A Database for Triticeae and Avena
Barley Genetics Newsletter 36:30-43 (2006)
New SSR markers for barley derived from the EST database
Karen A. Beaubien and
Kevin P. Smith*
Agronomy and Plant Genetics, University
of Minnesota, St. Paul, MN
author: E-mail: firstname.lastname@example.org
There are currently 1,196 microsatellite
[or simple sequence repeat (SSR)] marker primer sets that have been developed
for barley (from both genomic libraries and EST databases) of which 504 have
been mapped (Saghai Maroof et al., 1994; Becker and Heun, 1995; Liu et al., 1996;
Struss and Plieske, 1998; Ramsay et al., 2000; Pillen et al., 2000; Holton et
al., 2002; Thiel et al., 2003; Li et al., 2003; Yu et al., 2005). Unfortunately, the available SSR markers provide
uneven coverage of the barley genome and are concentrated near the centromeres. We have compared the available mapped SSRs
and identified 62 BINs out of the total of 99 barley BINs (http://barleygenomics.wsu.edu/;
http://rye.pw.usda.gov/cgi-bin/gbrowse/BarleyBinMaps) that have poor coverage. Although this represents, 63% of the barley
genome, only 31% of the available SSRs map to these BINs. Additional SSR markers are needed to increase
coverage in these BINs. Moreover, some
of the SSR markers recently published are restricted from being used to develop
new barley varieties, thus there is still a need for additional publicly
available SSR markers that can be used without restrictions.
Materials and Methods
Barley ESTs used for primer
development were selected by using either rice BAC or wheat EST sequences in a
BLASTn search for publicly available barley ESTs with the low complexity filter
turned off (BLASTn searches were completed between October, 2002 and October,
2004) (http://www.ncbi.nih.gov/BLAST/). Matches
with e-values between 0 and 1e-2 were used.
The resulting barley ESTs were processed through the Tandem repeats
finder which measures the rate at which the actual EST sequence matches a
perfect repeat sequence (Benson, 1999; http://tandem.bu.edu/trf/trf.submit.options.html). Primers were designed for SSRs with 85-100%
matches to the perfect repeat sequence.
Primer pairs were designed to flank SSR motifs using Primer3 software (Rozen
and Skaletsky, 2000; http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and
screened on a set of mapping parents to identify which population(s) were
appropriate for mapping. Primer pairs
were tested using two to four
PCR protocols to identify a protocol that resulted in clear products (Tables 1
and 2). Products were separated on 6%
polyacrylamide gels and visualized using silver staining.
Newly developed SSR markers were
mapped on the appropriate mapping population(s) including: Steptoe x Morex (Kleinhofs et
al., 1993), Chevron x M69 (Canci et al., 2003), Frederickson x Stander (Mesfin
et al., 2003), or Atahualpa x M81 (unpublished). We used JoinMap 3.0 for map construction (Van
Ooijen and Voorrips, 2001). Assignment
to BINs was based on adjacent mapped markers that have been previously assigned
to BINs (http://barleygenomics.wsu.edu/; http://rye.pw.usda.gov/cgi-bin/gbrowse/BarleyBinMaps). Wheat STS markers were mapped on the barley
populations to increase the coverage of chromosome 3 (3H) (Liu and Anderson,
Results and Discussion
A total of 76 new markers were
developed that produce between two to six alleles per locus among the twelve
mapping parents: Atahualpa, M81, Chevron, M69, Frederickson, Stander,
Harrington, OUH602, Hor211, Lacey, Steptoe and Morex (Table 3). Sixty of the markers were mapped using the Steptoe
x Morex, Chevron x M69, Frederickson x Stander, and Atahualpa x M81populations
(Figure 1 and Table 3). Of the 60 mapped
markers, 41 (68%) have mapped to BIN positions that were previously identified
as being poorly covered with the currently available SSR markers. These markers should provide additional tools
for barley genetic mapping and marker-assisted selection.
The authors would like to thank
Charles Gustus and Danielle Wojdyla for their assistance in STS and SSR marker
data collection. This work was carried
out in part using software provided by the University of Minnesota
Supercomputing Center. This research was
supported by U. S. Barley Genome Project.
J., and M. Heun. 1995. Barley microsatellites: allele variation and
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1. PCR recipes for UMB SSR markers
dNTPs (1.25 mM each)
10 X buffer *
Forward Primer (5μM)
Reverse Primer (5μM)
Betaine (5 M)
DNA (10 ng/μL)
* 10 X buffer is provided with purchase of Taq
2. PCR programs for UMB SSR markers
* Programs A, F and J are as in Ramsay et
Table 3. PCR and mapping
information for UMB markers.
A/M; F/S; H/O; H/L; S/M
A/M; F/S; S/M
A/M; H/O; H/L; S/M
C/M; F/S; H/O; H/L; S/M
A/M; C/M; H/O
C/M; H/L; S/M
C/M; F/S; S/M
A/M; C/M; F/S; H/O; S/M
A/M; C/M; F/S; S/M
C/M; F/S; H/L; S/M
A/M; C/M; F/S; H/L; S/M
A/M; C/M; S/M
A/M; H/O; H/L
CM; F/S; H/L
H/O; H/L; S/M
Table 3 cont. PCR and mapping information for UMB markers.
A/M; C/M; H/O; H/L; S/M
C/M; H/O; S/M
A/M; F/S; H/O
A/M; C/M; F/S; H/O; H/L; S/M
C/M; F/S; H/O; H/L
A/M; C/M; F/S; H/O; H/L
A/M; C/M; F/S
C/M; H/O; H/L
A/M; F/S; H/O; H/L
A/M; C/M; H/O; H/L
A/M; F/S; H/O; S/M
A/M; H/O; S/M
C/M; H/O; H/L; S/M
1 Bold type indicates
BINs with poor SSR marker coverage
2 Mapping populations:
A/M=Atahualpa x M81; C/M=Chevron x M69; F/S=Frederickson x Stander;
H/O=Harrington x OUH602; H/L=Hor211 x Lacey;
S/M=Steptoe x Morex
3 Scale from 1-5 where
1=Very easy to score and 5=Very hard to score
4 Number of Alleles
based on the twelve mapping parents
4. Design information for UMB primers.
cont. Ordering and design information for UMB primers.
4 cont. Design information for UMB primers.
1 EST accession from which the primers were
Figure 1. Map locations of the UMB markers on consensus
maps of the Steptoe x Morex (S/M), Frederickson x Stander (F/S) and Chevron x
M69 (C/M) populations. Chromosome 7 (5H)
is presented as a consensus map of the F/S and C/M populations with the S/M
population separate. UMB508 (denoted
with 'Ş') has had its location inferred from
the C/M population (C/M is included in the consensus but the inferred marker
did not map in the consensus). Markers
denoted with 'u' have had their location inferred from the
Atahualpa x M81 (A/M) population. Marker
"ABG497D" [chr. 1 (7H), BIN 11], was named "ABG497B" in
Canci et al. (2003), but the "ABG497B" locus should map to chr. 1 (7H),
BIN 4, therefore we have designated a new locus name.