Developing new SSR markers for barley derived from the EST database
Karen A. Beaubien and Kevin P. Smith*
Dept of Agronomy & Plant Genetics, University of Minnesota, St Paul, MN, *Corresponding Author: PH: (612) 624-1211; E-mail: smith376@umn.edu
INTRODUCTION
Genetic mapping of germplasm relevant to plant breeding programs with
molecular markers serves to connect the wide array of valuable germplasm
and genetic variation used in agriculture to the expanding frontier of plant
genomics. Progress in mapping genes for important traits in barley and
utilizing markers to improve the efficiency of breeding is dependent on the
availability, cost, and ease of use of molecular markers. There are currently
632 SSR marker primer sets that have been developed of which 242 have
been mapped in barley (Saghai Maroof et al., 1994; Liu et al., 1996; Ramsay
et al., 2000). More recently, several studies have reported the development
of an additional 202 SSR markers (Li et al., 2003, Thiel et al., 2003),
however, most of these markers mapped to regions of the barley genome
that already have good coverage with SSR markers (Table 1). There is still a
need for additional publicly available SSR markers to improve coverage of
the barley genome and facilitate breeding and genetics research.
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53
58
61
65
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120
122
123
ABG704
MWG036B
RISIC10A Bmag0767
BCD129
MWG851A
MWG555A
ABG320
HVM004
iEst5
His3A
Bmag0206
WG789A
MWG089
MWG530
ABC167A
dRcs1
ABG380
Mad1 MWG564
ABC158
ABC151A
KSUA1A
ABC154A
MWG836
UMB106
UMB107
Brz
ABC156D
BCD098
MWG649A
HVM014b
MWG911B
BCD147
ABC308
ABG476
BCD349
ABC154B UMB104
Bmag0110a KFP194
Bmag0321
Amy2
ABC255
Bmac0187
Bmac0064
Bmag0482b
ABG156B
Ubi1
MWG571D TLM1
ABC310B
KFP190
RISP103
bBE54E
ABG608
ABC305
UMB105 Bmac0156
ABG497B
ABG461A UMB101
UMB102
WG420 RZ682
ABG652A
ABC253
135
137
138
139
140
HVM005
HVPRP1B
MWG635B
UMB103
PSR106B
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RESULTS
Developed and evaluated 4
methods for designing SSR markers
which differed in efficiency (Table 2).
238 different primer pairs were
designed and screened.
44 new markers developed that
produce 2-6 alleles among 12
mapping parents (Figure 2).
31 markers mapped using the
Chevron x M69, Fredrickson x
Stander, and Steptoe x Morex
mapping populations (Figure 1).
Markers are distributed
throughout the barley genome. Most
do not map to the target region for
which they were designed.
16 of the 31 mapped markers map
to BINs identified as having poor
coverage with currently available
SSR markers.
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5
7
9
Integrate new EST-derived barley
SSR markers on a barley consensus
map derived from three mapping
populations.
ABG058
ABG703B
CDO057A
16
SC1373
22
25
27
28
29
30
31
33
36
40
43
44
45
49
51
ABG008
UMB205a RbcS
UMB203
BCD351F
ABG318
ABG002
ABC156A HVM036
UMB205b
MWG858
ABG358
ABC311
ABG459
Bmag0174c
Pox
ABG005MWG520A
Adh8 ABG156A
Bmac0218b
cMWG663A
UMB202
MWG887A
MWG557
Bmac0140ABG316C
UMB201 Bmac0093
EBmac0557
HVBKASI ABC167B
Bmac0129b EBmac0558
EBmac0521b GMS003
HVM023
UMB204 GBM1024
EBmac0521c
EBmac0521a
ABC306 EBmac0615
bBE54D
Bmag0140 Bmag0378
CDO588ABG014
BCD1087
TLM3 ABC256
ABG619MWG950
His3C
ABC152DBmag0003c
Bmag0003d
MWG865
Rrn5S1
Vrs1
KSUF15
Bmag0125
MWG503
GBM1062
MWG503B
Bmac0144g
MWG882B
MWG503A
Bmac0144b
Crg3A
ABG072
ABG497C
MWG649C
STS3B-142
ABC252
EBmac0415
HVM054
ABC157
ABC153
ABG317B
ABG317A
ABG010B
ABG316E
ABG010CPcr1
cMWG720
BG123A
bBE54C
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165
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13
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30
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43
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ASE1A
MWG878A
57
58
60
62
64
65
66
GBM1036
Develop PCR primers flanking
SSRs in barley ESTs and screen for
polymorphisms on a set of barley
mapping parents.
We evaluated four methods of designing EST-SSR markers. Methods 1, 2,
and 3 were based on identifying syntenous regions in barley using rice
genome sequence. Method 4 used expression specific ESTs.
3 (3H)
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MATERIALS AND METHODS
Increase the number of available
SSR markers in BINs with poor
coverage (Table 1).
2 (2H)
1 (7H)
OBJECTIVES
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161
4 (4H)
ABA307B
MWG571C
STS3B-94
STS3B-47
UMB301
STS3B-65
STS3B-80
STS3B-52
ABC171
CDO395
MWG798B
MWG584
ABG471
ABC171A
Bmag0828
UMB302
Bmag0138 UMB304
Bmag0010c
UMB303 Bmag0482a
HVDHN7 BCD828
HVM009 Bmag0131
ABG399A Bmag0122
Bmac0067 Bmac0129a
Bmag0136 Bmag0905
Bmag0603
Bmag0006 Bmag0482c
MWG680
Bmag0131
Bmag0828
PSR156A
CDO105C MWG571B
MWG2227C
ABG377
Bmag0225
MWG555B HVM060
ABG315
Bmag0010b
ABG453
PSR78
MWG571A
CDO345
ABG499
HVM070
CDO113B
Bmag0606
His4B Bmag0482d
ABG004
MWG803
WG110
ABC151C mPub
BCD147B
Bmag0013
ABC161
Bmag0877 MWG902
ABG654A
Glb4
iBgl
ABG495B
EBmac0541
MWG838
ABG319B
ABC172
Figure 2. Sample gel
images from 4
markers. Markers are
screened on 12
mapping parents
(from left): Atahualpa;
M81; Chevron; M69;
Fredrickson; Stander;
Harrington; OUH602;
Hor211; Lacey;
Steptoe; Morex.
Methods 1-3: Sequenced markers (RFLP and STS) from barley BINs with
poor SSR coverage were BLASTed against all Rice BAC sequences.
Contiguous BAC sequences with multiple strong hits to markers from the
same barley BIN were BLASTed against all barley ESTs with the low
complexity filter turned off. The resulting barley ESTs were processed
through the Tandem Repeats Finder (TRF) (Benson, 1999) to locate SSR
motifs (1-6 bases in the repeat unit). Primer pairs were designed to flank
SSR motifs using Primer3 software. All three methods used the same
steps, but differed in the level of homology between the rice BAC and
barley EST and the degree to which the SSR region in the EST matched the
pattern of a perfect repeat unit.
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ABG313B
MWG077
CDO669A
HVM040
B32E
BCD402B
BCD351D
ABG705C
MWG635A
UMB401
TubA1 BCD1087
Dhn6
ABG715 WG1026B
ABC303
ABA003
Adh4 ABG484
BCD1087a
ABC321
STS3B142
MWG058
Bmac0181
KFP195
EBmac0906 HVM003
Bmag0808 Bmag0740
WG464 Bmag0306
bBE54A
HVM068
MWG632D
Bmac0310
BCD453B
EBmac0775
ABG472
ABG319A
EBmac0635
EBmac0701
EBmac0788
CDO020
iHxk2
ABG498
ABG500B
HVMLOH1A
bAP91
ABG366
ABG397
ABG319C
Bmag0138b
ABC305B
HVM067
Bmy1
ksuH11
ASE1C
ABA307A
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Method 4 (Expression specific) One-hundred –seventy-three barley ESTs
were identified from a Fusarium-infected library that do not have homology
to any other publicly available barley and wheat ESTs in Genbank (as of
April 9, 2003). These Fusarium-specific barley ESTs were processed through
TRF to locate SSR motifs. Primer pairs were designed to flank SSR motifs
with 60-100% matches to the repeat unit using Primer3.
7 (5H)
6 (6H)
5 (1H)
MWG634
WG622
•Method 1 (Stringent) Barley ESTs processed through TRF had e-value between
0 and 1e-35. Primers were designed for SSRs with 85-100% matches to the
repeat unit.
•Method 2 (Relaxed) Barley ESTs processed through TRF had e-value between
0 and 1e-100. Primers were designed for SSRs with 50-100% matches to the
repeat unit.
•Method 3 (BLAST repeats) Isolated small repeats in the Rice BACs using TRF
and BLASTed only the portion of the BAC that contained the SSR motif and
some flanking sequence to the barley ESTs. Barley ESTs processed through TRF
had e-value between 1e-150 and 1e-2. Primers were designed for SSRs with 85100% matches to the repeat unit.
UMB502
MWG835A
MWG938
MWG036A
ACT008
MWG837
Hor1
Bmac0144a
MWG2077 ABA004
MWG801B
SC4-5B-1
BCD098
BCD738
ABG053
HVM043
Bmac0032
Ica1
MWG2040D
ABC706C
MWG2040C
Bmac0144i
ABG500A
CDO580
UMB501b
UMB501a
ABC152B Bmac0144h
ABC164A
Bmag0770 WG789B
HVM020
Bmac0090
ABR337
ABG074
Glb1
Bmag0872
ABC160
ABG452
BCD098
Bmac0213
ABG464
ABC156B
ABC165C
His3B
ABC307A
cMWG706A
ABC257
cMWG733A
AtpbA
ABG702A
ABC322B
ABC261
Cab2
Aga7
MWG912
ABG387A
0
2
ASE1B
ABG062
0
Act8D
MWG620
Nar1
7
Act8B
14
ABG378
16
MWG502
23
ABC152A
36
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56
60
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64
73
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78
86
87
90
93
97
UMB704
Bmac0163
Bmac0306
EBmac0970
Bmag0323 UMB705
Bmag0337
CDO348D
MWG635G
MWG635F
MWG635D ABG497A
MWG635H
MWG2227B
MWG635E
ABC302A
MWG503B
ABG497B
ABC168
ABC717
Bmag0812
UMB706
Bmag0113
UMB701
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107
115
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cMWG652A
MWG2040E
MWG2218
Bmag0500
PSR106
ABG387B
MWG916
Bmg0001
ABG458
MWG887B
Bmag0807
ABC169B
CDO497
Bmag0173
Bmac0018
Bmag0003e
Bmag0870 BCD340E
Bmag0003a MWG2227A
ksuD17
Bmac0218a
Bmag0613
ABC175
EBmac0806a
MWG820
EBmac0602
Bmac0251
EBmac0806b
Nar7
Amy1
bBE54B
MWG934
ABC170A ABG461B
UMB602
Bmac0040
MWG549A
131
134
137
138
140
ABG713
MWG514
UMB603
UMB601
MWG798A MWG2196
149
cMWG684A
S/M
C/M & F/S Consensus
0
2
7
9
11
Mad2
MWG502
ABC483
MWG920A
ABG316B
39
44
47
48
51
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57
60
62
64
68
73
77
ABG705A
ABG708
ABG395
CDO749
Ubi2
ksuA3A
WG541
WG530
UMB707
WG889
ABC324
UMB703
ABC302A
89
CDO57B
95
mSrh
100
WG364
0
CDO400B
15
CDO059B
25
28
33
37
40
43
44
UMB702
MWG2227B
ABG010D
GMS061
EBmatc0003
ABG495A
GMS027
134
139
143
145
149
152
MWG514B
CDO504
iEst9
WG908
MWG877
ABG495A
54
ABG496
159
ABC155
166
ABC482
172
174
ABG391
ABG390
181
183
187
190
194
CDO484
ABG463
ABG314B
MWG851C
MWG851B
67
ABG391
73
ABG392
89
90
MWG851B
MWG632C GMS001
Figure 1. Barley consensus maps developed from Chevron x M69 (C/M), Fredrickson x
Stander (F/S), and Steptoe x Morex (S/M) mapping populations. Previously available SSR
markers are in Maroon. New UMB SSR markers are in Blue.
Maps and marker information are available at http://agronomy.coafes.umn.edu/barley.
UMB704
CONCLUSIONS
Method 3 was the most efficient
method of producing useful SSR
markers.
UMB104
Approximately half of the
barley genome has good coverage
with SSR markers.
UMB603
UMB702
Table 1. SSR marker coverage in barley by
chromosome.
Chromosome
1
Bins with good
SSR coverage
1--2; 5--13
Bins with poor
SSR coverage
3--4
2
3
4
5
6
4--12
6--8
6--9
5--7
6--8
1--3;13--15
1--4;8--16
1--4;10--13
1--4;9--14
1--5;9--14
7
3--5
1--2;6--15
Table 2. Comparison of four methods for designing
EST based SSR markers.
Method
1
2
3
Pairs
tried
44
51
79
4
Overall
64
238
Produced Product
Polymorphism
Number
%
Number
%
27
61.4
6
13.6
15
29.4
3
5.9
51
64.5
35
44.3
15
108
23.4
45.3
0
44
0.0
18.5
New methods must be
developed to develop markers for
underrepresented regions of the
genome.
ACKNOWLEDGEMENTS
 This research was supported by
the North American Barley Genome
Project.
Special thanks to Charles Gustus
for help in constructing the linkage
maps.
REFERENCES
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chromosomal locations and population dynamics. Proc.
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characterization of gene-derived SSR-markers in barley (
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