U.S. patent application number 17/149521 was filed with the patent office on 2021-07-15 for compositions and methods for peronospora resistance in spinach.
The applicant listed for this patent is Seminis Vegetable Seeds, Inc.. Invention is credited to Bart Willem Brugmans, John Meeuwsen, Claudia Nooyen.
Application Number | 20210214742 17/149521 |
Document ID | / |
Family ID | 1000005480008 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210214742 |
Kind Code |
A1 |
Brugmans; Bart Willem ; et
al. |
July 15, 2021 |
COMPOSITIONS AND METHODS FOR PERONOSPORA RESISTANCE IN SPINACH
Abstract
The invention provides for spinach plants with broad-spectrum
resistance to downy mildew disease and their progeny. Such plants
may comprise unique combinations of alleles resulting in the
broad-spectrum resistance to downy mildew. In certain aspects,
compositions, including distinct polymorphic molecular markers, and
methods for producing, using, identifying, selecting, and the like
of plants or germplasm with resistance to downy mildew are
provided.
Inventors: |
Brugmans; Bart Willem; (Beek
en Donk, NL) ; Meeuwsen; John; (Bennekom, NL)
; Nooyen; Claudia; (Wageningen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seminis Vegetable Seeds, Inc. |
St. Louis |
MO |
US |
|
|
Family ID: |
1000005480008 |
Appl. No.: |
17/149521 |
Filed: |
January 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14632871 |
Feb 26, 2015 |
10927386 |
|
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17149521 |
|
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61945675 |
Feb 27, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8279 20130101;
C12Q 2600/13 20130101; C12Q 2600/156 20130101; C12Q 1/6895
20130101; A01H 5/12 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/12 20060101 A01H005/12; C12Q 1/6895 20060101
C12Q001/6895 |
Claims
1. A Spinacia oleracea spinach plant of a cultivated variety
comprising in its genome allele A, wherein said plant comprises a
DNA sequence selected from the group consisting of SEQ ID NO:2, SEQ
ID NO:19, and SEQ ID NO:22, and comprises the DNA sequence of SEQ
ID NO:5 or SEQ ID NO:10, and wherein a sample of seed comprising
said allele A has been deposited under ATCC Accession No.
PTA-120472.
2. A Spinacia oleracea spinach plant of a cultivated variety
comprising in its genome a heterozygous combination of alleles that
confers broad-spectrum resistance to Peronospora farinosa f. sp.
Spinaciae (Pfs), wherein the combination of alleles comprises two
alleles selected from the group consisting of allele A, allele Vt,
and allele C, and wherein a plant comprising allele A comprises a
DNA sequence selected from the group consisting of SEQ ID NO:2, SEQ
ID NO:19, and SEQ ID NO:22, and comprises the DNA sequence of SEQ
ID NO:5 or SEQ ID NO:10, wherein a plant comprising allele Vt
comprises a DNA sequence selected from the group consisting of SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17
and comprises the DNA sequence of SEQ ID NO: 20 or SEQ ID NO: 23,
and wherein a plant comprising allele C comprises a DNA sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:18,
and SEQ ID NO:21 and comprises the DNA sequence of SEQ ID NO:4, SEQ
ID NO:9, SEQ ID NO:12, and SEQ ID NO: 15, and wherein a sample of
seed comprising said allele A, allele Vt, and allele C has been
deposited under ATCC Accession No. PTA-120472, ATCC Accession No.
PTA-12041, and ATCC Accession No. PTA-12486, respectively.
3. The spinach plant of claim 2, wherein the broad-spectrum
resistance comprises resistance to at least 10 races of Peronospora
farinosa f. sp. spinaciae (Pfs).
4. (canceled)
5. The spinach plant of claim 2, wherein the combination of alleles
comprises: (a) alleles A and C; (b) alleles A and Vt; or (c)
alleles C and Vt.
6. The spinach plant of claim 3, wherein the plant is resistant to:
(a) Peronospora farinosa f. sp. Spinaciae races, 7, 8, 10, 11, 12,
14, and isolate UA4712; (b) Peronospora farinosa f. sp. Spinaciae
races, 7, 8, 10, 11, 12, 13, and 14; or (c) Peronospora farinosa f.
sp. Spinaciae races, 7, 8, 10, 11, 12, 13, and isolate UA4712.
7.-8. (canceled)
9. The spinach plant of claim 1, wherein said plant is an
inbred.
10. The spinach plant of claim 1, wherein said plant is a
hybrid.
11. The spinach plant of claim 1, comprising recessive resistance
to Peronospora farinosa f. sp. Spinaciae races 7 and 13.
12. A seed that produces the plant of claim 2.
13. A plant part of the plant of claim 2, wherein the plant is a
leaf, stem, root, flower or cell.
14. A method of producing a seed of a spinach plant with broad
spectrum resistance to Peronospora farinosa f. sp. Spinaciae
comprising: (a) crossing a first spinach plant and a second spinach
plant, wherein the first and second spinach plants collectively
comprise at least two alleles selected from the group consisting of
allele A, allele Vt, and allele C, wherein a plant comprising
allele A comprises a DNA sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:19, and SEQ ID NO:22, and
comprises the DNA sequence of SEQ ID NO:5 or SEQ ID NO:10, wherein
a plant comprising allele Vt comprises a DNA sequence selected from
the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ
ID NO:14, and SEQ ID NO:17 and comprises the DNA sequence of SEQ ID
NO: 20 or SEQ ID NO: 23, and wherein a plant comprising allele C
comprises a DNA sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:18, and SEQ ID NO:21 and comprises the DNA
sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:
15, and wherein a sample of seed comprising said allele A, allele
Vt, and allele C has been deposited under ATCC Accession No.
PTA-120472, ATCC Accession No. PTA-12041, and ATCC Accession No.
PTA-12486, respectively; (b) obtaining at least a first seed
resulting from said crossing that comprises a heterozygous
combination of said alleles that confers broad-spectrum resistance
to Peronospora farinosa f. sp. Spinaciae.
15. The method of claim 14, wherein the method further comprises
obtaining a population of seed resulting from said crossing that
comprises a heterozygous combination of said alleles that confers
broad-spectrum resistance to Peronospora farinosa f. sp.
Spinaciae.
16. The method of claim 14, wherein: (a) the first spinach plant
comprises allele A and the second spinach plant comprises allele C;
(b) the first spinach plant comprises allele A and the second
spinach plant comprises allele Vt; (c) the first spinach plant
comprises allele C and the second spinach plant comprises allele
Vt; (d) the first spinach plant or the second spinach plant is
homozygous for said allele A, allele Vt, or allele C; or (e) the
first spinach plant and the second spinach plant are homozygous for
said two alleles selected from the group consisting of allele A,
allele Vt, and allele C.
17. A seed produced by the method of claim 14.
18. The seed of claim 17, wherein: (a) the first allele is allele A
and the second allele is allele C; (b) the first allele is allele A
and the second allele is allele Vt; or (c) the first allele is
allele C and the second allele is allele Vt.
19. A DNA sequence selected from the group consisting of SEQ ID
NOs:1-25.
20. A method of introducing resistance to Peronospora farinosa f.
sp. Spinaciae into a spinach plant comprising: (a) crossing a first
spinach plant comprising in its genome a first allele selected from
the group consisting of A, Vt, and C, with a second spinach plant,
wherein a plant comprising allele A comprises a DNA sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:19,
and SEQ ID NO:22, and comprises the DNA sequence of SEQ ID NO:5 or
SEQ ID NO:10, wherein a plant comprising allele Vt comprises a DNA
sequence selected from the group consisting of SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 and comprises
the DNA sequence of SEQ ID NO: 20 or SEQ ID NO: 23, and wherein a
plant comprising allele C comprises a DNA sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:18, and SEQ ID NO:21
and comprises the DNA sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:12, and SEQ ID NO: 15, and wherein a sample of seed comprising
said allele A, allele Vt, and allele C has been deposited under
ATCC Accession No. PTA-120472, ATCC Accession No. PTA-12041, and
ATCC Accession No. PTA-12486, respectively; (b) selecting at least
one progeny plant that comprises said first allele for resistance
to Peronospora farinosa f. sp. Spinaciae, by detecting the presence
in the genome of said progeny plant the DNA sequence of claim
19.
21. The method of claim 20, wherein said crossing comprises
producing a population of progeny plants.
22. The method of claim 20, comprising screening the progeny plants
for the presence of at least two nucleic acid sequences selected
from the group consisting of SEQ ID NOs: 1-25.
23. A plant produced by the method of claim 20.
24. A method of identifying a plant comprising a desired allele
conferring resistance to Peronospora farinosa f. sp. Spinaciae, the
method comprising detecting in the genome of said plant the DNA
sequence of claim 19.
25. The method of claim 24, further defined as comprising detecting
in the genome of said plant at least two polymorphic nucleic acid
sequences selected from the group consisting of SEQ ID NOs:1-25,
wherein the presence of the polymorphic nucleic acid sequences are
indicative of the presence in the plant of at least two alleles
conferring resistance to Peronospora farinosa f. sp. Spinaciae
selected from of alleles A, Vt, and C, wherein a plant comprising
allele A comprises a DNA sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:19, and SEQ ID NO:22, and
comprises the DNA sequence of SEQ ID NO:5 or SEQ ID NO:10, wherein
a plant comprising allele Vt comprises a DNA sequence selected from
the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ
ID NO:14, and SEQ ID NO:17 and comprises the DNA sequence of SEQ ID
NO: 20 or SEQ ID NO: 23, and wherein a plant comprising allele C
comprises a DNA sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:18, and SEQ ID NO:21 and comprises the DNA
sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:
15, and wherein a sample of seed comprising said allele A, allele
Vt, and allele C has been deposited under ATCC Accession No.
PTA-120472, ATCC Accession No. PTA-12041, and ATCC Accession No.
PTA-12486, respectively.
26. A plant obtained by the method of claim 24.
27. A spinach plant, cell or cell containing the plant part of
claim 2.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/632,871 filed Feb. 26, 2015, which claims
the benefit of U.S. Provisional Application No. 61/945,675, filed
Feb. 27, 2014, each of which are herein incorporated by reference
in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing that is contained in the file named
"SEMB015US_ST25.txt," which is 6.2 kilobytes as measured in
Microsoft Windows operating system and was created on Feb. 25,
2015, is filed electronically herewith and incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to the field of plant breeding and,
more specifically, to methods and compositions for producing
spinach plants with resistance to downy mildew.
BACKGROUND OF THE INVENTION
[0004] Downy mildew, caused by the plant pathogen Peronospora
farinosa f. sp. Spinaciae (Pfs), is an economically important
disease of spinach worldwide, particularly for Spinacia oleracea,
the most commonly cultivated spinach species. Currently, fourteen
races of the downy mildew (DM)-causing pathogen are officially
recognized, although new isolates are currently being discovered
and named each year. To date, it has been believed that resistance
to DM in spinach was incomplete and race-specific. The ability of
new strains of the pathogen to overcome resistance in spinach
plants therefore makes the development of spinach varieties with
effective levels of resistance to Peronospora farinosa f. sp.
spinaciae challenging and increasingly important.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention provides a Spinacia oleracea
spinach plant comprising in its genome allele A, as described
herein. In another aspect, the invention provides a Spinacia
oleracea spinach plant comprising in its genome a heterozygous
combination of alleles that confers broad-spectrum resistance to
Peronospora farinosa f. sp. Spinaciae. In an embodiment, the
broad-spectrum resistance comprises resistance to at least 10 races
of Peronospora farinosa f. sp. spinaciae (Pfs). In another
embodiment, the allele that confers broad-spectrum resistance is a
combination of alleles which is selected from the group consisting
of allele A, allele Vt, and allele C. In other embodiments, the
combination of alleles comprises alleles A and C and the plant is
resistant to at least Peronospora farinosa f. sp. Spinaciae races
7, 8, 10, 11, 12, 14, and isolate UA4712; the combination of
alleles comprises alleles A and Vt, and the plant is resistant to
at least Peronospora farinosa f. sp. Spinaciae races 7, 8, 10, 11,
12, 13, and 14; or the combination of alleles comprises alleles C
and Vt, and the plant is resistant to at least Peronospora farinosa
f. sp. Spinaciae races 7, 8, 10, 11, 12, 13, and isolate UA4712. In
another embodiment, representative samples of seed comprising
allele A, allele C, and allele Vt have been deposited under ATCC
Accession No. PTA-120472, ATCC Accession No. PTA-12486, and ATCC
Accession No. PTA-12041, respectively. In further embodiments, the
allele A, allele C, and/or allele Vt is genetically linked to at
least one sequence selected from the group SEQ ID NOs:1-25.
[0006] In another embodiment, a plant of the invention comprises an
allele A, allele C, and/or allele Vt that shares a genetic source
for said allele with seed deposited under ATCC Accession Nos.
PTA-120472, ATCC Accession No. PTA-12486, or ATCC Accession No.
PTA-12041. In other embodiments, a plant of the invention may be an
inbred or a hybrid. In still further embodiments, the invention
provides a seed that produces such a plant, or a plant part of such
a plant. In another embodiment, the plant part is selected from the
group consisting of an embryo, meristem, cotyledon, pollen, leaf,
anther, root, pistil, flower, cell, and stalk. In a still further
embodiment, the invention provides a food product comprising the
harvested leaves of such a spinach plant.
[0007] In another aspect, the invention provides a Spinacia
oleracea spinach plant comprising in its genome a heterozygous
combination of alleles that confers broad-spectrum resistance to
Peronospora farinosa f. sp. Spinaciae, wherein one allele confers
recessive resistance to Peronospora farinosa f. sp. Spinaciae races
7 and 13. In one embodiment, the broad-spectrum resistance
comprises resistance to at least 10 races of Peronospora farinosa
f. sp. spinaciae (Pfs). In other embodiments, one allele is allele
Vt and the other allele is allele C, one allele is allele A and the
other is allele Vt; or one allele is allele A and the other is
allele C.
[0008] In another aspect, the invention provides a method of
producing a spinach plant with broad spectrum resistance to
Peronospora farinosa f. sp. Spinaciae comprising: (a) crossing a
first spinach plant comprising in its genome a first allele
selected from the group consisting of A, Vt, and C, with a second
spinach plant comprising in its genome a second allele selected
from the group consisting of A, Vt, and C, to produce a population
of hybrid progeny plants; and (b) selecting at least one hybrid
progeny plant from said population that comprises a combination of
said first allele and said second allele that confers
broad-spectrum resistance to Peronospora farinosa f. sp. Spinaciae.
In one embodiment, the method further comprises production of a
population of hybrid plants. In other embodiments, the first
spinach plant comprises allele A and the second spinach plant
comprises allele C; or the first spinach plant comprises allele A
and the second spinach plant comprises allele Vt; or the first
spinach plant comprises allele C and the second spinach plant
comprises allele Vt. In another embodiment, the invention provides
a hybrid progeny plant produced by such a method. In still other
embodiments, the first allele of the plant is allele A and the
second allele is allele C; or the first allele is allele A and the
second allele is allele Vt; or the first allele is allele C and the
second allele is allele Vt.
[0009] In another aspect, the invention provides a method of
introducing resistance to Peronospora farinosa f. sp. Spinaciae in
a spinach plant comprising: (a) crossing a first spinach plant
comprising in its genome a first allele selected from the group
consisting of A, Vt, and C, with a second spinach plant comprising
in its genome a second, distinct allele; (b) selecting at least one
progeny plant that comprises said first allele for resistance to
Peronospora farinosa f. sp. Spinaciae based on the presence in the
genome of the plant of a sequence selected from SEQ ID NOs:1-25. In
another embodiment, the method comprises detecting in the genome of
said plant at least two polymorphic nucleic acid sequences selected
from the group consisting of SEQ ID NOs:1-25, wherein the presence
of the polymorphic nucleic acid sequences are indicative of the
presence in the plant of at least two alleles conferring resistance
to Peronospora farinosa f. sp. Spinaciae selected from of alleles
A, Vt, and C.
[0010] In yet another aspect, the invention provides a spinach
plant, cell or cell containing plant part comprising at least two
polymorphic DNA sequences which are associated with different
alleles from among alleles A, Vt, and C. In one embodiment, a
spinach plant, cell or cell containing plant part are provided
comprising at least two DNA sequences that are represented in
different groups from among those designated (a), (b) and (c), said
groups being made up as follows: (a) a DNA sequence comprising SEQ
ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ
ID NO:16, SEQ ID NO:19, or SEQ ID NO:22; (b) a DNA sequence
comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:12, SEQ
ID NO:15, SEQ ID NO:18, SEQ ID NO:21, or SEQ ID NO:22; and (c) a
DNA sequence comprising SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID
NO:23. The invention also provides a DNA sequence selected from the
group consisting of SEQ ID NOs:1-25.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0011] SEQ ID NO:1--DNA sequence corresponding to scaffold SF63815
and diagnostic for allele C.
[0012] SEQ ID NO:2--DNA sequence corresponding to scaffold SF63815
and diagnostic for allele A.
[0013] SEQ ID NO:3--DNA sequence corresponding to scaffold SF63815
and diagnostic for allele Vt.
[0014] SEQ ID NO:4--DNA sequence corresponding to scaffold SF59002
and diagnostic for allele C.
[0015] SEQ ID NO:5--DNA sequence corresponding to scaffold SF59002
and diagnostic for allele A.
[0016] SEQ ID NO:6--DNA sequence corresponding to scaffold SF59002
and diagnostic for allele Vt.
[0017] SEQ ID NO:7--DNA sequence corresponding to scaffold SF59002
and diagnostic for allele A.
[0018] SEQ ID NO:8--DNA sequence corresponding to scaffold SF59002
and diagnostic for allele Vt.
[0019] SEQ ID NO:9--DNA sequence corresponding to scaffold SF95487
and diagnostic for allele C.
[0020] SEQ ID NO:10--DNA sequence corresponding to scaffold SF95487
and diagnostic for allele A.
[0021] SEQ ID NO:11--DNA sequence corresponding to scaffold SF95487
and diagnostic for allele Vt.
[0022] SEQ ID NO:12--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele C.
[0023] SEQ ID NO:13--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele A.
[0024] SEQ ID NO:14--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele Vt.
[0025] SEQ ID NO:15--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele C.
[0026] SEQ ID NO:16--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele A.
[0027] SEQ ID NO:17--DNA sequence corresponding to scaffold SF90906
and diagnostic for allele Vt.
[0028] SEQ ID NO:18--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele C.
[0029] SEQ ID NO:19--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele A.
[0030] SEQ ID NO:20--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele Vt.
[0031] SEQ ID NO:21--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele C.
[0032] SEQ ID NO:22--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele A.
[0033] SEQ ID NO:23--DNA sequence corresponding to scaffold SF34732
and diagnostic for allele Vt.
[0034] SEQ ID NO:24--DNA sequence corresponding to alleles A, C,
and Vt of scaffold SF62749.
[0035] SEQ ID NO:25--DNA sequence corresponding to alleles A, C,
and Vt of scaffold SF178637.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1: Shows alleles A, Vt, and C, which represent three
linkage groups assembled from genotypes and phenotypes collected
for three mapping populations, as described in Example 3.
[0037] FIG. 2A and FIG. 2B: Show possible breeding methods for
development of hybrids and three-way hybrids.
[0038] FIG. 3: Shows sequence alignments of scaffolds containing
polymorphisms corresponding to alleles C, A, and Vt. Polymorphisms
between alleles are indicated by shading, and can be used to
identify and/or diagnose the presence of downy mildew resistance
alleles C, A, and/or Vt in spinach.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention provides methods and compositions for
development of spinach varieties with resistance to downy mildew
(DM). The invention provides the identification of three distinct
alleles from Spinacia oleracea, which have been named A, Vt, and C.
These alleles can be used in various combinations to obtain spinach
plants with a unique broad-spectrum resistance to DM.
[0040] The three alleles, designated A from spinach line
SMBS011-1162M, C from SMB-66-1143M, and Vt from SSB-66-1131M, were
identified in Spinacia oleracea and found to provide resistance to
existing and emerging DM strains. Each allele was found to have
resistance to a specific set of races and/or isolates. The alleles
can be used in various combinations to make hybrids with desired DM
resistance.
[0041] As shown in further detail herein below, allele C was found
to confer resistance to races 1, 2, 3, 4, 5, 6, 7, 8, 10 and newly
occurring Pfs isolate UA4712, which is the candidate strain for
race 15. Allele A was found to confer resistance to Peronospora
farinosa (Pfs) races 1, 3, 5, 7, 8, 11, 12, 13 and 14. The
resistance to races 7 and 13, unlike most DM races, is inherited in
a recessive manner. Allele Vt was found to confer resistance to
races 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 and 13. These newly
characterized alleles can be used in novel heterozygous
combinations to obtain spinach plants with broad DM resistance. The
spinach plants may be inbred plants and/or hybrid plants and can
include various combinations of the alleles as described
herein.
[0042] For example, in accordance with one embodiment of the
invention, alleles C and Vt can be present in a spinach plant
(i.e., a heterozygous spinach plant) and provide resistance to Pfs
races 1-8, 10-13, and to newly occurring Pfs isolate UA4712. In
another embodiment, alleles A and Vt can be present in a spinach
plant and provide resistance to Pfs races 1-8 and 10-14. Allele Vt
complements the recessive resistances from allele A. Similarly,
when alleles A and C are present together in a spinach plant, the
plant is resistant to Pfs races 1-8, 10-12, 14 and UA4712. The
alleles can be utilized in any combination to provide desired
resistance for a particular market or region. For example, a hybrid
with alleles C and Vt exhibits resistance to all Pfs races except
Pfs 14. Since Pfs race 14 does not occur in Europe, this hybrid
would be fully resistant in that geography. As a further example, a
hybrid with alleles A and Vt exhibits resistance to all Pfs races
except isolate UA4712. New isolate UA4712 is found in limited
locations, so this hybrid would be fully resistant in many
areas.
[0043] In yet another embodiment, a three-way hybrid is
contemplated that is resistant to all described DM races and
isolate UA4712. In accordance with the invention, a spinach plant
comprising one or two alleles described herein, including, but not
limited to, A, C, and Vt, can be crossed with a second, distinct
spinach plant comprising a second or third allele including, but
not limited to, A, C, and Vt to produce a hybrid spinach plant
comprising a beneficial set of alleles as described herein.
[0044] As further described herein, alleles can be introgressed
into selected spinach varieties in any combination to provide a
desired resistance to DM. In accordance with the invention, such
alleles conferring resistance to DM may be introgressed into any
desired genomic background of a specific spinach variety or
cultivar. For example, a starting spinach plant containing a given
DM resistance allele in accordance with the invention can be
self-fertilized a sufficient number of generations to produce an
inbred spinach variety that is homozygous for the allele conferring
resistance to DM. Such an inbred plant may then be crossed with
another spinach plant that comprises a distinct DM resistance
allele to consistently produce spinach inbreds and/or hybrids that
comprise a combination of alleles conferring a desirable resistance
to DM as described herein. A spinach plant exhibiting resistance to
DM according to the invention may further be crossed to other
spinach plants and selections carried out according to the
invention to obtain new DM-resistant spinach inbreds and hybrids
with any desired combination of alleles described herein.
[0045] Although other alleles conferring resistance to certain
races of DM have previously been identified in spinach, such
alleles have conferred resistance only to a subset of races of
Peronospora farinose (Pfs), even in a hybrid combination. Non-host
resistance to DM may also be found in wild relatives of spinach,
such as Spinacia turkestanica and Spinacia tetrandra. However,
genes from wild relatives often result in negative drag for
commercial characteristics including yield and quality. The
unfavorable alleles found in the wild relatives are often
introduced into the elite germplasm together with DM resistance
alleles. The present invention describes novel S. oleracea
resistance sources, alleles, and markers that provide race-specific
and broad-spectrum DM resistance.
[0046] In one embodiment of the invention, alleles A, C, and Vt,
conferring resistance to DM may be defined as being on spinach
linkage group 6 by common markers in sequence scaffolds SF34732 and
SF63815 on the distal position and SFSF59002, SF95487, and SF90906
on the proximal position. Nucleotide sequences associated with and
diagnostic for the resistance alleles are provided in SEQ ID
NOs:1-25. Polymorphisms between alleles A, C, and Vt are shown in
FIG. 3. In other embodiments, alleles providing broad-spectrum
resistance to DM may be defined as from, or sharing, a genetic
source selected from accessions SMBS011-1162M, SMB-66-1143M, and
SSB-66-1131M, representative deposits of seed of which were made
with the ATCC under accession numbers PTA-120472, PTA-12486, and
PTA-12041, respectively.
[0047] The invention further provides methods of producing spinach
plants with broad-spectrum resistance to DM, as well as spinach
plants and parts thereof made by such methods. In one embodiment,
nucleic acid sequences or genomic markers may be used to identify
alleles according to the present invention. These nucleic acid
sequences may be used in the identification of polymorphisms or
markers genetically linked in a spinach genome to the DM-resistance
conferring alleles in accordance with the invention. The invention
also provides food products derived from such plants and their
method of production.
[0048] Genetic markers in linkage disequilibrium with DM-resistance
alleles of the present invention may permit efficient introduction
of DM-resistance into essentially any spinach genome. This also
results in significant economization by permitting substitution of
costly, time-intensive, and potentially unreliable phenotypic
assays. Further, breeding programs can be designed to explicitly
drive the frequency of specific favorable phenotypes by targeting
particular genotypes. Fidelity of these associations may be
monitored continuously to ensure maintained predictive ability and,
therefore, informed breeding decisions.
[0049] In accordance with the invention, one of skill in the art
may identify a candidate germplasm source possessing a desirable
DM-resistant phenotype, such as from an accession described herein.
One embodiment of the invention comprises using the materials and
methods of the invention to obtain an allele conferring
broad-spectrum resistance to DM from any additional spinach
accessions. Using the information set forth herein, including, but
not limited to, a sequence scaffold from spinach chromosome 6, DM
resistance can be introgressed into any other spinach
varieties.
[0050] The development of spinach varieties in a spinach breeding
program requires, in general, the development of homozygous inbred
lines, the crossing of these lines, and the evaluation of the
resulting hybrid plants. Spinach breeding programs combine the
genetic backgrounds from two or more inbred lines or various other
germplasm sources into breeding populations from which new inbred
lines are developed by selfing and selection of desired phenotypes.
Hybrids also can be used as a source of plant breeding material or
as source populations from which to develop or derive new spinach
lines. Plant breeding techniques known in the art and used in a
spinach breeding program include, but are not limited to, recurrent
selection, backcrossing, double haploids, pedigree breeding,
genetic marker enhanced selection, and transformation. Often a
combination of these techniques is used. Thus, inbred lines derived
from hybrids can be developed using plant breeding techniques as
described above. New inbred lines can be crossed with other inbred
lines and the hybrids from these crosses are evaluated to determine
which of those have commercial potential.
[0051] The techniques of the present invention may be used to
identify desirable disease-resistant phenotypes by identifying
genetic markers genetically linked to an allele or locus conferring
such a phenotype. In accordance with the invention, one of skill in
the art may develop molecular marker assays based on, for example,
SNPs and/or Indels in the spinach genome. In an embodiment,
molecular marker assays useful to identify DM-resistant spinach
plants according to the invention may be designed based on a
sequence scaffold from spinach chromosome 6. Such techniques may
also involve phenotypic assays to identify desired plants either
alone or in combination with genetic assays, thereby also
identifying a marker genotype associated with the trait that may be
used for production of new varieties with the methods described
herein.
[0052] The invention provides for the production of cultivated
spinach plants comprising a combination of alleles conferring
resistance to DM. Successful spinach production depends on
attention to various horticultural practices. These include soil
management with special attention to proper fertilization, crop
establishment with appropriate spacing, weed control, and the
introduction of bees or other insects for pollination, irrigation,
and pest management.
[0053] Spinach crops can be established from seed or from
transplants. Transplanting can result in an earlier crop compared
to a crop produced from direct seeding. Transplanting helps achieve
complete plant stands rapidly, especially where higher seed costs,
as with triploid seeds, make direct-seeding risky.
Development of Spinach Plants With Resistance to Downy Mildew
[0054] The present disclosure identifies alleles from cultivated
spinach and combinations thereof conferring broad-spectrum
resistance to DM, as well as sequence scaffolds from spinach
chromosome 6 that can be used for the tracking and introgression of
the loci into desirable germplasm, such as by marker-assisted
selection and/or marker-assisted backcrossing.
[0055] The invention provides for the tracking and introduction of
any such alleles and/or any combination of such alleles with other
resistance loci into a given genetic background. One of ordinary
skill will understand that resistance to DM conferred by alleles
described herein may be introgressed from one genotype to another
via marker-assisted selection. Accordingly, a germplasm source can
be selected that has resistance to DM. Using these alleles, a
breeder may select a spinach plant with resistance to DM, or track
such a phenotype during breeding using marker-assisted selection
for the region described herein. According to the invention,
screens with flanking markers may be sufficient to select progeny
carrying desired DM resistance in pedigrees that segregate for a
single resistance allele. One of skill in the art will also
understand that markers can be complemented by phenotypic screens,
for example with differential races showing a compatible
interaction with one allele and an incompatible interaction with
another, to select for individuals with desired resistance in
populations segregating for two or more haplotypes.
Development of Spinach Hybrids With Resistance to Downy Mildew
[0056] As described herein, spinach plants in accordance with the
present invention may comprise any heterozygous combinations of
allele C, allele A, and allele Vt to confer broad DM resistance.
For example, in one embodiment, alleles C and Vt can be present in
a heterozygous spinach plant and confer resistance to Pfs races
1-8, 10-13, and to newly occurring Pfs isolate UA4712. In other
embodiments, alleles A and Vt can be present in a spinach plant and
confer resistance to Pfs races 1-8 and 10-14, or alleles A and C
can be present together in a spinach plant and confer resistance to
Pfs races 1-8, 10-12, 14 and UA4712. Also in accordance with the
present invention is a three-way hybrid that is resistant to all
described DM races and isolate UA4712. Such a spinach plant
comprising one or two alleles as described herein may be crossed
with a second, distinct spinach plant comprising a second or third
allele as described herein to produce a hybrid spinach plant
comprising a beneficial set of alleles as described herein.
[0057] The process of introgressing a novel resistance gene into
acceptable commercial types can be a difficult process and may be
complicated by factors such as linkage drag, epistasis, and low
heritability. The heritability of a trait is the proportion of the
phenotypic variation attributed to the genetic variance, which
varies between 0 and 1.0. Thus, a trait with heritability near 1.0
is not greatly affected by the environment. Those skilled in the
art recognize the importance of creating commercial lines with
horticultural traits having high heritability because these
cultivars will allow growers to produce a crop with uniform market
specifications.
[0058] One of ordinary skill will understand that the resistance
alleles provided herein can be combined for improved resistance,
particularly in fields where mixed populations of DM races may
occur. According to the invention, the alleles described herein may
be introgressed into parents of a hybrid via marker-assisted and/or
phenotypic selection. Breeders may select alleles that mutually
complement race-specificity to achieve broad resistance to DM. The
described resistance donors are crossed to inbreds with
demonstrated combining ability. The F1 resulting from each initial
cross is then backcrossed with the recipient genotype, i.e. with
the recurrent parent. According to the invention, individuals
carrying the resistance in the heterozygous phase are selected
using marker-assisted and/or phenotypic selection. This backcross
and selection step is repeated, for example, three times.
Individuals carrying the desired resistance allele are then
self-pollinated, for example, for two generations through single
seed descent. A breeder may recover inbred spinach plants carrying
the desired resistance allele from the progeny of each pedigree,
introduced into the same genetic background as respective recurrent
parents, preferably at least 95%, 96%, 97%, 98%, or 99% identical.
Commercial, resistant cultivars are generated by crossing parental
lines, each with a complementary resistance allele, thereby
creating hybrids with superior resistance.
[0059] According to the invention, multiple resistance donors may
be individually crossed to the same recipient genotype. Following
the backcross and selection steps described above, an inbred
spinach plant carrying a distinct resistance allele may be obtained
that has been introduced into the same genetic background as the
recurrent parent, preferably at least 95%, 96%, 97%, 98%, or 99%
identical. One of skill in the art will understand that different
resistance alleles may be maintained in the same, fixed genetic
background, i.e. in near-isogenic lines. The steps to create
inbreds that are near-isogenic for resistance alleles can be
applied to both inbred parents of any hybrid. According to the
invention, breeders may create three-way hybrids by crossing two
near-isogenic lines derived from one parental line to generate an
F1 seed parent. The F1 seed parent is crossed with the third parent
carrying the complementary resistance allele. The resulting
three-way hybrid is uniform, except for a complementary combination
of DM resistance alleles. Hybrid plants consistently share the same
genetic background as the recurrent parent, preferably at least
95%, 96%, 97%, 98%, or 99% identical, and carry various
combinations of resistance alleles. The alleles provided herein
allow for the consistent production of commercial varieties with
broad DM resistance. Furthermore, the present invention provides
alleles that can be used in different combinations to provide
spinach plants for each area and/or region with distinct DM
populations. In addition, a single variety can be obtained with a
mix of resistance alleles to reduce pathogen pressure when
resistance-breaking races or newly emerging isolates exist.
Genomic Region, Polymorphic Nucleic Acids, and Alleles Associated
With DM Resistance
[0060] Applicants have discovered three alleles from cultivated
spinach, S. oleracea, that when present together in particular
combinations in a hybrid (heterozygous) spinach plant, confer
broad-spectrum resistance to DM. Using the methods outlined herein,
these alleles were found to be located on spinach chromosome 6 in a
locus that may be defined by sequence scaffolds SF34732 and SF63815
on the distal position and SF59002, SF95487, and SF90906 on the
proximal position, or sequences at least 95% identical thereto,
including sequences at least 96%, 97%, 98%, 99%, or 100% identical
thereto, as one of skill in the art would understand that
polymorphisms may exist in such regions in different populations.
Polymorphisms that may be used to identify or diagnose the presence
of resistance alleles C, A, and/or Vt are shown in FIG. 3. Examples
of such nucleotide sequences are provided in SEQ ID NOs:1-25. These
sequences may be used in accordance with the invention to identify
or diagnose the presence or identity of a particular allele in a
plant and thus identify plants that carry resistance to DM. One of
skill in the art will further appreciate that many genetic markers
can be located throughout the S. oleracea genome, and markers may
be developed from SNPs and/or Indels in flanking sequences of the
sequences described herein and in other fragments located
throughout the S. oleracea genome. Such markers are useful in
identifying the presence or absence of a resistance allele in
accordance with the invention. Examples of markers in the spinach
genome are described in, for example, Khattak et al. (Euphytica
148:311-318, 2006). The identification of alleles and DM-resistance
conferring alleles as set forth herein, allows the use of any other
such markers in the same region and genetically linked (in linkage
disequilibrium) therewith.
[0061] The genomic region, alleles, and polymorphic markers
identified herein can be mapped relative to any publicly available
physical or genetic map to place the region described herein on
such map. One of skill in the art would also understand that
additional polymorphic nucleic acids as described herein that are
genetically linked to an allele associated with resistance to DM in
spinach and that map within about 40 cM, 20 cM, 10 cM, 5 cM, or 1
cM of an allele or a markers associated with resistance to DM in
spinach may also be used.
[0062] The above markers and allelic states are therefore
exemplary. One of skill in the art would recognize how to identify
spinach plants with other polymorphic nucleic acid markers and
allelic states thereof related to resistance to DM in spinach
consistent with the present disclosure. One of skill the art would
also know how to identify the allelic state of other polymorphic
nucleic acid markers located in the genomic region(s) or linked to
an allele or other markers identified herein, to determine their
association with resistance to DM in spinach.
Genomic Regions, Polymorphic Nucleic Acids, and Genetic Background
Associated with Recipient Parent.
[0063] Described herein are markers for use in identifying the
presence or absence of a resistance allele. In accordance with the
invention, markers in the spinach genome that are not associated
with or lack significant genetic linkage to the resistance locus
allow the selection of genetic background. One example of
background selection is recovery of the genome of a recipient
parent in a recurring backcross scheme, an example of which is
provided herein. In recurrent selection, multiple rounds of mating
between sibs carrying favorable characteristics are carried out and
selections made of progeny, allowing introduction of DM
resistance-conferring alleles along with genetic diversity into a
pedigree while maintaining favorable attributes of one or more
elite parent. Examples of markers for spinach distributed
genome-wide and that may be used in production of plants according
to the methods of the invention are described in, for example,
Khattak et al. (Euphytica 148:311-318, 2006). The identification of
DM resistance-conferring alleles set forth herein, concurrent with
background selection, allows the use of any marker in the same
genetic interval and any marker in other genetic intervals of
genomic fragments.
Introgression of a Genomic Locus Associated with Resistance to DM
in Spinach
[0064] Provided herein are spinach plants (S. oleracea) comprising
a combination of alleles that confer broad-spectrum resistance to
DM and methods of obtaining the same. In accordance with the
invention, marker-assisted introgression involves the transfer of a
chromosomal region, defined by one or more markers, from one
germplasm to a second germplasm. Offspring of a cross that contain
an introgressed genomic region can be identified by the combination
of markers characteristic of the desired introgressed genomic
region from a first germplasm (e.g., germplasm with resistance to
DM) and both linked and unlinked markers characteristic of the
desired genetic background of a second germplasm.
[0065] Markers that are linked and/or either immediately adjacent
to a locus comprising an identified DM-resistance allele or locus
as described herein that permit introgression of an allele or locus
in the absence of extraneous linked DNA from the source germplasm
containing the allele or locus are provided herewith. Those of
skill in the art will appreciate that when seeking to introgress a
smaller genomic region comprising an allele or locus associated
with resistance to DM described herein, any of the telomere
proximal or centromere proximal markers that are immediately
adjacent to a larger genomic region comprising the allele or locus
can be used to introgress that smaller genomic region.
[0066] Spinach plants or germplasm comprising such an introgressed
region that is associated with resistance to DM wherein at least
10%, 25%, 50%, 75%, 90%, or 99% of the remaining genomic sequences
carry markers characteristic of plant or germplasm that otherwise
or ordinarily comprise a genomic region associated with another
phenotype, are therefore provided in specific embodiments.
Furthermore, spinach plants comprising an introgressed region where
closely linked regions adjacent and/or immediately adjacent to a
genomic region, allele or locus, and/or markers provided herewith
that comprise genomic sequences carrying markers characteristic of
spinach plants or germplasm that otherwise or ordinarily comprise a
genomic region associated with the phenotype are also provided.
Molecular Assisted Breeding Techniques
[0067] Genetic markers that can be used in the practice of the
present invention include, but are not limited to, Restriction
Fragment Length Polymorphisms (RFLP), Amplified Fragment Length
Polymorphisms (AFLP), Simple Sequence Repeats (SSR), simple
sequence length polymorphisms (SSLPs), Single Nucleotide
Polymorphisms (SNP), Insertion/Deletion Polymorphisms (Indels),
Variable Number Tandem Repeats (VNTR), Random Amplified Polymorphic
DNA (RAPD), isozymes, and others known to those skilled in the art.
Marker discovery and development in crops provides the initial
framework for applications to marker-assisted breeding activities
(U.S. Patent Pub. Nos.: 2005/0204780; 2005/0216545; 2005/0218305;
and 2006/00504538). The resulting "genetic map" is the
representation of the relative position of characterized loci
(polymorphic nucleic acid markers or any other locus for which
alleles can be identified) to each other.
[0068] Polymorphisms comprising as little as a single nucleotide
change can be assayed in a number of ways. For example, detection
can be made by electrophoretic techniques including a single-strand
conformational polymorphism (Orita et al. Genomics, 8(2):271-278,
1989), denaturing gradient gel electrophoresis (Myers EPO 0273085,
1985), or cleavage fragment length polymorphisms (Life
Technologies, Inc., Gathersberg, Md. 20877), although the
widespread availability of DNA sequencing machines often makes it
easier to just sequence amplified products directly. Once the
polymorphic sequence difference is known, rapid assays can be
designed for progeny testing, typically involving some version of
PCR amplification of specific alleles (PASA, Sommer et al.,
Biotechniques 12(1):82-87, 1992), or PCR amplification of multiple
specific alleles (PAMSA, Dutton et al., Biotechniques
11(6):700-702, 1991).
[0069] As a set, polymorphic markers serve as a useful tool for
fingerprinting plants to inform the degree of identity of lines or
varieties (U.S. Pat. No. 6,207,367). These markers form the basis
for determining associations with phenotypes and can be used to
drive genetic gain. In certain embodiments of methods of the
invention, polymorphic nucleic acids can be used to detect in a
spinach plant a genotype associated with resistance to DM, identify
a spinach plant with a genotype associated with resistance to DM,
and to select a spinach plant with a genotype associated with
resistance to DM. In certain embodiments of methods of the
invention, polymorphic nucleic acids can be used to produce a
spinach plant that comprises in its genome a combination of alleles
associated with resistance to DM. In certain embodiments of the
invention, polymorphic nucleic acids can be used to breed progeny
spinach plants comprising a combination of alleles associated with
resistance to DM.
[0070] Certain genetic markers may include "dominant" or
"codominant" markers. "Codominant" markers reveal the presence of
two or more alleles (two per diploid individual). "Dominant"
markers reveal the presence of only a single allele. Markers are
preferably inherited in a codominant fashion so that the presence
of both alleles at a diploid locus, or multiple alleles in triploid
or tetraploid loci, are readily detectable, and they are free of
environmental variation, i.e., their heritability is 1. A marker
genotype typically comprises two marker alleles at each locus in a
diploid organism. The marker allelic composition of each locus can
be either homozygous or heterozygous. Homozygosity is a condition
where both alleles at a locus are characterized by the same
nucleotide sequence. Heterozygosity refers to different conditions
of the allele at a locus.
[0071] Nucleic acid-based analyses for determining the presence or
absence of the genetic polymorphism (i.e., for genotyping) can be
used in breeding programs for identification, selection,
introgression, and the like. A wide variety of genetic markers for
the analysis of genetic polymorphisms are available and known to
those of skill in the art. The analysis may be used to select for
genes, portions of genes, QTL, alleles, or genomic regions that
comprise or are linked to a genetic marker that is linked to or
associated with a DM resistance phenotype.
[0072] As used herein, nucleic acid analysis methods include, but
are not limited to, PCR-based detection methods (e.g., TaqMan
assays), microarray methods, mass spectrometry-based methods and/or
nucleic acid sequencing methods, including whole-genome sequencing.
In certain embodiments, the detection of polymorphic sites in a
sample of DNA, RNA, or cDNA may be facilitated through the use of
nucleic acid amplification methods. Such methods specifically
increase the concentration of polynucleotides that span the
polymorphic site, or include that site and sequences located either
distal or proximal to it. Such amplified molecules can be readily
detected by gel electrophoresis, fluorescence detection methods, or
other means.
[0073] One method of achieving such amplification employs the
polymerase chain reaction (PCR) (Mullis et al. Cold Spring Harbor
Symp. Quant. Biol. 51:263-273, 1986; European Patent No. 50,424;
European Patent No. 84,796; European Patent No. 258,017; European
Patent No. 237,362; European Patent No. 201,184; U.S. Pat. No.
4,683,202; 4,582,788; and 4,683,194), using primer pairs that are
capable of hybridizing to the proximal sequences that define a
polymorphism in its double-stranded form. Methods for typing DNA
based on mass spectrometry can also be used. Such methods are
disclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and references
found therein.
[0074] Polymorphisms in DNA sequences can be detected or typed by a
variety of effective methods well known in the art including, but
not limited to, those disclosed in U.S. Pat. Nos. 5,468,613;
5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431;
5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944;
5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981;
and 7,250,252 all of which are incorporated herein by reference in
their entireties. However, the compositions and methods of the
present invention can be used in conjunction with any polymorphism
typing method to type polymorphisms in genomic DNA samples. These
genomic DNA samples used include, but are not limited to, genomic
DNA isolated directly from a plant, cloned genomic DNA, or
amplified genomic DNA.
[0075] For instance, polymorphisms in DNA sequences can be detected
by hybridization to allele-specific oligonucleotide (ASO) probes as
disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.
5,468,613 discloses allele specific oligonucleotide hybridizations
where single or multiple nucleotide variations in nucleic acid
sequence can be detected in nucleic acids by a process in which the
sequence containing the nucleotide variation is amplified, spotted
on a membrane and treated with a labeled sequence-specific
oligonucleotide probe.
[0076] Target nucleic acid sequence can also be detected by probe
ligation methods as disclosed in U.S. Pat. No. 5,800,944, where a
sequence of interest is amplified and hybridized to probes,
followed by ligation to detect a labeled part of the probe.
[0077] Microarrays can also be used for polymorphism detection,
wherein oligonucleotide probe sets are assembled in an overlapping
fashion to represent a single sequence such that a difference in
the target sequence at one point would result in partial probe
hybridization (Borevitz et al., Genome Res. 13:513-523, 2003; Cui
et al., Bioinformatics 21:3852-3858, 2005). On any one microarray,
it is expected there will be a plurality of target sequences, which
may represent genes and/or noncoding regions wherein each target
sequence is represented by a series of overlapping
oligonucleotides, rather than by a single probe. This platform
provides for high throughput screening of a plurality of
polymorphisms. Typing of target sequences by microarray-based
methods is disclosed in U.S. Pat. Nos. 6,799,122; 6,913,879; and
6,996,476.
[0078] Target nucleic acid sequence can also be detected by probe
linking methods as disclosed in U.S. Pat. No. 5,616,464, employing
at least one pair of probes having sequences homologous to adjacent
portions of the target nucleic acid sequence and having side chains
which non-covalently bind to form a stem upon base pairing of the
probes to the target nucleic acid sequence. At least one of the
side chains has a photoactivatable group that can form a covalent
cross-link with the other side chain member of the stem.
[0079] Other methods for detecting SNPs and Indels include single
base extension (SBE) methods. Examples of SBE methods include, but
are not limited to, those disclosed in U.S. Pat. Nos. 6,004,744;
6,013,431; 5,595,890; 5,762,876; and 5,945,283. SBE methods are
based on extension of a nucleotide primer that is adjacent to a
polymorphism to incorporate a detectable nucleotide residue upon
extension of the primer. In certain embodiments, the SBE method
uses three synthetic oligonucleotides. Two of the oligonucleotides
serve as PCR primers and are complementary to the sequence of the
locus of genomic DNA which flanks a region containing the
polymorphism to be assayed. Following amplification of the region
of the genome containing the polymorphism, the PCR product is mixed
with the third oligonucleotide (called an extension primer), which
is designed to hybridize to the amplified DNA adjacent to the
polymorphism in the presence of DNA polymerase and two
differentially labeled dideoxynucleoside triphosphates. If the
polymorphism is present on the template, one of the labeled
dideoxynucleoside triphosphates can be added to the primer in a
single base chain extension. The allele present is then inferred by
determining which of the two differential labels was added to the
extension primer. Homozygous samples will result in only one of the
two labeled bases being incorporated, and only one of the two
labels will be detected. Heterozygous samples have both alleles
present and will direct incorporation of both labels (into
different molecules of the extension primer), and thus both labels
will be detected.
[0080] In another method for detecting polymorphisms, SNPs and
Indels can be detected by methods disclosed in U.S. Pat. Nos.
5,210,015; 5,876,930; and 6,030,787, in which an oligonucleotide
probe having a 5' fluorescent reporter dye and a 3' quencher dye
covalently linked to the 5' and 3' ends of the probe. When the
probe is intact, the proximity of the reporter dye to the quencher
dye results in the suppression of the reporter dye fluorescence,
e.g. by Forster-type energy transfer. During PCR, forward and
reverse primers hybridize to a specific sequence of the target DNA
flanking a polymorphism, while the hybridization probe hybridizes
to polymorphism-containing sequence within the amplified PCR
product. In the subsequent PCR cycle, a DNA polymerase with
5'.fwdarw.3' exonuclease activity cleaves the probe and separates
the reporter dye from the quencher dye resulting in increased
fluorescence of the reporter.
[0081] In another embodiment, an allele or locus of interest can be
directly sequenced using nucleic acid sequencing technologies.
Methods for nucleic acid sequencing are known in the art and
include technologies provided by 454 Life Sciences (Branford,
Conn.), Agencourt Bioscience (Beverly, Mass.), Applied Biosystems
(Foster City, Calif.), LI-COR Biosciences (Lincoln, Nebr.),
NimbleGen Systems (Madison, Wis.), Illumina (San Diego, Calif.),
and VisiGen Biotechnologies (Houston, Tex.). Such nucleic acid
sequencing technologies comprise formats such as parallel bead
arrays, sequencing by ligation, capillary electrophoresis,
electronic microchips, "biochips," microarrays, parallel
microchips, and single-molecule arrays, as reviewed by R.F. Service
Science 311:1544-1546, 2006 .
[0082] Markers used in accordance with the present invention should
preferably be diagnostic of origin in order for inferences to be
made about subsequent populations. Experience to date suggests that
SNP markers may be ideal for mapping because the likelihood that a
particular SNP allele is derived from independent origins in the
extant populations of a particular species is very low. As such,
SNP markers appear to be useful for tracking and assisting
introgression of alleles or loci.
Definitions
[0083] The following definitions are provided to better define the
present invention and to guide those of ordinary skill in the art
in the practice of the present invention. Unless otherwise noted,
terms are to be understood according to conventional usage by those
of ordinary skill in the relevant art.
[0084] As used herein, the term "plant" includes plant cells, plant
protoplasts, plant cells of tissue culture from which spinach
plants can be regenerated, plant calli, plant clumps and plant
cells that are intact in plants or parts of plants such as pollen,
flowers, seeds, leaves, stems, and the like.
[0085] As used herein, "DM" or "downy mildew" refers to a disease
of plants, such as spinach, caused by a pathogen from the genus
Peronospora, in particular Peronospora farinosa f. sp. Spinaciae
(Pfs).
[0086] As used herein, "race" refers to an officially designated
strain of Peronospora farinosa f. sp. spinaciae (Pfs) that can
cause DM. As used herein, "isolate" refers to a newly occurring
strain of Peronospora farinosa f. sp. spinaciae (Pfs) that can
cause DM, and has not yet been officially named. A spinach plant
with resistance to DM according to the present invention carries a
combination of alleles selected from A, C, and Vt. The DM
resistance may be to one or more known races of Peronospora
farinosa f. sp. spinaciae, or may be to one or more isolates of
Peronospora farinosa f. sp. spinaciae. In another embodiment, a
plant of the invention may be defined as resistant to at least
Peronospora farinosa f. sp. spinaciae Pfs 7, 8, 10, 11, 12, 13,
and/or 14.
[0087] As used herein, the terms "pedigree," "population," and
"progeny" mean a collection of plants that share a common parental
derivation.
[0088] As used herein, the terms "variety" and "cultivar" mean a
group of similar plants that by their genetic pedigrees and
performance can be identified from other varieties within the same
species.
[0089] As used herein, an "allele" refers to one of two or more
alternative forms of a genomic sequence at a given locus on a
chromosome.
[0090] A "Quantitative Trait Locus (QTL)" is a chromosomal location
that encodes for at least a first allele that affects the
expressivity of a phenotype.
[0091] As used herein, a "marker" means a detectable characteristic
that can be used to discriminate between organisms. Examples of
such characteristics include, but are not limited to, genetic
markers, biochemical markers, metabolites, morphological
characteristics, and agronomic characteristics.
[0092] As used herein, the term "phenotype" means the detectable
characteristics of a cell or organism that can be influenced by
gene expression.
[0093] As used herein, the term "resistance" means evasion and/or
reduction of pathogen infection by plant innate immunity, which can
be shown by the absence and/or reduction of disease symptoms when
compared to a susceptible plant.
[0094] As used herein, the term "susceptible" means the infection
of a plant by a pathogen, resulting in disease symptoms.
[0095] As used herein, the term "compatible interaction" means the
infection of a susceptible plant by a pathogen, resulting in
disease symptoms.
[0096] As used herein, the term "incompatible interaction" means
the evasion and/or reduction of infection of a resistant plant by a
pathogen, which can be shown by the absence and/or a reduction of
disease symptoms.
[0097] As used herein, the term "genotype" means the specific
allelic makeup of a plant.
[0098] As used herein, the term "heterozygous phase" means a
diploid plant, such as spinach, that carries two distinct copies of
an allele.
[0099] As used herein, the term "homozygous phase" means a diploid
plant, such as spinach, that carries two identical copies of an
allele.
[0100] As used herein, the term "introgressed," when used in
reference to a genetic locus, refers to a genetic locus that has
been introduced into a new genetic background, such as through
backcrossing. Introgression of a genetic locus can therefore be
achieved through plant breeding methods and/or by molecular genetic
methods. Such molecular genetic methods include, but are not
limited to, various plant transformation techniques and/or methods
that provide for homologous recombination, non-homologous
recombination, site-specific recombination, and/or genomic
modifications that provide for locus substitution or locus
conversion.
[0101] As used herein, the term "linked," when used in the context
of nucleic acid markers and/or genomic regions, means that the
markers and/or genomic regions are located on the same linkage
group or chromosome such that they tend to segregate together at
meiosis.
[0102] As used herein, the term "denoting" when used in reference
to a plant genotype refers to any method whereby a plant is
indicated to have a certain genotype. This includes any means of
identification of a plant having a certain genotype. Indication of
a certain genotype may include, but is not limited to, any entry
into any type of written or electronic medium or database whereby
the plant's genotype is provided. Indications of a certain genotype
may also include, but are not limited to, any method where a plant
is physically marked or tagged. Illustrative examples of physical
marking or tags useful in the invention include, but are not
limited to, a barcode, a radio-frequency identification (RFID), a
label, or the like.
Deposit Information
[0103] A deposit was made of at least 2500 seeds of Spinacia
oleracea accessions designated SMBS011-1162M, SMB-66-1143M, and
SSB-66-1131M. The deposits were made with the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va.
20110-2209 USA. The deposits were assigned ATCC Accession Nos.
PTA-120472, PTA-12486, and PTA-12041, respectively. The dates of
deposit of these accessions were Jul. 17, 2013, Feb. 2, 2012, and
Aug. 19, 2011, respectively. Access to the deposits will be
available during the pendency of the application to persons
entitled thereto upon request. The deposits will be maintained in
the ATCC Depository, which is a public depository, for a period of
30 years, or 5 years after the most recent request, or for the
enforceable life of the patent, whichever is longer, and will be
replaced if nonviable during that period. Applicant does not waive
any infringement of their rights granted under this patent or any
other form of variety protection, including the Plant Variety
Protection Act (7 U.S.C. 2321 et seq.).
EXAMPLES
[0104] The following disclosed embodiments are merely
representative of the invention which may be embodied in various
forms. Thus, specific structural, functional, and procedural
details disclosed in the following examples are not to be
interpreted as limiting.
Example 1
Identification of Novel Resistance to DM Races in Spinacia
oleracea
[0105] A screen was performed on S. oleracea germplasm using the
disease assay described in Example 5. S. oleracea accessions in an
internal genebank were screened. In addition, publicly available
spinach hybrids that were either fully susceptible, or carried a
combination of resistance specificities were included in the trial
as susceptible and resistance checks. Infection was performed with
Peronospora farinosa f. sp. spinaciae (Pfs) races 7, 8, and 10-14
to complement historic data on races 1-6. Race 9 is no longer
present in the field. In addition to strains with a race
designation, a resistance-breaking isolate, collected in California
(US) in the spring of 2013 was included. This isolate is currently
referred to as UA4712 by the International Working Group on
Peronospora and is a probable candidate for race Pfs 15.
[0106] The response of control entries to infection is presented in
Table 1 and is consistent with data published elsewhere (Correll et
al., Eur J Plant Pathol 129:193-205, 2011). Despite the
contribution of resistance specificities by both parents, all
commercial hybrid checks included in the trial failed to respond
with resistance to all isolates. For example, variety Lion is
susceptible to race 10, Lazio is susceptible to races 11-14, and
Pigeon is susceptible to race 14. Resistance to both races 10 and
14 was not observed in any single hybrid, and a combination of
resistance to races 13 and 14 was only observed for the hybrid
Lion.
[0107] Among the S. oleracea accessions in the internal genebank,
three lines were found to have a complementary combination of
resistances. Inbred line SMBS011-1162M, carrying an allele
identified as allele A, was resistant to multiple races, including
races 13 and 14. A second inbred line, SSB-66-1131M, included an
allele designated Vt, was found to be resistant to races 1-13. A
final inbred line, SMB-66-1143M, which includes an allele
identified as allele C, was shown to be resistant to new isolate
UA4712, in addition to races 1-10. Each of the identified alleles
were found to confer DM resistance to different combinations of
races.
TABLE-US-00001 TABLE 1 Phenotypic response of control and
experimental entries to infection by Pfs races 1-14 and isolate
UA4712. LINES A Vt C Accession COMMERCIAL HYBRIDS (SMBS011-
(SSB-66- (SMB-66- Race Viroflay Resistoflay Clermont Campania
Boeing Califlay Whale Lion Lazio Pigeon 1162M) 1131M) 1143M) Pfs 1
S R R R R R R R R R R R R Pfs 2 S R R R R S S R R R S R R Pfs 3 S S
R R R R R R R R R R R Pfs 4 S S R R R S S R R R S R R Pfs 5 S S S R
R R R R R R R R R Pfs 6 S S S S R S S R R R S R R Pfs 7 S S S R R S
S R R R R R R Pfs 8 S S S S S R R R R R R R R Pfs 10 S S S S S S S
S R R S R R Pfs 11 S S S R R R R R S R R R S Pfs 12 S S S S S R R R
S R R R S Pfs 13 S S S S R S S R S R R R S Pfs 14 S S S S S R R R S
S R S S UA4712 S S R S, susceptible response, with an average
percentage of sporulating leaf surface estimated above 85%; R,
resistance, demonstrated by no or little (<3%) sporulation.
Example 2
Mapping Resistance to DM
[0108] Irish et al. (2008) identified a co-dominant marker,
designated Dm-1, which was closely linked to the resistance locus
RPF1 from S. oleracea. The genetic distance between Dm-1 and RPF1
was estimated to be 1.7 cM. Further efforts to fine-map the Dm-1
marker relative to the RPF1 locus generated sequence resources in
the vicinity of the Dm-1 marker, but did not result in cloning RPF1
(Yang et al., Initial fine-mapping of the spinach downy mildew
resistance locus RPF1, University of Arkansas, 2013, 102 pages;
1536814). Given the absence of a causal polymorphism and the
distance between Dm-1 and RPF1, association may be lost during
backcrossing or recurrent selection. Therefore, markers flanking
resistance on both sides were needed for tracking resistance
alleles and further fine-mapping.
[0109] Spinach lines carrying one of the three resistance alleles,
A, Vt, or C, were individually crossed to a line that exclusively
harbored resistance to races Pfs 1-4. Resistance to most races is
inherited in a dominant fashion, based on the absence of symptoms
upon infection of F1 individuals with races that differentiate
between resistance from both parents. In contrast, resistance from
allele A to races 7 and 13 appears to be recessive. F3 families
were obtained through single seed descent from the separate
bi-parental F1 crosses and screened phenotypically. The disease
assay included 74 F3 families segregating for allele A, which were
infected with Pfs race 8; 107 F3 families segregating for allele Vt
challenged with races 7, 10, and 12; and 40 F3 families segregating
for allele C, which were inoculated with race 10. Interestingly,
when Vt-derived families demonstrated resistance to one race,
immunity to the other two races was also observed.
[0110] Based on public information, the likely position of sequence
scaffolds designated SF34732, SF59002, SF62749, SF63815, SF90906,
SF95487, and SF178637 co-located with DM resistance. Examples of
nucleotide sequences corresponding to alleles C, A, and Vt
conferring resistance to DM are provided as SEQ ID NOs:1-25.
Additionally, polymorphisms between individual alleles of scaffolds
are shown in FIG. 3. Single nucleotide polymorphisms (SNPs) derived
from these scaffolds were selected based on being polymorphic in
one or more mapping populations. The SNPs were converted to
TaqMan.TM. assays and used for genotyping F2 individuals of the
respective F3 families of each population. Linkage disequilibrium
was tested, and the genetic distances among SNPs and phenotypes
were estimated using Joinmap (Stam, Plant J 3:739-744, 1993).
Association analysis confirmed linkage of sequence scaffolds and
resistance by assembly of single groups. This result was found to
be independent of the source or race used for mapping (FIG. 1).
Observed recombination frequencies and derived genetic distances
varied due to the distinct sample size of each population.
Scaffolds SF59002 and SF95487 appeared to co-segregate in every
population. The order of scaffolds and genetic markers was
consistent for populations segregating for allele Vt or allele C.
However, the exact position of SF62749 relative to, for example,
scaffolds SF59002 and SF90906 remains ambiguous. Finally, converted
and mapped SSR markers, which were published by Khattak et al.
(Euphytica 148:311-318, 2006), identified the linkage group as
public LG6.
Example 3
Quantitative Effects of DM Resistance Alleles
[0111] The genotype and phenotypes collected in Example 2 were used
in a quantitative analysis with mapQTL, applying standard interval
mapping settings (Van Ooijen, 1996). A major locus for resistance
to Peronospora farinosa f. sp. spinaciae race 8 was detected in the
mapping population segregating for allele A. Evidenced by a LOD
peak of 57.51, the trait was highly associated with markers derived
from scaffolds SF59002, SF95487, and SF63815 (Table 2). The locus
appeared to have a major effect, explaining 97.2% of the observed
variance.
TABLE-US-00002 TABLE 2 Quantitative analysis of resistance allele A
to Pfs race 8. map lod iter mu_A mu_H mu_B var % expl add dom locus
-- -- -- -- -- -- -- -- -- -- -- 0 40.53 4 94.4444 50 2.63158
92.0657 92 45.9064 1.46199 SF90906a 0.7 46.42 4 97.0588 50 2.63158
63.8022 94.4 47.2136 0.154799 SF90906b 1.4 57.51 4 100 50 2.63158
32.0057 97.2 48.6842 -1.31579 SF59002 1.4 57.51 4 100 50 2.63158
32.0057 97.2 48.6842 -1.31579 SF95487 1.4 57.51 4 100 50 2.63158
32.0057 97.2 48.6842 -1.31579 SF63815 5.7 31.74 4 94.1176 50
2.77778 159.093 86.1 45.6699 1.55229 SF62749
[0112] A second analysis with source Vt (SSB-66-1131M) for
resistance to Pfs races 7, 10, and 12 also identified a resistance
locus (Tables 3, 4, and 5). Independent of the isolate used, the
locus appeared to be strongly linked to scaffolds SF95487 and
SF59002, evident by a LOD peak of 91.81. This locus had a major
effect, explaining nearly all phenotypic variance. Strikingly, the
loci identified in resistant sources A (SMBS011-1162M) and Vt
(SSB-66-1131M) were collinear.
TABLE-US-00003 TABLE 3 Quantitative analysis of resistance allele
Vt to Pfs race 7. map lod iter mu_A mu_H mu_B var % expl add dom
locus -- -- -- -- -- -- -- -- -- -- -- 0 24.43 4 88 52.7273 9.25926
408.188 65.1 39.3704 4.09764 SF34732a 0.6 25.6 4 88.4615 51.8519
9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.7 91.81 4 98.0769
50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 4 98.0769 50
0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.4444
50.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 4
89.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749
TABLE-US-00004 TABLE 4 Quantitative analysis of resistance allele
Vt to Pfs race 10. map lod iter mu_A mu_H mu_B var % expl add dom
locus -- -- -- -- -- -- -- -- -- -- -- 0 24.43 4 88 52.7273 9.25926
408.188 65.1 39.3704 4.09764 SF34732a 0.6 25.6 4 88.4615 51.8519
9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.7 91.81 4 98.0769
50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 4 98.0769 50
0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.4444
50.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 4
89.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749
TABLE-US-00005 TABLE 5 Quantitative analysis of resistance allele
Vt to Pfs race 12. map lod iter mu_A mu_H mu_B var % expl add dom
locus -- -- -- -- -- -- -- -- -- -- -- 0 24.43 4 88 52.7273 9.25926
408.188 65.1 39.3704 4.09764 SF34732a 0.6 25.6 4 88.4615 51.8519
9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.7 91.81 4 98.0769
50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 4 98.0769 50
0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.4444
50.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 4
89.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749
[0113] A study for resistance from allele C was performed with Pfs
race 10. Similar to previous results, a major locus for resistance
appeared to be significantly associated with scaffolds SF34732 and
SF6381 (Table 6).
TABLE-US-00006 TABLE 6 Quantitative analysis of resistance allele C
to Pfs race 10. map lod iter mu_A mu_H mu_B var % expl add dom
locus -- -- -- -- -- -- -- -- -- -- -- 0 perfect fit 3 100 50 0 0
100 50 0 SF34732 0 perfect fit 3 100 50 0 0 100 50 0 SF63815 1.3
28.02 4 100 46.6667 0 58.3333 96 50 -3.33333 SF95487 1.3 28.02 4
100 46.6667 0 58.3333 96 50 -3.33333 SF59002 2.6 22.3 4 100 46.4286
6.25 112.723 92.3 46.875 -6.69643 SF90906 8.4 12.19 4 100 47.2222
8.33333 361.111 75.4 45.8333 -6.94444 SF62749 22.1 6.14 4 96.6667
47.2222 28.5714 724.504 50.7 34.0476 -15.3968 SF178637
[0114] DM resistance mapped to Chromosome 6 independent of the
source or isolate used. The resistance loci were delineated by
common markers, on the distal position typically sequence scaffolds
SF34732 and SF63815 and on the proximal position typically sequence
scaffolds SF59002, SF95487 and SF90906.
Example 4
Deployment of Resistance Alleles A, Vt, and C in Hybrids
[0115] The resistance alleles A, C and Vt were introduced into a
susceptible hybrid. The inbred SMBS011-1162M was used as a donors
for allele A, the line SSB-66-1131M for Vt, and for allele C,
SMB-66-1143M was used as a source. Each of the alleles was
introgressed separately into both inbred parents of the susceptible
hybrid, following breeding methods known in the art. Next, a first
spinach plant which is homozygous for allele A (A/A) was crossed to
a second spinach plant (C/C) to generate a hybrid F 1 which was
heterozygous for alleles A and C (FIG. 2A). This hybrid was
referred to as Single Hybrid A, or as F1 (A/C). F 1(A/C) was
screened with known races and isolate UA4712 of Peronospora
farinosa f. sp. spinaciae, as described in Example 5. The hybrid
was found to be resistant to all Pfs races, with the exception of
race 13 (Table 7). Hybrid A is also resistant to the newly emerging
isolate UA4712.
[0116] Hybrid B carrying alleles C and Vt (FIG. 2B) was screened as
described and found to be resistant to all Pfs races except Pfs 14
(Table 7). This is important, as Pfs race 14 does not occur in
Europe. A combination of alleles C and Vt in a spinach plant
provides a fully resistant variety for the European market.
[0117] Hybrid C with alleles A and Vt was resistant to all Pfs
races except isolate UA4712 (Table 7). This is also significant,
since the distribution of a new isolate is initially limited to few
locations. F1(A/Vt) is resistant to all currently named Pfs
races.
[0118] In addition to biparental hybrids, three-way hybrids were
generated. Three-way hybrids are produced by crossing an F1 seed
parent to an inbred parent P2. The F1 seed parent results from a
cross between two near-isogenic lines of P1, each carrying a
distinct resistance allele. The inbred parent P2 carries a third,
complementary allele. A three-way hybrid allows the deployment of
all three identified alleles in a mixed population of diploid
hybrid plants. Three versions of the 3-way hybrid are possible
(FIG. 2B). Each cross results in a population of hybrid plants with
resistance to all known Pfs races and isolate UA4712. However, the
frequency of each allele will depend on how the three-way cross is
structured.
[0119] Novel resistance originating from the genetic variety
contained within cultivated spinach, Spinacia oleracea was
identified. This resistance is surprising because genetic diversity
in cultivated crops is typically narrow (Fernie et al., Curr.
Opinion Pl. Biol. 9:196-202, 2006). The identification of the three
unique resistance alleles, A, Vt, and/or C, in the three spinach
lines provides for race-specific DM resistance breeding, with only
intra-specific S. oleracea crosses. Additionally, these alleles
allow stacking of resistance in single hybrid combinations that are
unsurpassed in combining novel attributes, including resistance to
all known Pfs races, to the new isolate UA4712, or resistance to
all described European DM populations. Markers associated with the
three resistance alleles allow introduction of the described and
other DM-resistance alleles in any cultivated spinach hybrid.
Finally, a three-way hybrid method is disclosed to allow for the
development of additional DM resistant varieties.
TABLE-US-00007 TABLE 7 Phenotypic response of testcrosses to
infection of leaf discs by Pfs races 1-8, 10-14 and isolate UA4712
TEST CROSS Pfs 1 Pfs 2 Pfs 3 Pfs 4 Pfs 5 Pfs 6 Pfs 7 Pfs 8 Pfs 10
Pfs 11 Pfs 12 Pfs 13 Pfs 14 UA4712 F1 (A/C) R R R R R R R R R R R S
R R F1 (C/Vt) R R R R R R R R R R R R S R F1(AxVt) R R R R R R R R
R R R R R S "S" indicates a susceptible response. "R" indicates
resistance
Example 5
Assays for Screening Spinach Accessions for Resistance to DM
[0120] A test was utilized for screening spinach accessions for
resistance to downy mildew (DM) that originated from the
International Union for the Protection of New Varieties of Plants
(UPOV). The "Protocol for Tests on Distinctness, Uniformity, and
Stability of Spinacia oleracea L. Spinach," UPOV Code: SPINA_OLE,
CPVO-TP/055/5 was adopted and into force on Feb. 27, 2013. The
protocol is as follows:
[0121] Races of Peronospora farinosa f. sp. spinaciae are
maintained on living host plants, obtainable from Naktuinbouw (P.O.
Box 40, NL-2370 AA, Roelofarendsveen, Netherlands,
naktuinbouw.com), or plant material with spores stored at
-20.degree. C. for a maximum of one year.
[0122] Execution of test: Growth stage of plants: First
cotyledons/leaf, eleven-day-old plants; Temperature: 15.degree. C.
during day/12.degree. C. during night; Light: 15 hours per day,
after emergence; Growing method: In soil in pots or trays in a
glasshouse or growth chamber.
[0123] Method of inoculation: Sporulating leaves, taken from host
plants that were infected seven days before, are thoroughly rinsed
with sterile tap water (maximum 150 ml water per 224 plants). The
spore suspension is filtered through cheesecloth and sprayed on
test plants until the inoculum covers the leaves but does not run
off. 150 ml of suspension is enough for up to 3.times.224 plants.
Spore density should be 20,000 to 100,000 conidia/ml water. The
spore suspension should be used fresh. As spinach downy mildew is
wind-borne, sporulating plants should be kept in closed containers
or isolated chambers to prevent any cross-contamination.
[0124] Resistant controls are needed in each multiplication and in
each test to ensure the race identity. Light and humidity
conditions during seedling development and incubation are critical.
Optimal humidity of approximately 80-90% RH allows plant growth and
fungal growth; strong light inhibits spore germination and
infection. The test should be carried out in wintertime with
protection against direct sunshine. After inoculation, the plants
should remain under plastic for three days. After this time, the
plastic should be slightly raised during the daytime.
[0125] Duration of test: Multiplication harvest spores 7 days after
inoculation; Sowing to inoculation: 11 days; Inoculation to
reading: 10 days; Number of plants tested: 56 plants; Evaluation of
infection: Resistance is usually complete; sometimes necrotic spots
are visible as a result of infection. Susceptible plants show
varying degrees of sporulation. Sporulation is visible as a grey
covering on leaves, starting on the more humid abaxial side.
[0126] Differential varieties to identify races: Races Pfs: 1-8 and
10-13 of Peronospora farinosa f. sp. spinaciae are defined with a
standard set of "differential varieties" according to Table 8.
TABLE-US-00008 TABLE 8 Differential varieties to identify races:
Races Pfs: 1-8 and 10-13 of Peronospora farinosa f. sp. spinaciae.
Differential variety Pfs: 1 Pfs: 2 Pfs: 3 Pfs: 4 Pfs: 5 Pfs: 6 Pfs:
7 Pfs: 8 Pfs: 10 Pfs: 11 Pfs: 12 Pfs: 13 Viroflay S S S S S S S S S
S S S Resistoflay R R S S S S S S S S S S Califlay R S R S R S S R
S R R S Clermont R R R R S S S S S S S S Campania R R R R R S R S S
R S S Boeing R R R R R R R S S R S R Lion R R R R R R R R S R R R
Lazio R R R R R R R R R S S S R, resistance present; S, resistance
absent (susceptible)
Sequence CWU 1
1
25193DNASpinacia oleracea 1taggggtaat taaccaaatt ggtattaaat
tatacccatt tgccctgttg gtgtaaaggt 60cgatggatga gtataaatat tactctctcc
gtc 93270DNASpinacia oleracea 2tggggctaat taaccagatt ggtattaact
tatacccatt tgccacgttg gtgtaaaggt 60cgatggatgg 70393DNASpinacia
oleracea 3taggggtaat taaccagatt ggtattaaat tatacccatt tgccacgttg
gtgtaaaggt 60cgatggatga gtataaatat tactctctct gtc 934102DNASpinacia
oleracea 4aaggtttgat gctgcaagag aaaagtagat ttagaaacgg gtaaacagtg
aaaaaaagat 60ggaatattac tcatactata acatttgttt caaggaaacc at
1025100DNASpinacia oleracea 5aaggtttgac gctgcaagac aaaggtagat
ttagaaacgg gtcaacagta aaaaaaagat 60ggaattactc atactataac atttgtttca
aggaaaccat 1006100DNASpinacia oleracea 6aaggtttgac gctgcaagac
aaaggtagat ttagaaacgg gtcaacagta aaaaaaagat 60ggaattactc atactataac
atttgtttca aggaaaccat 1007101DNASpinacia oleracea 7aagttatgtt
aggcttggga atggaaggtt attcactggg acgtctattt ataaagggag 60ggtgaatttg
tccgtcaaga agttgtaccc gattgtgtat a 1018101DNASpinacia oleracea
8aagttatgtt aggcttggga atggaaggtt attcactggg acgtctattt ataaagggag
60gatgaatttg tccgtcaaga agttgtaccc gattgtgtat a 1019100DNASpinacia
oleracea 9taatatcaat attttttata taaaccattt taataaatta ttcccttcgt
cccttaatat 60tcgacccgat ttgacttttt gcactgttac ataattcaat
10010101DNASpinacia oleracea 10taatatcaat attttttata aaaaccattt
taataaatta ctccctccgt ctcttaatac 60tcgactcgct ttgacttttt gcactattta
cataattcaa t 10111101DNASpinacia oleracea 11taatatcaat attttttata
aaaaccattt taataaatta ctccctccgt ctcttaatac 60tcgactcgct ttgacttttt
gcactattta cataattcaa t 10112103DNASpinacia oleracea 12ttgaatgaga
actttgattt tagaaaggaa gataacaaca agttttctgt ttttcacaaa 60attaaaaaat
caaaatataa aaatcacaaa aagtaatttt cag 10313104DNASpinacia oleracea
13ttgaatgaga actttgattt tagaaaggaa gacaacaaca agttttctgt tttttacaaa
60attaaaaaat caaaatataa aaatcacgaa aagtaatttt tcag
10414100DNASpinacia oleracea 14ttgaatgaga actttgattt tagaaaggaa
gacaacaaca agttttctgt ttttcacaaa 60attaaaaatc aaaatataaa aatcacgaaa
agtaattttc 10015101DNASpinacia oleracea 15aactaacact actaaaaaat
gatgtgattt tttattttat tttttcatct aaaaaaagaa 60aagaacaaga aacccccaat
cacaccgtaa cccttaaaaa g 10116101DNASpinacia oleracea 16aactaacact
actaaaaaat gatgtgattt tttattttat tttttcatct aagaaaagaa 60aagaacaaga
aacccccaat cacaccgtaa cccttaaaaa g 10117101DNASpinacia oleracea
17aactaacact actaaaaaat gatgtgattt tttattttat tttttcatct aagaaaagaa
60aagaacaaga aacccccaat cacaccgtaa cccttaaaaa g 10118101DNASpinacia
oleracea 18ggctcaatgt catgttttct acaaaatggc acccataact cggcaaagct
agctgcctca 60gccattgcct cgaaagttag gagagcgccg ccatcatcgg a
10119101DNASpinacia oleracea 19ggctcaatgt catgttttct acaaaatggc
acccataact cggcaaagct agctgcctca 60gccattgcct cgaaagttag gagagcgccg
ccatcatcgg a 10120101DNASpinacia oleracea 20ggctcaatgt catgttttct
acaaaatggc acccataact cggcaaagct agctgcttca 60gccattgcct cgaaagttag
gagagcgccg ccatcatcgg a 10121102DNASpinacia oleracea 21gcaatcgtta
catattgtaa atctgcatat aataaaaatt ataaaaaaat aaattgatat 60tctaaaacat
tttaattgtc gcaacttacg aacctttatc at 10222102DNASpinacia oleracea
22gcaatcgtta catattgtaa atctgcatat aataaaaatt gtaaaaaaat aaattgatat
60tctaaaacat tttaattgtc gcaacttacg aacctttatc at
10223102DNASpinacia oleracea 23gcaatcgtta catattgtaa atctgcatat
aataaaaatt ataaaaaaat aaattgatat 60tctaaaacat tttaattgtg gcaacttacg
aacctttatc at 10224239DNASpinacia oleracea 24aaaatgcaac acaatctatc
ttaacctaat cattaagttg aataatcaac tattaaccca 60aaaaatgact gctcttatca
ttaagttgaa taatcagtag atattgccta gtgaaccatc 120aaacaaatta
aaaatgcaac acaatctatc ttaacctaat cattaagttg aataatcaac
180tattaacccg aaaaatggct gctcttttaa acctttgaaa cccgttcatc tttctcaac
23925198DNASpinacia oleracea 25agaatcgtcc tgttaatcga tctaaaccct
cttctccacc tccaaaaccc taaatcttac 60atcacttcaa tcctcacttc cgccaaaatt
ctcctctcat ttccccctct ttctctttcc 120ctatcctcct tcaagctctt
cttctcttct ctatctcctc tcaaatcgtc atcctcgctc 180cccaacttcc caatttca
198
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