
[0335] Plants or plant cells or the invention, or used in the
methods of the invention, or used to source naturally occurring
ARF3 sequences, may be from any species.
[0336] In one embodiment the plant cell or plant, is or is derived
from a gymnosperm plant species.
[0337] In a further embodiment the plant cell or plant, is or is
derived from an angiosperm plant species.
[0338] In a further embodiment the plant cell or plant, is or is
derived from a from dicotyledonous plant species.
[0339] In a further embodiment the plant cell or plant, is or is
derived from a monocotyledonous plant species.
[0340] Preferred plants in which to introduce dwarfing associated
pheotypes include those from any species that produces fruit.
[0341] Preferred plants from which to source naturally occurring
ARF3 sequences include those from any species that produces
fruit.
[0342] Preferred fruit producing plants include apple, avocado,
pear, peach, cherry, plum, kiwifruit, grape, mango, and orange
plants.
[0343] A preferred apple genus is Malus.
[0344] Preferred apple species include: Malus angustifolia, Malus
asiatica, Malus baccata, Malus coronaria, Malus doumeri, Malus
florentina, Malus floribunda, Malus fusca, Malus halliana, Malus
honanensis, Malus hupehensis, Malus ioensis, Malus kansuensis,
Malus mandshurica, Malus micromalus, Malus niedzwetzkyana, Malus
ombrophilia, Malus orientalis, Malus prattii, Malus prunifolia,
Malus pumila, Malus sargentii, Malus sieboldii, Malus sieversii,
Malus sylvestris, Malus toringoides, Malus transitoria, Malus
trilobata, Malus tschonoskii, Malus.times.domestica,
Malus.times.domestica.times.Malus sieversii,
Malus.times.domestica.times.Pyrus communis, Malus xiaojinensis, and
Malus yunnanensis.
[0345] A particularly preferred apple species is
Malus.times.domestica.
[0346] A preferred pear genus is Pyrus.
[0347] Preferred pear species include: Pyrus calleryana, Pyrus
caucasica, Pyrus communis, Pyrus elaeagrifolia, Pyrus hybrid
cultivar, Pyrus pyrifolia, Pyrus salicifolia, Pyrus ussuriensis and
Pyrus.times.bretschneideri.
[0348] A particularly preferred pear species are Pyrus communis and
Asian pear Pyrus.times.bretschneideri.
[0349] A preferred avocado genus is Persea.
[0350] Preferred avacado species include Persea americana and
Persea gratissima.
[0351] A preferred peach genus is Prunus.
[0352] Preferred peach species include: Prunus africana, Prunus
apetala, Prunus arborea, Prunus armeniaca, Prunus avium, Prunus
bifrons, Prunus buergeriana, Prunus campanulata, Prunus canescens,
Prunus cerasifera, Prunus cerasoides, Prunus cerasus, Prunus
ceylanica, Prunus cocomilia, Prunus cornuta, Prunus crassifolia,
Prunus davidiana, Prunus domestica, Prunus dulcis, Prunus
fruticosa, Prunus geniculata, Prunus glandulosa, Prunus gracilis,
Prunus grayana, Prunus incana, Prunus incisa, Prunus jacquemontii,
Prunus japonica, Prunus korshinskyi, Prunus kotschyi, Prunus
laurocerasus, Prunus laxinervis, Prunus lusitanica, Prunus maackii,
Prunus mahaleb, Prunus mandshurica, Prunus maximowiczii, Prunus
minutiflora, Prunus mume, Prunus murrayana, Prunus myrtifolia,
Prunus nipponica, Prunus occidentalis, Prunus padus, Prunus
persica, Prunus pleuradenia, Prunus pseudocerasus, Prunus
prostrata, Prunus salicina, Prunus sargentii, Prunus scoparia,
Prunus serrula, Prunus serrulata, Prunus sibirica, Prunus simonii,
Prunus sogdiana, Prunus speciosa, Prunus spinosa, Prunus spinulosa,
Prunus ssiori, Prunus subhirtella, Prunus tenella, Prunus
tomentosa, Prunus triloba, Prunus turneriana, Prunus ursina, Prunus
vachuschtii, Prunus verecunda, Prunus.times.yedoensis, Prunus
zippeliana, Prunus alabamensis, Prunus alleghaniensis, Prunus
americana, Prunus andersonii, Prunus angustifolia, Prunus
brigantina, Prunus buxifolia, Prunus caroliniana, Prunus
cuthbertii, Prunus emarginata, Prunus eremophila, Prunus
fasciculata, Prunus fremontii, Prunus geniculata, Prunus gentryi,
Prunus havardii, Prunus hortulana, Prunus huantensis, Prunus
ilicifolia, Prunus integrifolia, Prunus maritima, Prunus mexicana,
Prunus munsoniana, Prunus nigra, Prunus pensylvanica, Prunus
pumila, Prunus rigida, Prunus rivularis, Prunus serotina, Prunus
sphaerocarpa, Prunus subcordata, Prunus texana, Prunus umbellate
and Prunus virginiana.
[0353] A particularly preferred peach species is Prunus
persica.
[0354] A preferred kiwifruit genus is Actinidia.
[0355] Preferred kiwifruit species include: Actinidia arguta,
Actinidia arisanensis, Actinidia callosa, Actinidia camosifolia,
Actinidia chengkouensis, Actinidia chinensis, Actinidia chrysantha,
Actinidia cinerascens, Actinidia cordifolia, Actinidia coriacea,
Actinidia cylindrica, Actinidia deliciosa, Actinidia eriantha,
Actinidia farinosa, Actinidia fasciculoides, Actinidia fortunatii,
Actinidia foveolata, Actinidia fulvicoma, Actinidia
glauco-callosa-callosa, Actinidia glaucophylla, Actinidia globosa,
Actinidia gracilis, Actinidia grandiflora, Actinidia hemsleyana,
Actinidia henryi, Actinidia holotricha, Actinidia hubeiensis,
Actinidia indochinensis, Actinidia kolomikta, Actinidia laevissima,
Actinidia lanceolata, Actinidia latifolia, Actinidia leptophylla,
Actinidia liangguangensis, Actinidia lijiangensis, Actinidia
linguiensis, Actinidia longicarpa, Actinidia macrosperma, Actinidia
maloides, Actinidia melanandra, Actinidia melliana, Actinidia
obovata, Actinidia oregonensis, Actinidia persicina, Actinidia
pilosula, Actinidia polygama, Actinidia purpurea, Actinidia
rongshuiensis, Actinidia rubricaulis, Actinidia rubus, Actinidia
rudis, Actinidia rufa, Actinidia rufotricha, Actinidia sabiaefolia,
Actinidia sorbifolia, Actinidia stellato-pilosa-pilosa, Actinidia
styracifolia, Actinidia suberifolia, Actinidia tetramera, Actinidia
trichogyna, Actinidia ulmifolia, Actinidia umbelloides, Actinidia
valvata, Actinidia venosa, Actinidia vitifolia and Actinidia
zhejiangensis.
[0356] Particularly preferred kiwifruit species are Actinidia
arguta, Actinidia chinensis and Actinidia deliciosa.
[0357] A preferred orange genus is Citrus.
[0358] Preferred orange species include: Citrus aurantiifolia,
Citrus crenatifolia, Citrus maxima, Citrus medica, Citrus
reticulata, Citrus trifoliata, Australian limes Citrus
australasica, Citrus australis, Citrus glauca, Citrus garrawayae,
Citrus gracilis, Citrus inodora, Citrus warburgiana, Citrus
wintersii, Citrus japonica, Citrus indica and Citrus xsinensis.
[0359] Particularly preferred orange species are: Citrus maxima,
Citrus reticulate, Citrus.times.sinensis.
[0360] A preferred grape genus is Vitis.
[0361] Preferred grape species include: Vitis vinifera, Vitis
labrusca, Vitis riparia, Vitis aestivalis, Vitis rotundifolia,
Vitis rupestris, Vitis coignetiae, Vitis amurensis, Vitis
vulpine.
[0362] A particularly preferred grape species is Vitis
vinifera.
[0363] A preferred avocado genus is Persea.
[0364] Preferred avacado species include Persea americana and
Persea gratissima. A preferred mango genus is Mangifera.
[0365] Preferred mango species include: Mangifera foetida and
Mangifera indica.
[0366] A particularly preferred grape species is Mangifera
indica.
[0367] A preferred plum genus is Prunus.
[0368] Preferred plum species include: P. cerasifera, P. cocomilia,
P. consociiflora, P. domestica, P. domestica ssp. insititia, P.
simonii, P. spinosa, P. alleghaniensis, P. americana, P.
angustifolia, P. hortulana, P. maritima, P. mexicana, P. nigra, and
P.
[0369] subcordata.
[0370] A particularly preferred plum species is Prunus
domestica.
[0371] Plant Parts, Propagues and Progeny
[0372] The term "plant part" or grammatical equivalents thereof is
intended to include any part of a plant, a tissue, an organ, a
seed, a fruit, propagules and progeny of a plant.
[0373] The term `propagule` means any part of a plant that may be
used in reproduction or propagation, either sexual or asexual,
including seeds and cuttings.
[0374] The plants of the invention may be grown and either self-ed
or crossed with a different plant strain and the resulting progeny,
comprising the polynucleotides or constructs of the invention,
and/or expressing the ARF3 sequences of the invention, also form an
part of the present invention.
[0375] Preferably the plants, plant parts, propagules and progeny
comprise a polynucleotide or construct of the invention, and/or
express a ARF3 sequence of the invention.
[0376] Marker Assisted Selection
[0377] Marker assisted selection (MAS) is an approach that is often
used to identify plants that possess a particular trait using a
genetic marker, or markers, associated with that trait. MAS may
allow breeders to identify and select plants at a young age and is
particularly valuable for fruit traits that are hard to measure at
a young stage. The best markers for MAS are the causal mutations,
but where these are not available, a marker that is in strong
linkage disequilibrium with the causal mutation can also be used.
Such information can be used to accelerate genetic gain, or reduce
trait measurement costs, and thereby has utility in commercial
breeding programs.
[0378] Methods for marker assisted selection are well known to
those skilled in the art, for example: (Collard, B. C. Y. and D. J.
Mackill, Marker-assisted selection: an approach for precision plant
breeding in the twenty-first century. Philosophical Transactions of
the Royal Society B-Biological Sciences, 2008. 363(1491): p.
557-572.)
[0379] Markers
[0380] Markers for use in the methods of the invention may include
nucleic acid markers, such as single nucleotide polymorphisms
(SNPs), simple sequence repeats (SSRs or microsatellites),
insertions, substitutions, indels and deletions.
[0381] Preferably the marker is in linkage disequilibrium (LD) with
the trait.
[0382] Preferably the marker is in LD with the trait at a D' value
of at least 0.1, more preferably at least 0.2, more preferably at
least 0.3, more preferably at least 0.4, more preferably at least
0.5.
[0383] Preferably the marker is in LD with the trait at a R.sup.2
value of at least 0.05, more preferably at least 0.075, more
preferably at least 0.1, more preferably at least 0.2, more
preferably at least 0.3, more preferably at least 0.4, more
preferably at least 0.5.
[0384] The term "linkage disequilibrium" or LD as used herein,
refers to a derived statistical measure of the strength of the
association or co-occurrence of two independent genetic markers.
Various statistical methods can be used to summarize linkage
disequilibrium (LD) between two markers but in practice only two,
termed D' and R.sup.2, are widely used.
[0385] Markers linked, and or in LD, with the trait may be of any
type including but not limited to, SNPs, substitutions, insertions,
deletions, indels, simple sequence repeats (SSRs).
[0386] In the present invention, markers are associated with [0387]
a) altered expression of at least one ARF3 polypeptide, [0388] b)
altered expression of at least one ARF3 polynucleotide, [0389] c)
altered activity of at least one ARF3 polypeptide,
[0390] One marker associated with altered activity of at least one
ARF3 polypeptide identified by the applicant is the presence of a
hydrophobic amino acid residue at a position corresponding the
amino acid residue 72 in SEQ ID NO:1 (MdARF3).
[0391] In one embodiment, the hydrophobic amino acid residue is a
Leucine residue.
[0392] Thus, in one embodiment, the invention the method involves
identifying presence of a Leucine residue at a position
corresponding the amino acid residue 72 in SEQ ID NO:1
(MdARF3).
[0393] A further marker associated with altered activity of at
least one ARF3 polypeptide identified by the applicant is the
presence of a codon encoding the Leucine residue.
[0394] In one embodiment the codon is found at a position
corresponding to nucleotides 214 to 216 in the ARF3 polynucleotide
of SEQ ID NO:12.
[0395] In one embodiment the codon is selected from: TTA, TTG, CTT,
CTC, CTA and CTG.
[0396] In a preferred embodiment the codon is TTG.
[0397] Thus in a preferred embodiment, the marker is a T nucleotide
at a position corresponding to nucleotide 215 in the ARF3
polynucleotide of SEQ ID NO:12.
[0398] This marker defines the M9 allele of ARF3.
[0399] Other Markers Linked to the M9 Allele of ARF3.
[0400] It would be most desirable to identify the presence of the
M9 allele of ARF3 discussed above when selecting for at least one
dwarfing associated phenotype. However, following the applicants
present disclosure, those skilled in the art would know that it
would also be possible to select for at least one dwarfing
associated phenotype by identifying the presence of a marker linked
to the M9 allele of ARF3. Selection methods utilising such linked
markers also form part of the present invention. Methods for
identify such linked markers are known to those skilled in the
art.
[0401] Two other preferred markers for use in the marker assisted
selection methods of the invention are Hi01c04 and Hi04a08.
[0402] The applicants have now shown that these are the closest
markers defining the Dw1 QTL interval.
[0403] Hi01c04
[0404] Hi01c04 is an SSR marker. Suitable primers for amplifying
the Hi01c04 marker (and hybridising to the flanking sequences) are
shown below.
TABLE-US-00002 Hi01c04 foward primer: 5'-GCTGCCGTTGACGTTAGAG-3'
Hi01c04 reverse primer: 5'-GTTTGTAGAAGTGGCGTTTGAGG-3'
[0405] The variable region between the flanking sequences is
defined by the formula (CTC).sub.n
[0406] The whole sequence of the Hi01c04 is shown in SEQ ID
NO:26
[0407] Hi04a08
[0408] Hi04a08 is also an SSR marker. Suitable primers for
amplifying the Hi04a08 marker (and hybridising to the flanking
sequences) are shown below.
TABLE-US-00003 Hi04a08 foward primer: 5'-TTGAAGGAGTTTCCGGTTTG-3'
Hi04a08 reverse primer: 5'-GTTTCACTCTGTGCTGGATTATGC-3'
[0409] The variable region between the flanking sequences is
defined by the formula (CTC).sub.n
[0410] The whole sequence of the Hi04a08 is shown in SEQ ID
NO:27
[0411] Methods for Modifying Endogenous Polynucleotides
[0412] Some embodiments of the invention involve modifying and
endogenous polynucleotide to induce a dwarfing associated phenotype
in a plant, or scion grafted onto the plant.
[0413] Methods for modifying endogenous genomic DNA sequences in
plants are known to those skilled in the art. Such methods may
involve the use of sequence-specific nucleases that generate
targeted double-stranded DNA breaks in genes of interest. Examples
of such methods for use in plants include: zinc finger nucleases
(Curtin et al., 2011. Plant Physiol. 156:466-473; Sander, et al.,
2011. Nat. Methods 8:67-69.), transcription activator-like effector
nucleases or "TALENs" (Cermak et al., 2011, Nucleic Acids Res.
39:e82; Mahfouz et al., 2011 Proc. Natl. Acad. Sci. USA
108:2623-2628; Li et al., 2012 Nat. Biotechnol. 30:390-392), and
LAGLIDADG homing endonucleases, also termed "meganucleases" (Tzfira
et al., 2012. Plant Biotechnol. J. 10:373-389).
[0414] Targeted genome editing using engineered nucleases such as
clustered, regularly interspaced, short palindromic repeat (CRISPR)
technology, is an important new approach for generating RNA-guided
nucleases, such as Cas9, with customizable specificities. Genome
editing mediated by these nucleases has been used to rapidly,
easily and efficiently modify endogenous genes in a wide variety of
biomedically important cell types and in organisms that have
traditionally been challenging to manipulate genetically. A
modified version of the CRISPR-Cas9 system has been developed to
recruit heterologous domains that can regulate endogenous gene
expression or label specific genomic loci in living cells (Nature
Biotechnology 32,347-355 (2014). The system is applicable to
plants, and can be used to regulate expression of target genes.
(Bortesi and Fischer, Biotechnology Advances Volume 33, Issue 1,
January-February 2015, Pages 41-52).
[0415] Those skilled in the art will thus appreciate that there are
numerous ways in which the expression or activity of MdARF3 can be
reduced or eliminated. Any such method is modified within the scope
of the invention.
[0416] In certain embodiments of the invention, a genome editing
technology (e.g. TALENs, a Zinc finger nuclease or CRISPR-Cas9
technology) can be used to modify one or more base pairs in a
target ARF3 gene to create a codon encoding a hydrophobic amino
acid, such as a Leucine residue at a position corresponding the
amino acid residue 72 in SEQ ID NO:1 (MdARF3). This approach
effectively creates an M9 type ARF3 allele in the target plant.
[0417] Polynucleotides and Fragments
[0418] The term "polynucleotide(s)," as used herein, means a single
or double-stranded deoxyribonucleotide or ribonucleotide polymer of
any length but preferably at least 15 nucleotides, and include as
non-limiting examples, coding and non-coding sequences of a gene,
sense and antisense sequences complements, exons, introns, genomic
DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes,
recombinant polypeptides, isolated and purified naturally occurring
DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid
probes, primers and fragments.
[0419] A "fragment" of a polynucleotide sequence provided herein is
a subsequence of contiguous nucleotides.
[0420] The term "primer" refers to a short polynucleotide, usually
having a free 3'OH group, that is hybridized to a template and used
for priming polymerization of a polynucleotide complementary to the
target. The primer may consist of a "fragment" of a polynucleotide
as defined herein.
[0421] The term "probe" refers to a short polynucleotide that is
used to detect a polynucleotide sequence that is complementary to
the probe, in a hybridization-based assay. The probe may consist of
a "fragment" of a polynucleotide as defined herein.
[0422] Polypeptides and Fragments
[0423] The term "polypeptide", as used herein, encompasses amino
acid chains of any length but preferably at least 5 amino acids,
including full-length proteins, in which amino acid residues are
linked by covalent peptide bonds. Polypeptides of the present
invention, or used in the methods of the invention, may be purified
natural products, or may be produced partially or wholly using
recombinant or synthetic techniques.
[0424] A "fragment" of a polypeptide is a subsequence of the
polypeptide that in some embodiments performs a function/activity
of and/or influences three dimensional structure of the
polypeptide.
[0425] The term "isolated" as applied to the polynucleotide or
polypeptide sequences disclosed herein is used to refer to
sequences that are removed from their natural cellular environment.
The isolated sequence is preferably separated from the sequences
that may be found flanking the sequence in its naturally occurring
environment. An isolated molecule may be obtained by any method or
combination of methods including biochemical, recombinant, and
synthetic techniques.
[0426] The term "recombinant" refers to a polynucleotide sequence
that is removed from sequences that surround it in its natural
context and/or is recombined with sequences that are not present in
its natural context.
[0427] A "recombinant" polypeptide sequence is produced by
translation from a "recombinant" polynucleotide sequence.
[0428] The term "derived from" with respect to polynucleotides or
polypeptides of the invention being derived from a particular
genera or species, means that the polynucleotide or polypeptide has
the same sequence as a polynucleotide or polypeptide found
naturally in that genera or species. The polynucleotide or
polypeptide, derived from a particular genera or species, may
therefore be produced synthetically or recombinantly.
[0429] Variants
[0430] As used herein, the term "variant" refers to polynucleotide
or polypeptide sequences different from the specifically identified
sequences, wherein one or more nucleotides or amino acid residues
is deleted, substituted, or added. Variants may be naturally
occurring allelic variants, or non-naturally occurring variants.
Variants may be from the same or from other species and may
encompass homologues, paralogues and orthologues. In certain
embodiments, variants of the inventive polypeptides and
polypeptides possess biological activities that are the same or
similar to those of the inventive polypeptides or polypeptides. The
term "variant" with reference to polypeptides and polypeptides
encompasses all forms of polypeptides and polypeptides as defined
herein.
[0431] Polynucleotide Variants
[0432] Variant polynucleotide sequences preferably exhibit at least
50%, more preferably at least 51%, more preferably at least 52%,
more preferably at least 53%, more preferably at least 54%, more
preferably at least 55%, more preferably at least 56%, more
preferably at least 57%, more preferably at least 58%, more
preferably at least 59%, more preferably at least 60%, more
preferably at least 61%, more preferably at least 62%, more
preferably at least 63%, more preferably at least 64%, more
preferably at least 65%, more preferably at least 66%, more
preferably at least 67%, more preferably at least 68%, more
preferably at least 69%, more preferably at least 70%, more
preferably at least 71%, more preferably at least 72%, more
preferably at least 73%, more preferably at least 74%, more
preferably at least 75%, more preferably at least 76%, more
preferably at least 77%, more preferably at least 78%, more
preferably at least 79%, more preferably at least 80%, more
preferably at least 81%, more preferably at least 82%, more
preferably at least 83%, more preferably at least 84%, more
preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, and most
preferably at least 99% identity to a sequence of the present
invention. Identity is found over a comparison window of at least
20 nucleotide positions, preferably at least 50 nucleotide
positions, more preferably at least 100 nucleotide positions, and
most preferably over the entire length of a polynucleotide of the
invention.
[0433] Polynucleotide sequence identity can be determined in the
following manner. The subject polynucleotide sequence is compared
to a candidate polynucleotide sequence using BLASTN (from the BLAST
suite of programs, version 2.2.5 [November 2002]) in bl2seq
(Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2
sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250), which is publicly
available from the NCBI website on the World Wide Web at
ftp://ftp.ncbi.nih.gov/blast/. The default parameters of bl2seq are
utilized except that filtering of low complexity parts should be
turned off.
[0434] The identity of polynucleotide sequences may be examined
using the following unix command line parameters: [0435] bl2seq-i
nucleotideseq1-j nucleotideseq2-F F-p blastn
[0436] The parameter-F F turns off filtering of low complexity
sections. The parameter-p selects the appropriate algorithm for the
pair of sequences. The bl2seq program reports sequence identity as
both the number and percentage of identical nucleotides in a line
"Identities=".
[0437] Polynucleotide sequence identity may also be calculated over
the entire length of the overlap between a candidate and subject
polynucleotide sequences using global sequence alignment programs
(e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48,
443-453). A full implementation of the Needleman-Wunsch global
alignment algorithm is found in the needle program in the EMBOSS
package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European
Molecular Biology Open Software Suite, Trends in Genetics June
2000, vol 16, No 6. pp. 276-277) which can be obtained from the
World Wide Web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The
European Bioinformatics Institute server also provides the facility
to perform EMBOSS-needle global alignments between two sequences on
line at http:/www.ebi.ac.uk/emboss/align/.
[0438] Alternatively the GAP program may be used which computes an
optimal global alignment of two sequences without penalizing
terminal gaps. GAP is described in the following paper: Huang, X.
(1994) On Global Sequence Alignment. Computer Applications in the
Biosciences 10, 227-235.
[0439] A preferred method for calculating polynucleotide % sequence
identity is based on aligning sequences to be compared using
Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23,
403-5.)
[0440] Polynucleotide variants of the present invention also
encompass those which exhibit a similarity to one or more of the
specifically identified sequences that is likely to preserve the
functional equivalence of those sequences and which could not
reasonably be expected to have occurred by random chance. Such
sequence similarity with respect to polypeptides may be determined
using the publicly available bl2seq program from the BLAST suite of
programs (version 2.2.5 [November 2002]) from the NCBI website on
the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/.
[0441] The similarity of polynucleotide sequences may be examined
using the following unix command line parameters: [0442] bl2seq
nucleotideseq1-j nucleotideseq2-F F-p tblastx
[0443] The parameter-F F turns off filtering of low complexity
sections. The parameter-p selects the appropriate algorithm for the
pair of sequences. This program finds regions of similarity between
the sequences and for each such region reports an "E value" which
is the expected number of times one could expect to see such a
match by chance in a database of a fixed reference size containing
random sequences. The size of this database is set by default in
the bl2seq program. For small E values, much less than one, the E
value is approximately the probability of such a random match.
[0444] Variant polynucleotide sequences preferably exhibit an E
value of less than 1.times.10-6 more preferably less than
1.times.10-9, more preferably less than 1.times.10-12, more
preferably less than 1.times.10-15, more preferably less than
1.times.10-18, more preferably less than 1.times.10-21, more
preferably less than 1.times.10-30, more preferably less than
1.times.10-40, more preferably less than 1.times.10-50, more
preferably less than 1.times.10-60, more preferably less than
1.times.10-70, more preferably less than 1.times.10-80, more
preferably less than 1.times.10-90 and most preferably less than
1.times.10-100 when compared with any one of the specifically
identified sequences.
[0445] Alternatively, variant polynucleotides of the present
invention, or used in the methods of the invention, hybridize to
the specified polynucleotide sequences, or complements thereof
under stringent conditions.
[0446] The term "hybridize under stringent conditions", and
grammatical equivalents thereof, refers to the ability of a
polynucleotide molecule to hybridize to a target polynucleotide
molecule (such as a target polynucleotide molecule immobilized on a
DNA or RNA blot, such as a Southern blot or Northern blot) under
defined conditions of temperature and salt concentration. The
ability to hybridize under stringent hybridization conditions can
be determined by initially hybridizing under less stringent
conditions then increasing the stringency to the desired
stringency.
[0447] With respect to polynucleotide molecules greater than about
100 bases in length, typical stringent hybridization conditions are
no more than 25 to 30.degree. C. (for example, 10.degree. C.) below
the melting temperature (Tm) of the native duplex (see generally,
Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual,
2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current
Protocols in Molecular Biology, Greene Publishing,). Tm for
polynucleotide molecules greater than about 100 bases can be
calculated by the formula Tm=81. 5+0.41% (G+C-log (Na+). (Sambrook
et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390).
Typical stringent conditions for polynucleotide of greater than 100
bases in length would be hybridization conditions such as
prewashing in a solution of 6.times.SSC, 0.2% SDS; hybridizing at
65.degree. C., 6.times.SSC, 0.2% SDS overnight; followed by two
washes of 30 minutes each in 1.times.SSC, 0.1% SDS at 65.degree. C.
and two washes of 30 minutes each in 0.2.times.SSC, 0.1% SDS at
65.degree. C.
[0448] With respect to polynucleotide molecules having a length
less than 100 bases, exemplary stringent hybridization conditions
are 5 to 10.degree. C. below Tm. On average, the Tm of a
polynucleotide molecule of length less than 100 bp is reduced by
approximately (500/oligonucleotide length).degree. C.
[0449] With respect to the DNA mimics known as peptide nucleic
acids (PNAs) (Nielsen et al., Science. 1991 Dec. 6;
254(5037):1497-500) Tm values are higher than those for DNA-DNA or
DNA-RNA hybrids, and can be calculated using the formula described
in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6.
Exemplary stringent hybridization conditions for a DNA-PNA hybrid
having a length less than 100 bases are 5 to 10.degree. C. below
the Tm.
[0450] Variant polynucleotides of the present invention, or used in
the methods of the invention, also encompasses polynucleotides that
differ from the sequences of the invention but that, as a
consequence of the degeneracy of the genetic code, encode a
polypeptide having similar activity to a polypeptide encoded by a
polynucleotide of the present invention. A sequence alteration that
does not change the amino acid sequence of the polypeptide is a
"silent variation". Except for ATG (methionine) and TGG
(tryptophan), other codons for the same amino acid may be changed
by art recognized techniques, e.g., to optimize codon expression in
a particular host organism.
[0451] Polynucleotide sequence alterations resulting in
conservative substitutions of one or several amino acids in the
encoded polypeptide sequence without significantly altering its
biological activity are also included in the invention. A skilled
artisan will be aware of methods for making phenotypically silent
amino acid substitutions (see, e.g., Bowie et al., 1990, Science
247, 1306).
[0452] Variant polynucleotides due to silent variations and
conservative substitutions in the encoded polypeptide sequence may
be determined using the publicly available bl2seq program from the
BLAST suite of programs (version 2.2.5 [Nov. 2002]) from the NCBI
website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/ via
the tblastx algorithm as previously described.
[0453] Polypeptide Variants
[0454] The term "variant" with reference to polypeptides
encompasses naturally occurring, recombinantly and synthetically
produced polypeptides. Variant polypeptide sequences preferably
exhibit at least 50%, more preferably at least 51%, more preferably
at least 52%, more preferably at least 53%, more preferably at
least 54%, more preferably at least 55%, more preferably at least
56%, more preferably at least 57%, more preferably at least 58%,
more preferably at least 59%, more preferably at least 60%, more
preferably at least 61%, more preferably at least 62%, more
preferably at least 63%, more preferably at least 64%, more
preferably at least 65%, more preferably at least 66%, more
preferably at least 67%, more preferably at least 68%, more
preferably at least 69%, more preferably at least 70%, more
preferably at least 71%, more preferably at least 72%, more
preferably at least 73%, more preferably at least 74%, more
preferably at least 75%, more preferably at least 76%, more
preferably at least 77%, more preferably at least 78%, more
preferably at least 79%, more preferably at least 80%, more
preferably at least 81%, more preferably at least 82%, more
preferably at least 83%, more preferably at least 84%, more
preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, and most
preferably at least 99% identity to a sequences of the present
invention. Identity is found over a comparison window of at least
20 amino acid positions, preferably at least 50 amino acid
positions, more preferably at least 100 amino acid positions, and
most preferably over the entire length of a polypeptide of the
invention.
[0455] Polypeptide sequence identity can be determined in the
following manner. The subject polypeptide sequence is compared to a
candidate polypeptide sequence using BLASTP (from the BLAST suite
of programs, version 2.2.5 [November 2002]) in bl2seq, which is
publicly available from the NCBI website on the World Wide Web at
ftp://ftp.ncbi.nih.gov/blast/. The default parameters of bl2seq are
utilized except that filtering of low complexity regions should be
turned off.
[0456] Polypeptide sequence identity may also be calculated over
the entire length of the overlap between a candidate and subject
polynucleotide sequences using global sequence alignment programs.
EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and
GAP (Huang, X. (1994) On Global Sequence Alignment. Computer
Applications in the Biosciences 10, 227-235.) as discussed above
are also suitable global sequence alignment programs for
calculating polypeptide sequence identity.
[0457] A preferred method for calculating polypeptide % sequence
identity is based on aligning sequences to be compared using
Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23,
403-5.)
[0458] Polypeptide variants of the present invention, or used in
the methods of the invention, also encompass those which exhibit a
similarity to one or more of the specifically identified sequences
that is likely to preserve the functional equivalence of those
sequences and which could not reasonably be expected to have
occurred by random chance. Such sequence similarity with respect to
polypeptides may be determined using the publicly available bl2seq
program from the BLAST suite of programs (version 2.2.5 [November
2002]) from the NCBI website on the World Wide Web at
ftp://ftp.ncbi.nih.gov/blast/. The similarity of polypeptide
sequences may be examined using the following unix command line
parameters: [0459] bl2seq-i peptideseq1-j peptideseq2-F F-p
blastp
[0460] Variant polypeptide sequences preferably exhibit an E value
of less than 1.times.10-6 more preferably less than 1.times.10-9,
more preferably less than 1.times.10-12, more preferably less than
1.times.10-15, more preferably less than 1.times.10-18, more
preferably less than 1.times.10-21, more preferably less than
1.times.10-30, more preferably less than 1.times.10-40, more
preferably less than 1.times.10-50, more preferably less than
1.times.10-60, more preferably less than 1.times.10-70, more
preferably less than 1.times.10-80, more preferably less than
1.times.10-90 and most preferably 1.times.10-100 when compared with
any one of the specifically identified sequences.
[0461] The parameter-F F turns off filtering of low complexity
sections. The parameter-p selects the appropriate algorithm for the
pair of sequences. This program finds regions of similarity between
the sequences and for each such region reports an "E value" which
is the expected number of times one could expect to see such a
match by chance in a database of a fixed reference size containing
random sequences. For small E values, much less than one, this is
approximately the probability of such a random match.
[0462] Conservative substitutions of one or several amino acids of
a described polypeptide sequence without significantly altering its
biological activity are also included in the invention. A skilled
artisan will be aware of methods for making phenotypically silent
amino acid substitutions (see, e.g., Bowie et al., 1990, Science
247, 1306).
[0463] Constructs, Vectors and Components Thereof
[0464] The term "genetic construct" refers to a polynucleotide
molecule, usually double-stranded DNA, which may have inserted into
it another polynucleotide molecule (the insert polynucleotide
molecule) such as, but not limited to, a cDNA molecule. A genetic
construct may contain the necessary elements that permit
transcribing the insert polynucleotide molecule, and, optionally,
translating the transcript into a polypeptide. The insert
polynucleotide molecule may be derived from the host cell, or may
be derived from a different cell or organism and/or may be a
recombinant polynucleotide. Once inside the host cell the genetic
construct may become integrated in the host chromosomal DNA. The
genetic construct may be linked to a vector.
[0465] The term "vector" refers to a polynucleotide molecule,
usually double stranded DNA, which is used to transport the genetic
construct into a host cell. The vector may be capable of
replication in at least one additional host system, such as E.
coli.
[0466] The term "expression construct" refers to a genetic
construct that includes the necessary elements that permit
transcribing the insert polynucleotide molecule, and, optionally,
translating the transcript into a polypeptide. An expression
construct typically comprises in a 5' to 3' direction: [0467] a) a
promoter functional in the host cell into which the construct will
be transformed, [0468] b) the polynucleotide to be expressed, and
[0469] c) a terminator functional in the host cell into which the
construct will be transformed.
[0470] The term "coding region" or "open reading frame" (ORF)
refers to the sense strand of a genomic DNA sequence or a cDNA
sequence that is capable of producing a transcription product
and/or a polypeptide under the control of appropriate regulatory
sequences. The coding sequence may, in some cases, identified by
the presence of a 5' translation start codon and a 3' translation
stop codon. When inserted into a genetic construct, a "coding
sequence" is capable of being expressed when it is operably linked
to promoter and terminator sequences.
[0471] "Operably-linked" means that the sequenced to be expressed
is placed under the control of regulatory elements that include
promoters, tissue-specific regulatory elements, temporal regulatory
elements, enhancers, repressors and terminators.
[0472] The term "noncoding region" refers to untranslated sequences
that are upstream of the translational start site and downstream of
the translational stop site. These sequences are also referred to
respectively as the 5' UTR and the 3' UTR. These regions include
elements required for transcription initiation and termination,
mRNA stability, and for regulation of translation efficiency.
[0473] Terminators are sequences, which terminate transcription,
and are found in the 3' untranslated ends of genes downstream of
the translated sequence. Terminators are important determinants of
mRNA stability and in some cases have been found to have spatial
regulatory functions.
[0474] The term "promoter" refers to nontranscribed cis-regulatory
elements upstream of the coding region that regulate gene
transcription. Promoters comprise cis-initiator elements which
specify the transcription initiation site and conserved boxes such
as the TATA box, and motifs that are bound by transcription
factors. Introns within coding sequences can also regulate
transcription and influence post-transcriptional processing
(including splicing, capping and polyadenylation).
[0475] A promoter may be homologous with respect to the
polynucleotide to be expressed. This means that the promoter and
polynucleotide are found operably linked in nature.
[0476] Alternatively the promoter may be heterologous with respect
to the polynucleotide to be expressed. This means that the promoter
and the polynucleotide are not found operably linked in nature.
[0477] In certain embodiments the ARF3 polynucleotides/polypeptides
of the invention may be andvantageously expessed under the contol
of selected promoter sequences as described below.
[0478] Vegetative Tissue Specific Promoters
[0479] An example of a vegetative specific promoter is found in
U.S. Pat. No. 6,229,067; and U.S. Pat. No. 7,629,454; and U.S. Pat.
No. 7,153,953; and U.S. Pat. No. 6,228,643.
[0480] Pollen Specific Promoters
[0481] An example of a pollen specific promoter is found in U.S.
Pat. No. 7,141,424; and U.S. Pat. No. 5,545,546; and U.S. Pat. No.
5,412,085; and U.S. Pat. No. 5,086,169; and U.S. Pat. No.
7,667,097.
[0482] Seed Specific Promoters
[0483] An example of a seed specific promoter is found in U.S. Pat.
No. 6,342,657; and U.S. Pat. No. 7,081,565; and U.S. Pat. No.
7,405,345; and U.S. Pat. No. 7,642,346; and U.S. Pat. No.
7,371,928. A preferred seed specific promoter is the napin promoter
of Brassica napus (Josefsson et al., 1987, J Biol Chem.
262(25):12196-201; Ellerstrom et al., 1996, Plant Molecular
Biology, Volume 32, Issue 6, pp 1019-1027).
[0484] Fruit Specific Promoters
[0485] An example of a fruit specific promoter is found in U.S.
Pat. No. 5,536,653; and U.S. Pat. No. 6,127,179; and U.S. Pat. No.
5,608,150; and U.S. Pat. No. 4,943,674.
[0486] Non-Photosynthetic Tissue Preferred Promoters
[0487] Non-photosynthetic tissue preferred promoters include those
preferentially expressed in non-photosynthetic tissues/organs of
the plant.
[0488] Non-photosynthetic tissue preferred promoters may also
include light repressed promoters.
[0489] Light Repressed Promoters
[0490] An example of a light repressed promoter is found in U.S.
Pat. No. 5,639,952 and in U.S. Pat. No. 5,656,496.
[0491] Root Specific Promoters
[0492] An example of a root specific promoter is found in U.S. Pat.
No. 5,837,848; and US 2004/0067506 and US 2001/0047525.
[0493] Tuber Specific Promoters
[0494] An example of a tuber specific promoter is found in U.S.
Pat. No. 6,184,443.
[0495] Bulb Specific Promoters
[0496] An example of a bulb specific promoter is found in Smeets et
al., (1997) Plant Physiol. 113:765-771.
[0497] Rhizome Preferred Promoters
[0498] An example of a rhizome preferred promoter is found Seong
Jang et al., (2006) Plant Physiol. 142:1148-1159.
[0499] Endosperm Specific Promoters
[0500] An example of an endosperm specific promoter is found in
U.S. Pat. No. 7,745,697.
[0501] Corm Promoters
[0502] An example of a promoter capable of driving expression in a
corm is found in Schenk et al., (2001) Plant Molecular Biology,
47:399-412.
[0503] Photosythetic Tissue Preferred Promoters
[0504] Photosythetic tissue preferred promoters include those that
are preferrentially expressed in photosynthetic tissues of the
plants. Photosynthetic tissues of the plant include leaves, stems,
shoots and above ground parts of the plant. Photosythetic tissue
preferred promoters include light regulated promoters.
[0505] Light Regulated Promoters
[0506] Numerous light regulated promoters are known to those
skilled in the art and include for example chlorophyll a/b (Cab)
binding protein promoters and Rubisco Small Subunit (SSU)
promoters. An example of a light regulated promoter is found in
U.S. Pat. No. 5,750,385. Light regulated in this context means
light inducible or light induced.
[0507] Transgene
[0508] A "transgene" is a polynucleotide that is introduced into an
organism by transformation. The transgene may be derived from the
same species or from a different species to the organism into which
the transgene is introduced. In one embodiment the transgene is a
naturally occurring sequence. In a further embodiment the transgene
is a non-naturally occurring sequence. The transgene may be
synthesized or produced by recombinant methods.
[0509] Host Cells
[0510] Host cells may be derived from, for example, bacterial,
fungal, yeast, insect, mammalian, algal or plant organisms. Host
cells may also be synthetic cells. Preferred host cells are
eukaryotic cells. A particularly preferred host cell is a plant
cell, particularly a plant cell in a tissue of a plant.
[0511] A "transgenic plant" refers to a plant which contains new
genetic material as a result of genetic manipulation or
transformation. The new genetic material may be derived from a
plant of the same species as the resulting transgenic plant or from
a different species. Subsequent offspring or generations of the
plant that still contain the new genetic material are also
transgenic plants according to the invention.
[0512] Methods for Isolating or Producing Polynucleotides
[0513] The polynucleotide molecules of the invention can be
isolated by using a variety of techniques known to those of
ordinary skill in the art. By way of example, such polypeptides can
be isolated through use of the polymerase chain reaction (PCR)
described in Mullis et al., Eds. 1994 The Polymerase Chain
Reaction, Birkhauser, incorporated herein by reference. The
polypeptides of the invention can be amplified using primers, as
defined herein, derived from the polynucleotide sequences of the
invention.
[0514] Further methods for isolating polynucleotides of the
invention include use of all, or portions of, the polypeptides
having the sequence set forth herein as hybridization probes. The
technique of hybridizing labelled polynucleotide probes to
polynucleotides immobilized on solid supports such as
nitrocellulose filters or nylon membranes, can be used to screen
the genomic or cDNA libraries. Exemplary hybridization and wash
conditions are: hybridization for 20 hours at 65.degree. C. in
5.0.times.SSC, 0.5% sodium dodecyl sulfate, 1.times.Denhardt's
solution; washing (three washes of twenty minutes each at
55.degree. C.) in 1.0.times.SSC, 1% (w/v) sodium dodecyl sulfate,
and optionally one wash (for twenty minutes) in 0.5.times.SSC, 1%
(w/v) sodium dodecyl sulfate, at 60.degree. C. An optional further
wash (for twenty minutes) can be conducted under conditions of
0.1.times.SSC, 1% (w/v) sodium dodecyl sulfate, at 60.degree.
C.
[0515] The polynucleotide fragments of the invention may be
produced by techniques well-known in the art such as restriction
endonuclease digestion, oligonucleotide synthesis and PCR
amplification.
[0516] A partial polynucleotide sequence may be used, in methods
well-known in the art to identify the corresponding full length
polynucleotide sequence. Such methods include PCR-based methods,
5'RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and
hybridization-based method, computer/database--based methods.
Further, by way of example, inverse PCR permits acquisition of
unknown sequences, flanking the polynucleotide sequences disclosed
herein, starting with primers based on a known region (Triglia et
al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by
reference). The method uses several restriction enzymes to generate
a suitable fragment in the known region of a gene. The fragment is
then circularized by intramolecular ligation and used as a PCR
template. Divergent primers are designed from the known region. In
order to physically assemble full-length clones, standard molecular
biology approaches can be utilized (Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press,
1987).
[0517] It may be beneficial, when producing a transgenic plant from
a particular species, to transform such a plant with a sequence or
sequences derived from that species. The benefit may be to
alleviate public concerns regarding cross-species transformation in
generating transgenic organisms. For these reasons among others, it
is desirable to be able to identify and isolate orthologues of a
particular gene in several different plant species.
[0518] Variants (including orthologues) may be identified by the
methods described.
[0519] Methods for Identifying Variants
[0520] Physical Methods
[0521] Variant polypeptides may be identified using PCR-based
methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction,
Birkhauser). Typically, the polynucleotide sequence of a primer,
useful to amplify variants of polynucleotide molecules of the
invention by PCR, may be based on a sequence encoding a conserved
region of the corresponding amino acid sequence.
[0522] Alternatively library screening methods, well known to those
skilled in the art, may be employed (Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press,
1987). When identifying variants of the probe sequence,
hybridization and/or wash stringency will typically be reduced
relatively to when exact sequence matches are sought.
[0523] Polypeptide variants may also be identified by physical
methods, for example by screening expression libraries using
antibodies raised against polypeptides of the invention (Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring
Harbor Press, 1987) or by identifying polypeptides from natural
sources with the aid of such antibodies.
[0524] Computer Based Methods
[0525] The variant sequences of the invention, including both
polynucleotide and polypeptide variants, may also be identified by
computer-based methods well-known to those skilled in the art,
using public domain sequence alignment algorithms and sequence
similarity search tools to search sequence databases (public domain
databases include Genbank, EMBL, Swiss-Prot, PIR and others). See,
e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of
online resources. Similarity searches retrieve and align target
sequences for comparison with a sequence to be analyzed (i.e., a
query sequence). Sequence comparison algorithms use scoring
matrices to assign an overall score to each of the alignments.
[0526] An exemplary family of programs useful for identifying
variants in sequence databases is the BLAST suite of programs
(version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX,
tBLASTN and tBLASTX, which are publicly available from
(ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for
Biotechnology Information (NCBI), National Library of Medicine,
Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server
also provides the facility to use the programs to screen a number
of publicly available sequence databases. BLASTN compares a
nucleotide query sequence against a nucleotide sequence database.
BLASTP compares an amino acid query sequence against a protein
sequence database.
[0527] BLASTX compares a nucleotide query sequence translated in
all reading frames against a protein sequence database. tBLASTN
compares a protein query sequence against a nucleotide sequence
database dynamically translated in all reading frames. tBLASTX
compares the six-frame translations of a nucleotide query sequence
against the six-frame translations of a nucleotide sequence
database. The BLAST programs may be used with default parameters or
the parameters may be altered as required to refine the screen.
[0528] The use of the BLAST family of algorithms, including BLASTN,
BLASTP, and BLASTX, is described in the publication of Altschul et
al., Nucleic Acids Res. 25: 3389-3402, 1997.
[0529] The "hits" to one or more database sequences by a queried
sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a
similar algorithm, align and identify similar portions of
sequences. The hits are arranged in order of the degree of
similarity and the length of sequence overlap. Hits to a database
sequence generally represent an overlap over only a fraction of the
sequence length of the queried sequence.
[0530] The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms
also produce "Expect" values for alignments. The Expect value (E)
indicates the number of hits one can "expect" to see by chance when
searching a database of the same size containing random contiguous
sequences. The Expect value is used as a significance threshold for
determining whether the hit to a database indicates true
similarity. For example, an E value of 0.1 assigned to a
polynucleotide hit is interpreted as meaning that in a database of
the size of the database screened, one might expect to see 0.1
matches over the aligned portion of the sequence with a similar
score simply by chance. For sequences having an E value of 0.01 or
less over aligned and matched portions, the probability of finding
a match by chance in that database is 1% or less using the BLASTN,
BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
[0531] Multiple sequence alignments of a group of related sequences
can be carried out with CLUSTALW (Thompson, J. D., Higgins, D. G.
and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix choice. Nucleic
Acids Research, 22:4673-4680,
http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or
T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa,
T-Coffee: A novel method for fast and accurate multiple sequence
alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which
uses progressive, pairwise alignments. (Feng and Doolittle, 1987,
J. Mol. Evol. 25, 351).
[0532] Pattern recognition software applications are available for
finding motifs or signature sequences. For example, MEME (Multiple
Em for Motif Elicitation) finds motifs and signature sequences in a
set of sequences, and MAST (Motif Alignment and Search Tool) uses
these motifs to identify similar or the same motifs in query
sequences. The MAST results are provided as a series of alignments
with appropriate statistical data and a visual overview of the
motifs found. MEME and MAST were developed at the University of
California, San Diego.
[0533] PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22,
3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method
of identifying the functions of uncharacterized proteins translated
from genomic or cDNA sequences. The PROSITE database
(www.expasy.org/prosite) contains biologically significant patterns
and profiles and is designed so that it can be used with
appropriate computational tools to assign a new sequence to a known
family of proteins or to determine which known domain(s) are
present in the sequence (Falquet et al., 2002, Nucleic Acids Res.
30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL
databases with a given sequence pattern or signature.
[0534] Methods for Isolating Polypeptides
[0535] The polypeptides of the invention, or used in the methods of
the invention, including variant polypeptides, may be prepared
using peptide synthesis methods well known in the art such as
direct peptide synthesis using solid phase techniques (e.g. Stewart
et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San
Francisco Calif., or automated synthesis, for example using an
Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.).
Mutated forms of the polypeptides may also be produced during such
syntheses.
[0536] The polypeptides and variant polypeptides of the invention,
or used in the methods of the invention, may also be purified from
natural sources using a variety of techniques that are well known
in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol.
182, Guide to Protein Purification,).
[0537] Alternatively the polypeptides and variant polypeptides of
the invention, or used in the methods of the invention, may be
expressed recombinantly in suitable host cells and separated from
the cells as discussed below.
[0538] Methods for Producing Constructs and Vectors
[0539] The genetic constructs of the present invention comprise one
or more polynucleotide sequences of the invention and/or
polynucleotides encoding polypeptides of the invention, and may be
useful for transforming, for example, bacterial, fungal, insect,
mammalian or plant organisms. The genetic constructs of the
invention are intended to include expression constructs as herein
defined.
[0540] Methods for producing and using genetic constructs and
vectors are well known in the art and are described generally in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols
in Molecular Biology, Greene Publishing, 1987).
[0541] Methods for Producing Host Cells Comprising Polynucleotides,
Constructs or Vectors
[0542] The invention provides a host cell which comprises a genetic
construct or vector of the invention.
[0543] Host cells comprising genetic constructs, such as expression
constructs, of the invention are useful in methods well known in
the art (e.g. Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing, 1987)
for recombinant production of polypeptides of the invention. Such
methods may involve the culture of host cells in an appropriate
medium in conditions suitable for or conducive to expression of a
polypeptide of the invention. The expressed recombinant
polypeptide, which may optionally be secreted into the culture, may
then be separated from the medium, host cells or culture medium by
methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in
Enzymology, Vol 182, Guide to Protein Purification).
[0544] Methods for Producing Plant Cells and Plants Comprising
Constructs and Vectors
[0545] The invention further provides plant cells which comprise a
genetic construct of the invention, and plant cells modified to
alter expression of a polynucleotide or polypeptide of the
invention, or used in the methods of the invention. Plants
comprising such cells also form an aspect of the invention.
[0546] Methods for transforming plant cells, plants and portions
thereof with polypeptides are described in Draper et al., 1988,
Plant Genetic Transformation and Gene Expression. A Laboratory
Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and
Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag,
Berlin; and Gelvin et al., 1993, Plant Molecular Biol. Manual.
Kluwer Acad. Pub. Dordrecht. A review of transgenic plants,
including transformation techniques, is provided in Galun and
Breiman, 1997, Transgenic Plants. Imperial College Press,
London.
[0547] Methods for Genetic Manipulation of Plants
[0548] A number of plant transformation strategies are available
(e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297,
Hellens et al., (2000) Plant Mol Biol 42: 819-32, Hellens et al.,
Plant Meth 1: 13). For example, strategies may be designed to
increase expression of a polynucleotide/polypeptide in a plant
cell, organ and/or at a particular developmental stage where/when
it is normally expressed or to ectopically express a
polynucleotide/polypeptide in a cell, tissue, organ and/or at a
particular developmental stage which/when it is not normally
expressed. The expressed polynucleotide/polypeptide may be derived
from the plant species to be transformed or may be derived from a
different plant species.
[0549] Transformation strategies may be designed to reduce
expression of a polynucleotide/polypeptide in a plant cell, tissue,
organ or at a particular developmental stage which/when it is
normally expressed. Such strategies are known as gene silencing
strategies.
[0550] Genetic constructs for expression of genes in transgenic
plants typically include promoters for driving the expression of
one or more cloned polynucleotide, terminators and selectable
marker sequences to detect presence of the genetic construct in the
transformed plant.
[0551] The promoters suitable for use in the constructs of this
invention are functional in a cell, tissue or organ of a monocot or
dicot plant and include cell-, tissue- and organ-specific
promoters, cell cycle specific promoters, temporal promoters,
inducible promoters, constitutive promoters that are active in most
plant tissues, and recombinant promoters. Choice of promoter will
depend upon the temporal and spatial expression of the cloned
polynucleotide, so desired. The promoters may be those normally
associated with a transgene of interest. In one embodiment the
promoter is not normally associated with a transgene of interest.
Such a promoter may be described as a heterologous promoter, with
respect to the transgene.
[0552] The promoters may be derived from genes of other plants,
viruses, and plant pathogenic bacteria and fungi. Those skilled in
the art will, without undue experimentation, be able to select
promoters that are suitable for use in modifying and modulating
plant traits using genetic constructs comprising the polynucleotide
sequences of the invention. Examples of constitutive plant
promoters include the CaMV 35S promoter, the nopaline synthase
promoter and the octopine synthase promoter, and the Ubi 1 promoter
from maize. Plant promoters which are active in specific tissues
respond to internal developmental signals or external abiotic or
biotic stresses are described in the scientific literature.
Exemplary promoters are described, e.g., in WO 02/00894 and
WO2011/053169, which is herein incorporated by reference.
[0553] Exemplary terminators that are commonly used in plant
transformation genetic construct include, e.g., the cauliflower
mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens
nopaline synthase or octopine synthase terminators, the Zea mays
zein gene terminator, the Oryza sativa ADP-glucose
pyrophosphorylase terminator and the Solanum tuberosum PI-II
terminator.
[0554] Selectable markers commonly used in plant transformation
include the neomycin phophotransferase II gene (NPT II) which
confers kanamycin resistance, the aadA gene, which confers
spectinomycin and streptomycin resistance, the phosphinothricin
acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta
(Hoechst) resistance, and the hygromycin phosphotransferase gene
(hpt) for hygromycin resistance.
[0555] Use of genetic constructs comprising reporter genes (coding
sequences which express an activity that is foreign to the host,
usually an enzymatic activity and/or a visible signal (e.g.,
luciferase, GUS, GFP) which may be used for promoter expression
analysis in plants and plant tissues are also contemplated. The
reporter gene literature is reviewed in Herrera-Estrella et al.,
1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to
Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline,
pp. 325-336.
[0556] The following are representative publications disclosing
genetic transformation protocols that can be used to genetically
transform the following plant species: Rice (Alam et al., 1999,
Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell
Reports 14, 407-412); maize (U.S. Pat. Nos. 5,177,010 and
5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996,
877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996
Plant J. 9:821); cassava (Li et al., 1996 Nat. Biotechnology 14,
736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439);
tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat.
Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073
and 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17,
165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183);
caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S.
Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834;
5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No.
5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general
(U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos.
5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. No.
6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep.
24(1):45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep.
25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006; 25(2):117-23;
Gonzalez Padilla et al., 2003 Plant Cell Rep.22(1):38-45);
strawberry (Oosumi et al., 2006 Planta. 223(6):1219-30; Folta et
al., 2006 Planta April 14; PMID: 16614818), rose (Li et al., 2003),
Rubus (Graham et al., 1995 Methods Mol Biol. 1995; 44:129-33),
tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple
(Yao et al., 1995, Plant Cell Rep. 14, 407-412), Canola (Brassica
napus L.). (Cardoza and Stewart, 2006 Methods Mol Biol.
343:257-66), safflower (Orlikowska et al., 1995, Plant Cell Tissue
and Organ Culture 40:85-91), ryegrass (Altpeter et al., 2004
Developments in Plant Breeding 11(7):255-250), rice (Christou et
al., 1991 Nature Biotech. 9:957-962), maize (Wang et al., 2009 In:
Handbook of Maize pp. 609-639) and Actinidia eriantha (Wang et al.,
2006, Plant Cell Rep. 25, 5: 425-31). Transformation of other
species is also contemplated by the invention. Suitable methods and
protocols are available in the scientific literature.
[0557] In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art.
[0558] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting each statement
in this specification that includes the term "comprising", features
other than that or those prefaced by the term may also be present.
Related terms such as "comprise" and "comprises" are to be
interpreted in the same manner. In some embodiments, the term
"comprising" (and related terms such as "comprise and "comprises")
can be replaced by "consisting of" (and related terms "consist" and
"consists").
BRIEF DESCRIPTION OF THE FIGURES
[0559] FIG. 1 shows identification of the rootstock dwarfing loci,
Dw1. a) Using a bulked segregant analysis, a major dwarfing locus
(Dw1) from `M9` was identified at the top of linkage group (LG) 5.
The markers flanking Dw1 were NZraAM18-700 (developed by Plant
& Food Research, not publically avaliable) and CH03a09
(publically available). b) A multi-trait QTL analysis identified
Dw1 as having a very strong influence on rootstock induced
dwarfing. The markers flanking Dw1 are Hi01c04a and CH03a09.
[0560] FIG. 2 shows genetic markers flanking o Dw1 according to the
applicant, and that described by Fazio et al. a) Markers flanking
our Dw1 are shown in red and extend from 4.72 Mb to 7.62 Mb. b)
Markers flanking the Fazio et al Dw1 are shown in green. The distal
marker CH05b06z is not mapped. c) The proximal marker CH05b06z maps
elsewhere, and the distal most maps incorrectly. d) The distal
marker C3843 does not map to LG5. Based on the markers that do map,
this would place the Fazio et al Dw1 more distal than ours.
[0561] FIG. 3 shows recombinant Dwarf & Semi-Dwarf individuals
narrow the genomic interval containing Dw1 to <1.1 Mb. Parents
and progeny are listed along the left most column, phenotypes in
the next column over, each the remaining columns are genotypes for
genetic markers sequentially ordered along LG5. Pink indicates the
`M9` allele and green the `R5` allele. Individuals highlighted in
yellow are recombinant over the interval. Only dwarfed (D) and
semi-dwarfed (SD) individuals are informative, as some intermediate
(I) and vigorous (V) individuals carry Dw1.
[0562] FIG. 4 shows the number of trees in each flowering class and
composition of classes by Dw1 and Dw2 genotype. Flowering was
assessed by estimating the total number of flower clusters on each
tree in the spring of year two, and placing them into quartiles
relative to the most highly floral trees, ie, 1%-25%, 26-50%,
51-75%, 76-100%. Trees with no flowers were also recorded. Data is
from 109 trees from the first population, replicate 1.
[0563] FIG. 5 shows the average year seven TCA of trees in each
genotypic class. The number of individuals in each class is given
in parentheses, error bars indicate standard error. Average TCAs
were compared to the group with neither Dw1 nor Dw2 by ANOVA,
asterisks indicate the means are significantly different with a p
value of .ltoreq.0.001. Data is from 303 trees from the second
population.
[0564] FIG. 6 shows the composition of each phenotypic class by Dw1
and Dw2 genotype. Trees from both populations (449 trees in total)
were visually assessed after seven years of growth and placed into
one of five phenotypic classes, D=dwarf, SD=semi-dwarf,
I=intermediate, V=vigorous, and VV=very vigorous.
[0565] FIG. 7 shows quantitative RT-PCR of ARF3. For each time
point, RNA was isolated and analysed from vascular-enriched tissue
from 4-6 separate biological replicates of each genotype. Error
bars indicate standard error for biological replicates.
[0566] FIG. 8 shows an amino acid line up of ARF3 proteins from
plants. ARF3 proteins have a highly conserved B3 DNA binding
domain, an auxin response element and a tasi-ARF recognition site.
`M9` is heterozygous for a non-synonymous SNP that changes a
conserved Serine/Proline to a Leucine (indicated by red box)
[0567] FIG. 9 shows a table demonstrating % similarity between ARF
3 proteins. Proteins were aligned using MUSCLE and the phylogenetic
tree used to generate this table was constructed with PHYML, using
JTT substitution model and 1,000 bootstrap interations
[0568] FIG. 10 shows over-expression of `M9` ARF3 in petunia. a)
Non-transformed and b-f) 355:'M9' ARF3 flowers. Three independent
lines showed incomplete petal fusion at the tube (b-c), irregular
petal margins (d), and vascular patterning defects (e). (f) shows a
close up of the abaxial (outside) of the flower, revealing
incomplete petal fusion and vascular patterning defects.
[0569] FIG. 11 shows over-expression of `M9` ARF3 in petunia. a)
untransfomed and b) 35S:M9 ARF3 flower showing petaloid stamen that
appear in two lines.
[0570] FIG. 12 shows over-expression of `M9` ARF3 in tobacco. (a)
un-transformed and (b-c) 35S: M9 ARF3. Vascular patterning defects
were observed in several lines (arrows in b and c). One line showed
an asymmetric leaf phenotype (arrowheads in c).
[0571] FIG. 13 shows the vascular patterning defects in the `M9`
ARF3 overexpression tobacco plants.
[0572] FIG. 14 shows `M9` overexpression plants exhibiting reduced
height, thick stems, shorter internodes and more axillary outgrowth
compared to wild-type tobacco.
[0573] FIG. 15 shows floral phenotypes of 35S:ARF3 in tobacco.
Extra petaloid organs are common (arrows in a, c, e) as well as
patterning defects, irregular vascular patterning (arrows in a, b)
and unfused tube (arrow in d).
[0574] FIG. 16 shows irregular vascular development in 35S:ARF3 in
tobacco. Sections of (a) untransformed and (b-d) 35S: M9 ARF3
tobacco petioles. Tobacco has a co-lateral arrangement of xylem
surrounded by phloem on both abaxial (AB) and adaxial (AD) sides.
The M9 ARF3 over-expression lines show irregular vascular
patterning, with more inner phloem cells (red arrows in b-d).
[0575] FIG. 17 shows a summary of Dw1 and Dw2 genotyping of
rootstock accessions. SSR makers were used to genotype rootstock
accessions for the presence of Dw1 and Dw2. A green square
indicates the presence of a single allele of Dw1, yellow represents
Dw2. The very dwarfing rootstock `M27` is homozygous for Dw1,
suggesting that Dw1 is a semi-dominant mutation.
[0576] FIG. 18 shows that a pear rootstock QTL maps to the same
position as Dw1. a) A rootstock QTL affecting scion flowering,
shoot growth and TCA (Trunk Cross-sectional Area) was detected on
LG5, in the same position as Dw1. One major difference between the
two QTLs, the pear QTL controlling early flowering is on the same
position, but on the other chromosome, ie derived from the other
parent. An HRM marker detecting the ARF3 SNP in apple was screened
over the pear population. In b-d, individuals scored as "AA" were
statistically different than siblings scored as "AB" for b)
flowering, c) primary axis growth and d) TCA. *=p value<0.001,
very significant.
[0577] FIG. 19 illustrates a grafting experiment to demonstrate
effect on scion. A--illustrates that one apical meristem is allowed
to grow out. B--shows the grafted non-transformed wild-type stem.
C--shows thwe graft junction. D--shows the "rootstock" which can be
35S:Dw1 (M9 mutant allele), 35S:dw1 (M793 non-dwarf allele) or
non-transformed (WT).
[0578] FIG. 20 shows the phenotypic characteristics of scions
grafted onto 4 different "rootstocks" as indicated. Panel A (left
side) shows shoot length of the grafted scion. Panel B (right side)
shows days to flowering of the grafted scion. Values were compared
to WT/WT by ANOVA, **=p-value<0.01, *=p-value<0.05.
[0579] FIG. 21 shows the phenotypic characteristics of scions
grafted onto 4 different "rootstocks" as indicated. Panel A (left
side) shows number of nodes on the grafted scion. Panel B (right
side) shows Trunk Cross-sectional Area (TCA) of the grafted scion.
Values were compared to WT/WT by ANOVA, **=p-value<0.01,
*=p-value<0.05.
[0580] FIG. 22 shows the total scion dry weight of scions grafted
onto 4 different "rootstocks" (same root stocks as in FIGS. 21 and
22). Values were compared to WT/WT by ANOVA, **=p-value<0.01,
*=p-value<0.05.
[0581] FIG. 23 shows the total leaf area of scions grafted onto 4
different "rootstocks" (same root stocks as in FIGS. 21 and 22).
Values were compared to WT/WT by ANOVA, *=p-value<0.05.
[0582] FIG. 24 shows tree dry weight accumulation during the first
year of growth. `Royal Gala` scions were grafted to `M793`
(vigorous), `M9` (dwarfing) or `M27` (very dwarfing). At each time
point, six composite trees of each rootstock genotype were severed
at the graft junction, a) scion and b) rootstock were dried and
weighed. Values were compared by ANOVA and the only significant
differences detected between vigorous and dwarfing rootstocks was
at the final time point (*=p-value<0.001). Error bars are
SE.
[0583] FIG. 25 shows average primary and total lateral root length
of two week old seedlings. Seedlings were germinated on media,
grown for two weeks, then harvested for photography. Digital images
were measured using Image J. Error bars are standard error.
BRIEF DESCRIPTION OF THE SEQUENCES
TABLE-US-00004 [0584] SEQ ID NO: Sequence type Common name Species
Reference 1 Polypeptide Apple Malus domestica MdARF3 2 Polypeptide
Apple Malus domestica MdARF3 `M9` 3 Polypeptide Arabidopsis
Arabidopsis thaliana ARF3/ETTIN 4 Polypeptide Bean Phaseolus
vulgaris PvARF3 5 Polypeptide Tomato Solanum lycopersicum SIARF3 6
Polypeptide Mandarin orange Citrus clemantina CcARF3 7 Polypeptide
Strawberry Frageria vesca FvARF3 8 Polypeptide Plum Prunus persica
PpARF3 9 Polypeptide Pear Pyrus communis PcARF3 10 Polypeptide
Poplar Populus tremula PtARF3 11 Polypeptide Grape Vitis vinefera
VvARF3 12 Polynucleotide Apple Malus domestica MdARF3 (cDNA) 13
Polynucleotide Apple Malus domestica MdARF3 (gDNA) 14
Polynucleotide Apple Malus domestica MdARF3 `M9`(cDNA) 15
Polynucleotide Apple Malus domestica MdARF3 `M9`(gDNA) 16
Polynucleotide Arabidopsis Arabidopsis thaliana ARF3/ETTIN (cDNA)
17 Polynucleotide Arabidopsis Arabidopsis thaliana ARF3/ETTIN
(gDNA) 18 Polynucleotide Bean Phaseolus vulgaris PvARF3 (cDNA) 19
Polynucleotide Tomato Lycopersicum esculentum LeARF3 (cDNA) 20
Polynucleotide Mandarin orange Citrus clemantina CcARF3 (cDNA) 21
Polynucleotide Strawberry Frageria vesca FvARF3 (cDNA) 22
Polynucleotide Plum Prunus persica PpARF3 (cDNA) 23 Polynucleotide
Pear Pyrus communis PcARF3 (cDNA) 24 Polynucleotide Poplar Populus
tremula PtARF3 (cDNA) 25 Polynucleotide Grape Vitis vinefera VvARF3
(cDNA) 26 Polynucleotide Apple Malus domestica Hi01C04 marker 27
Polynucleotide Apple Malus domestica Hi04A08 marker
EXAMPLES
[0585] The invention will now be illustrated with reference to the
following non-limiting examples.
[0586] It is not the intention to limit the scope of the invention
to the present example only. As would be appreciated by a skilled
person in the art, many variations are possible without departing
from the scope of the invention.
Example 1: Refining the Genomic Region Containing the Dw1 Loci
BACKGROUND
[0587] In a previous QTL study, the closest genetic markers that
defined Dw1 were Hi01c04 and Ch03a09 (FIG. 1), which are located at
4.72 and 7.62 Mb respectively on the reference golden delicious
genome (Celton et al 2009). More recently Fazio and co-workers
(Fazio et al 2014) found a more distal position for Dw1, between
Hi22f12 (2.69 Mb) and Hi04a08 (5.15 Mb) (FIG. 2).
[0588] In the present work, the applicants developed genetic
markers based on genomic sequence from the interval between 4.5 Mb
and 7.2 Mb on linkage group 5 (LG5). By screening these markers
over the parents and progeny of their rootstock population, the
applicants were able to identify recombinants within this interval
(i.e. had a chromosomal cross over between `M9` and `R5`).
Intermediate and vigorous recombinants were not informative,
because some of the individuals carried Dw1. However, all dwarfed
and semi-dwarfed individuals carried Dw1, so these recombinants
were informative in defining the interval that contains Dw1. Based
on four dwarfed and two semi-dwarfed recombinant individuals, the
applicants were able to narrow the genomic interval containing Dw1
to a smaller region, between 4.75 Mb and 5.80 Mb (FIG. 3).
[0589] This region defines an interval of 1.05 Mb (5.80-4.75
Mb).
[0590] Although this is a smaller interval, this region could still
contain over 100 genes. It is also possible that the genetic
determinant of dwarfing at the Dw1 locus would be a micro RNA
(miRNA) or other non-protein encoding gene. Furthermore, prior to
the present application, there were no obvious candidate gene/s, or
even classes of candidate genes that might be responsible for the
dwarfing effect of the the Dw1 locus.
[0591] Dw1 has a More Significant Effect than Dw2 on
Rootstock-Induced Dwarfing
[0592] To elucidate the relative contributions of Dw1 and Dw2 to
dwarfing of the scion, the applicants examined three of the most
robust phenotypes associated with dwarfing, i.e. early flowering
(spring of year two), final TCA (year seven), and overall visual
assessment (year seven) of scions grafted to rootstocks carrying
various combinations of Dw1 and Dw2.
[0593] Early flowering was assessed in the spring of year two by
estimating the number of floral clusters on 109 trees from the
first population. The majority of the trees with the highest degree
of flowering had been grafted onto rootstocks carrying both Dw1 and
Dw2 (50%), or Dw1 alone (41.7%) (FIG. 4). Conversely, the trees
with no flowers or the fewest flowers were predominantly grafted
onto rootstocks carrying Dw2 alone (33.9%), or neither dwarfing
locus (44.6%).
[0594] After seven years of growth, the TCA of 303 trees from the
second population were measured. Trees grafted onto rootstocks
carrying both Dw1 and Dw2 exhibited the lowest average TCA, only
23% of that of scions on rootstocks with neither loci. Rootstocks
with Dw1 alone reduced scion TCA to 73% of those with neither
rootstock loci. Surprisingly, trees grafted onto rootstocks with
Dw2 alone had the highest TCA of all (FIG. 5).
[0595] As rootstock-induced dwarfing becomes more pronounced over
successive growth cycles, an expert visual assessment of the whole
tree phenotype after seven years provided an overall measure of
scion vigour. When 449 grafted trees from both populations were
compared, a clear trend relating rootstock genotype to phenotypic
class was observed. All the dwarfed and semi-dwarfed trees were
grafted onto rootstocks with Dw1 and Dw2 or Dw1 alone, whereas the
vigorous and very vigorous trees had rootstocks carrying Dw2 alone,
Dw1 alone, or neither locus (FIG. 6). Nearly 40% of the vigorous
trees were on rootstocks carrying Dw2, indicating that this locus
alone is not sufficient to dwarf the scion.
[0596] However in contrast to the recent work of Fazio et al
(Fazio, Wan et al. 2014) the present study does indicate that the
Dw1 loci can influence dwarfing alone (i.e. even in the absence of
Dw2).
[0597] Other Dwarfing and Semi-Dwarfing Rootstocks Carry Dw1 and
Dw2
[0598] Genetic markers linked to Dw1 and Dw2 were screened over 41
rootstock accessions that confer a range of effects on scion
growth. The majority of dwarfing and semi-dwarfing rootstock
accessions screened carried marker alleles linked to both Dw1 and
Dw2 (Foster et al, 2015 and FIG. 17). This suggests that most apple
dwarfing rootstocks have been derived from the same genetic
source.
Example 2: A Pear Rootstock QTL Influencing Scion Size and
Flowering
[0599] Pear does not have a true dwarfing rootstock, such as `M9`,
although some rootstocks are known to influence scion size and
flowering. A pear segregating rootstock population was generated by
crossing `Old Home` to `Louis Bon Jersey`. The progeny were grafted
with `Cornice`, and scions were phenotyped for 4 years. A QTL
influencing scion size and flowering was identified at the top of
LG5, in the exact location as Dw1 (FIG. 18, PFR, unpublished). No
QTL corresponding to Dw2 was identified. Pear and apple are very
closely related and show strong synteny of gene order along their
orthologous chromosomes. This finding raises the exciting
possibility that Dw1 predates the divergence of apple and pear and
that the same gene may be influencing both the apple and pear
QTL.
Example 3: Identification of ARF3 as a Candidate Gene for Dw1
[0600] The applicants found that there are approximately 168
annotated genes within the 1.1 Mb interval (unpublished). Based on
expressed sequence ESTs from the Plant and Food proprietary Malus
database (Newcomb, Crowhurst et al. 2006) and RNA seq experiments
(unpublished), the applicants estimated the number of expressed
genes is about 100.
[0601] The applicants identified an Auxin Response Factor 3 (ARF3)
transcription factor gene present in the refined Dw1 interval,
which they showed to be upregulated in M9 rootstock, as a candidate
gene for the Dw1 QTL effect.
[0602] Many hypotheses to explain the mechanism of dwarfing
rootstocks implicate auxin, but the genetic basis of any auxin
effect is completely unknown. ARF3 is a member of a large family of
Auxin Response Factors, transcription factors that activate or
repress downstream genes in response to auxin. ARF3/ETTIN was first
discovered as a gene required for normal patterning of floral
organs in Arabidopsis (Sessions and Zambryski 1995; Sessions,
Nemhauser et al. 1997). It was later discovered that ARF3 and the
transcription factor KANADI mediate both auxin flow and organ
polarity, which includes vascular patterning (Pekker, Alvarez et
al. 2005; Izhakia and Bowman 2007; Kelley, Arreola et al. 2012).
ARF3 also has a key role in promoting phase change (transition to
flowering), increased ARF3 expression leads to earlier flowering,
loss of ARF3 function delays flowering. (Fahlgren, Montgomery et
al. 2006; Hunter, Willmann et al. 2006).
[0603] ARF3 is Up-Regulated in `M9` and `M27` Relative to Vigorous
Rootstocks
[0604] The applicants used quantitative real time PCR (qRT-PCR) to
compare ARF3 expression in vascular-enriched tissue from `M9` and
another dwarfing rootstock `M27` with a vigorous rootstock, `M793`
(FIG. 7). ARF3 expression was about four times higher in `M9` than
`M793` at all time points. In `M27`, ARF3 expression was 2-4 times
higher levels than `M793`.
[0605] `M9` has a Mutation in the ARF3 Gene
[0606] To identify any `M9`-specific DNA changes that might alter
gene expression or function/activity the applicants performed
genomic sequencing of `M9`. This revealed that the `M9` MdARF3
(MDP000173151) carried a single nucleotide polymorphism (SNP) that
changed a conserved Serine to a Leucine. FIG. 8 shows an amino acid
line-up with the `M9`, the reference MdARF3 proteins and ARF3
proteins from a variety of plants. This SNP alter the function of
the ARF3 protein.
[0607] The `M9` ARF3 SNP as a Genetic Marker in Apple and Pear
[0608] To test if the SNP identified in the `M9`ARF3 segregates
with dwarfing individuals, the applicants used primers that amplify
the SNP in a High Resolution Melting (HRM) analysis over the entire
`M9`.times.`R5` rootstock population. The results showed clear
segregation of a distinct melting curve with all individuals that
were previously identified as having Dw1. The same marker was also
tested on the pear rootstock population and showed clear
segregation with one curve associated with high flowering
individuals, another with low or no flowering trees.
Example 4: Transgenic Expression of ARF3 in Petunia and Tobacco
[0609] To test if the higher expression and/or the non-synonymous
SNP in the `M9` ARF3 cause phenotypes associated with dwarfing
rootstocks, the applicants made transgenic lines of both tobacco
and petunia that over-express either the `M9` or the reference
allele of ARF3. These are hence referred to as M9 ARF3 and wt ARF3
respectively. Petunia and tobacco were chosen as models because
they are both amenable to grafting.
[0610] The applicants generated 10 independent lines expressing
35S: M9 ARF3, but the applicants were unable to recover 35S: wt
ARF3 petunias. The applicants verified that the plants were
expressing the construct by q-RT-PCR. Three independent lines of
the 35S:M9 ARF3 had a floral phenotype, ranging from irregular
petal margins, incomplete tube fusion, vascular defects, and
petaloid stamens (FIGS. 10, 11). Microscopic analysis of the
irregular petal margins revealed small patches of inverted petal
polarity, which is consistent with the known function of ARF3 in
adaxial-abaxial patterning.
[0611] The applicants generated 10 M9 ARF3 and 10 wt ARF3
over-expression lines in tobacco. The applicants verified that all
T.sub.0 plants were expressing the construct. Preliminary analysis
indicates that several of the plants exhibit irregular vascular
patterning in the leaves (FIG. 13). Two plants have asymmetric
leaves, with half of the blade missing entirely or double midveins
(FIG. 12 b, c). The most extreme line of 35S: M9 ARF 3 (#6) is much
shorter than wild-type with thick stems, and decreased apical
dominance, creating a bushy phenotype (FIG. 14). The lines with the
highest ARF3 expression flowered earlier than the others. Early
flowering is also seen in dwarfed scions in apple. Many of the M9
and wt ARF3 plants have floral phenotypes. These include incomplete
fusion of the tube, patterning defects, and extra petaloid organs
(FIG. 15).
[0612] To examine the vascular patterning defects in more detail,
petioles from untransformed and ARF3 over-expression plants were
fixed, sectioned and stained with safranin fast green. FIG. 16
shows representative micrographs illustrating that 35S:M9 ARF3
plants have irregular vascular patterning, with more inner phloem
cells, consistent with the similar phenotype seen in M9 apple
rootstock.
[0613] Phenotypic analysis of the ARF3 over-expression tobacco
plants, can also be carried out on plants produced from T.sub.1
seed.
[0614] Plants transformed to express ARF3 and M9 ARF3 can be
phenotyped, as can scions grafted onto the transgenic, and control
plants.
[0615] Such phenotyping can involve a detailed architectural
analysis to document metamer initiation rate, the outgrowth and
size of axillary brances, the size and node number of the primary
shoot, and time to flowering.
[0616] Growth chambers can also be used to test if the transgenic
plants have an altered sensitivity to long days or short days.
[0617] Further histological analysis can also be undertaken to
compare vascular development between transgenic lines and
untrasformed controls.
Example 5: Transgenic Expression of ARF3 in Apple
[0618] The constructs described in Example 4 above were transformed
into apple, to further assess the phenotypic effect of higher
expression and/or the non-synonymous SNP.
[0619] Plantlettes generated, can be tested to verify that ARF3 is
over-expressed using qRT-PCR. Transgenic lines can be assessed for
dwarfing-associated phenotypes by comparing the overall plant
architecture (main axis hight, outgrowth of axillary branches, etc)
with un-transformed controls. To examine any changes to the
vasculature, tissue can be fixed, sectioned, stained and
photographed on a microscope to compare with untransformed
controls.
[0620] Once plantlettes have generated roots and are large enough,
they can be grafted with un-transformed controls. Scions can be
assessed for dwarfing-associated phenotypes by comparing the number
of growth units on the primary and secondary axis, comparing the
number and size of sylleptic and prolleptic shoots, and eventually
the number of flowers.
Example 6: Transgenic Expression of ARF3 in Pear
[0621] The constructs described in Example 4 above were transformed
into pear, to further assess the phenotypic effect of higher
expression and/or the non-synonymous SNP.
[0622] Plantlettes generated, can be tested to verify that ARF3 is
over-expressed using qRT-PCR. Transgenic lines can be assessed for
dwarfing-associated phenotypes by comparing the overall plant
architecture (main axis hight, outgrowth of axillary branches, etc)
with un-transformed controls. To examine any changes to the
vasculature, tissue can be fixed, sectioned, stained and
photographed on a microscope to compare with untransformed
controls.
[0623] Once plantlettes have generated roots and are large enough,
they can be grafted with un-transformed controls. Scions can be
assessed for dwarfing-associated phenotypes by comparing the number
of growth units on the primary and secondary axis, comparing the
number and size of sylleptic and prolleptic shoots, and eventually
the number of flowers.
Example 7: Determine if the `M9` SNP Alters Protein Function
[0624] Transient expression experiments in Nicotiana benthamiana
(Martin, Kopperud et al. 2009), can be used to further assess the
function of the non-synonomous SNP in the `M9` ARF3. First an an
auxin responsive reporter line, DR5:LUC (Ulmasov, Murfett et al.
1997) can be generated. This reporter will result in an enzyme that
generates fluorescent compound in response to auxin.
[0625] The reporter construct can be co-expressed with either the
`M9` or wt ARF3 and the fluorescent compound measured after 1-3
days. These experiments can also be repeated with application of
exogenous auxin to compare auxin sensitivity.
Example 8: Determine if Pear has Altered ARF3 Sequence and/or
Expression
[0626] ARF3 expression in pear can be assessed by qRT-PCR to
determine if "dwarfish" individuals from the pear rootstock
population have higher expression of ARF3 than vigorous
individuals. To determine if the same non-synonomous SNP exists
"dwarfish" individuals, the pear ARF3 gene can be amplified and
sequenced.
Example 9: Examination of the Phenotype of Apple Seedlings
Genotyped for Dw1 and Dw2
[0627] Seedlings derived from controlled crosses can be genotyed
for Dw1 and Dw2 to identify individuals that have zero, one or two
copies of Dw1, and either zero or one copy of Dw2. ARF3 expression
in apple seedlings and young trees can be assessed. Seedlings/trees
can be measured for differences in metamer number of primary and
secondary axes, the outgrowth of axillary shoots, and the time to
flowering. Stem vascular development can also be assessed
histologically.
Example 10: Tree Dry Weight Accumulation During the First Year of
Growth
[0628] `Royal Gala` scions were grafted to `M793` (vigorous), `M9`
(dwarfing) or `M27` (very dwarfing). At each time point (60, 120,
180 and 300 days after bud break [DABB]), four to six composite
trees of each rootstock genotype were severed at the graft
junction. Scion and rootstock material was oven dried at 60.degree.
C. to a constant mass and weighed. Dry weights of scion include
scion budwood, primary axis, sylleptic shoots and leaves, whilst
dry weights of rootstock include roots and rootstock stem. Values
were compared by ANOVA and the only significant differences
detected between vigorous and dwarfing rootstocks was at the final
time point (*=p-value<0.001). The results are shown in FIG. 24.
Error bars are SE.
Example 11: Grafting Experiments
[0629] Methods of Grafting
[0630] Tobacco plants were grown in pots until plants had 10-15
leaves. In this experiment, all scions were wild-type tobacco, the
"rootstocks" were wild-type, M9 ARF3 (2 independent lines, 2 and 6)
and 35S: 793 (wt) ARF3 (line 4). We note M27 has the same ARF3
allele as M9, thus M27 contains the M9 allele of ARF3. In FIGS. 20
to 23, the M9 ARF3 rootstock lines are labelled M27 2-1 and M27
6-16 and the WT ARF3 rootstock line is labelled M793 4-3.
[0631] At the time of grafting, a horizontal cut was made through
the "rootstock" stem at the very top of node 4-5. A "V"-shaped
notch was cut vertically into the stem, 5-10 mm deep. The wild-type
scion was cut from the base of the plant such that the base was
approximately the diameter of the "rootstock". Leaves and shoot tip
were removed and a piece of stem containing 2 nodes (each with an
axillary meristem) was cut into a wedge shape at the bottom end.
The scion was inserted into the "rootstock" notch and the junction
was secured with a small piece of parafilm. Plants were placed in a
mist tent to recover. After one week, all leaves from the
"rootstock" were removed. Once it became apparent that one or more
axillary meristems of the scion was growing out, the other was
removed.
[0632] The scion shoots were grown until the first flower was fully
extended, this date was considered the flowering date. The time
between grafting date and the flowering date is the days to
flowering. Once plants had flowered, architectural data was
collected from the scion. The shoot length and node number was
measured from the axil to the uppermost leaf base, this does not
include the original scion stem segment, only the shoot that grew
from the axillary meristem. The scion shoot diameter was measured
at the base of the shoot using an electronic calliper. Trunk
circumference area (TCA) was calculated with the formula:
(diameter/2).sup.2 and is given in mm.sup.2. The area of each leaf
was measured with an electronic leaf scanner, total leaf area is
the sum of all leaves on a plant and is given in cm.sup.2. The
scions were dried and weighed to determine dry weight (gm). Each
line was compared to WT/WT by one way-ANOVA to determine
significant differences.
[0633] Results
[0634] As ungrafted plants, 35S: M9 ARF3 line 6, hereafter referred
to as line 6, show the most extreme phenotype. 35S: M9 ARF3 line 2
(line 2) has the mildest phenotype and 35S:793 ARF3 line 4 is
undistinguishable from wild-type.
[0635] Relative to the WT/WT homografts, the WT scions on line 6
rootstocks were significantly shorter (FIG. 20). Scions on line 2
and line 4 had slightly shorter lengths, but these were not
significant.
[0636] Scions on all three transgenic rootstocks flowered slightly
earlier than the WT/WT (FIG. 21).
[0637] Line 6 had significantly fewer nodes than WT/WT (FIG.
20).
[0638] Scions on both line 2 and line 6 had a smaller TCA than
WT/WT. Line 6 was significantly different than WT/WT (FIG. 21).
[0639] Scions on line 2 and line 6 had a smaller dry weight than
WT/WT. Line 6 was significantly different than WT/WT (FIG. 22).
[0640] Although lines 2, 6 and 4 had less total leaf area, only
line 6 was significantly different from WT/WT (FIG. 23).
[0641] To our knowledge, there has been no report of dwarfing
rootstocks causing smaller leaf size in scions.
[0642] Seedling Root Measurements
[0643] 35S: M9 ARF3, 35S: wt ARF3 and wild-type tobacco seeds were
sterilized in 2% bleach for 30 minutes, rinsed in distilled H2O,
3.times., for 10 minutes each, then plated on MS media containing
Kanamycin (for the transgenic seeds) or just MS (wild-type). Two
weeks after plating, seedlings were removed from the media, excess
media was removed and seedlings were photographed on a grid using a
stereo microscope equipped with a digital camera. Primary and
lateral root length were measured from digital images using Image
J, total lateral root length is the sum of all lateral root
lengths. (see FIG. 25).
[0644] In terms of shoot length, node number, TCA, scion dry
weight, and scion mass, the effect of line 6 on the scion appears
to replicate the effect of the `M9` dwarfing rootstock.
[0645] Summary of Data Shown in Transgenic Plants, and Grafted
Scions.
[0646] The phenotypes shown in transgenic plants over-expressing M9
ARF1 or WT ARF1, and in WT plants grafted onto transgenic plants
over-expressing M9 ARF1 or WT ARF1, in comparison to the known
phenotypes in known root stock and dwarfed grafted scions are
summarised in the tables below.
TABLE-US-00005 TABLE 2 Phenotypes shown in transgenic plants
over-expressing M9 ARF1 or WT ARF1 Known dwarfing- associated
phenotypes Shown in plants Shown in plants found in dwarfing
over-expressing over-expressing rootstock plants M9 ARF1 WT ARF1
(previous data) (this study) (this study) bushier Yes No altered
xylem/phloem ratio Yes No more phloem elements Yes No reduced
apical dominance Yes No reduced root mass Yes Yes
TABLE-US-00006 TABLE 3 Phenotypes shown in WT plants grafted onto
transgenic plants over-expressing M9 ARF1 or WT ARF1 Known
dwarfing- Shown in WT "scions" Shown in WT "scions" associated
phenotypes grafted on to "root- grafted on to "root- found in
scions grafted stock" plants over- stock" plants over- onto
dwarfing rootstock expressing M9 ARF1 expressing M9 ARF1 plants
(previous data) (this study) (this study) reduced vigour Yes Yes
less vegetative growth Yes Yes earlier termination of Yes Yes shoot
growth smaller canopy Yes No reduced stem Yes No circumference
reduced scion mass Yes No
[0647] Materials and Methods
[0648] Plant Material
[0649] A rootstock population derived from crosses between
Malus.times.domestica `Malling9` (`M9`) and Malus robusta 5 (`R5`)
was used for QTL analysis. For the first population, 135 seedlings
were planted in 1998 and grown as stoolbeds to produce multiple
rooted stocks of each genotype. The rootstocks were cleft grafted
with `Braeburn` scions, grown in the nursery for two years, then
transplanted into the Plant & Food Research orchard (Havelock
North, New Zealand) as described by Pilcher et al. (Pilcher, Celton
et al. 2008) Replicates of the original 135 rootstocks were
propagated in 2000 and planted in the orchard as one-year-old
grafted trees. Of the replicated trees, 112 individuals from
replicate two, and 57 individuals from replicate three were
phenotyped for QTL analysis. The second population consisted of 350
seedlings, which were grafted as described above and planted in the
orchard as one-year-old trees in 2004. From the second population,
81 individuals were evaluated for the QTL analysis and 314 survived
until final phenotypic assessment in year seven. Trees were grown
with in-row spacing of 1.5 m between trees and a double wire
trellis as support, in a complete randomized block design. Scions
grafted onto `M9` and `R5` were planted throughout as controls.
Trees were not pruned, to allow full expression of the rootstock
effects on scion growth. Once trees began fruiting, chemical
thinning sprays were applied to avoid over-cropping and limb
breakage.
[0650] Forty-one (41) apple rootstock accessions (Malus spp.)
representing rootstock varieties used in major apple-growing
regions in the world were used for pedigree analysis of Dw1 and
Dw2.
[0651] Phenotypic Analysis
[0652] Rootstock effects on the development of `Braeburn` scions
were assessed using multiple methods, over seven years, within the
two populations. Table 1 presents the specific traits that were
assessed for the QTL analysis in each population/replicate and the
sample size phenotyped. Height, internode number, and average
internode length of the scion were recorded at the end of the first
year of growth after grafting (year one). Flowering was scored by
estimating the total number of flower clusters on each tree in the
spring of year two, and placing them into quartiles relative to the
most highly floral trees, i.e., 1-25% had the fewest flowers,
75-100% had the most flowers. Trees without any flowers in year two
were recorded as "0". Trunk Cross-sectional Area (TCA) was measured
20 cm above the graft junction at the end of each year from year
two to year seven. From year two to year seven, the overall vigour
of each tree was assessed annually by comparing trunk size, crown
height and spread, branch density and vigour. For the QTL analysis,
an overall dwarfing phenotype (DW %) was assigned in year seven,
with 100%=very vigorous, 80%=vigorous, 60%=intermediate,
40%=semi-dwarfed, and 20%=dwarfed.
[0653] The 41 rootstocks accessions used for the pedigree analysis
were classified according to their dwarfing effect in accordance
with the literature and in-house Plant & Food Research
professional expertise.
[0654] DNA Isolation and Genotyping of `M9`.times.`R5` Rootstock
Population and Rootstock Accessions
[0655] Total genomic DNA was extracted from leaves and quantified
according to Gardiner et al. (Gardiner, Bassett et al. 1996) Leaf
material was collected from 135 seedlings from the first
`M9`.times.`R5` population and 350 from the second population.
Leaves of the rootstock accessions were collected from the Plant
& Food Research germplasm collection in Havelock North, NZ, or
from the USDA-ARS collection in Geneva, N.Y., USA.
[0656] For Dw1 and Dw2 genotyping of the entire population of
`M9`.times.`R5` rootstocks, polymerase chain reaction (PCR)
products containing single nucleotide polymorphisms (SNP) were
amplified on a LightCycler480 instrument (Roche Diagnostics) and
screened using the High Resolution Melting (HRM) technique as
described by Chagne et al. (Chagne, Gasic et al. 2008)
Supplementary Table 1 lists the position of markers on the `Golden
Delicious` genome (Velasco, Zharkikh et al. 2010) and primer
sequence.
[0657] Markers detecting SSRs located on LG5 and LG11 were employed
to genotype the 41 rootstock accessions. Hi01c04, Hi04a08, CH03a09
and CH02d08 were developed by Silfverberg-Dilworth et al.
(Silfverberg-Dilworth, Matasci et al. 2006) and Liebhard et al.
(Liebhard, Gianfranceschi et al. 2002) Two new SSR markers
(MDP0000365711 and MDP00024370) located at the top of LG11 were
developed using the Plant & Food Research Malus genome database
(Newcomb, Crowhurst et al. 2006), with the programmes Sputnik and
Primer3. The M13 sequence TGTAAAACGACGGCCAGT was added to the 5'
end of the forward primer to enable the use of Schuelke's (Schuelke
2000) approach to fluorescent labelling. PCR reactions were
performed and analysed on an ABI 3500 Genetic Analyzer (Applied
Biosystems) as described by Hayden et al. (Hayden, Nguyen et al.
2008)
[0658] QTL Analysis
[0659] The parental genetic maps for `M9` and `R5` were constructed
using a total of 316 loci amplified from 296 primer pairs as
described in Celton et al. (Celton, Tustin et al. 2009) The maps
span a total of 1,175.7 and 1,086.7 cM for `M9` and `R5`
respectively. (Celton, Tustin et al. 2009) The linkage phase of the
markers was determined using JoinMap.RTM. 3.0 (Kyazma, NL). QTL
analysis was performed for all growth traits using MapQTL.RTM. 5
Software (Kyazma, NL). Traits evaluated over multiple years and
replicates were analysed separately. Interval mapping (IM),
followed by multiple QTL model (MQM) analysis using the best
markers obtained by IM as co-factors, was used for normally
quantitative traits. Only additive models were considered for the
QTL analysis. The threshold for QTL genome-wide significance was
calculated after 1,000 permutations. Kruskal-Wallis analysis was
used for ordinal traits such as the estimated number of flower
clusters and expert assessment of dwarfing.
[0660] RNA Purification
[0661] For RNA-seq, tissue was collected from the rootstock stem of
two M.793 and two M.9 individuals in November (60 DABB, .about.90
days after grafting). M.27 was not included in the RNA-seq
experiment because suitable material was not available. For qRT-PCR
expression analysis, 30 `Royal Gala` trees grafted onto M.9, M.27
and M.793 rootstocks were grown as previously described. Tissue was
collected for RNA purification in November, January, March and July
(60, 120, 180 and 300 DABB respectively). For each time point, four
to six trees of each genotype were selected for uniform scion
growth to minimize any effects due to differential tree size. RNA
was pooled from four shoots from each of the rootstock accessions
shown in FIG. 5. For all other experiments, RNA from each
individual was extracted and analysed separately. For all
collections, the outer bark was removed, vascular tissue was
scraped off with a scalpel, and snap frozen in liquid nitrogen.
Tissue was harvested between four and five hours after sunrise for
all time points. Total RNA was isolated and cDNA generated as
described in (Janssen et al. 2008). The quality and concentration
of the RNA samples was assessed with an RNA Nano kit (Agilent) and
only samples with a RIN value of 8 or higher were further analyzed
by sequencing or qRT-PCR.
[0662] RNA Sequencing and Data Processing
[0663] RNA was sent to Axeq/Macrogen for library preparation and
sequencing using an Illumina Hiseq 2000 instrument. Individual
samples were run as a multiplexed sample on one lane to produce 100
nucleotide paired end sequence reads. The first 13 bases of all
RNAseq reads were trimmed using an in-house perl script. Adapters
were removed using fastq-mcf from the ea-utils package (Aronesty
2011) using a minimum read retention length of 50 and a minimum
quality score threshold of 20. Quality score analysis was performed
using fastqc
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) both
before and after trimming. Trimmed reads were mapped to the
reference using bowtie2 (Langmead and Salzberg 2012) using the
following settings: -a--end_to_end--sensitive. SAM file to BAM file
conversion was undertaken using samtools (Li et al. 2009). Raw read
counts and reads per kilobase per million (RPKM) values were
extracted from BAM files using the multicov option of bedtools
(Quinlan and Hall 2010) and either an in-house R script or
cufflinks (Trapnell et al. 2010). Apple homologues of Arabidopsis
flowering genes were determined by BLASP value and tested by
reciprocal BLASTP. Differentially expressed genes were selected
using the Limma package (Smyth 2005) in BioConductor, genes were
selected using an adjusted P value of <0.05 and fold change
cutoff>6 (Smyth 2005).
[0664] Transformation of ARF3 into Plants
[0665] Primers were designed to amplify the MdARF3 gene, from from
100 bp upstream of the start codon to 50 bp 5' of the stop codon.
Single products were amplified from cDNA derived from `Royal Gala`
or `M9` meristem enriched tissue. These products were cloned into
an expression vector (pHEX), which uses the cauliflower mosaic
virus (CaMV) 35S promoter to drive expression and contains the
neomycin phoshotransferase II gene (NPTII) to confer kanamycin
resistance. Agrobacterium tumefaciens strain GV3-101 transformed
with either the `Royal Gala` ("wt") or the `M9` ARF3 was used to
transform leaf discs from N. tabacum (`Samsun`), petunia
(`Mitchell`) or apple transformation cell lines. Callus formation
and regeneration of plantlettes are as described in (Kotoda and
Wada 2005).
[0666] Histology
[0667] Stem and petiole sections were fixed overnight in FAA (3.7%
Formaldehyde, 50% EtOH, 5% Acetic Acid), processed and embedded in
paraffin as described in Ruzin (Ruzin 1999). Tissue was sectioned
to 10.quadrature.m on a rotary microtome, and slides were stained
using a safranin/fast green procedure to distinguish xylem from
phloem.
[0668] Grafting
[0669] Scions can be grafted onto rootstocks using cleft grafting
or chip-budding depending on the material (Stoltz and Strang 1982;
Webster and Wertheim 2003; Crasweller 2005).
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Sequence CWU 1
1
291708PRTMalus domesticamisc_feature(106)..(106)Xaa can be any
naturally occurring amino acidmisc_feature(359)..(359)Xaa can be
any naturally occurring amino acidmisc_feature(403)..(403)Xaa can
be any naturally occurring amino acidmisc_feature(405)..(405)Xaa
can be any naturally occurring amino
acidmisc_feature(412)..(412)Xaa can be any naturally occurring
amino acidmisc_feature(469)..(469)Xaa can be any naturally
occurring amino acidmisc_feature(540)..(540)Xaa can be any
naturally occurring amino acid 1Met Ala Gly Leu Ile Asp Leu Asn Ser
Ala Thr Glu Asp Glu Glu Thr 1 5 10 15 Pro Ser Ser Gly Ser Pro Ser
Ser Ala Ser Ser Val Ser Asp Ala Leu 20 25 30 Gly Ser Ser Ala Ser
Val Cys Met Glu Leu Trp His Ala Cys Ala Gly 35 40 45 Pro Leu Ile
Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr Leu Pro 50 55 60 Gln
Gly His Leu Glu Gln Val Ser Asp Phe Pro Thr Ser Ala Tyr Asp 65 70
75 80 Leu Pro Pro His Leu Phe Cys Arg Val Val Asp Val Lys Leu His
Ala 85 90 95 Glu Thr Gly Thr Asp Asp Val Phe Ala Xaa Val Ser Leu
Val Pro Glu 100 105 110 Ser Glu Glu Ile Glu His Arg Leu Arg Glu Gly
Val Thr Asp Ala Asp 115 120 125 Ala Glu Glu Asp Val Glu Ala Met Gly
Thr Ser Thr Thr Pro His Met 130 135 140 Phe Cys Lys Thr Leu Thr Ala
Ser Asp Thr Ser Thr His Gly Gly Phe 145 150 155 160 Ser Val Pro Arg
Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr 165 170 175 Thr Gln
Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu His Gly 180 185 190
Leu Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro Arg Arg His 195
200 205 Leu Leu Thr Thr Gly Trp Ser Ala Phe Val Asn Lys Lys Lys Leu
Val 210 215 220 Ser Gly Asp Ala Val Leu Phe Leu Arg Gly Asp Asp Gly
Glu Leu Arg 225 230 235 240 Leu Gly Ile Arg Arg Ala Ala Gln Phe Lys
Ser Ser Ala Thr Cys Pro 245 250 255 Thr Leu Cys Ser Gln Gln Leu Asn
Tyr Ser Thr Ile Thr Asp Val Val 260 265 270 Asn Ala Ile Phe Ala Lys
Asn Ala Phe Asn Val Tyr Tyr Asn Pro Arg 275 280 285 Ser Ser Ser Ser
Glu Phe Ile Ile Pro Ser His Lys Phe Leu Arg Ser 290 295 300 Leu Asp
His Cys Phe Cys Ala Gly Met Arg Ile Lys Met Arg Phe Glu 305 310 315
320 Thr Glu Asp Ala Ala Glu Arg Arg Tyr Thr Gly Leu Ile Thr Gly Ile
325 330 335 Ser Glu Leu Asp Pro Val Arg Trp Pro Gly Ser Lys Trp Arg
Cys Leu 340 345 350 Val Val Arg Trp Asp Asp Xaa Asp Thr Ser Lys His
Gly Arg Val Ser 355 360 365 Pro Trp Glu Val Glu Arg Ser Gly Ser Val
Ser Ser Ser His Thr Leu 370 375 380 Met Thr Thr Gly Leu Lys Arg Ser
Arg Ile Gly Leu Ser Ala Thr Lys 385 390 395 400 Pro Glu Xaa Pro Xaa
Pro Ser Met Ser Cys Asn Xaa Gly Ile Gly Thr 405 410 415 Ser Asp Phe
Gly Glu Ser Leu Arg Phe Gln Lys Val Leu Gln Gly Gln 420 425 430 Glu
Ile Ser Gly Phe Asp Thr Pro Phe Ser Gly Leu Gly Gly Leu Asn 435 440
445 Ser His Pro Ser Glu Ala Arg Arg Val Phe His Gly Ser Gly Gly Ser
450 455 460 Gly Ile Ala Ala Xaa Gly Asn Gly Leu Arg Gln Ser Leu Val
Asp Ser 465 470 475 480 Glu Ile Ala Ser Lys Gly Ile Gly Phe Gly Glu
Ser Phe Arg Phe His 485 490 495 Lys Val Leu Gln Gly Gln Glu Ile Phe
Pro Ser Ser Pro Tyr Gly Arg 500 505 510 Ala Pro Ala Ser Asn Glu Ala
His Glu Tyr Gly Gly Pro Gly Leu Tyr 515 520 525 Asp Gly Phe Gln Val
Pro Gly Phe Arg Asn Gly Xaa Ser Thr Met Met 530 535 540 Gln Ser Asn
Asn Thr Asn Val His Ser Ser Ala Pro Ser Val Gln Val 545 550 555 560
Ser Ser Pro Ser Ser Val Leu Met Phe Gln Gln Ala Met Asn Pro Val 565
570 575 Ala Glu Phe Asn Ser Val Tyr Asn Gly His Asn Gln Glu Asp His
Arg 580 585 590 Val Asn Arg Thr Pro His Val Leu Glu His Asp Gly Gly
Arg Gln Thr 595 600 605 Ser Ser Ser Phe Gly Glu Arg Asn Phe Ser Arg
Glu Asp Arg Gly Gly 610 615 620 Thr His Ser Tyr Asn Gln His Gly Ile
Ser Pro His Pro Val Ile Ser 625 630 635 640 Gln Ser Thr Ile Ser Gly
Ser Gln Asp Ser Val Ser Pro Ile Lys Gly 645 650 655 Ser Cys Arg Leu
Phe Gly Phe Ser Leu Ser Glu Asp Lys Cys Val Pro 660 665 670 Asp Gln
Glu Gly Asn Pro Asn Val Gly Val Gln Phe His Ser Lys Pro 675 680 685
Pro Leu Met Thr Ser Thr Val Gly Ile Thr Cys Thr Lys Val Ser Asn 690
695 700 Leu Phe Ala Ala 705 2708PRTMalus
domesticamisc_feature(106)..(106)Xaa can be any naturally occurring
amino acidmisc_feature(359)..(359)Xaa can be any naturally
occurring amino acidmisc_feature(403)..(403)Xaa can be any
naturally occurring amino acidmisc_feature(405)..(405)Xaa can be
any naturally occurring amino acidmisc_feature(412)..(412)Xaa can
be any naturally occurring amino acidmisc_feature(469)..(469)Xaa
can be any naturally occurring amino
acidmisc_feature(540)..(540)Xaa can be any naturally occurring
amino acid 2Met Ala Gly Leu Ile Asp Leu Asn Ser Ala Thr Glu Asp Glu
Glu Thr 1 5 10 15 Pro Ser Ser Gly Ser Pro Ser Ser Ala Ser Ser Val
Ser Asp Ala Leu 20 25 30 Gly Ser Ser Ala Ser Val Cys Met Glu Leu
Trp His Ala Cys Ala Gly 35 40 45 Pro Leu Ile Ser Leu Pro Lys Lys
Gly Ser Val Val Val Tyr Leu Pro 50 55 60 Gln Gly His Leu Glu Gln
Val Leu Asp Phe Pro Thr Ser Ala Tyr Asp 65 70 75 80 Leu Pro Pro His
Leu Phe Cys Arg Val Val Asp Val Lys Leu His Ala 85 90 95 Glu Thr
Gly Thr Asp Asp Val Phe Ala Xaa Val Ser Leu Val Pro Glu 100 105 110
Ser Glu Glu Ile Glu His Arg Leu Arg Glu Gly Val Thr Asp Ala Asp 115
120 125 Ala Glu Glu Asp Val Glu Ala Met Gly Thr Ser Thr Thr Pro His
Met 130 135 140 Phe Cys Lys Thr Leu Thr Ala Ser Asp Thr Ser Thr His
Gly Gly Phe 145 150 155 160 Ser Val Pro Arg Arg Ala Ala Glu Asp Cys
Phe Pro Pro Leu Asp Tyr 165 170 175 Thr Gln Gln Arg Pro Ser Gln Glu
Leu Val Ala Lys Asp Leu His Gly 180 185 190 Leu Glu Trp Arg Phe Arg
His Ile Tyr Arg Gly Gln Pro Arg Arg His 195 200 205 Leu Leu Thr Thr
Gly Trp Ser Ala Phe Val Asn Lys Lys Lys Leu Val 210 215 220 Ser Gly
Asp Ala Val Leu Phe Leu Arg Gly Asp Asp Gly Glu Leu Arg 225 230 235
240 Leu Gly Ile Arg Arg Ala Ala Gln Phe Lys Ser Ser Ala Thr Cys Pro
245 250 255 Thr Leu Cys Ser Gln Gln Leu Asn Tyr Ser Thr Ile Thr Asp
Val Val 260 265 270 Asn Ala Ile Phe Ala Lys Asn Ala Phe Asn Val Tyr
Tyr Asn Pro Arg 275 280 285 Ser Ser Ser Ser Glu Phe Ile Ile Pro Ser
His Lys Phe Leu Arg Ser 290 295 300 Leu Asp His Cys Phe Cys Ala Gly
Met Arg Ile Lys Met Arg Phe Glu 305 310 315 320 Thr Glu Asp Ala Ala
Glu Arg Arg Tyr Thr Gly Leu Ile Thr Gly Ile 325 330 335 Ser Glu Leu
Asp Pro Val Arg Trp Pro Gly Ser Lys Trp Arg Cys Leu 340 345 350 Val
Val Arg Trp Asp Asp Xaa Asp Thr Ser Lys His Gly Arg Val Ser 355 360
365 Pro Trp Glu Val Glu Arg Ser Gly Ser Val Ser Ser Ser His Thr Leu
370 375 380 Met Thr Thr Gly Leu Lys Arg Ser Arg Ile Gly Leu Ser Ala
Thr Lys 385 390 395 400 Pro Glu Xaa Pro Xaa Pro Ser Met Ser Cys Asn
Xaa Gly Ile Gly Thr 405 410 415 Ser Asp Phe Gly Glu Ser Leu Arg Phe
Gln Lys Val Leu Gln Gly Gln 420 425 430 Glu Ile Ser Gly Phe Asp Thr
Pro Phe Ser Gly Leu Gly Gly Leu Asn 435 440 445 Ser His Pro Ser Glu
Ala Arg Arg Val Phe His Gly Ser Gly Gly Ser 450 455 460 Gly Ile Ala
Ala Xaa Gly Asn Gly Leu Arg Gln Ser Leu Val Asp Ser 465 470 475 480
Glu Ile Ala Ser Lys Gly Ile Gly Phe Gly Glu Ser Phe Arg Phe His 485
490 495 Lys Val Leu Gln Gly Gln Glu Ile Phe Pro Ser Ser Pro Tyr Gly
Arg 500 505 510 Ala Pro Ala Ser Asn Glu Ala His Glu Tyr Gly Gly Pro
Gly Leu Tyr 515 520 525 Asp Gly Phe Gln Val Pro Gly Phe Arg Asn Gly
Xaa Ser Thr Met Met 530 535 540 Gln Ser Asn Asn Thr Asn Val His Ser
Ser Ala Pro Ser Val Gln Val 545 550 555 560 Ser Ser Pro Ser Ser Val
Leu Met Phe Gln Gln Ala Met Asn Pro Val 565 570 575 Ala Glu Phe Asn
Ser Val Tyr Asn Gly His Asn Gln Glu Asp His Arg 580 585 590 Val Asn
Arg Thr Pro His Val Leu Glu His Asp Gly Gly Arg Gln Thr 595 600 605
Ser Ser Ser Phe Gly Glu Arg Asn Phe Ser Arg Glu Asp Arg Gly Gly 610
615 620 Thr His Ser Tyr Asn Gln His Gly Ile Ser Pro His Pro Val Ile
Ser 625 630 635 640 Gln Ser Thr Ile Ser Gly Ser Gln Asp Ser Val Ser
Pro Ile Lys Gly 645 650 655 Ser Cys Arg Leu Phe Gly Phe Ser Leu Ser
Glu Asp Lys Cys Val Pro 660 665 670 Asp Gln Glu Gly Asn Pro Asn Val
Gly Val Gln Phe His Ser Lys Pro 675 680 685 Pro Leu Met Thr Ser Thr
Val Gly Ile Thr Cys Thr Lys Val Ser Asn 690 695 700 Leu Phe Ala Ala
705 3608PRTArabidopsis thaliana 3Met Gly Gly Leu Ile Asp Leu Asn
Val Met Glu Thr Glu Glu Asp Glu 1 5 10 15 Thr Gln Thr Gln Thr Pro
Ser Ser Ala Ser Gly Ser Val Ser Pro Thr 20 25 30 Ser Ser Ser Ser
Ala Ser Val Ser Val Val Ser Ser Asn Ser Ala Gly 35 40 45 Gly Gly
Val Cys Leu Glu Leu Trp His Ala Cys Ala Gly Pro Leu Ile 50 55 60
Ser Leu Pro Lys Arg Gly Ser Leu Val Leu Tyr Phe Pro Gln Gly His 65
70 75 80 Leu Glu Gln Ala Pro Asp Phe Ser Ala Ala Ile Tyr Gly Leu
Pro Pro 85 90 95 His Val Phe Cys Arg Ile Leu Asp Val Lys Leu His
Ala Glu Thr Thr 100 105 110 Thr Asp Glu Val Tyr Ala Gln Val Ser Leu
Leu Pro Glu Ser Glu Asp 115 120 125 Ile Glu Arg Lys Val Arg Glu Gly
Ile Ile Asp Val Asp Gly Gly Glu 130 135 140 Glu Asp Tyr Glu Val Leu
Lys Arg Ser Asn Thr Pro His Met Phe Cys 145 150 155 160 Lys Thr Leu
Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser Val 165 170 175 Pro
Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr Ser Gln 180 185
190 Pro Arg Pro Ser Gln Glu Leu Leu Ala Arg Asp Leu His Gly Leu Glu
195 200 205 Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro Arg Arg His
Leu Leu 210 215 220 Thr Thr Gly Trp Ser Ala Phe Val Asn Lys Lys Lys
Leu Val Ser Gly 225 230 235 240 Asp Ala Val Leu Phe Leu Arg Gly Asp
Asp Gly Lys Leu Arg Leu Gly 245 250 255 Val Arg Arg Ala Ser Gln Ile
Glu Gly Thr Ala Ala Leu Ser Ala Gln 260 265 270 Tyr Asn Gln Asn Met
Asn His Asn Asn Phe Ser Glu Val Ala His Ala 275 280 285 Ile Ser Thr
His Ser Val Phe Ser Ile Ser Tyr Asn Pro Lys Ala Ser 290 295 300 Trp
Ser Asn Phe Ile Ile Pro Ala Pro Lys Phe Leu Lys Val Val Asp 305 310
315 320 Tyr Pro Phe Cys Ile Gly Met Arg Phe Lys Ala Arg Val Glu Ser
Glu 325 330 335 Asp Ala Ser Glu Arg Arg Ser Pro Gly Ile Ile Ser Gly
Ile Ser Asp 340 345 350 Leu Asp Pro Ile Arg Trp Pro Gly Ser Lys Trp
Arg Cys Leu Leu Val 355 360 365 Arg Trp Asp Asp Ile Val Ala Asn Gly
His Gln Gln Arg Val Ser Pro 370 375 380 Trp Glu Ile Glu Pro Ser Gly
Ser Ile Ser Asn Ser Gly Ser Phe Val 385 390 395 400 Thr Thr Gly Pro
Lys Arg Ser Arg Ile Gly Phe Ser Ser Gly Lys Pro 405 410 415 Asp Ile
Pro Val Ser Glu Gly Ile Arg Ala Thr Asp Phe Glu Glu Ser 420 425 430
Leu Arg Phe Gln Arg Val Leu Gln Gly Gln Glu Ile Phe Pro Gly Phe 435
440 445 Ile Asn Thr Cys Ser Asp Gly Gly Ala Gly Ala Arg Arg Gly Arg
Phe 450 455 460 Lys Gly Thr Glu Phe Gly Asp Ser Tyr Gly Phe His Lys
Val Leu Gln 465 470 475 480 Gly Gln Glu Thr Val Pro Ala Tyr Ser Ile
Thr Asp His Arg Gln Gln 485 490 495 His Gly Leu Ser Gln Arg Asn Ile
Trp Cys Gly Pro Phe Gln Asn Phe 500 505 510 Ser Thr Arg Ile Leu Pro
Pro Ser Val Ser Ser Ser Pro Ser Ser Val 515 520 525 Leu Leu Thr Asn
Ser Asn Ser Pro Asn Gly Arg Leu Glu Asp His His 530 535 540 Gly Gly
Ser Gly Arg Cys Arg Leu Phe Gly Phe Pro Leu Thr Asp Glu 545 550 555
560 Thr Thr Ala Val Ala Ser Ala Thr Ala Val Pro Cys Val Glu Gly Asn
565 570 575 Ser Met Lys Gly Ala Ser Ala Val Gln Ser Asn His His His
Ser Gln 580 585 590 Gly Arg Asp Ile Tyr Ala Met Arg Asp Met Leu Leu
Asp Ile Ala Leu 595 600 605 4 730PRTPhaseolus vulgaris 4Met Val Gly
Ile Ile Asp Leu Asn Thr Thr Glu Glu Asp Glu Lys Thr 1 5 10 15 Thr
Pro Ser Ser Gly Ser Phe Ser Ser Pro Ser Ser Ser Ser Ser Thr 20 25
30 Ser Ala Ala Leu Ser Ala Thr Asn Leu Ser Ser Ala Pro Val Ser Gly
35 40 45 Ser Val Cys Leu Glu Leu Trp His Ala Cys Ala Gly Pro Leu
Ile Ser 50 55 60 Leu Pro Lys Lys Gly Ser Val Val Val Tyr Phe Pro
Gln Gly His Leu 65 70 75 80 Glu Gln Leu Pro Asp Leu Pro Leu Ala Val
Tyr Asp Leu Pro Ser Tyr 85 90 95 Ile Phe Cys Arg Val Val Asp Val
Lys Leu His Ala Glu Thr Ala Asn 100 105 110 Asp Glu Val Tyr Ala Gln
Val Ser Leu Val Pro Asp Ser Glu Gln Ile 115 120 125 Glu Gln Lys Leu
Lys Gln Gly Lys Leu Glu Gly His Cys Glu Glu Glu 130 135 140
Asp Val Glu Ala Val Val Lys Ser Thr Thr Thr His Met Phe Cys Lys 145
150 155 160 Thr Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser
Val Pro 165 170 175 Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp
Tyr Ser Gln Gln 180 185 190 Arg Pro Ser Gln Glu Leu Val Ala Lys Asp
Leu His Gly Phe Glu Trp 195 200 205 Lys Phe Arg His Ile Tyr Arg Gly
Gln Pro Arg Arg His Leu Leu Thr 210 215 220 Thr Gly Trp Ser Ala Phe
Val Asn Lys Lys Lys Leu Val Ser Gly Asp 225 230 235 240 Ala Val Leu
Phe Leu Arg Gly Asp Asp Gly Glu Leu Arg Leu Gly Ile 245 250 255 Arg
Arg Ala Ala Gln Val Lys Cys Gly Ala Ser Phe Pro Ala Leu Cys 260 265
270 Ser Gln Gln Leu Asn Gln Ser Thr Leu Thr Asp Val Val His Ala Met
275 280 285 Ser Met Arg Ser Leu Phe Asn Ile Cys Tyr Asn Pro Arg Ala
Ser Ser 290 295 300 Ser Glu Phe Ile Ile Pro Leu His Lys Phe Leu Lys
Ser Leu Asp Tyr 305 310 315 320 Ser Phe Ser Val Gly Met Arg Phe Lys
Met Arg Phe Glu Thr Glu Asp 325 330 335 Ala Ala Glu Arg Arg Tyr Met
Gly Leu Ile Thr Gly Ile Ser Asp Leu 340 345 350 Asp Pro Ala Arg Trp
Pro Gly Ser Lys Trp Arg Cys Leu Val Val Arg 355 360 365 Trp Asp Asp
Met Glu Thr Asn Arg His Ser Arg Val Ser Pro Trp Glu 370 375 380 Ile
Glu Pro Ser Gly Ser Val Ser Ser Cys Asn Ser Phe Met Thr Pro 385 390
395 400 Gly Leu Lys Arg Ser Arg Ser Gly Phe Pro Ser Ser Lys Pro Glu
Phe 405 410 415 Pro Val Pro Asp Gly Ile Gly Ala Ser Asp Phe Gly Glu
Pro Ser Arg 420 425 430 Phe Gln Lys Val Leu Gln Gly Gln Glu Ile Leu
Asn Phe Asn Thr Leu 435 440 445 Tyr Asp Gly Val Asp Gln Asn Arg His
Pro Ser Asp Ile Arg Arg Cys 450 455 460 Phe Pro Gly Ser Arg Ser Ser
Met Ile Ala Thr Thr Arg Asn Gly Ala 465 470 475 480 Arg Asp Pro Val
Val Asn Ser Asp Val Ser Tyr Lys Ser Ile Gly Phe 485 490 495 Ser Glu
Ser Leu Arg Phe His Lys Val Leu Gln Gly Gln Glu Ile Ile 500 505 510
Pro Ser Ser Pro Phe Gly Arg Ala Pro Ala Ser Thr Asn Glu Ala Cys 515
520 525 Glu Asn Gly Cys Phe Gly Ile Ser Asp Gly Val Gln Met Thr Ser
Ser 530 535 540 Arg Asn Gly Trp Ser Ser Met Met Gln Gly Tyr Asn Thr
Arg Ile Arg 545 550 555 560 Pro Pro Ala Gln Val Ser Ser Pro Cys Ser
Val Leu Met Phe Gln Gln 565 570 575 Ala Ser Asn Gln Val Ser Asn Pro
Ser Pro Arg Tyr Gly Phe Asn Asp 580 585 590 Leu Glu Glu Gln Gly Val
Asn Thr Gln Ser Trp Phe His Asn Pro Glu 595 600 605 Thr Cys Gly Glu
Lys Arg Met Ser Ser Ser Arg Ser Glu His Ile Phe 610 615 620 Arg Arg
Asn Asn Gln Trp Gly Met Asp Ser Phe Ser Leu Ser His Glu 625 630 635
640 His Ser Gln His Gly Leu Leu Gln Pro Leu Val Ala Gln Pro Pro Cys
645 650 655 Lys Gly Gly Gln Asp Leu Val Ser Ser Cys Lys Ser Ser Cys
Arg Leu 660 665 670 Phe Gly Phe Gln Leu Thr Glu Asp Arg His Val Ala
Asn Lys Asp Asp 675 680 685 Ser Ser Ile Pro Met Ala Ser Leu Asn Ala
Gly Ser Phe Met Pro His 690 695 700 Ala Gly Glu Gln Phe His Leu Lys
Pro Pro Ala Ile Thr Asn Ala Val 705 710 715 720 Gly Ser Ser Cys Thr
Lys Val Ser Val Leu 725 730 5747PRTSolanum lycopersicum 5Met Met
Cys Gly Leu Ile Asp Leu Asn Thr Val Asp Asn Asp Asp Ala 1 5 10 15
Gly Glu Glu Thr Thr Ala Pro Val Ser Leu Asp Ser Pro Ala Ser Ser 20
25 30 Ser Ala Ala Ser Gly Ser Ser Asp Leu Thr Ser Ser Thr Thr Pro
Ala 35 40 45 Val Ala Ser Val Cys Met Glu Leu Trp His Ala Cys Ala
Gly Pro Leu 50 55 60 Ile Ser Leu Pro Lys Lys Gly Ser Ala Val Val
Tyr Leu Pro Gln Gly 65 70 75 80 His Leu Glu His Leu Ser Glu Tyr Pro
Ser Ile Ala Cys Asn Leu Pro 85 90 95 Pro His Val Phe Cys Arg Val
Val Asp Val Lys Leu Gln Ala Asp Ala 100 105 110 Ala Thr Asp Glu Val
Tyr Ala Gln Val Ser Leu Val Pro Asp Asn Gln 115 120 125 Gln Ile Glu
Gln Lys Trp Lys Asp Gly Asp Ile Asp Ala Asp Ile Glu 130 135 140 Glu
Glu Glu Ile Glu Gly Ala Gly Lys Ser Ile Thr Pro His Met Phe 145 150
155 160 Cys Lys Thr Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe
Ser 165 170 175 Val Pro Arg Arg Ala Ala Glu Asp Cys Phe Ala Pro Leu
Asp Tyr Arg 180 185 190 Gln Gln Arg Pro Ser Gln Glu Leu Val Ala Lys
Asp Leu His Gly Ile 195 200 205 Glu Trp Lys Phe Arg His Ile Tyr Arg
Gly Gln Pro Arg Arg His Leu 210 215 220 Leu Thr Thr Gly Trp Ser Ala
Phe Val Asn Lys Lys Lys Leu Val Ser 225 230 235 240 Gly Asp Ala Val
Leu Phe Leu Arg Thr Gly Asp Gly Glu Leu Arg Leu 245 250 255 Gly Val
Arg Arg Ala Ala Gln Ala Lys Thr Cys Ser Ser Tyr Leu Ala 260 265 270
Pro Cys Ser Lys Pro Leu Asn Val Ser Gly Ile Val Asp Ala Val Asn 275
280 285 Val Ile Ser Ser Arg Asn Ala Phe Asn Ile Cys Tyr Asn Pro Arg
Asp 290 295 300 Ser Ser Ser Asp Phe Ile Val Pro Tyr His Lys Phe Ser
Lys Thr Leu 305 310 315 320 Ala His Pro Phe Ser Ala Gly Met Arg Phe
Lys Met Arg Val Glu Thr 325 330 335 Glu Asp Ala Ala Glu Gln Arg Phe
Thr Gly Leu Val Val Gly Val Ser 340 345 350 Asn Val Asp Pro Val Arg
Trp Pro Gly Ser Lys Trp Arg Cys Leu Leu 355 360 365 Val Arg Trp Asp
Asp Leu Asp Val Ser Arg His Asn Arg Val Ser Pro 370 375 380 Trp Glu
Ile Glu Pro Ser Gly Ser Ala Pro Val Pro Ser Ser Leu Val 385 390 395
400 Met Pro Ser Ala Lys Arg Thr Arg Val Gly Phe Pro Ile Ser Lys Ala
405 410 415 Asp Phe Pro Ile Pro Arg Glu Gly Ile Ala Val Ser Asp Phe
Gly Glu 420 425 430 Pro Ser Arg Phe Gln Lys Val Leu Gln Gly Gln Glu
Ile Leu Arg Met 435 440 445 His Ala Pro Tyr Gly Gly Leu Asp Ala Arg
Ser Pro Arg Pro Ala Gly 450 455 460 Thr Arg Cys Phe Pro Gly Phe Pro
Ser Ser Gly Ile Ser Arg Met Gly 465 470 475 480 Asn Ser Ile Arg Pro
Leu Phe Gly Asp Thr Asp Lys Ser His Glu Ser 485 490 495 Ile Gly Phe
Ser Glu Ser Leu Arg Phe Asn Lys Val Leu Gln Gly Gln 500 505 510 Glu
Ile Phe Thr Ser Pro Pro Tyr Gly Arg Ala Gln Ala Gly Ile Gln 515 520
525 Met Gln Glu Lys Ser Arg Thr Gly Ile Phe Val Gly Ile Gln Val Pro
530 535 540 Asn His Gly Asn Arg Trp Pro Ala Pro Asn Gln Asp Asn Asn
Thr Pro 545 550 555 560 Cys Lys Pro Ile Asn Pro Val Ser Ala Ser Ser
Pro Pro Ser Ala Leu 565 570 575 Asn Phe Gln His Pro Ser Pro Pro Ala
Ser Lys Phe Gln Ala Met Phe 580 585 590 Asn His Lys His Asp Leu Val
Asn Gln Ala Ser Leu Asp Leu Ser Glu 595 600 605 Asn Cys Cys Arg Tyr
Pro Tyr Leu Ser Ser Gly Ser His Thr Glu Asp 610 615 620 Ile Ser Gln
Lys Glu Gly Thr Gln Gly Ile Ser Ser Phe Gly Phe Leu 625 630 635 640
Lys Glu Gln Lys Gln Thr Gly Leu Ser Tyr Leu Ser Pro Gly Thr Gln 645
650 655 Ser Ser Phe Lys Gly Asn Gln Asn Leu Val Ser Thr Cys Lys Thr
Gly 660 665 670 Cys Arg Ile Phe Gly Phe Pro Leu Thr Glu Ser Lys Ile
Ser Ala Thr 675 680 685 Arg Ala Asp Thr Pro Ser Glu Ala Val Tyr Ser
His Gly Leu Glu Thr 690 695 700 Thr Phe Leu Pro Ser Ser Asp Gly Lys
Leu Gln Pro Gly Pro Pro Leu 705 710 715 720 Met Thr Asn Val Val Gly
Thr Asn Phe Thr Lys Val Asn Asp Leu Tyr 725 730 735 Ala Ala Arg Asp
Val Leu Leu Asp Ile Ala Leu 740 745 6 743PRTCitrus clemantina 6Met
Val Gly Leu Ile Asp Leu Asn Thr Thr Glu Asp Asp Glu Asn Pro 1 5 10
15 Ser Ser Gly Ser Leu Ser Pro Ser Ser Ser Ser Ala Ser Ala Leu Ser
20 25 30 Ala Ser Gly Phe Ala Leu Ala Pro Ala Ser Ala Ser Ala Ser
Gly Val 35 40 45 Ser Leu Glu Leu Trp His Ala Cys Ala Gly Pro Leu
Ile Ser Leu Pro 50 55 60 Lys Arg Gly Ser Val Val Val Tyr Phe Pro
Gln Gly His Leu Glu His 65 70 75 80 Val Ser Asp Phe Ser Ala Ala Ala
Ser Ala Ala Tyr Asp Leu Pro Pro 85 90 95 His Leu Phe Cys Arg Val
Ala Asp Val Lys Leu His Ala Glu Ala Ala 100 105 110 Ser Asp Glu Val
Tyr Ala Gln Val Ser Leu Val Pro Asp Glu Leu Ile 115 120 125 Glu Gln
Lys Val Arg Glu Gly Lys Ile Glu Glu Asp Gly Asp Glu Glu 130 135 140
Ser Val Glu Val Val Ala Lys Ser Ser Thr Pro His Met Phe Cys Lys 145
150 155 160 Thr Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser
Val Pro 165 170 175 Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp
Tyr Ser Gln Gln 180 185 190 Arg Pro Ser Gln Glu Leu Val Ala Lys Asp
Leu His Gly Leu Glu Trp 195 200 205 Arg Phe Arg His Ile Tyr Arg Gly
Gln Pro Arg Arg His Leu Leu Thr 210 215 220 Thr Gly Trp Ser Ala Phe
Val Asn Lys Lys Lys Leu Val Ser Gly Asp 225 230 235 240 Ala Val Leu
Phe Leu Arg Gly Glu Asp Gly Glu Leu Arg Leu Gly Ile 245 250 255 Arg
Arg Ala Pro His Val Lys Ser Gly Ala Thr Phe Pro Ser Phe Cys 260 265
270 Ser Gln Gln Ser Ser Pro Asn Ser Val Thr Glu Val Val Asp Ala Ile
275 280 285 Ala Arg Lys Arg Ala Phe Ser Ile Ser Tyr Asn Pro Arg Ala
Ser Ala 290 295 300 Ser Glu Phe Ile Ile Pro Val Asn Lys Phe Leu Lys
Ser Leu Gly His 305 310 315 320 Ser Phe Ala Val Gly Met Arg Phe Lys
Met Arg Phe Glu Thr Glu Asp 325 330 335 Ala Ala Glu Arg Arg Tyr Thr
Gly Val Ile Met Gly Val Gly Asp Met 340 345 350 Asp Pro Val Arg Trp
Pro Gly Ser Lys Trp Arg Cys Leu Leu Val Arg 355 360 365 Trp Asp Asp
Val Glu Ser Asn Arg His Thr Arg Val Ser Pro Trp Glu 370 375 380 Ile
Glu Pro Ser Gly Ser Val Cys Gly Ser Asn Asn Leu Ile Thr Ser 385 390
395 400 Gly Leu Lys Arg Thr Arg Ile Gly Leu Pro Ser Gly Lys Pro Glu
Phe 405 410 415 Pro Val Pro Asp Gly Ile Gly Val Thr Asp Phe Gly Glu
Ser Leu Arg 420 425 430 Phe Gln Lys Val Leu Gln Gly Gln Glu Ile Leu
Gly Phe Asn Thr Leu 435 440 445 Tyr Asp Gly Gly Asp Cys Gln Asn Leu
His Pro Ser Glu Val Arg Arg 450 455 460 Gly Ile Pro Gly Ser Asn Gly
Ser Gly Ile Ala Ala Ile Gly Asp Gly 465 470 475 480 Ser Arg Asn Leu
Gln Val Lys Ser Asp Ile Ser Tyr Lys Gly Ile Gly 485 490 495 Ile Gly
Phe Gly Glu Ser Phe Arg Phe His Lys Val Leu Gln Gly Gln 500 505 510
Glu Ile Phe Pro Lys Ser Pro Tyr Gly Arg Ala Pro Thr Asn Asn Glu 515
520 525 Ala Arg Ser Ile Gly Ser Leu Gly Ile Ser Asp Gly Val Pro Val
Ser 530 535 540 Gly Ser Arg Asn Arg Trp Ser Ala Val Val Pro Gly Tyr
Asn Thr His 545 550 555 560 Met Ser Pro Ser Ala Pro Pro Val Gln Val
Ser Ser Pro Ser Ser Val 565 570 575 Leu Met Phe Gln Leu Ala Ser Asn
Pro Ile Ser Asn Tyr Asn Pro Pro 580 585 590 Tyr Ser Leu Asn Asp Gln
Glu Lys Glu Gln Arg Val Asn Cys Gln Ser 595 600 605 Phe Phe His Asn
Ser Glu Ile Tyr Gly Gly Lys His Ala Ser Ser Ser 610 615 620 Phe Leu
Asp His Ser Phe Val Gly Gly Asp Gln Glu Val Met Asp Ser 625 630 635
640 Ile Gly Gln Ser Asn Glu His Ile Ser Pro Pro Leu Val Gly Gln Pro
645 650 655 Thr Val Arg Gly Ser Gln Asp Leu Val Ser Ser Cys Lys Gly
Ser Cys 660 665 670 Arg Leu Phe Gly Phe Ser Leu Thr Glu Glu Arg His
Val Ala Asn Ile 675 680 685 Glu Asp Asn Ala Ala Pro Val Ala Ser Pro
Leu Asn Pro Arg Ser Ser 690 695 700 Phe Leu Ser His Val Gly Gln Gln
Phe His Pro Lys Pro Pro Val Met 705 710 715 720 Ser Lys Ala Thr Gly
Ser Asn Cys Thr Asn Gly Ile Met Gln His Cys 725 730 735 Leu Gly Asn
Tyr Asp Ile Tyr 740 7 720PRTFragaria vesca 7Met Ala Gly Leu Ile Asp
Leu Asn Ser Thr Thr Glu Glu Glu Glu Glu 1 5 10 15 Thr Pro Ser Ser
Gly Ser Ser Ser Asn Ser Ser Gly Ser Asn Gly Leu 20 25 30 Ile Ser
Gly Ser Val Cys Leu Glu Leu Trp His Ala Cys Ala Gly Pro 35 40 45
Leu Ile Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr Leu Pro Gln 50
55 60 Gly His Leu Glu Gln Val Ser Asp Phe Pro Ala Ser Val Tyr Asp
Leu 65 70 75 80 Pro Ala His Leu Phe Cys Arg Val Leu Asp Val Lys Leu
His Ala Glu 85 90 95 Ser Gly Ser Asp Glu Val Tyr Ala Gln Val Gln
Leu Val Pro Glu Ser 100 105 110 Glu Glu Phe Glu His Lys Leu Gly Glu
Arg Glu Thr Val Ala Asp Gly 115 120 125 Asp Glu Asp Ala Glu Gly Ser
Glu Lys Ser Thr Thr Pro His Met Phe 130 135 140 Cys Lys Thr Leu Thr
Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser 145 150 155 160 Val Pro
Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr Ser 165 170 175
Gln Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu His Gly Leu 180
185 190 Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro Arg Arg His
Leu 195 200 205 Leu Thr Thr Gly Trp Ser Ala Phe Val Asn Lys Lys Lys
Leu Val Ser 210
215 220 Gly Asp Ala Val Leu Phe Leu Arg Gly Glu Asp Gly Glu Leu Arg
Leu 225 230 235 240 Gly Val Arg Arg Ala Ala Gln Val Lys Ala Ser Ala
Thr Tyr Pro Thr 245 250 255 Pro Gly Ser Gln His Leu Asn Tyr Asn Ser
Val Thr Glu Leu Val Asp 260 265 270 Ala Ile Ser Thr Lys Thr Ala Phe
Asn Ala Tyr Tyr Asn Pro Arg Ala 275 280 285 Ser Ser Ser Glu Phe Ile
Ile Pro Phe Arg Lys Phe Leu Arg Ser Leu 290 295 300 Gly His Ser Phe
Cys Ala Gly Met Arg Phe Lys Met Arg Phe Glu Thr 305 310 315 320 Glu
Asp Ala Ala Glu Gln Arg Tyr Thr Gly Leu Val Thr Gly Ile Ser 325 330
335 Glu Leu Asp Pro Leu Arg Trp Pro Gly Ser Lys Trp Lys Cys Val Ala
340 345 350 Val Arg Trp Asp Asp Ile Asp Thr Ser Lys Gln His Gly Arg
Val Ser 355 360 365 Pro Trp Glu Ile Glu Pro Ser Gly Ser Ile Ser Asn
Ser Ser Gly Leu 370 375 380 Met Ala Ser Gly Leu Lys Arg Ser Arg Met
Gly Leu Ser Ala Glu Lys 385 390 395 400 Gln Glu Phe Pro Val Pro His
Gly Ile Gly Ala Ser Asp Phe Gly Glu 405 410 415 Ser Leu Arg Phe Gln
Lys Val Leu Gln Gly Gln Glu Val Ser Gly Phe 420 425 430 Asp Thr Pro
Phe Gly Ser Ile Gly Gly Gln Asn Gln His Pro Ser Glu 435 440 445 Ser
Arg Arg Val Phe His Gly Ser Ile Gly Ser Arg Gly Asn Asp Leu 450 455
460 Arg Asn Ser Phe Val Asn Ser Glu Ile Ala Ser Lys Gly Phe Gly Glu
465 470 475 480 Ser Phe Arg Phe Gln Lys Val Leu Gln Gly Gln Glu Ile
Phe Pro Ser 485 490 495 Thr Pro Tyr Gly Arg Ala Pro Ala Thr Asn Glu
Ala Arg Glu Tyr Gly 500 505 510 Cys Pro Gly Ile Phe Asp Gly Phe Gln
Val Pro Ser Phe Arg Asn Gly 515 520 525 Trp Ser Thr Met Met Gln Gly
Ser Asn Thr Pro Met His Arg Ala Ala 530 535 540 Pro Val Gln Val Ser
Ser Pro Ser Ser Val Leu Met Phe Gln Gln Ala 545 550 555 560 Ile Asn
Ala Gly Ala Glu Phe Asn Ser Val Tyr Asn Gly His Asn Gln 565 570 575
Gln Glu Gln Arg Ile Met Gln Arg Thr His Ser Glu Ser Asp Gly Gly 580
585 590 Lys Gln Thr Ser Ala Ser Phe Cys Glu Arg Ser Phe Thr Arg Glu
Gly 595 600 605 His Gly Gly Met Asn Ser Phe Asp Gln His Gly Ile Ser
His Pro Pro 610 615 620 Leu Leu Ser Gln Ser Ser Leu Arg Gly Ser Gln
Asp Met Val Ser Ser 625 630 635 640 Cys Lys Ser Ser Cys Arg Leu Phe
Gly Phe Ser Leu Ser Glu Glu Thr 645 650 655 His Ala Pro Asn Lys Val
Asp Asn Ser Thr Ser Val Thr Ser Ala Leu 660 665 670 Glu Ser Gly Ala
Ser Met Phe Pro Asn Val Glu Pro Arg Phe His Ser 675 680 685 Lys Pro
Pro Ser Met Ser Ala Ala Val Gly Ile Pro Cys Thr Lys Glu 690 695 700
Trp Ala Phe Asn Trp Arg Gly Glu Arg Met Glu Ser Cys Leu Gln Gly 705
710 715 720 8722PRTPrunus persica 8Met Gly Gly Leu Ile Asp Leu Asn
Ser Ala Thr Glu Asp Glu Glu Thr 1 5 10 15 Pro Ser Ser Gly Ser Ser
Ser Thr Ser Ser Ala Ser Asp Ala Ser Ala 20 25 30 Ser Ala Ser Ala
Ser Val Cys Leu Glu Leu Trp His Ala Cys Ala Gly 35 40 45 Pro Leu
Ile Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr Leu Pro 50 55 60
Gln Gly His Leu Glu Gln Val Ser Asp Phe Pro Ala Ser Ala Tyr Asn 65
70 75 80 Leu Pro Pro His Leu Phe Cys Arg Val Val Asp Val Lys Leu
His Ala 85 90 95 Glu Thr Gly Thr Asp Asp Val Tyr Ala Gln Val Ser
Leu Val Pro Glu 100 105 110 Ser Glu Glu Ile Glu His Lys Leu Arg Glu
Gly Glu Thr Asp Ala Tyr 115 120 125 Gly Glu Glu Glu Asp Val Glu Ala
Ile Gly Lys Ser Thr Thr Pro His 130 135 140 Met Phe Cys Lys Thr Leu
Thr Ala Ser Asp Thr Ser Thr His Gly Gly 145 150 155 160 Phe Ser Val
Pro Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp 165 170 175 Tyr
Asn Gln Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu His 180 185
190 Gly Leu Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro Arg Arg
195 200 205 His Leu Leu Thr Thr Gly Trp Ser Ala Phe Val Asn Lys Lys
Lys Leu 210 215 220 Val Ser Gly Asp Ala Val Leu Phe Leu Arg Gly Asp
Asp Gly Glu Leu 225 230 235 240 Arg Leu Gly Ile Arg Arg Ala Ala Gln
Val Lys Gly Ser Ala Thr Tyr 245 250 255 Pro Thr Leu Cys Ser Gln Gln
Leu Asn Tyr Asn Thr Ile Thr Asp Val 260 265 270 Val Asn Ala Ile Ser
Met Lys Asn Ala Phe Asn Ile Phe Tyr Asn Pro 275 280 285 Arg Ala Ser
Ser Ser Glu Phe Ile Ile Pro Ser Arg Lys Phe Leu Arg 290 295 300 Ser
Leu Asp His Ser Phe Ser Pro Gly Met Arg Phe Lys Met Arg Phe 305 310
315 320 Glu Thr Glu Asp Ala Ala Glu Arg Arg Tyr Thr Gly Leu Ile Thr
Gly 325 330 335 Ile Ser Glu Leu Asp Pro Val Arg Trp Pro Gly Ser Lys
Trp Arg Cys 340 345 350 Leu Val Val Arg Trp Asp Asp Ile Asp Thr Ser
Lys His Gly Arg Val 355 360 365 Ser Pro Trp Glu Ile Glu Pro Ser Gly
Ser Val Ser Ser Ser His Ser 370 375 380 Leu Met Ala Ala Gly Leu Lys
Arg Ala Arg Ser Gly Leu Ser Ala Ala 385 390 395 400 Lys Thr Glu Phe
Pro Val Pro Asn Gly Ile Gly Ala Ser Asp Phe Gly 405 410 415 Glu Ser
Leu Arg Phe Gln Lys Val Leu Gln Gly Gln Glu Ile Leu Gly 420 425 430
Phe Asp Thr His Phe Gly Gly Leu Gly Gly Gln Asn Gln His Pro Ser 435
440 445 Glu Pro Arg Arg Gly Phe His Gly Ser Ser Gly Ser Gly Ile Ala
Ala 450 455 460 Gly Gly Asn Gly Leu Arg Lys Ser Leu Ala His Ser Glu
Ile Thr Ser 465 470 475 480 Thr Gly Ile Gly Phe Gly Glu Ser Phe Arg
Phe His Lys Val Leu Gln 485 490 495 Gly Gln Glu Ile Phe Pro Ser Pro
Pro Tyr Gly Arg Ala Ser Thr Asn 500 505 510 Asn Glu Ala His Glu Tyr
Gly Gly Pro Gly Ile Tyr Asp Gly Phe Gln 515 520 525 Val Pro Ser Phe
Arg Asn Gly Trp Pro Ala Met Met Gln Ser Asn Asn 530 535 540 Ala His
Val Arg Pro Ser Ala Ser Ser Val Gln Val Ser Ser Pro Ser 545 550 555
560 Ser Val Leu Met Phe Gln Gln Ala Met Asn Pro Gly Pro Glu Phe Asn
565 570 575 Ser Val Tyr Asn Gly His Asn Gln Glu Glu Gln Arg Val Ile
Lys Arg 580 585 590 Thr Pro Tyr Val Ser Glu Ser Asp Gly Gly Lys Gln
Ala Ser Ser Ser 595 600 605 Phe Cys Glu Arg Ser Phe Ser Arg Glu Asp
His Gly Gly Met Asn Ser 610 615 620 Tyr Asn Gln His Gly Ile Ser Asn
His Pro Val Ile Ser Gln Ser Thr 625 630 635 640 Phe Ser Gly Ser Gln
Asp Ala Val Ser Pro Tyr Lys Gly Ser Cys Arg 645 650 655 Leu Phe Gly
Phe Ser Leu Ser Glu Glu Lys Arg Val Pro Asp Arg Glu 660 665 670 Ser
Asn Ser Thr Ser Thr Ala Ser Thr Leu Asn Pro Gly Val Gln Phe 675 680
685 His Ser Lys Pro Ala Leu Met Thr Ser Ala Val Gly Ile Thr Cys Thr
690 695 700 Lys Glu Trp Ala Phe Asp Trp Arg Gly Glu Arg Met Glu Ser
Cys Leu 705 710 715 720 Gln Gly 9704PRTPyrus communis 9Met Ala Gly
Leu Ile Asp Leu Asn Ser Ala Thr Glu Asp Glu Gln Thr 1 5 10 15 Pro
Ser Ser Gly Ser Pro Ser Ser Ala Ser Ser Val Ser Asp Ala Leu 20 25
30 Gly Ser Ser Ala Ser Val Cys Met Glu Leu Trp His Ala Cys Ala Gly
35 40 45 Pro Leu Ile Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr
Leu Pro 50 55 60 Gln Gly His Leu Glu Gln Val Ser Asp Phe Pro Thr
Ser Ala Tyr Asp 65 70 75 80 Leu Pro Pro His Leu Phe Cys Arg Val Val
Asp Val Lys Leu His Ala 85 90 95 Glu Thr Gly Thr Asp Asp Val Phe
Ala Arg Val Ser Leu Val Pro Glu 100 105 110 Ser Glu Glu Ile Glu His
Arg Leu Arg Glu Gly Glu Thr Asp Ala Asp 115 120 125 Ala Glu Asp Asp
Val Glu Ala Met Gly Thr Ser Ala Thr Pro His Met 130 135 140 Phe Cys
Lys Thr Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe 145 150 155
160 Ser Val Pro Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr
165 170 175 Thr Gln Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu
His Gly 180 185 190 Leu Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln
Pro Arg Arg His 195 200 205 Leu Leu Thr Thr Gly Trp Ser Ala Phe Val
Asn Lys Lys Lys Leu Val 210 215 220 Ser Gly Asp Ala Val Leu Phe Leu
Arg Gly Asp Asp Gly Glu Leu Arg 225 230 235 240 Leu Gly Ile Arg Arg
Ala Ala Gln Phe Lys Ser Ser Ala Thr Cys Pro 245 250 255 Thr Leu Cys
Ser Gln Gln Leu Asn Cys Ser Ala Ile Thr Asp Val Leu 260 265 270 Asn
Ala Ile Phe Ala Lys Asn Ala Phe Asn Val Tyr Tyr Asn Pro Arg 275 280
285 Ser Ser Ser Ser Glu Phe Ile Ile Pro Ser His Lys Phe Leu Arg Ser
290 295 300 Leu Asp His Cys Phe Ser Ala Gly Met Arg Ile Lys Met Arg
Phe Glu 305 310 315 320 Thr Glu Asp Ala Ala Glu Arg Arg Tyr Ile Gly
Phe Ile Thr Arg Ile 325 330 335 Ser Glu Leu Asp Pro Val Arg Trp Pro
Gly Ser Lys Trp Arg Cys Leu 340 345 350 Val Val Arg Trp Asp Asp Ile
Asp Thr Ser Lys His Ser Arg Val Ser 355 360 365 Pro Trp Glu Val Glu
Pro Ser Gly Ser Val Ser Ser Ser His Thr Leu 370 375 380 Met Ala Thr
Gly Leu Lys Arg Ser Arg Ile Gly Leu Ser Ala Thr Lys 385 390 395 400
Pro Glu Cys Ser Val Pro Asn Gly Gly Ile Gly Thr Ser Asp Phe Gly 405
410 415 Glu Ser Leu Arg Phe Gln Lys Val Leu Gln Gly Gln Glu Ile Ser
Gly 420 425 430 Phe Asp Thr Pro Phe Ser Gly Leu Gly Gly Leu Asn Ser
Leu Pro Ser 435 440 445 Glu Ala Arg Arg Val Phe His Gly Ser Gly Gly
Ser Gly Ile Ala Ala 450 455 460 Gly Gly Asn Gly Leu Arg Gln Ser Leu
Val Asp Ser Glu Ile Ala Ser 465 470 475 480 Lys Gly Ile Gly Phe Gly
Glu Ser Phe Arg Phe Arg Lys Val Leu Gln 485 490 495 Gly Gln Glu Ile
Leu Pro Ser Ser Pro Tyr Gly Arg Ala Pro Ala Ser 500 505 510 Asn Glu
Ala His Glu Tyr Gly Gly Pro Gly Ile Tyr Asp Gly Phe His 515 520 525
Val Pro Gly Phe Arg Asn Gly Trp Ser Thr Met Met Gln Ser Asn Asn 530
535 540 Thr His Val His Ser Ser Ala Pro Ser Val Gln Val Ser Ser Pro
Ser 545 550 555 560 Ser Val Leu Met Phe Gln Gln Ala Val Asn Pro Val
Val Glu Phe Asn 565 570 575 Ser Val Tyr Asn Gly His Asn Pro Glu Asp
His Arg Val Asn Arg Thr 580 585 590 Leu His Val Ser Glu His Asp Gly
Gly Arg Gln Thr Ser Ser Ser Phe 595 600 605 Gly Glu Leu Asn Phe Ser
Arg Glu Asp Arg Gly Gly Thr His Ser Tyr 610 615 620 Asn Gln His Gly
Ile Ser Pro His Pro Gly Thr Ser Gln Ser Thr Ile 625 630 635 640 Ser
Gly Ser Gln Asp Ser Ile Ser Pro Ile Lys Gly Ser Cys Arg Leu 645 650
655 Phe Gly Phe Ser Leu Ser Glu Asp Lys Cys Val Pro Asp Gln Glu Gly
660 665 670 Asn Pro Asn Val Gly Val Arg Phe His Ser Lys Pro Ser Leu
Met Thr 675 680 685 Ser Thr Val Gly Ile Thr Cys Thr Lys Val Ser Asn
Leu Phe Ala Ala 690 695 700 10709PRTPopulus tremula 10Met Val Gly
Met Ile Asp Leu Asn Thr Thr Glu Glu Asp Glu Thr Thr 1 5 10 15 Pro
Ser Ser Gly Ser Leu Ser Ser Pro Ser Ser Ser Ser Ala Ala Ser 20 25
30 Ala Leu Ser Ala Ser Gly Ser Gly Ser Gly Thr Ser Pro Val Cys Leu
35 40 45 Glu Leu Trp His Ala Cys Ala Gly Pro Leu Ile Ser Leu Pro
Lys Arg 50 55 60 Gly Ser Ile Val Val Tyr Val Pro Gln Gly His Leu
Glu Gln Leu Pro 65 70 75 80 Asp Leu Pro Leu Gly Ile Tyr Asp Leu Pro
Pro His Val Phe Cys Arg 85 90 95 Val Val Asp Val Lys Leu His Ala
Glu Ala Ala Ser Asp Asp Val Tyr 100 105 110 Ala Gln Val Ser Leu Val
Pro Glu Ser Glu Glu Ile Glu Gln Lys Leu 115 120 125 Arg Glu Gly Val
Phe Glu Gly Asp Gly Glu Glu Glu Asp Val Glu Ala 130 135 140 Thr Val
Lys Thr Thr Thr Pro His Met Phe Cys Lys Thr Leu Thr Ala 145 150 155
160 Ser Asp Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala
165 170 175 Glu Asp Cys Phe Pro Pro Leu Asp Tyr Thr Gln Gln Arg Pro
Ser Gln 180 185 190 Glu Leu Val Ala Lys Asp Leu His Gly Ser Glu Trp
Lys Phe Arg His 195 200 205 Ile Tyr Arg Gly Gln Pro Arg Arg His Leu
Leu Thr Thr Gly Trp Ser 210 215 220 Ala Phe Val Asn Lys Lys Lys Leu
Val Ser Gly Asp Ala Val Leu Phe 225 230 235 240 Leu Arg Gly Glu Asp
Gly Glu Leu Arg Leu Gly Val Arg Arg Ala Ala 245 250 255 Gln Val Lys
Cys Gly Pro Thr Phe Pro Ala Leu Trp Asn Gln Gln Leu 260 265 270 Asn
Gln Ser Ser Leu Ala Asp Val Ala Asn Ala Ile Ser Met Arg Ser 275 280
285 Ala Phe Arg Ile Tyr Tyr Asn Pro Arg Ala Ser Ser Ser Glu Phe Ile
290 295 300 Ile Pro Phe Asn Lys Phe Leu Lys Ser Leu Asp Gln Ser Phe
Ser Ala 305 310 315 320 Gly Met Arg Val Lys Met Arg Phe Glu Thr Glu
Asp Ala Ala Glu Arg 325 330 335 Arg Tyr Thr Gly Leu Ile Thr Gly Ile
Ser Glu Leu Asp Pro Thr Arg 340 345 350 Trp Pro Gly Ser Lys Trp Lys
Cys Leu Leu Val Arg Trp Asp Asp Thr 355 360 365
Glu Ala Asn Arg His Ser Arg Val Ser Pro Trp Glu Val Glu Pro Ser 370
375 380 Gly Ser Val Ser Gly Ser Gly Ser Gly Ser Ile Ser Ser Ser Asn
Asn 385 390 395 400 Ser Met Ala Pro Gly Leu Lys Arg Ser Arg Ser Gly
Leu Pro Ser Leu 405 410 415 Lys Ala Glu Phe Pro Ile Pro Asp Gly Ile
Gly Ala Ser Asp Phe Arg 420 425 430 Val Ser Ser Arg Phe Gln Glu Val
Leu Gln Gly Gln Glu Ile Met Arg 435 440 445 Ser Gly Ile Arg Gly Ser
Ile Pro Thr Ser Glu Asn Ser Phe Lys Gly 450 455 460 Ile Gly Phe Asn
Glu Ser Tyr Arg Phe His Lys Val Leu Gln Gly Gln 465 470 475 480 Glu
Ile Phe Pro Arg Ser Pro Tyr Arg Arg Ile Pro Asn Ala Asn Lys 485 490
495 Ala Arg Glu Asn Cys Gly Leu Gly Leu Ser Asp Gly Val Gln Arg Ser
500 505 510 Ser Ser Arg Asn Gly Trp Ser Thr Met Met Gln Gly Tyr Asn
Thr Gln 515 520 525 Met Arg Pro Pro Thr Gln Val Ser Ser Pro Ser Ser
Val Leu Met Phe 530 535 540 Gln His Ala Ser Asn Gln Val Ser Asn Pro
Thr Ser Ile Phe Asn Ser 545 550 555 560 Asn Asp His Glu Glu Gln Thr
Thr Asn Thr Gln Ser Trp Phe Tyr Pro 565 570 575 Glu Thr His Gly Gly
Lys Phe Lys Leu Ser Ser His Ser Asp Pro Gly 580 585 590 Leu Arg Gly
Asp Ser Gln Cys Ser Thr Asn Pro Tyr Val Leu Ser His 595 600 605 Glu
His Leu Gln His Gly Ile Ser Gln Pro Val Val Ala Gln Ser Ala 610 615
620 Phe Arg Ser Ser Gln Asp Met Val Leu Cys Lys Ser Ser Cys Arg Leu
625 630 635 640 Phe Gly Phe Ser Leu Thr Glu Asp Arg His Val Val Asn
Lys Glu Asp 645 650 655 Asn Ile Ala Ser Ile Thr Ser Pro Leu Asn Pro
Glu Ser Ser Phe Leu 660 665 670 Pro Arg Val Gly Glu Gln Leu His Pro
Lys Pro Pro Ala Ile Asn Asn 675 680 685 Ala Val Gly Ser Ser Cys Thr
Lys Ala Ile Arg Gln His His Ala Glu 690 695 700 Asn Tyr Arg Ile Tyr
705 11786PRTVitis vinifera 11Met Val Ala Met Ile Asp Leu Asn Thr
Val Asp Asp Asp Glu Thr Pro 1 5 10 15 Ser Ser Gly Ser Ser Ser Ser
Ser Ser Ser Ser Ala Ser Ala Ser Ala 20 25 30 Ser Thr Val Cys Gly
Ser Leu Leu Ser Ala Ala Ser Ser Val Cys Leu 35 40 45 Glu Leu Trp
His Ala Cys Ala Gly Pro Leu Ile Ser Leu Pro Lys Lys 50 55 60 Gly
Ser Leu Val Val Tyr Phe Pro Gln Gly His Leu Glu Gln Leu Ser 65 70
75 80 Asp Tyr Pro Ala Val Ala Tyr Asp Leu Pro Pro His Val Phe Cys
Arg 85 90 95 Val Val Asp Val Lys Leu His Ala Glu Val Val Thr Asp
Glu Val Tyr 100 105 110 Ala Gln Val Ser Leu Val Pro Glu Thr Lys Gln
Ile Lys Gln Lys Leu 115 120 125 Gln Glu Gly Glu Ile Glu Ala Asp Gly
Gly Glu Glu Glu Asp Ile Glu 130 135 140 Gly Ser Ile Lys Ser Met Thr
Pro His Met Phe Cys Lys Thr Leu Thr 145 150 155 160 Ala Ser Asp Thr
Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala 165 170 175 Ala Glu
Asp Cys Phe Pro Pro Leu Asp Tyr Lys Gln Gln Arg Pro Ser 180 185 190
Gln Glu Leu Val Ala Lys Asp Leu His Gly Phe Glu Trp Arg Phe Arg 195
200 205 His Ile Tyr Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly
Trp 210 215 220 Ser Ala Phe Val Asn Lys Lys Lys Leu Val Ser Gly Asp
Ala Val Leu 225 230 235 240 Phe Leu Arg Gly Gly Asp Gly Glu Leu Arg
Leu Gly Ile Arg Arg Ala 245 250 255 Ala Gln Ile Lys Gly Ser Ser Pro
Phe Pro Ala Leu Cys Ser Gln Gln 260 265 270 Leu Asn Leu Asn Thr Leu
Thr Ala Val Val Asn Ala Ile Ser Thr Arg 275 280 285 Ser Val Phe Asn
Ile Cys Tyr Asn Pro Arg Ala Ser Ser Ser Glu Phe 290 295 300 Ile Ile
Pro Leu Arg Lys Phe Ser Lys Ser Ile Asp His Ser Phe Ser 305 310 315
320 Ala Gly Met Arg Phe Lys Met Arg Val Glu Thr Glu Asp Ala Ala Glu
325 330 335 Arg Arg Tyr Thr Gly Leu Ile Thr Gly Ile Ser Asp Met Asp
Pro Val 340 345 350 Arg Trp Pro Gly Ser Lys Trp Arg Cys Leu Leu Val
Arg Trp Asp Asp 355 360 365 Ile Glu Ala Asn Arg His Asn Arg Val Ser
Pro Trp Glu Ile Glu Leu 370 375 380 Ser Gly Ser Leu Ser Gly Ser Gly
Ser Leu Thr Val Pro Gly Ser Lys 385 390 395 400 Arg Thr Arg Ile Gly
Leu Pro Gly Thr Arg Pro Asp Phe Ser Val Pro 405 410 415 Asn Gly Met
Gly Val Ser Asp Phe Gly Glu Ser Ser Arg Phe Gln Lys 420 425 430 Val
Leu Gln Gly Gln Glu Ile Phe Gly Phe Asn Thr Pro Tyr Asp Gly 435 440
445 Val Asp Thr Gln Asp His His Pro Ser Glu Ile Arg Cys Phe Pro Gly
450 455 460 Ser Ser Cys Ser Gly Ile Ala Ala Ile Gly Asn Gly Val Arg
Asn Pro 465 470 475 480 Leu Gly Asn Ser Asp Ile Ser Tyr Lys Gly Ile
Gly Phe Gly Glu Ser 485 490 495 Phe Arg Phe His Lys Val Leu Gln Gly
Gln Glu Thr Phe Pro Ser Pro 500 505 510 Pro Cys Gly Arg Ala Leu Ser
Ala Asn Gln Ala His Glu Asn Gly Ser 515 520 525 Phe Gly Ile Phe Asp
Gly Val Gln Val Pro Thr Ser Arg Asn Gly Trp 530 535 540 Pro Ala Leu
Val Gln Gly Tyr Asn Ala His Thr His Leu Ser Thr Pro 545 550 555 560
Ser Val Gln Val Ser Ser Pro Ser Ser Val Leu Met Phe Gln Gln Ala 565
570 575 Ser Thr Ala Ala Pro Asn Ile Tyr Ser Met His Ser Ala Asn Asn
Gln 580 585 590 Glu Lys Glu Gln Glu Ile Ser Asn Arg Ser Ser Phe Asp
Ile Pro Glu 595 600 605 Val Tyr Gly Glu Lys Leu Thr Pro Ser Arg Cys
Glu Leu Ser Val Arg 610 615 620 Gly Gly Gly Gln Gly Gly Met Asn Phe
Phe Gly Leu Leu Asn Glu His 625 630 635 640 Asn Gln Leu Ala Val Pro
His Pro Leu Val Thr Gln Ser Ala Phe Arg 645 650 655 Gly Ser Gln Asp
Leu Val Pro Thr Cys Lys Ser Ser Cys Arg Leu Phe 660 665 670 Gly Phe
Ser Leu Thr Glu Glu Arg Ser Ile Gly Asn Lys Val Asp Asn 675 680 685
Pro Thr Pro Val Thr Ser Ser Leu Ile Pro Gly Thr Ser Phe Leu Pro 690
695 700 Gln Gln Leu His Ser Glu Pro Pro Val Met Thr Lys Ala Ile Gly
Ser 705 710 715 720 Asn Cys Thr Lys Arg Thr Ala Val Val Arg Ser Lys
Leu Gln Phe His 725 730 735 Lys Leu Gly Ser Val Val Asp Gln Ala Ile
Asn Arg Trp Lys Leu Asp 740 745 750 Arg His Asp Asp Leu Ile Cys Ala
Leu Lys His Leu Phe Asp Met Glu 755 760 765 Gly Gly Leu Leu His Gly
Glu Gly Lys Leu Phe Thr Arg Ile Met Arg 770 775 780 Met Leu 785
122127DNAMalus domestica 12atggcgggtc taattgatct gaacagtgcg
acggaggacg aggaaacgcc atcgtccggc 60tcgccgtctt cggcttcctc tgtttccgac
gctctgggtt cgtcggcgtc ggtgtgcatg 120gagctctggc acgcctgcgc
gggcccactg atttcgctgc cgaagaaagg gagtgtggtg 180gtgtatctgc
cgcagggcca cctggagcaa gtctcggatt ttccgacctc ggcttatgat
240ctcccgcccc acctcttctg tcgggttgtc gatgtcaagc tccatgctga
gactggcact 300gacgatgtct tcgctcrggt ttcccttgtt cctgaaagtg
aggaaattga gcacagattg 360cgggaagggg taaccgatgc agatgccgag
gaggacgttg aggcaatggg gacgtcaacc 420acaccccaca tgttctgcaa
aacccttact gcttctgata ctagcactca cggaggcttc 480tctgtgcctc
gtcgtgctgc cgaggattgc tttcctcccc tggattacac tcaacaaagg
540ccttcacaag agcttgtagc aaaggatctg catggcctgg agtggaggtt
ccggcatatc 600tatagggggc agccgcggag gcatttgctc accactgggt
ggagtgcgtt tgtgaacaag 660aagaagctcg tctctggaga tgcagtgctg
tttcttaggg gtgacgatgg agaactgagg 720ctaggaatta gaagggcagc
ccagtttaaa agttctgcta cttgtccaac tctttgtagc 780cagcaattga
actatagcac tatcactgat gtggtgaatg ctatattcgc gaagaatgct
840tttaatgtgt actacaatcc aaggtccagc tcttctgaat tcataatacc
ttcccataag 900tttttgagga gccttgatca ttgtttttgt gctggaatga
ggatcaaaat gcgttttgaa 960actgaagatg cagcagagcg aagatacact
gggttgataa cggggattag tgaattggat 1020cctgtaagat ggcctggttc
aaaatggaga tgcctagttg tcaggtggga tgatrtagac 1080acaagcaagc
atggcagggt ttccccatgg gaagttgagc gatctggttc tgtttctagt
1140tcccataccc taatgacaac tggcttgaag cggtccagga ttggcttgtc
tgcaacaaaa 1200ccagaatktc cagytcctag tatgtcctgc aatyatggga
ttggaacatc agactttggg 1260gaatctttaa ggttccagaa ggtcttgcaa
ggtcaagaaa tttcggggtt tgatactcct 1320ttcagtggtt taggtggtct
gaattcgcat ccatctgaag caaggagagt cttccacggt 1380tccggtggtt
ctgggattgc tgctggrggt aatggtctca gacagtcact tgtggattct
1440gagattgcct caaaaggcat aggctttggt gaatcattcc gattccataa
ggtcttgcaa 1500ggtcaagaaa tatttccaag ctcaccatat ggaagagctc
ccgcttctaa tgaagctcat 1560gaatatggtg gacctggact ctatgatggt
tttcaggtgc ctggctttag gaatggatgs 1620tccaccatga tgcagagcaa
taatacaaat gtgcactcat ctgccccatc tgtgcaagtt 1680tcatcacctt
cgtctgtgtt aatgttccag caagcaatga atccagttgc ggaattcaac
1740tcggtataca atggccataa ccaagaggac catagagtaa atcggactcc
acatgtcttg 1800gaacatgatg gtggaaggca aacatcatcc tcattcggtg
aacgtaactt cagcagggaa 1860gatcgtggag gcacacattc ttacaatcag
catggtattt cacctcatcc agttataagt 1920caatcaacaa ttagtggcag
ccaggattct gtttcaccaa tcaaaggtag ctgtagactc 1980tttggtttct
cattgtccga ggacaaatgt gtcccggatc aagagggcaa ccccaatgtt
2040ggagtgcagt ttcattcaaa gcctcctttg atgacctcaa cagttggaat
aacctgtact 2100aaagtaagca acctctttgc tgcatga 2127135500DNAMalus
domestica 13actctcccac gatacccacc tagcaaatgc taaattytct cgctccccaa
atgttctgca 60atcaggcagg cargggttta gtaattagtc gggtcaaaga ctcggataca
ctaatttcaa 120aataaagaaa tgttatagat cggagkattg tgccttggca
gacaagccct ctttcaacag 180gkttacatat gagggwtaag caatattaaa
tatagattct acaaactttt gttctcaaag 240ctgaattaac aatacaaatc
aaagtcccct ggagcgctta gttacaactt ctgcctcaaa 300attaagttac
ataaatgaca gcacacacat aatcccgaag aaaacctccg cctgtcgaga
360gttttcttca tcggtacaac gcaaaactat tgatattaca gtaacatcgc
ctgccaaaag 420cgaaattcaa acaaataaat gaccggaaca caagcatctt
cttcatgttc atccggttta 480cttaaaacga ctttggaagg atatagtgcg
gcgacaaaca tggccatgat gagttccatg 540ggtagggttt ataagcggac
ttcraggcat accatatgta agttttgaca atagacgggc 600aatgaagaat
gccatgtaga ttccgatgct aggttgcaaa cagaacaaaa atagrcaaat
660acaataagta aacgtacgag gacttactag ttactgcctc ggatcatctg
caacaagcac 720aggatgatca ctatccttgt aaacagcttt ccatcctttc
tccgcgccag tcaaaagccc 780aatcctagac ataaacatcc cacttgctaa
tcagataatc atttgcttcc gtcaaataga 840agattaaata ccccagtcgt
aacatttgaa aattgtattt catgcagcaa agaggttgct 900tactttagta
caggttattc caactgttga ggtcatcaaa ggaggctttg aatgaaactg
960cactccaaca ttggggttgc cctcttgatc cgggacacat ttgtcctcgg
acaatgagaa 1020accaaagagt ctacagctac ctttgattgg tgaaacagaa
tcctggctgc cactaattgt 1080tgattgactt ataactggat gaggtgaaat
accatgctga ttgtaagaat gtgtgcctcc 1140acgatcttcc ctgctgaagt
tacgttcacc gaatgaggat gatgtttgcc ttccaccatc 1200atgttccaag
acatgtggag tccgatttac tctatggtcc tcttggttat ggccattgta
1260taccgagttg aattccgcaa ctggattcat tgcttgctgg aacattaaca
cagacgaagg 1320tgatgaaact tgcacagatg gggcagatga gtgcacattt
gtattattgc tctgcatcat 1380ggtggascat ccattcctaa agccaggcac
ctgaaaacca tcatagagtc caggtccacc 1440atattcatga gcttcattag
aagcgggagc tcttccatat ggtgagcttg gaaatatttc 1500ttgaccttgc
aagaccttat ggaatcggaa tgattcacca aagcctatgc cttttgaggc
1560aatctcagaa tccacaagtg actgtctgag accattaccy ccagcagcaa
tcccagaacc 1620accggaaccg tggaagactc tccttgcttc agatggatgc
gaattcagac cacctaaacc 1680actgaaagga gtatcaaacc ccgaaatttc
ttgaccttgc aagaccttct ggaaccttaa 1740agattcccca aagtctgatg
ttccaatccc atctgaaaag gaaacactca aataaatcaa 1800ttatgttgca
gagaccagtg ttatgatgac atgtcgtggt acrattgcag gacatactag
1860garctggama ttctggtttt gttgcagaca agccaatcct ggaccgcttc
aagccagttg 1920tcattagggt atgggaacta gaaacagaac cagatcgctc
aacttcccat ggggaaaccc 1980tgccatgctt gcttgtgtct ayatcatccc
acctgacctg caatagcaaa agaaatgttc 2040aaacctatat atctagtgct
aatggcatga gcgaataccg gaagtcaata tggacatgct 2100acagaataga
ggaaacatag tttatgatat ttaggtaaac gactttcaag acagtgaaaa
2160ttaactctca ctatcaagac ataattgctc ataaaccctg agtttctact
ccgtaggact 2220atgagtaggg tggcttgatc aacaattaag ataaaactgt
gtagctaatg cgggagaaaa 2280tttatgcttc cagtttaaat ttagccagaa
attctaccaa attgtgacat ttcacatgac 2340atatgaagtg tgcaattcat
cctagttaga atacggtgaa gccggctaca ttttttttgt 2400taccttttgc
actctcatgc ttcaactatt gttgagaaaa gcttatcata gatttgttca
2460tgacaatggt tttgggaatc tcaaagtaaa ataaccaata cgtatagtaa
gtctcccata 2520aagaagataa tacagaattt atggtacgct aaagataact
tttggtttag tacattggca 2580ttcaccaccc gaaccttctc aagcagatac
ttataagtgc aaacaaaggg gaaaaaatag 2640tacataaaag ttttgtgtag
aaaatacgta caactaggca tctccatttt gaaccaggcc 2700atcttacagg
atccaattca ctaatccccg ttatcaaccc agtgtatctg ccaaaaagaa
2760aaatttaata aaaattttgc acaaaactac ttcaactatt tcaaaagtaa
gaatgccaaa 2820ccttcgctct gctgcatctt cagtttcaaa acgcattttg
atcctcattc cagcacaaaa 2880acaatgatca aggctcctca aaaacttatg
ggaaggtatt atgaattcag aagagctgga 2940cctacaagat catggacgat
tataaggaaa aaagatgcag aaggaagaaa ggtctaaagt 3000ctcaagcacg
ataactttca acattacttt aggaacatgt taaatgatga gcaccagatt
3060accttggatt gtagtacaca ttaaaagcat tcttcgcgaa tatagcattc
accacatcag 3120tgatagtgct atagttcaat tgctggctac aaagagttgg
acaagtagca gaacttttaa 3180actgggctgc ccttctaatt cctagcctca
gttctccatc gtcaccccta ttataaatac 3240tggatgagaa ttataaataw
ggggatccaa aatctcttca atcgaagggt ttatatacct 3300aagaaacagc
actgcatctc cagagacgag cttcttcttg ttcacaaacg cactccaccc
3360agtggtgagc aaatgcctcc gcggctgccc tactcaaacc aattagaaaa
aaggaataag 3420aaacagaaaa acgttgaatc taaagcatcc aatttacaaa
gttcattata aaactctaat 3480aagattgcta ccgtatcatg gaaagataaa
cccccaacta atgaagcaca aaagggatca 3540tattttttgt attaccccta
tagatatgcc ggaacctcca ctccaggcca tgcagatcct 3600ttgctacaag
ctcttgtgaa ggcctttgtt gagtgtaatc ctaaaagcac aatctattcg
3660tctcaaaaac aaaatactaa tatgtacttg attagactaa taatttagtc
tcaaaaaagt 3720tatgcagaag ctaaamcaaa agttggggga gaacatgaac
taggaatacc aggggaggaa 3780agcaatcctc ggcagcacga cgaggcacag
agaagcctcc gtgagtgcta gtatcagaag 3840cagtaagggt tttgcagaac
atgtggggtg tggttgacgt ccccattgcc tcaacgtcct 3900cctcggcatc
tgcatcggtt accccttccc gcaatctgtg ctcaatttcc tacaccgatt
3960aaagcattag tgaaatcacc tcaatgtcaa tgtttaacag acaaaaacat
cacamgatca 4020ccaggaagag caaagaaaat taaaaaacay caaccccgaa
atcgaattgt ataaattata 4080agtaactaat tcataaattt gtgaaatgac
taattgtaag garacagaga tgctaacata 4140taatcttcga taatgccaaa
gtgggaaaga aagaaagtga aattctaaag cttgcgtttt 4200taattgtttc
ggtttctacg gaaagggagg attttctcag aattttttta cttttcgagc
4260attttctcag gaaccaaaca aaaaatcaaa ccttgtacaa ataaacccac
ctcactttca 4320ggaacaaggg aaaccygagc gaagacatcg tcagtgccag
tctcagcctg cgaccaaaaa 4380agacctcaag aaattgagaa cccagatagc
aaaaccccaa aagctatcaa atttcatcaa 4440aaatccaaaa ccccaaaagc
atggagaaag agaggctcat tgtatagaga gagactgaca 4500tggagcttga
catcgacaac ccgacagaag aggtggggcg ggagatcata agccgaggtc
4560ggaaaatccg agacttgctc caggtggccc tgcggcagat acaccaccac
actccctttc 4620ttcggcagcg aaatcagtgg gcccgcgcag gcgtgccaga
gctccatgca caccgacgcc 4680gacgaaccca gagcgtcgga aacagaggaa
gccgaagacg gcgagccgga cgatggcgtt 4740tcctcgtcct ccgtcgcact
gttcagatca attagacccg ccatgtgaaa ttaaacagaa 4800actgtaagga
cccaacaagc gaaagaggaa gaagaaggag aaaagcactt tgcttttttg
4860ctttttgctt ctgcttctgc ttaagcttgt ttataatatg gagaagagaa
aaaaragagc 4920agatgaggtt tcaattctcc tgcattttga gagatgggtt
tgatggcatt tttgctaaaa 4980gaagctcaga aaagcacttc tctctctttt
ctgcttaatt cttaactttt atttattttt 5040cttttggacg ggcttttggg
tatcaaatca aaagctcaaa gcttttacaa tttgggagat 5100gagaaaaaga
aaatggaaat taataccaaa aaaaatcaga taatttaata tgggacttct
5160tttggtgttg aattgaatcc ctttttgggt ttttaaatta caaaagatta
aaccttttct 5220ctctgtctgc aggctgaact gcaacactgc aattgctggg
tgtgatgtgt ggcagaaaca 5280gtgtgtcaga gagtgagaga gagaggaaga
gagagagtgg aggtcttgta cggcaccccc 5340tgtatttccg ggcggatgtc
tattctgtcc ccttctactc cgtcaaatcc cactcgtatc 5400cccccccccc
tcttccttaa tcttcattct ctccttcctc ccatatttaa tttattttaa
5460ttaaggagga
tgtagttaaa ctaagatttt aaaagatttt 5500142127DNAMalus domestica
14atggcgggtc taattgatct gaacagtgcg acggaggacg aggaaacgcc atcgtccggc
60tcgccgtctt cggcttcctc tgtttccgac gctctgggtt cgtcggcgtc ggtgtgcatg
120gagctctggc acgcctgcgc gggcccactg atttcgctgc cgaagaaagg
gagtgtggtg 180gtgtatctgc cgcagggcca cctggagcaa gtcttggatt
ttccgacctc ggcttatgat 240ctcccgcccc acctcttctg tcgggttgtc
gatgtcaagc tccatgctga gactggcact 300gacgatgtct tcgctcrggt
ttcccttgtt cctgaaagtg aggaaattga gcacagattg 360cgggaagggg
taaccgatgc agatgccgag gaggacgttg aggcaatggg gacgtcaacc
420acaccccaca tgttctgcaa aacccttact gcttctgata ctagcactca
cggaggcttc 480tctgtgcctc gtcgtgctgc cgaggattgc tttcctcccc
tggattacac tcaacaaagg 540ccttcacaag agcttgtagc aaaggatctg
catggcctgg agtggaggtt ccggcatatc 600tatagggggc agccgcggag
gcatttgctc accactgggt ggagtgcgtt tgtgaacaag 660aagaagctcg
tctctggaga tgcagtgctg tttcttaggg gtgacgatgg agaactgagg
720ctaggaatta gaagggcagc ccagtttaaa agttctgcta cttgtccaac
tctttgtagc 780cagcaattga actatagcac tatcactgat gtggtgaatg
ctatattcgc gaagaatgct 840tttaatgtgt actacaatcc aaggtccagc
tcttctgaat tcataatacc ttcccataag 900tttttgagga gccttgatca
ttgtttttgt gctggaatga ggatcaaaat gcgttttgaa 960actgaagatg
cagcagagcg aagatacact gggttgataa cggggattag tgaattggat
1020cctgtaagat ggcctggttc aaaatggaga tgcctagttg tcaggtggga
tgatrtagac 1080acaagcaagc atggcagggt ttccccatgg gaagttgagc
gatctggttc tgtttctagt 1140tcccataccc taatgacaac tggcttgaag
cggtccagga ttggcttgtc tgcaacaaaa 1200ccagaatktc cagytcctag
tatgtcctgc aatyatggga ttggaacatc agactttggg 1260gaatctttaa
ggttccagaa ggtcttgcaa ggtcaagaaa tttcggggtt tgatactcct
1320ttcagtggtt taggtggtct gaattcgcat ccatctgaag caaggagagt
cttccacggt 1380tccggtggtt ctgggattgc tgctggrggt aatggtctca
gacagtcact tgtggattct 1440gagattgcct caaaaggcat aggctttggt
gaatcattcc gattccataa ggtcttgcaa 1500ggtcaagaaa tatttccaag
ctcaccatat ggaagagctc ccgcttctaa tgaagctcat 1560gaatatggtg
gacctggact ctatgatggt tttcaggtgc ctggctttag gaatggatgs
1620tccaccatga tgcagagcaa taatacaaat gtgcactcat ctgccccatc
tgtgcaagtt 1680tcatcacctt cgtctgtgtt aatgttccag caagcaatga
atccagttgc ggaattcaac 1740tcggtataca atggccataa ccaagaggac
catagagtaa atcggactcc acatgtcttg 1800gaacatgatg gtggaaggca
aacatcatcc tcattcggtg aacgtaactt cagcagggaa 1860gatcgtggag
gcacacattc ttacaatcag catggtattt cacctcatcc agttataagt
1920caatcaacaa ttagtggcag ccaggattct gtttcaccaa tcaaaggtag
ctgtagactc 1980tttggtttct cattgtccga ggacaaatgt gtcccggatc
aagagggcaa ccccaatgtt 2040ggagtgcagt ttcattcaaa gcctcctttg
atgacctcaa cagttggaat aacctgtact 2100aaagtaagca acctctttgc tgcatga
2127155500DNAMalus domestica 15aaaatctttt aaaatcttag tttaactaca
tcctccttaa ttaaaataaa ttaaatatgg 60gaggaaggag agaatgaaga ttaaggaaga
gggggggggg gatacgagtg ggatttgacg 120gagtagaagg ggacagaata
gacatccgcc cggaaataca gggggtgccg tacaagacct 180ccactctctc
tcttcctctc tctctcactc tctgacacac tgtttctgcc acacatcaca
240cccagcaatt gcagtgttgc agttcagcct gcagacagag agaaaaggtt
taatcttttg 300taatttaaaa acccaaaaag ggattcaatt caacaccaaa
agaagtccca tattaaatta 360tctgattttt tttggtatta atttccattt
tctttttctc atctcccaaa ttgtaaaagc 420tttgagcttt tgatttgata
cccaaaagcc cgtccaaaag aaaaataaat aaaagttaag 480aattaagcag
aaaagagaga gaagtgcttt tctgagcttc ttttagcaaa aatgccatca
540aacccatctc tcaaaatgca ggagaattga aacctcatct gctctytttt
ttctcttctc 600catattataa acaagcttaa gcagaagcag aagcaaaaag
caaaaaagca aagtgctttt 660ctccttcttc ttcctctttc gcttgttggg
tccttacagt ttctgtttaa tttcacatgg 720cgggtctaat tgatctgaac
agtgcgacgg aggacgagga aacgccatcg tccggctcgc 780cgtcttcggc
ttcctctgtt tccgacgctc tgggttcgtc ggcgtcggtg tgcatggagc
840tctggcacgc ctgcgcgggc ccactgattt cgctgccgaa gaaagggagt
gtggtggtgt 900atctgccgca gggccacctg gagcaagtct tggattttcc
gacctcggct tatgatctcc 960cgccccacct cttctgtcgg gttgtcgatg
tcaagctcca tgtcagtctc tctctataca 1020atgagcctct ctttctccat
gcttttgggg ttttggattt ttgatgaaat ttgatagctt 1080ttggggtttt
gctatctggg ttctcaattt cttgaggtct tttttggtcg caggctgaga
1140ctggcactga cgatgtcttc gctcrggttt cccttgttcc tgaaagtgag
gtgggtttat 1200ttgtacaagg tttgattttt tgtttggttc ctgagaaaat
gctcgaaaag taaaaaaatt 1260ctgagaaaat cctccctttc cgtagaaacc
gaaacaatta aaaacgcaag ctttagaatt 1320tcactttctt tctttcccac
tttggcatta tcgaagatta tatgttagca tctctgtytc 1380cttacaatta
gtcatttcac aaatttatga attagttact tataatttat acaattcgat
1440ttcggggttg rtgtttttta attttctttg ctcttcctgg tgatcktgtg
atgtttttgt 1500ctgttaaaca ttgacattga ggtgatttca ctaatgcttt
aatcggtgta ggaaattgag 1560cacagattgc gggaaggggt aaccgatgca
gatgccgagg aggacgttga ggcaatgggg 1620acgtcaacca caccccacat
gttctgcaaa acccttactg cttctgatac tagcactcac 1680ggaggcttct
ctgtgcctcg tcgtgctgcc gaggattgct ttcctcccct ggtattccta
1740gttcatgttc tcccccaact tttgktttag cttctgcata acttttttga
gactaaatta 1800ttagtctaat caagtacata ttagtatttt gtttttgaga
cgaatagatt gtgcttttag 1860gattacactc aacaaaggcc ttcacaagag
cttgtagcaa aggatctgca tggcctggag 1920tggaggttcc ggcatatcta
taggggtaat acaaaaaata tgatcccttt tgtgcttcat 1980tagttggggg
tttatctttc catgatacgg tagcaatctt attagagttt tataatgaac
2040tttgtaaatt ggatgcttta gattcaacgt ttttctgttt cttattcctt
ttttctaatt 2100ggtttgagta gggcagccgc ggaggcattt gctcaccact
gggtggagtg cgtttgtgaa 2160caagaagaag ctcgtctctg gagatgcagt
gctgtttctt aggtatataa acccttcgat 2220tgaagagatt ttggatcccc
wtatttataa ttctcatcca gtatttataa taggggtgac 2280gatggagaac
tgaggctagg aattagaagg gcagcccagt ttaaaagttc tgctacttgt
2340ccaactcttt gtagccagca attgaactat agcactatca ctgatgtggt
gaatgctata 2400ttcgcgaaga atgcttttaa tgtgtactac aatccaaggt
aatctggtgc tcatcattta 2460acatgttcct aaagtaatgt tgaaagttat
cgtgcttgag actttagacc tttcttcctt 2520ctgcatcttt tttccttata
atcgtccatg atcttgtagg tccagctctt ctgaattcat 2580aataccttcc
cataagtttt tgaggagcct tgatcattgt ttttgtgctg gaatgaggat
2640caaaatgcgt tttgaaactg aagatgcagc agagcgaagg tttggcattc
ttacttttga 2700aatagttgaa gtagttttgt gcaaaatttt tattaaattt
ttctttttgg cagatacact 2760gggttgataa cggggattag tgaattggat
cctgtaagat ggcctggttc aaaatggaga 2820tgcctagttg tacgtatttt
ctacacaaaa cttttatgta ctattttttc ccctttgttt 2880gcacttataa
gtatctgctt gagaaggttc gggtggtgaa tgccaatgta ctaaaccaaa
2940agttatcttt agcgtaccat aaattctgta ttatcttctt tatgggagac
ttactatacg 3000tattggttat tttactttga gattcccaaa accattgtca
tgaacaaatc tatgataagc 3060ttttctcaac aatagttgaa gcatgagagt
gcaaaaggta acaaaaaaaa tgtagccggc 3120ttcaccgtat tctaactagg
atgaattgca cacttcatat gtcatgtgaa atgtcacaat 3180ttggtagaat
ttctggctaa atttaaactg gaagcataaa ttttctcccg cattagctac
3240acagttttat cttaattgtt gatcaagcca ccctactcat agtcctacgg
agtagaaact 3300cagggtttat gagcaattat gtcttgatag tgagagttaa
ttttcactgt cttgaaagtc 3360gtttacctaa atatcataaa ctatgtttcc
tctattctgt agcatgtcca tattgacttc 3420cggtattcgc tcatgccatt
agcactagat atataggttt gaacatttct tttgctattg 3480caggtcaggt
gggatgatrt agacacaagc aagcatggca gggtttcccc atgggaagtt
3540gagcgatctg gttctgtttc tagttcccat accctaatga caactggctt
gaagcggtcc 3600aggattggct tgtctgcaac aaaaccagaa tktccagytc
ctagtatgtc ctgcaatygt 3660accacgacat gtcatcataa cactggtctc
tgcaacataa ttgatttatt tgagtgtttc 3720cttttcagat gggattggaa
catcagactt tggggaatct ttaaggttcc agaaggtctt 3780gcaaggtcaa
gaaatttcgg ggtttgatac tcctttcagt ggtttaggtg gtctgaattc
3840gcatccatct gaagcaagga gagtcttcca cggttccggt ggttctggga
ttgctgctgg 3900rggtaatggt ctcagacagt cacttgtgga ttctgagatt
gcctcaaaag gcataggctt 3960tggtgaatca ttccgattcc ataaggtctt
gcaaggtcaa gaaatatttc caagctcacc 4020atatggaaga gctcccgctt
ctaatgaagc tcatgaatat ggtggacctg gactctatga 4080tggttttcag
gtgcctggct ttaggaatgg atgstccacc atgatgcaga gcaataatac
4140aaatgtgcac tcatctgccc catctgtgca agtttcatca ccttcgtctg
tgttaatgtt 4200ccagcaagca atgaatccag ttgcggaatt caactcggta
tacaatggcc ataaccaaga 4260ggaccataga gtaaatcgga ctccacatgt
cttggaacat gatggtggaa ggcaaacatc 4320atcctcattc ggtgaacgta
acttcagcag ggaagatcgt ggaggcacac attcttacaa 4380tcagcatggt
atttcacctc atccagttat aagtcaatca acaattagtg gcagccagga
4440ttctgtttca ccaatcaaag gtagctgtag actctttggt ttctcattgt
ccgaggacaa 4500atgtgtcccg gatcaagagg gcaaccccaa tgttggagtg
cagtttcatt caaagcctcc 4560tttgatgacc tcaacagttg gaataacctg
tactaaagta agcaacctct ttgctgcatg 4620aaatacaatt ttcaaatgtt
acgactgggg tatttaatct tctatttgac ggaagcaaat 4680gattatctga
ttagcaagtg ggatgtttat gtctaggatt gggcttttga ctggcgcgga
4740gaaaggatgg aaagctgttt acaaggatag tgatcatcct gtgcttgttg
cagatgatcc 4800gaggcagtaa ctagtaagtc ctcgtacgtt tacttattgt
atttgyctat ttttgttctg 4860tttgcaacct agcatcggaa tctacatggc
attcttcatt gcccgtctat tgtcaaaact 4920tacatatggt atgcctygaa
gtccgcttat aaaccctacc catggaactc atcatggcca 4980tgtttgtcgc
cgcactatat ccttccaaag tcgttttaag taaaccggat gaacatgaag
5040aagatgcttg tgttccggtc atttatttgt ttgaatttcg cttttggcag
gcgatgttac 5100tgtaatatca atagttttgc gttgtaccga tgaagaaaac
tctcgacagg cggaggtttt 5160cttcgggatt atgtgtgtgc tgtcatttat
gtaacttaat tttgaggcag aagttgtaac 5220taagcgctcc aggggacttt
gatttgtatt gttaattcag ctttgagaac aaaagtttgt 5280agaatctata
tttaatattg cttawccctc atatgtaamc ctgttgaaag agggcttgtc
5340tgccaaggca caatmctccg atctataaca tttctttatt ttgaaattag
tgtatccgag 5400tctttgaccc gactaattac taaacccytg cctgcctgat
tgcagaacat ttggggagcg 5460agaraattta gcatttgcta ggtgggtatc
gtgggagagt 5500162367DNAArabidopsis thaliana 16taatgtctct
ctctccacgc acaaaaggtc taaaagccac accacacaca tcagtcacca 60gacgtagcag
agagcctcac tgttgcagag agcactcagt actgttctgt ttctctgata
120cctctctctc tcctctctct tttaacattg tccaaattaa aaatctaaac
tttttttcta 180gttttttttt tttctttaat agaaaagttt tttttctcca
cggcttaaag actcactcat 240cactgtgcta ctactctctc ttcttttggc
tgagagggta aaagtcatga agaaactcct 300ctgagttttt tttctttctt
tcttataata aagctcttat ctttatctct gtttctctct 360ctttaatggg
tggtttaatc gatctgaacg tgatggagac ggaggaagac gaaacgcaaa
420cgcaaacacc gtcttcagct tctgggtctg tctctcctac ttcgtcttct
tcagcttctg 480tgtctgtggt gtcttcgaat tctgctggtg gaggggtttg
tttggagctg tggcatgctt 540gtgctggacc ccttatctct ctaccaaaaa
gaggaagcct tgtgttgtat ttccctcagg 600gacatttgga acaagccccc
gatttctccg ccgcgattta cgggctccct cctcacgtgt 660tctgtcgtat
tctcgatgtt aagcttcacg cagagacgac tacagatgaa gtttatgctc
720aagtctctct tcttcctgag tcagaggaca ttgagaggaa ggtgcgtgaa
ggaattatag 780atgttgatgg tggagaggaa gattatgaag tgcttaagag
gtctaatact cctcacatgt 840tttgcaaaac ccttactgct tctgatacaa
gcacccatgg tggtttctct gttcctcgcc 900gagctgctga ggattgcttc
cctcctctgg actatagcca gccccggcct tctcaggagc 960ttcttgctag
ggatcttcat ggcctggagt ggcgatttcg ccacatttat cgagggcaac
1020ctaggaggca tttgctcact accgggtgga gtgcgtttgt gaacaagaag
aagcttgtct 1080ctggtgatgc tgtgcttttc cttagaggag atgatggcaa
actgcgactg ggagttagaa 1140gagcttctca aatcgaaggc accgctgctc
tctcggctca atataatcag aatatgaacc 1200acaacaattt ctctgaagta
gctcatgcca tatcgaccca tagcgttttc agcatttcct 1260acaaccccaa
ggcaagctgg tcaaacttca taatccctgc accaaagttc ttgaaggttg
1320ttgactatcc cttttgcatt gggatgagat ttaaagcgag ggttgaatct
gaagatgcat 1380ctgagagaag atcccctggg attataagtg gtatcagcga
cttggatcca atcaggtggc 1440ctggttcaaa atggagatgc cttttggtaa
ggtgggacga cattgtggca aatgggcatc 1500aacagcgtgt ctcgccatgg
gagatcgaac catctggttc catctccaat tcaggcagct 1560tcgtaacaac
tggtcccaag agaagcagga ttggcttttc ctcaggaaag cctgatatcc
1620ctgtctctga ggggattcgc gccacagact ttgaggaatc attgagattc
cagagggtct 1680tgcaaggtca agaaattttt ccgggtttta tcaacacttg
ttcggatggt ggagccggtg 1740ccaggagagg ccgcttcaaa ggaacagaat
ttggtgactc ttatggtttc cataaggtct 1800tgcaaggtca agaaacagtt
cccgcctact caataaccga tcatcggcag cagcacgggt 1860tgagccagag
gaacatttgg tgtgggccgt tccagaactt tagtacacgt atcctccccc
1920catctgtatc atcatcaccc tcttccgtct tgcttaccaa ctcgaacagt
cctaacggac 1980gtctggaaga ccatcacgga ggttcaggca gatgcaggct
gtttggtttc ccattaaccg 2040acgaaaccac agcagttgca tctgcgacgg
ctgtcccctg cgttgaaggg aattccatga 2100aaggtgcgtc agctgttcaa
agcaatcatc atcattcgca aggaagggac atctatgcaa 2160tgagagacat
gttgctagac attgctctct agaagggttc tttggtttct gtgttttatt
2220tgcttgtggc ttaagtaaag ttcttatttt agttgatgat gacttgctgc
taacttttgg 2280aatgtcacaa gttgtgactt atgagagact tgtaaacttg
gttcaagaat gttctgtgtt 2340aggttcaatt taaaaagtgt ttgcatc
2367173710DNAArabidopsis thaliana 17taatgtctct ctctccacgc
acaaaaggtc taaaagccac accacacaca tcagtcacca 60gacgtagcag agagcctcac
tgttgcagag agcactcagt actgttctgt ttctctgata 120cctctctctc
tcctctctct tttaacattg tccaaattaa aaatctaaac tttttttcta
180gttttttttt tttctttaat agaaaagttt tttttctcca cggcttaaag
actcactcat 240cactgtgcta ctactctctc ttcttttggc tgagagggta
aaagtcatga agaaactcct 300ctgagttttt tttctttctt tcttataata
aagctcttat ctttatctct gtttctctct 360ctttaatggg tggtttaatc
gatctgaacg tgatggagac ggaggaagac gaaacgcaaa 420cgcaaacacc
gtcttcagct tctgggtctg tctctcctac ttcgtcttct tcagcttctg
480tgtctgtggt gtcttcgaat tctgctggtg gaggggtttg tttggagctg
tggcatgctt 540gtgctggacc ccttatctct ctaccaaaaa gaggaagcct
tgtgttgtat ttccctcagg 600gacatttgga acaagccccc gatttctccg
ccgcgattta cgggctccct cctcacgtgt 660tctgtcgtat tctcgatgtt
aagcttcacg tatgtaacta actctctctt tctttctatt 720ttttgttttg
ttttgttttc ttcatttatg ttttctcctc tgctctcaaa gcagagagat
780atgggttttg ttctgttttt ctgattcttt gattttttta attgtttgtt
tggtgaatct 840gagttgggtt ttcgatacaa gtatggagat ttgtgccttt
ggtttattga attgtttgag 900acaaacgaat ttatgttggg agaaaagttt
cctcttttgc tccatttgca tttcttctcg 960tggcattttg atgacgaata
cttgaaatcc ccataaatta tcttcagttt tttctttgat 1020gataatgaat
ttgatttcaa agtttcgcct tttgctccat tttgatgaca attgatttca
1080gaacaattca attctctgta aaggtttaaa ctttttttgt tgttgtgagg
attaataaac 1140aaaatgtggg ggattttgat ttcgtaggca gagacgacta
cagatgaagt ttatgctcaa 1200gtctctcttc ttcctgagtc agaggtgagt
ttttctttag gctcttgagt tttgtaacaa 1260agagagagaa atttgctcga
gcttaggggg tttgagttga tttgttacag gacattgaga 1320ggaaggtgcg
tgaaggaatt atagatgttg atggtggaga ggaagattat gaagtgctta
1380agaggtctaa tactcctcac atgttttgca aaacccttac tgcttctgat
acaagcaccc 1440atggtggttt ctctgttcct cgccgagctg ctgaggattg
cttccctcct ctggtactaa 1500tccactctct gtagattctg taatcagctt
tgtacattgc acattgtgtt ctagagttct 1560cttattagct catattgaga
gattttaact acataatcat tttgttatgt aggactatag 1620ccagccccgg
ccttctcagg agcttcttgc tagggatctt catggcctgg agtggcgatt
1680tcgccacatt tatcgaggta agtttgttgc cttatgttgc aatttttctt
gcctggattt 1740agtagatgga aaatttgaat ggtttgagtg acttttaggg
caacctagga ggcatttgct 1800cactaccggg tggagtgcgt ttgtgaacaa
gaagaagctt gtctctggtg atgctgtgct 1860tttccttagg tagttaaaaa
tgcttctatg ttttctcact acacaacctc tttgattttc 1920tgtaagaggt
tttgtgatat atgggtttct ctgatacaga ggagatgatg gcaaactgcg
1980actgggagtt agaagagctt ctcaaatcga aggcaccgct gctctctcgg
ctcaatataa 2040tcagaatatg aaccacaaca atttctctga agtagctcat
gccatatcga cccatagcgt 2100tttcagcatt tcctacaacc ccaagtaagc
cccaaccaca atgacctttt ttcgttttca 2160gcatttaaaa tttcatttca
gaatcataat tagaatctcc tgaagcctta agttgtgtat 2220tgttctagtt
gattgttcct tagaagtttg ttaactccaa taaatatcag gaatttagca
2280ttaatactag ttcactggta aaacattttc agggcaagct ggtcaaactt
cataatccct 2340gcaccaaagt tcttgaaggt tgttgactat cccttttgca
ttgggatgag atttaaagcg 2400agggttgaat ctgaagatgc atctgagaga
aggttactta tagacttata attcaagctt 2460taagataacc ttgcacacct
gtgttttata tgcccaactt tctaacatgt ttcacctgtt 2520ttgtcagatc
ccctgggatt ataagtggta tcagcgactt ggatccaatc aggtggcctg
2580gttcaaaatg gagatgcctt ttggtaagct atgaattatg ttcttaagtc
attagtttgg 2640tctgagaggt cttctataag attgttgcct tttctatatc
cgtaagctca ctgctctgaa 2700actatgtttt gacgtcatat tcaggtaagg
tgggacgaca ttgtggcaaa tgggcatcaa 2760cagcgtgtct cgccatggga
gatcgaacca tctggttcca tctccaattc aggcagcttc 2820gtaacaactg
gtcccaagag aagcaggatt ggcttttcct caggaaagcc tgatatccct
2880gtctctggta cacatctact tagccaaaga cattgtacca ctcatataac
catttatcgc 2940ctgtaatata acgttttctg ctattattgc agaggggatt
cgcgccacag actttgagga 3000atcattgaga ttccagaggg tcttgcaagg
tcaagaaatt tttccgggtt ttatcaacac 3060ttgttcggat ggtggagccg
gtgccaggag aggccgcttc aaaggaacag aatttggtga 3120ctcttatggt
ttccataagg tcttgcaagg tcaagaaaca gttcccgcct actcaataac
3180cgatcatcgg cagcagcacg ggttgagcca gaggaacatt tggtgtgggc
cgttccagaa 3240ctttagtaca cgtatcctcc ccccatctgt atcatcatca
ccctcttccg tcttgcttac 3300caactcgaac agtcctaacg gacgtctgga
agaccatcac ggaggttcag gcagatgcag 3360gctgtttggt ttcccattaa
ccgacgaaac cacagcagtt gcatctgcga cggctgtccc 3420ctgcgttgaa
gggaattcca tgaaaggtgc gtcagctgtt caaagcaatc atcatcattc
3480gcaaggaagg gacatctatg caatgagaga catgttgcta gacattgctc
tctagaaggg 3540ttctttggtt tctgtgtttt atttgcttgt ggcttaagta
aagttcttat tttagttgat 3600gatgacttgc tgctaacttt tggaatgtca
caagttgtga cttatgagag acttgtaaac 3660ttggttcaag aatgttctgt
gttaggttca atttaaaaag tgtttgcatc 3710182935DNAPhaseolus vulgaris
18ccatgataaa ataacatttt gcttgtgaaa tgttactggt tgttttggtt acatccaact
60tcattgatgg tgtcagtgtt ctgtaagtga gaaaattgaa ttttggagtg gtttggttaa
120gagagatggt taaatgtgga ggataacggt agagtgagtt ggtaagagag
tgggaatgaa 180agggtttccc aaaaagcgaa ccccataatt gtgtgaggag
tgaagagccc aaaaagggtg 240ggaaaaatga agagagagtg attggtcagt
gggagagaaa gacagagaca gagaaagcgg 300gtctctttct ttttggagta
ttgggtcagg ttcttttggc aatgccaaag ttggtcacgc 360tctctgaaaa
tgcaggaggc atgaaaccac tccttcaaat atctcccacc actcactcgc
420tcctgtgctg cttcttcttc tccttctgtt tctgttcctt ctctctctat
ctccctcttt 480tctcctcctc ctcctcatgc ctgccctcat cgatctcaac
agcgccaccg aggaccatga 540aacgccgtcg tctcgcccat cctccgtctg
cctcgaactc tggcacgcct gcgcgggtcc 600tatgatctcc ttgcccaaga
aagggaccct tgtcgtgtac ttccctcaag gacacttgga 660acaacacctt
cacgattttc cgctccctgc ttctgctaac atcccctccc atctcttctg
720tcgcgttctc gatgtcaagc tccattctga ggaagggagc gatgaggtgt
attgccaggt 780ggtgctggtt cccgaaagtg agcaagggca tcagaagttg
cgggaagggg aaattgatgc 840tgatggtgaa gaggaggatg ctgaagctgt
gatgaagtcc accacacccc acatgttctg 900caagactttg acagcttctg
atactagcac tcatggcgga ttctctgtgc ctcgtcgtgc 960tgcggaagat
tgttttccac ctctggatta cagtcaacag agaccttcac aggagcttgt
1020ggcgaaggat ctgcatggcc aagaatggag
gttccgacat atttataggg ggcaaccacg 1080acgacacttg cttaccactg
ggtggagtgc atttgtgaac aagaagaaac ttgtatctag 1140agatgctgtt
ctgtttctta ggggtgagga tggagaactg agattgggaa ttcgtagggc
1200tgctcaattg aaaagtggca gtaccatttc aacttttgct ggccagcaat
tgaatcatag 1260cagtcttctg gatgtggtta atgctttatc agcaagatgt
gcctttagtg ttcactataa 1320tccaagggtc agttcatctg agttcatcat
acccattaag aaattcttga ggagccttga 1380ttattcttat tcagttggaa
caagatttag gatgcgtttt gaaactgaag atgctgcaga 1440gcgaagattt
acaggattga ttgttggaat tactgatgtg gatcctgtta gatggcctgg
1500atcaaaatgg agatgcctaa tggtaaggtg ggatgacctg gaagccacaa
ggcataatag 1560ggtttcaccc tgggagattg agccatctgg ttctgcatct
actgcaaata acatgatatc 1620agctggtttg aagaggacca agattggatt
gccttcaacc aagctagatt ttcaagtttc 1680caatgcaatt ggagcatcag
actttggcga atcactaagg ttccagaagg tcttgcaagg 1740tcaagaaatg
ttgggtgtta acacaacttt tgatagtact aatggtcagg gtcaccagct
1800atcagatttg aggagatgct atcctggctc aaactgttct aggattgctg
caaccggaaa 1860cagcattgga attccgcaag tgagttccaa tgtttcctgc
aatggcatag gcttcagtga 1920atctttcaga ttccagaagg tcttgcaagg
tcaagaaata cttccaagcc aaccatatgg 1980aagggccctg tctgttgatg
aggcttgtgg aaatggtcgc tttggacctt ttgatggtta 2040ccatacactg
agatccagaa atggatggtc ttcccacttg agtaacagtt cttcacattt
2100gcatccacct gttccatctg ggcaagtttc atctccatca tctgtgttaa
tgttccagca 2160agcacacaat ccagtttcaa actctgatta caacagcaaa
attagtcagg tgatggaagg 2220taaagtccag caacgatcat catacacttc
tgaagctaaa ggtggaaaat ttgtatcaac 2280cccttatgag cctcttcgtg
gactagctca ggaaggcaca aattcttatg gggtctcaaa 2340cttgcacaat
cagtttgaaa cttcacgttc acacgattct atttcagcac ttcgggctac
2400tcaagagttg gttcccacat gtaaaagtcg atgcagagtc tttggcttct
cattaactga 2460gggtgctcct gttgcaagta aagaagtagc cggcaccgac
ccatcggccg tcacatgttc 2520tggaccttcc tttgcaagac acgctgaaga
tgatttccat ccagtgcata gcaaggcagt 2580gggaagttat tgcaccaaag
gtgtgctgca atattgactt gaaaatcatg gtgtatggta 2640gtagttatgc
tgtcataagg tggcagaaga gaatgttcac tgttgtacta atgtggagaa
2700tatgataact attcgcctaa ctagatattt atcttgttaa attggctgtg
acacaatcaa 2760tttctgtatt aatctatgta ctctttattg acttgtaaaa
cgatgcatgt gtgttcactg 2820ttatggccat atgaggctct ggtggcactg
cataaccctt catttatctt gaattgggca 2880tgattacttt agggaactat
agctcatcat gtttcatgac cttaagttat ctcca 2935192762DNALycopersicum
esculentum 19aattcgccct tgagtgcgca gttgaactag caaaagggtt taaagatgat
gtgtggactt 60attgatctga atactgtgga taacgatgat gccggagaag aaacgacggc
gccggtgtca 120ttggattcac cggcgtcgtc gtcggcggca tcaggaagtt
cggatttaac gtcgtcaact 180acgccagcgg tggcatcggt gtgtatggag
ctttggcatg cgtgtgctgg accgttgatt 240tcgctgccga agaaaggaag
tgcggttgtg tacctgcctc aaggtcactt ggaacattta 300tctgagtacc
cgtccatagc ctgtaatctc cctcctcatg tgttttgtcg cgttgtagat
360gtgaagctac aagcagatgc ggctactgat gaggtctatg cacaagtctc
actagttcct 420gacaatcagc agattgagca gaaatggaag gatggagaca
ttgatgctga tattgaagaa 480gaggaaatag aaggtgctgg aaaatcaata
acaccacaca tgttctgcaa aactcttact 540gcatcggata ccagcactca
tggtggtttc tctgtccctc ggcgggcagc agaagattgt 600tttgctccct
tggattacag acaacagagg ccctcgcagg agctggtagc caaagatcta
660catggtatag agtggaaatt tcggcatatc tatcggggtc agccacggcg
gcatctgctc 720actacaggat ggagtgcatt tgtaaacaag aagaagcttg
tttctggtga cgctgttctt 780ttcttgagga ctggtgatgg agagcttagg
ttaggagtga gacgagctgc ccaagcaaaa 840acatgttcta gttatctggc
tccttgtagc aaaccgttga atgttagtgg cattgtagat 900gctgttaacg
ttatatctag cagaaatgct ttcaacattt gttacaaccc aagggatagc
960tcatcggatt tcattgtacc ttaccacaaa ttctctaaga ctcttgcaca
tcccttttca 1020gctggaatga ggtttaaaat gcgtgtcgaa acagaagatg
cagctgagca aaggttcact 1080ggactagttg tgggagtcag caatgtagat
ccagttcgat ggccaggttc taaatggagg 1140tgcctattgg tcagatggga
tgatcttgat gtttctagac ataatagggt ttcaccatgg 1200gagattgagc
catctggttc agctcctgtg cccagcagct tggtgatgcc ttctgctaag
1260aggaccaggg ttggcttccc tatttcaaag gcagattttc caattcctag
agaaggaatt 1320gcagtatcag actttgggga accttctagg ttccagaagg
tcttgcaagg tcaagaaatt 1380ttgaggatgc atgctcctta tggcggactt
gatgctcgga gtcctcgtcc agcaggcaca 1440agatgctttc ctggttttcc
tagttctggg atatctagaa tgggaaacag catcagaccc 1500ctgtttggtg
acactgacaa gtcccatgaa agcattggct ttagtgaatc tcttcgattc
1560aataaggtct tgcaaggtca agaaattttt acaagccctc cttatgggag
agctcaagct 1620ggtatccaaa tgcaggagaa aagtaggacc ggtatttttg
tcggtattca ggttccaaac 1680catggaaaca ggtggcctgc tccaaatcag
gataataaca ctccttgcaa gccaattaat 1740cctgtctcag catcatcacc
gccttctgca ctcaattttc agcatccgag ccctccagca 1800tcaaagttcc
aggctatgtt caatcataaa catgatcttg ttaaccaggc ttcgttagat
1860ctgtctgaga actgttgtag gtatccgtat ctctcatctg gttcacatac
cgaggacatc 1920agtcagaagg aaggtactca aggaatcagc tcgtttggtt
tcttaaagga gcaaaagcaa 1980acaggacttt catatctttc tcctgggaca
cagtcgtcat tcaaaggcaa tcaaaactta 2040gtttccactt gtaaaactgg
ttgcaggatc tttgggttcc ccttgaccga gagtaaaata 2100agtgcaacta
gagcggatac tccctctgaa gctgtatact cacatggtct agaaactaca
2160tttctccctt ccagtgatgg aaagttgcag ccggggccac cattgatgac
taacgttgtg 2220gggacaaatt ttaccaaagt aaatgacctc tatgcagcaa
gagatgtgct tcttgatatt 2280gctctgtagc aggtgtttgt tgtgaggttg
tgctagaata tgtagactga aggatgtgtg 2340tgcagcatta ttgattatta
gcttttagtt ggcgttgtaa tcttctggct gttgagtgcg 2400caagcatttg
gttgccagta gaatgcttat ccagagatga gaattgagag ttattaatga
2460agattgatac cgttgaggaa cgtatgttct tgaaaatttg gtgtatatgt
tcctgtgacg 2520ctgatgtact atgtaacaat tggaagctgt gtttgctgca
tcaaagatgt ctgtatgata 2580gttgtactct acttgagatg acttctgtat
ttgtatattt acctagtcta gatttgctgt 2640gaactaactc gagctcctat
aaatcggtaa gtttgttgta ggagctctcg tctcaggaac 2700acaatactgt
actgaatttt gtaaggaatt gtcatgtata ttcctgcaat taagggcgaa 2760tt
2762202622DNACitrus clemantina 20ttgacgaagt tgcagcagcc agcagcactt
aaaacacttg cctctaaaat gcagtcatga 60aactctctct ccttctcgct ctcaaaagca
cttgtttttc acaacttttt cttctcagct 120tccactacaa caccattgta
tcgttctaag catctctcta aaatggtggg tttgattgat 180cttaacacaa
cagaagacga tgagaatccg tcatcgggat ctttatctcc gtcctcttct
240tctgcttctg ccttgagtgc ttctggtttc gctttagctc ctgcttctgc
ttctgcttct 300ggggtgtctt tagagctatg gcacgcatgt gcagggccac
taatatctct gcccaagaga 360ggcagtgtgg tcgtttactt ccctcaggga
catttggagc atgtctccga tttttccgcc 420gctgcttcag ctgcttatga
tctcccccct catctgtttt gtcgggttgc tgatgtcaag 480ctccatgcag
aggcggcaag tgatgaggtt tatgcgcagg tctcactggt tccagatgag
540ctaattgagc agaaggtgcg tgaagggaaa attgaggagg atggtgatga
ggagagtgtt 600gaggtggttg ctaagtcttc aacaccccac atgttctgca
agaccctcac ggcttctgat 660actagcactc atggaggctt ctctgtacct
cgtcgagctg cagaagactg cttccctccc 720ctggactata gtcaacagag
gccttcacag gagcttgtgg caaaggatct ccatggcctg 780gaatggaggt
tccggcacat ttacaggggg caaccacgga ggcatttgct gactactgga
840tggagtgcat ttgttaataa gaagaagctt gtttctggag atgctgtgct
tttccttagg 900ggtgaagatg gtgaattgag acttggaatc cgaagagcac
ctcatgtaaa aagtggtgct 960actttccctt ctttctgcag ccaacagtcg
agtcccaatt ctgtcacaga ggtggttgat 1020gccatagcta ggaagcgtgc
tttcagcatt tcctacaatc caagggccag cgcctcagag 1080ttcataattc
ctgtcaataa gtttttgaag agccttggtc attctttcgc tgttggaatg
1140aggttcaaaa tgcgttttga aacagaagat gcagcagagc gaagatacac
tggagtgatt 1200atgggagtcg gtgacatgga tcctgtgaga tggcctggtt
caaaatggag atgcctgttg 1260gtgagatggg atgatgttga gtccaacagg
cacaccaggg tatctccatg ggaaattgag 1320ccatctggtt ctgtttgtgg
ttccaataac ctgatcacat ctggtttgaa gaggaccagg 1380attggattgc
cttctgggaa accagaattt ccagttcctg atggaattgg agtgacagac
1440tttggggaat ctttgaggtt ccagaaggtc ttgcaaggtc aagaaatatt
aggttttaac 1500actctttatg atggtggtga ttgtcagaat ctgcatccat
ctgaagtaag gaggggcatt 1560cctggttcaa atggttctgg gattgctgct
ataggagatg gtagcagaaa cctgcaggtg 1620aaatctgaca tttcctacaa
aggcataggc ataggctttg gtgaatcatt ccgattccat 1680aaggtcttgc
aaggtcaaga aatatttccg aagtctccat atggaagagc ccctactaat
1740aatgaggctc gtagtattgg cagccttgga atctctgatg gtgttccggt
atctggatca 1800agaaatagat ggtctgctgt ggtgccgggc tataacactc
atatgagccc atctgcaccg 1860cctgtacaag tgtcatcacc ttcttcggtg
ttaatgtttc agctggcaag caatccaatt 1920tctaactata atcctcctta
tagcttgaac gatcaggaga aagagcaacg tgtcaactgt 1980caaagctttt
ttcataattc tgaaatatat ggaggaaagc atgcatcatc ttcatttctt
2040gaccatagtt tcgtgggggg tgatcaggag gtcatggatt ctataggtca
gtcaaatgag 2100catatttcac cacctcttgt aggtcagcca acagttaggg
gcagccaaga tttagtttcc 2160tcgtgtaagg gtagctgcag actctttggt
ttttcattga ctgaggaaag acatgttgcc 2220aacatagagg acaatgcagc
tccagttgcg tctcctttga atcctagatc ttcttttctg 2280tctcatgttg
gacagcagtt ccatccaaag cctccagtaa tgtctaaggc aactggaagc
2340aactgtacca atggaatcat gcaacattgt cttggaaatt atgatatata
ctaaccaagg 2400tgcagaggta gcacctgggc ttgcagaaga aaatgcttgt
aattgctcta attatacata 2460tgctgtagta aatgatacaa tttaattagc
tggtgagaaa accaagtgta agtatttttt 2520tcaaagtaac ttgatgtcat
gattatgctt aactccacta tggaaagcaa acaacatatg 2580tattgtatta
atctattaag tttccagcgg atgttgtcta tt 2622212917DNAFragaria vesca
21ctttcactgt ctttctctgc cacacatcac acccagcagt tgcagcgttg cagaggctgc
60agcactgcag cagagctgca gagagagaca gactcaaaga aataatataa aaattcgcaa
120ggaaaaagat aaaagagaaa gaattcttta aaaaaaacaa atttttaagt
cttttgagta 180ttctattggg ttgggtttgg gtctgtcaaa gtttttaaga
ttaaagctct gagcttttta 240ttcacataca ccaaaaaaga gtgtgtcttg
gtttcttttg ggtgtgtttc ttttagcaaa 300tgccttaaaa atgcacaagt
gaaaccagtt gggttgtagt ttgagctttg acacataaaa 360aggcttgagc
tttagctttg tctgttgtgt tgtttagagt tttgggttat ggcgggtctg
420atcgatctga acagcacgac ggaggaggag gaggagacgc cgtcatctgg
gtcgtcgtcc 480aattcgtctg gctcaaatgg tttgatttct gggtctgttt
gcttggagct gtggcacgcc 540tgtgctgggc cactgatttc tttgcccaag
aaaggtagtg tggtggttta tcttccacaa 600gggcacttgg agcaagtgag
tgattttcca gcctcggttt atgatctccc tgctcatctg 660ttttgccgag
ttctggatgt taagcttcat gcggagagtg gtagtgatga agtgtacgca
720caggttcagt tggttcctga aagtgaggaa tttgagcaca aactagggga
aagagaaact 780gttgcagatg gggacgagga tgctgagggt tcagagaaat
caactacacc ccatatgttc 840tgcaaaaccc ttactgcttc tgatactagc
actcatgggg gcttctctgt ccctcgccgt 900gctgctgagg attgttttcc
tcccctggat tacagtcaac aaaggccttc acaggagcta 960gtggcaaagg
atctgcatgg cctggaatgg aggttcagac atatctatag ggggcagcca
1020cgcaggcatt tgcttaccac tggatggagt gcctttgtga acaagaagaa
gctcgtttct 1080ggagatgctg tgttgtttct caggggtgag gatggagaac
tgagacttgg agttagaagg 1140gcagcccaag taaaagcttc tgccacttat
ccaactcctg gtagccagca tttaaactat 1200aactctgtca cagagctggt
ggatgctata tctacgaaga ctgcttttaa cgcctattac 1260aatccaagag
ccagctcatc agaatttata atacctttcc gtaagttttt gaggagcctt
1320ggtcattcct tctgtgctgg aatgagattt aaaatgcgct ttgaaacaga
agacgccgca 1380gagcaaagat acactggact ggtaacgggg attagtgagt
tggatcccct aagatggcct 1440ggttccaagt ggaaatgtgt agctgtacgg
tgggatgata tagatactag caagcagcat 1500ggccgggttt ccccatggga
aattgagcca tctggttcta tttctaattc cagtggctta 1560atggcatctg
gtctgaagag gtccaggatg ggcttatctg cagaaaagca agaatttcca
1620gttcctcatg ggattggagc ctcagacttt ggggaatctt taagattcca
gaaggtcttg 1680caaggtcaag aagtttcggg ttttgatact ccttttggtt
ctataggggg tcaaaatcag 1740catccctctg aatcgaggag agtctttcac
ggctctattg gttctagagg taatgatctc 1800agaaactcat ttgtgaattc
tgagattgcc tcaaaaggct ttggtgaatc tttccgattc 1860cagaaggtct
tgcaaggtca agaaatattt ccaagcacac catacggaag agctccagct
1920actaatgagg ctcgtgaata tggttgccct ggaatctttg atggttttca
ggtgccaagc 1980tttagaaatg gatggtctac gatgatgcag ggcagtaata
cacctatgca ccgagctgcc 2040cctgtacagg tgtcatcacc atcatctgtg
ctgatgttcc agcaagcaat aaatgcagga 2100gccgagttca attcagtata
caatggtcat aaccaacagg aacagagaat aatgcaacgc 2160actcattctg
aatcagatgg tgggaagcaa acatcagcct cgttctgtga acgaagcttc
2220accagggagg gtcatggtgg catgaattct tttgatcaac atggtatttc
acatcctcct 2280cttttgagtc agtcttcatt gagaggcagt caagatatgg
tttcatcatg caaaagtagc 2340tgcagactgt ttggtttctc actgtctgag
gaaacacatg ccccaaataa agtggacaac 2400tccacctcag ttacatctgc
attagagtct ggagcttcta tgttccccaa tgttgaacca 2460cggtttcatt
caaagccgcc ttcgatgtct gcagctgttg ggattccttg taccaaagaa
2520tgggcattta actggcgtgg agaaaggatg gaaagttgtt tacaaggata
gtgacaacga 2580cacagagctt gttgtgacgt tccacccaga aatgctacca
caatattatg taagaaacac 2640taggctagaa gatgtagtct ttgcgcccga
ggattttcgc aggttatgcg tgtgttgtat 2700tttatgtaac tgaactacat
ttgaaggtaa aaaaaaagaa aaaaagaaga aggtttgatt 2760aagcgcttct
gggtgacttg attgtattat gatgaattaa gctgtaagaa acggtttgca
2820gaatctatga ttgcctcttg aaaaaggggc ctatccgaaa ctgaacaaat
tgtaccgatt 2880tgcaattgtt aaataatgtt attacttatc gtcccta
2917222215DNAPrunus persica 22atggggggtc taatcgatct gaacagtgca
acggaggacg aggaaacgcc gtcgtctggt 60tcgtcttcaa cttcctctgc ttctgacgct
tcggcttcgg cttcggcttc ggtgtgcttg 120gagctgtggc acgcgtgtgc
gggcccactg atttcgctgc caaagaaagg gagtgtggta 180gtgtatcttc
cacagggcca cttggagcaa gtctctgatt ttccagcttc ggcttataat
240ctcccacctc accttttctg tcgcgttgtt gatgtcaagc tccatgctga
gactggtacc 300gacgatgtgt atgcgcaggt ttcacttgtt cctgaaagtg
aggaaattga acacaaactg 360cgggaagggg aaactgatgc atatggtgag
gaggaggatg tcgaagcaat tgggaagtca 420accacacccc atatgttctg
caaaaccctt actgcttctg atactagcac tcatggaggc 480ttctccgtcc
ctcgtcgtgc tgctgaggat tgttttcctc ccctggatta caatcaacaa
540aggccttcac aagaactcgt agcaaaggat ctgcatggcc tggagtggag
gttcagacat 600atttataggg ggcagccacg aaggcatttg ctcaccactg
gatggagtgc atttgtgaac 660aagaagaagc tcgtctctgg agatgcagtg
ctgtttctca ggggtgatga tggagaactg 720aggctaggaa ttagaagggc
agcccaggtt aaagggtccg ctacttatcc aactctttgt 780agccagcaat
taaactataa cactatcacg gacgtggtga atgctatatc catgaagaat
840gcatttaaca tcttctacaa tccaagagcc agctcatcag aattcataat
accttcccgt 900aaatttttga ggtcccttga tcattccttt tcacctggaa
tgcggttcaa aatgcgtttt 960gaaacagaag atgcagcaga gcgaagatac
actgggctga taactggaat tagtgaattg 1020gatcctgtaa gatggcctgg
ttcaaaatgg agatgtctag ttgtaaggtg ggatgatata 1080gacacaagta
agcatggcag ggtttcccca tgggaaatcg agccatctgg ttctgtttca
1140agttcccata gcttaatggc agctggtttg aagagggcca ggagtggctt
gtctgcagca 1200aaaacagaat ttccagttcc taatgggatt ggagcatcag
actttgggga atctttaagg 1260ttccagaagg tcttgcaagg tcaagaaatt
ttaggttttg atactcattt tggtggttta 1320ggtggtcaga atcaacatcc
atctgaacca aggaggggtt ttcatggttc tagtggttct 1380gggattgctg
ctggaggtaa tggtctcaga aagtcacttg cgcactctga gattacctca
1440accggcatag gctttggtga atcattccga ttccataagg tcttgcaagg
tcaagaaata 1500tttccaagcc caccatatgg aagagcttcc actaataacg
aggctcatga atatggtggc 1560cctggaattt atgatggttt tcaggtgcca
agctttagaa atgggtggcc tgccatgatg 1620cagagcaata atgcacacgt
gcgcccatct gcctcgtctg tgcaagtttc atcaccatcg 1680tctgtgttaa
tgttccagca agcaatgaat ccaggcccgg aattcaattc agtatacaat
1740ggtcataacc aggaggaaca gagagttata aaacggactc catatgtctc
tgaatcagat 1800ggcggaaagc aagcatcatc ctcattttgt gaacgtagct
tcagcaggga agatcatgga 1860ggcatgaatt cttacaatca acatggtatc
tcaaatcatc ctgtaataag tcaatcaaca 1920tttagtggca gtcaggatgc
ggtttcacca tacaaaggca gctgtagact ctttggtttc 1980tcattgtctg
aggaaaaacg tgtcccagac agagagagca actccacctc aactgcatct
2040acattaaatc ctggagtgca gtttcattca aagcctgcat tgatgacatc
agcagttgga 2100attacctgta ccaaagaatg ggcttttgac tggcgtggag
aaaggatgga aagctgttta 2160caaggatagc gatgatacaa tggcttgttg
cagaagatca aaggcagggt cttaa 221523669DNAPyrus communis 23attgcctcaa
aaggcatagg ctttggtgaa tcactccgat tccataaggt cttgcaaggt 60caagaaatat
ttccaagctc accatatgga agagctccca cttctaacaa agctcatgaa
120tatggtggac ctggagtcta tgatggtttt caggtgcccg gctttagaaa
tggatggtcc 180accatgatgc agagcaataa tacacatgtg cacccatctg
ccacatctgt gcaagtttca 240tcaccatcgt ctgtgttaat gttccagcaa
gcaatcaacc cagttatgga attcaattcg 300gtatacaatg gtcataacca
agaggaacat acagttataa atcgaactcc atatgtctct 360gaatatgacg
gtggaaggca aacatcatcc tcatttggtg aacgtaactt cagcagggaa
420gataatggtg gcacgcattc ttacagtatt tcaaacgatc cagttataag
tcgatcaaca 480tttagtggca gtcaggattc agtttcacca accaaaggta
gctgtagact ctttggtttc 540tcattgtctg aggacaaatg tgtcccggat
caagccccta ctgctggagt gcggtttcat 600tcaaagcctc ctttgatgac
ttcagcagtt ggaattacct gtactaaagt aagcaacctc 660tttgctgca
669243045DNAPopulus tremula 24atggaaagga agaagcactt gaaaaagaaa
agataagaga gacagatata gttcccatta 60atatctcttt ttctgtctct ctctctaaca
ctactgccac acatcgcatc cttgcagggt 120cttcacagca tggcagcatt
gcggcaggca ctgcatctca gttttgcaga tcatgagcaa 180ggaaagaaac
ccatgaaaaa ttgagaagaa aataaataaa aagttgaaag ctttaattta
240atttaattta atactagtac cctttaaagc ctttgatttg atatcttaaa
aaagcagaga 300gagacaaagg gtctctcttt ttaagagtct tgactctaat
ctccttttag gcaattgcca 360aagttgcact ataatgcagt catgaaatct
ctcctcgctc acaaaagcac ttgtctttta 420ataaaccttc attattgtta
tcaacagtta ctccttgtta ttcttcaaga actctacact 480gttcctgttg
ttactgcctt tgtttaggaa aggctataga gctgatcaag gctaaaaatg
540gtgggtatga tagatctcaa cactattgaa gaagatgaaa ctacaccgtc
ttgtgggtct 600ttatcttctc catcatcatc ctctgctgct tctgctttga
gtgcttctgg ctctggttct 660agtacctctt ctgtttgttt ggagctttgg
catgcttgtg ctggcccact aatatctttg 720ccaaagagag ggagtgttgt
tgtgtatttc cctcaaggcc acttggaaca actccctgat 780ttgcctcttg
cagtttatga tctcccttct catgtcttct gtcgagttgt tgatgtcaag
840ctccatgccg aggcagcaag tgatgaggtg tatgcacagg tctccctggt
tcctgagagt 900gaggaaattg agcagaagtt gagggagggg atatttgagg
gggatggtga ggaggaggat 960ggtgaagcca ctgtgaagat gacaacaccc
catatgttct gtaagaccct aactgcttct 1020gacactagca ctcatggagg
cttttcagtc cctcgtcgag ctgctgagga ctgcttccct 1080cctctggatt
atactcaaca aaggccttca caagagcttg tggcaaagga tcttcatggc
1140tctgagtgga agtttcgaca tatctacagg ggtcagccac ggaggcattt
gctcactact 1200ggatggagtg cgtttgtcaa taagaaaaaa cttgtctctg
gggatgccgt tctctttctc 1260aggggtgagg atggggaatt gagactggga
gttcgaagag cagcacaagt taaatgtggc 1320cctacatttc cagctcaatg
gaatcatcag ctgaatcaga tctctcctgg ggatgtagct 1380aatgctattt
ctactagaag ttttttccac atttactaca atccaagggc cagctcatca
1440gagttcataa taccttttaa taaattcttg aagagccttg atcaatcctt
ctcttctgga 1500atgagattca aaatgcgttt tgaaacagaa gatgcagcag
agagaagata cactggaata 1560ataactggag tcagtgagct agatcctgct
agatggcctg gttcaaaatg gaaatgcctg 1620ttggtaaggt gggatgatat
ggaggctaac aggctcagca
gggtttctcc ttgggaagtt 1680gagccttctg gttctggttc tttttccagt
tccaataact ttacggcacc tggtttgaag 1740aggagcaggt ctggattgcc
ttcatcaaag gcagaatttc caattcctga tgggatagga 1800gcaccagact
ttagggaatc ttcaaggtcc caggaggtct tgcaaggtca agaaattatg
1860agttttaatg ctctttatga tggtgttgat ggtcagaacc agcacccatc
tgaaataagg 1920agttgttttc ctggttacca cagttctggg attgctgcat
taggaagtgg tatcagagac 1980tcgattgcca cttcaaataa ctcctacaag
ggcataggct ttaacgaatc ttatagattc 2040cataaggtct tccaaggtca
agaaattttt ccaagctcac catatggaag aatcccaaat 2100gctaatgagg
ctcgtgaaaa ttgtagtctt ggattctctg atggtgtcca aaggtcaagc
2160tcaagctcaa gaaatggatg gtctacattg atgcagggct ataatactca
aattcgacct 2220cctgcacaag tatcatcacc atcttcggtg ttaatgtttc
agcatgctag caatccagtt 2280ccaaagccat cttccaattt taatttcaat
gatcatgtgc agcagacagc taccacccga 2340agttggtttt gtggtcctga
aatgcagggg ggggatttca agttgcctgc acattctgag 2400cccagtgtaa
aaagaggcgg ccagtggagc aatagtcctt ttggtctgtc ccatgagcat
2460cttcaacatg gtgtttcaca acctattgta gctcaatcag cctttagggg
tagtcaagat 2520ttggtgtcgt gcaaaagcag ctgcagactc tttggtttct
cattgactga ggataaatgc 2580cttgttaata aggaggacaa tatgacctta
ataacatctc cattgaatcc tggatcctcc 2640tttctgcctc gcgcaggaga
gcacttccat ccaaagcctc cagcaataaa taatgcagtt 2700gggagcagtt
gtaccgaagc aattctgcaa acccgtgctg aaaattatcg aatatactaa
2760tgaggctcgc acaagggatg cttcctgttg cttggtttta tatgtattag
cttgtgagag 2820aatataatta ttctcctaag gtaacttggc tatatcctaa
ctcctttgac tatgcaacag 2880agctgtttgt acctggtact aatctctgtt
agatttccca tgataaccca cattcaagaa 2940tgttctcttc atacagtgca
caatccaatc tggaaatgta gttgtaatag cgccagatat 3000tttatatggt
tgtcatctct caatatgttt tgttctatgc tagcc 3045252790DNAVitis vinifera
25atggtggcta tgatcgatct caacaccgtc gacgacgacg agacaccctc gtctgggtcg
60tcgtcttcct cctcctcatc cgcctctgct tctgcttcca cagtttgtgg ttctttgttg
120tcggcggcgt cgtcggtatg tttggagctg tggcacgcgt gtgctggccc
gctcatatcg 180cttccgaaga aaggcagcct tgtggtgtac tttccacagg
gccacctgga gcagctttct 240gattatccgg ccgtagccta tgatctcccg
cctcacgtct tctgtcgagt ggttgatgtc 300aagctccatg ccgaggtagt
tacggatgaa gtttacgcac aggtctcgct ggttcctgaa 360accaagcaga
ttaagcagaa actgcaggaa ggggaaattg aagcagatgg tggtgaagaa
420gaggatattg agggttctat caagtccatg acaccccaca tgttctgcaa
aactcttact 480gcttcagata ctagcaccca tgggggtttt tctgtccccc
gccgagctgc agaggactgt 540tttcctcccc tggattacaa acagcagaga
ccttcacaag agcttgtggc caaagatttg 600catggcttcg aatggagatt
ccggcatatc tacagggggc agccaaggcg gcatttgctt 660actactggtt
ggagtgcatt tgtaaacaag aagaagcttg tgtctggaga tgctgtactc
720tttcttaggg gtggggatgg agaactaaga ctgggaatcc gaagagcagc
tcaaattaaa 780ggttcgtctc ctttcccagc tctttgtagc caacagttga
atctcaacac ccttacagct 840gtggtcaatg ctatatccac aagaagtgtt
ttcaacatat gctacaatcc gagggctagc 900tcatcagagt tcataatacc
gctccgtaaa ttctcaaaga gcattgatca ttcattttct 960gctgggatga
ggttcaaaat gcgtgttgaa acagaagatg cagcagaacg aagatatact
1020ggactgataa ctgggatcag tgacatggat cctgttagat ggcctggttc
taaatggagg 1080tgcctattgg taaggtggga cgatatagag gctaatcgac
ataacagggt ttctccatgg 1140gaaattgagc tatctggttc gctttctggt
tctggcagct tgacagttcc tggctcaaag 1200aggaccagga ttggtttgcc
gggaactaga ccagattttt cagttcccaa tgggatggga 1260gtgtcagact
ttggggaatc ttcaaggttc cagaaggtct tgcaaggtca agaaattttt
1320ggttttaaca ctccttatga tggtgttgat acccaggatc atcatccatc
tgaaataagg 1380tgttttcctg gttcaagttg ttctgggatt gctgcaatag
gaaatggtgt tagaaaccct 1440cttgggaatt ctgatatttc ctataaaggc
ataggctttg gtgaatcttt tcgattccat 1500aaggtcttgc aaggtcaaga
aacatttcca agcccaccat gtggaagagc tctgtctgct 1560aaccaggctc
atgaaaatgg tagctttgga atctttgatg gtgttcaagt gccgacttct
1620agaaatggat ggcctgccct tgtgcaggga tataatgccc acactcacct
gtccacacca 1680tcagtgcaag tgtcgtcacc atcatcggtg ttaatgttcc
agcaagcaag cactgctgct 1740cctaacattt actcaatgca tagcgccaat
aatcaggaga aggagcaaga aattagtaac 1800cggagttcat ttgatattcc
tgaagtgtat ggtgaaaagc tcacaccatc acgttgtgag 1860cttagtgtca
ggggaggagg tcagggaggt atgaatttct ttggtctgtt aaacgagcat
1920aatcaactag ctgttccaca tcctcttgta actcaatcag catttagagg
cagtcaagat 1980ttagttccta catgtaaaag tagctgcagg ctctttggct
tttccttaac ggaggaaaga 2040agcattggaa ataaagtgga caaccccact
cctgttacat cttcattgat tcctggaacc 2100tcttttctgc cccagcagtt
gcactcagag cctccggtga tgaccaaggc aattggaagc 2160aattgtacca
aagtaagtga cttctatgct gtaagggata tgctttttga tattgcgctg
2220tagcgtactg ctgttgtaag atcaaaattg caatttcaca agctggggag
tgttgtagac 2280caggcaatta atcgctggaa gcttgatagg catgatgatt
tgatttgtgc attgaagcat 2340ttatttgata tggagggagg gcttctgcat
ggtgagggaa agttgtttac caggatcatg 2400aggatgttgt gatgcttgtt
cgagatgact catggcagga aatctgcagc atttgatgaa 2460aaattatgat
atttactaat gaagacgtag tgatggcacc aaacatagat gcttatagtt
2520gctgagaggc acacatggca tcattgatat gtttttagct cttgggcgaa
agaactgtaa 2580ttattgccat aacagtaatg tatcttaacc tcccttgcta
tggagaacaa tttaaactaa 2640tttactaggt tccttggaat actcagttaa
gaaattactt ttaaaactgt atcaaaatat 2700tactctatgt tgttcatcag
ttgtgttact gtattgcagc tattgcttct gtatctctgc 2760ttaacattgt
tggcttaagg ctgtttccca 279026656DNAMalus domestica 26aacaatgcgc
ggctgccgtt gacgttagag tctacgctcc tctctctcct ctctctcctc 60tctctctctc
tctaccgttt ataaatcccg tcctcaaacg ccacttctac atcaccgccc
120ctccctctga aatcctctcc ctctctacgc tcctctctct cctctctctt
ctctctcttc 180tctctctcta ccgtttataa atcccctgtc ctcaaacgcc
acttctacat caccaccccc 240ccctctgaaa atcctctttc tctctctctc
tctctctctc tctctgtgaa aaagctctct 300ctctctctct ctgtgaaaaa
gttaaacttt aattgcaata tgcaatcaca aactatccca 360tatggaacat
ggattccggg cacccgagcc ggccacgcga cgccgtcctc acgcgacgcc
420tcacgtgagc aacacctagc ggcggccagc gtacaaaaaa tggtgtcgga
gaatgctgtc 480acggttgtcg gacgacgtgg ctgctgcatg tgccacgtcg
tcaagcggct gctcctcggt 540cacggggtca accctacggt tttcggcgat
gagccagatc tgaaacaagg gcgaattctg 600cagatatcca tcacactggc
ggccgctcga gcatgcatct agagggccca attcgc 65627421DNAMalus domestica
27actctcgctc ccggagctcc tcaaccagtc ggagcagctc cgtcacgtcc ggcggcttag
60gctgcacctg cgcggaggtg cgtgggaagt atacaccaaa ggagcgcgag aacaccgccc
120ccttctgggg tgtcttccca gagctcaacg aaggcgacgg agactttgaa
ggagtttccg 180gtttgggatt cgccggcgat ttctgtagcc ccattgccac
cctcaccttc ccagcaacca 240ttgcaaaacc ccctctccct ttcactcaac
accctgtttc tctcactctc tctctctctc 300tctctctctc tctctctctc
tctctctctc tctctctctc tctaaaagtg tgatgttcct 360ctctcaagga
tttggggtgt gtgattagct tgcataatcc agcacagagt gaaggggtta 420c
42128707PRTMalus domestica 28Met Ala Gly Leu Ile Asp Leu Asn Ser
Ala Thr Glu Asp Glu Glu Thr 1 5 10 15 Pro Ser Ser Gly Ser Pro Ser
Ser Ala Ser Ser Val Ser Asp Ala Leu 20 25 30 Gly Ser Ser Ala Ser
Val Cys Met Glu Leu Trp His Ala Cys Ala Gly 35 40 45 Pro Leu Ile
Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr Leu Pro 50 55 60 Gln
Gly His Leu Glu Gln Val Ser Asp Phe Pro Thr Ser Ala Tyr Asp 65 70
75 80 Leu Pro Pro His Leu Phe Cys Arg Val Val Asp Val Lys Leu His
Ala 85 90 95 Glu Thr Gly Thr Asp Asp Val Phe Ala Gln Val Ser Leu
Val Pro Glu 100 105 110 Ser Glu Glu Ile Glu His Arg Leu Arg Glu Gly
Val Thr Asp Ala Asp 115 120 125 Ala Glu Glu Asp Val Glu Ala Met Gly
Thr Ser Thr Thr Pro His Met 130 135 140 Phe Cys Lys Thr Leu Thr Ala
Ser Asp Thr Ser Thr His Gly Gly Phe 145 150 155 160 Ser Val Pro Arg
Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr 165 170 175 Thr Gln
Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu His Gly 180 185 190
Leu Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro Arg Arg His 195
200 205 Leu Leu Thr Thr Gly Trp Ser Ala Phe Val Asn Lys Lys Lys Leu
Val 210 215 220 Ser Gly Asp Ala Val Leu Phe Leu Arg Gly Asp Asp Gly
Glu Leu Arg 225 230 235 240 Leu Gly Ile Arg Arg Ala Ala Gln Phe Lys
Ser Ser Ala Thr Cys Pro 245 250 255 Thr Leu Cys Ser Gln Gln Leu Asn
Tyr Ser Thr Ile Thr Asp Val Val 260 265 270 Asn Ala Ile Phe Ala Lys
Asn Ala Phe Asn Val Tyr Tyr Asn Pro Arg 275 280 285 Ser Ser Ser Ser
Glu Phe Ile Ile Pro Ser His Lys Phe Leu Arg Ser 290 295 300 Leu Asp
His Cys Phe Cys Ala Gly Met Arg Ile Lys Met Arg Phe Glu 305 310 315
320 Thr Glu Asp Ala Ala Glu Arg Arg Tyr Thr Gly Leu Ile Thr Gly Ile
325 330 335 Ser Glu Leu Asp Pro Val Arg Trp Pro Gly Ser Lys Trp Arg
Cys Leu 340 345 350 Val Val Arg Trp Asp Asp Ile Asp Thr Ser Lys His
Gly Arg Val Ser 355 360 365 Pro Trp Glu Val Glu Arg Ser Gly Ser Val
Ser Ser Ser His Thr Leu 370 375 380 Met Thr Thr Gly Leu Lys Arg Ser
Arg Ile Gly Leu Ser Ala Thr Lys 385 390 395 400 Pro Glu Phe Pro Val
Pro Ser Met Ser Cys Asn Gly Ile Gly Thr Ser 405 410 415 Asp Phe Gly
Glu Ser Leu Arg Phe Gln Lys Val Leu Gln Gly Gln Glu 420 425 430 Ile
Ser Gly Phe Asp Thr Pro Phe Ser Gly Leu Gly Gly Leu Asn Ser 435 440
445 His Pro Ser Glu Ala Arg Arg Val Phe His Gly Ser Gly Gly Ser Gly
450 455 460 Ile Ala Ala Gly Gly Asn Gly Leu Arg Gln Ser Leu Val Asp
Ser Glu 465 470 475 480 Ile Ala Ser Lys Gly Ile Gly Phe Gly Glu Ser
Phe Arg Phe His Lys 485 490 495 Val Leu Gln Gly Gln Glu Ile Phe Pro
Ser Ser Pro Tyr Gly Arg Ala 500 505 510 Pro Ala Ser Asn Glu Ala His
Glu Tyr Gly Gly Pro Gly Leu Tyr Asp 515 520 525 Gly Phe Gln Val Pro
Gly Phe Arg Asn Gly Trp Ser Thr Met Met Gln 530 535 540 Ser Asn Asn
Thr Asn Val His Ser Ser Ala Pro Ser Val Gln Val Ser 545 550 555 560
Ser Pro Ser Ser Val Leu Met Phe Gln Gln Ala Met Asn Pro Val Ala 565
570 575 Glu Phe Asn Ser Val Tyr Asn Gly His Asn Gln Glu Asp His Arg
Val 580 585 590 Asn Arg Thr Pro His Val Leu Glu His Asp Gly Gly Arg
Gln Thr Ser 595 600 605 Ser Ser Phe Gly Glu Arg Asn Phe Ser Arg Glu
Asp Arg Gly Gly Thr 610 615 620 His Ser Tyr Asn Gln His Gly Ile Ser
Pro His Pro Val Ile Ser Gln 625 630 635 640 Ser Thr Ile Ser Gly Ser
Gln Asp Ser Val Ser Pro Ile Lys Gly Ser 645 650 655 Cys Arg Leu Phe
Gly Phe Ser Leu Ser Glu Asp Lys Cys Val Pro Asp 660 665 670 Gln Glu
Gly Asn Pro Asn Val Gly Val Gln Phe His Ser Lys Pro Pro 675 680 685
Leu Met Thr Ser Thr Val Gly Ile Thr Cys Thr Lys Val Ser Asn Leu 690
695 700 Phe Ala Ala 705 29707PRTMalus domestica 29Met Ala Gly Leu
Ile Asp Leu Asn Ser Ala Thr Glu Asp Glu Glu Thr 1 5 10 15 Pro Ser
Ser Gly Ser Pro Ser Ser Ala Ser Ser Val Ser Asp Ala Leu 20 25 30
Gly Ser Ser Ala Ser Val Cys Met Glu Leu Trp His Ala Cys Ala Gly 35
40 45 Pro Leu Ile Ser Leu Pro Lys Lys Gly Ser Val Val Val Tyr Leu
Pro 50 55 60 Gln Gly His Leu Glu Gln Val Leu Asp Phe Pro Thr Ser
Ala Tyr Asp 65 70 75 80 Leu Pro Pro His Leu Phe Cys Arg Val Val Asp
Val Lys Leu His Ala 85 90 95 Glu Thr Gly Thr Asp Asp Val Phe Ala
Gln Val Ser Leu Val Pro Glu 100 105 110 Ser Glu Glu Ile Glu His Arg
Leu Arg Glu Gly Val Thr Asp Ala Asp 115 120 125 Ala Glu Glu Asp Val
Glu Ala Met Gly Thr Ser Thr Thr Pro His Met 130 135 140 Phe Cys Lys
Thr Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe 145 150 155 160
Ser Val Pro Arg Arg Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr 165
170 175 Thr Gln Gln Arg Pro Ser Gln Glu Leu Val Ala Lys Asp Leu His
Gly 180 185 190 Leu Glu Trp Arg Phe Arg His Ile Tyr Arg Gly Gln Pro
Arg Arg His 195 200 205 Leu Leu Thr Thr Gly Trp Ser Ala Phe Val Asn
Lys Lys Lys Leu Val 210 215 220 Ser Gly Asp Ala Val Leu Phe Leu Arg
Gly Asp Asp Gly Glu Leu Arg 225 230 235 240 Leu Gly Ile Arg Arg Ala
Ala Gln Phe Lys Ser Ser Ala Thr Cys Pro 245 250 255 Thr Leu Cys Ser
Gln Gln Leu Asn Tyr Ser Thr Ile Thr Asp Val Val 260 265 270 Asn Ala
Ile Phe Ala Lys Asn Ala Phe Asn Val Tyr Tyr Asn Pro Arg 275 280 285
Ser Ser Ser Ser Glu Phe Ile Ile Pro Ser His Lys Phe Leu Arg Ser 290
295 300 Leu Asp His Cys Phe Cys Ala Gly Met Arg Ile Lys Met Arg Phe
Glu 305 310 315 320 Thr Glu Asp Ala Ala Glu Arg Arg Tyr Thr Gly Leu
Ile Thr Gly Ile 325 330 335 Ser Glu Leu Asp Pro Val Arg Trp Pro Gly
Ser Lys Trp Arg Cys Leu 340 345 350 Val Val Arg Trp Asp Asp Ile Asp
Thr Ser Lys His Gly Arg Val Ser 355 360 365 Pro Trp Glu Val Glu Arg
Ser Gly Ser Val Ser Ser Ser His Thr Leu 370 375 380 Met Thr Thr Gly
Leu Lys Arg Ser Arg Ile Gly Leu Ser Ala Thr Lys 385 390 395 400 Pro
Glu Phe Pro Val Pro Ser Met Ser Cys Asn Gly Ile Gly Thr Ser 405 410
415 Asp Phe Gly Glu Ser Leu Arg Phe Gln Lys Val Leu Gln Gly Gln Glu
420 425 430 Ile Ser Gly Phe Asp Thr Pro Phe Ser Gly Leu Gly Gly Leu
Asn Ser 435 440 445 His Pro Ser Glu Ala Arg Arg Val Phe His Gly Ser
Gly Gly Ser Gly 450 455 460 Ile Ala Ala Gly Gly Asn Gly Leu Arg Gln
Ser Leu Val Asp Ser Glu 465 470 475 480 Ile Ala Ser Lys Gly Ile Gly
Phe Gly Glu Ser Phe Arg Phe His Lys 485 490 495 Val Leu Gln Gly Gln
Glu Ile Phe Pro Ser Ser Pro Tyr Gly Arg Ala 500 505 510 Pro Ala Ser
Asn Glu Ala His Glu Tyr Gly Gly Pro Gly Leu Tyr Asp 515 520 525 Gly
Phe Gln Val Pro Gly Phe Arg Asn Gly Trp Ser Thr Met Met Gln 530 535
540 Ser Asn Asn Thr Asn Val His Ser Ser Ala Pro Ser Val Gln Val Ser
545 550 555 560 Ser Pro Ser Ser Val Leu Met Phe Gln Gln Ala Met Asn
Pro Val Ala 565 570 575 Glu Phe Asn Ser Val Tyr Asn Gly His Asn Gln
Glu Asp His Arg Val 580 585 590 Asn Arg Thr Pro His Val Leu Glu His
Asp Gly Gly Arg Gln Thr Ser 595 600 605 Ser Ser Phe Gly Glu Arg Asn
Phe Ser Arg Glu Asp Arg Gly Gly Thr 610 615 620 His Ser Tyr Asn Gln
His Gly Ile Ser Pro His Pro Val Ile Ser Gln 625 630 635 640 Ser Thr
Ile Ser Gly Ser Gln Asp Ser Val Ser Pro Ile Lys Gly Ser 645 650 655
Cys Arg Leu Phe Gly Phe Ser Leu Ser Glu Asp Lys Cys Val Pro Asp 660
665 670 Gln Glu Gly Asn Pro Asn Val Gly Val Gln Phe His Ser Lys Pro
Pro 675 680 685 Leu Met Thr Ser Thr Val Gly Ile Thr Cys Thr Lys Val
Ser Asn Leu 690 695 700 Phe Ala Ala 705
* * * * *
References