Barley Genetics Newsletter 36:30-43 (2006)

 

 

New SSR markers for barley derived from the EST database

 

Karen A. Beaubien and Kevin P. Smith*

Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN

*Corresponding author: E-mail: smith376@umn.edu

 

Introduction

            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, 2003).    

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.

 

Acknowledgements

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.

 

References:

 

Becker, J., and M. Heun.  1995.  Barley microsatellites: allele variation and mapping.  Plant Mol. Biol. 27: 835-845.

Benson, G.  1999.  Tandem repeats finder: a program to analyze DNA sequences.  Nucleic Acids Research 27: 573-580.

Canci, P.C., L.M. Nduulu, R. Dill-Macky, G.J. Muehlbauer, D.C. Rasmusson, and K.P. Smith.  2003.  Genetic relationship between kernel discoloration and grain protein concentration in barley.  Crop Sci. 43: 1671-1679.

Holton, T.A., J.T. Christopher, L. McClure, N. Harker, and R.J. Henry.  2002.  Identification and mapping of polymorphic SSr markers from expressed gene sequences of barley and wheat.  Mol. Breeding 9: 63-71. 

Kleinhofs, A., A. Kilian, M.A. Saghai Maroof, R.M. Biyashev, P. Hayes, F.Q. Chen, N. Lapitan, A. Fenwick, T.K. Blake, V. Kanazin, E. Ananiev, L. Dahleen, D. Kudrna, J. Bollinger, S.J. Knapp, B. Liu, M. Sorrells, M. Heun, J.D. Franckowiak, D. Hoffman, R. Skadsen, and B.J. Steffenson.  1993.  A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome.  Theor. Appl. Genet. 86: 705-712.

Li , J.Z., T.G. Sjakste, M.S. Röder and M.W. Ganal.  2003.  Development and genetic mapping of 127 new microsatellite markers in barley.  Theor. Appl. Genet. 107: 1021–1027.

Liu, S., and J.A. Anderson.  2003.  Targeted molecular mapping of a major wheat QTL for Fusarium head blight resistance using wheat ESTs and synteny with rice.  Genome 46: 817-823.

Liu, Z.-W., R.M. Biyashev and M.A. Saghai Maroof.  1996.  Development of simple sequence repeat markers and their integration into a barley linkage map. Theor. Appl. Genet. 93: 869–876.

Mesfin, A., K.P. Smith, R. Dill-Macky, C.K. Evans, R Waugh, C.D. Gustus, and G.J. Muehlbauer.  2003.  Quantitative trait loci for Fusarium head blight resistance in barley detected in a two-rowed by six-rowed population.  Crop Sci. 43: 307-318.

Pillen, K., A. Binder, B. Kreuzkam, L. Ramsay, R. Waugh, J. Förster, J. Léon.  2000.  Mapping new EMBL-derived barley microsatellites and their use in differentiating German barley cultivars.  Theor. Appl. Genet. 101: 652-660.

Ramsay, L., M. Macaulay, S. degli Ivanissevich, K. MacLean, L. Cardle, J. Fuller, K. J. Edwards, S. Tuvesson, M. Morgante, A. Massari, E. Maestri, N. Marmiroli, T. Sjakste, M. Ganal, W. Powell, and R. Waugh.  2000.  A Simple Sequence Repeat-Based Linkage Map of Barley.  Genetics 156: 1997-2005.

Rozen, S., and H. Skaletsky.  2000.  Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology. Eds Krawetz, S., and Misener, S.  Humana. Totowa, NJ.  pp 365-386.

Saghai Maroof, M.A., R.M. Biyashev, G.P. Yang, Q. Zhang and R.W. Allard.  1994.  Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations and population dynamics. Proc. Natl. Acad. Sci. USA 91: 5466–5470.

Struss, D., and J. Plieske.  1998.  The use of microsatellite markers for detection of genetic diversity in barley populations.  Theor.  Appl.  Genet. 97: 308-15.

Thiel, T, W. Michalek, R.K. Varshney, and A. Graner.  2003.  Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.).  Theor. Appl. Genet. 106: 411–422.

Van Ooijen, J.W., and R.E. Voorrips.  2001.  JoinMap 3.0, Software for the calculation of genetic linkage maps.  Plant Research International, Wageningen, the Netherlands.

 

 

 

 

                         Table 1. PCR recipes for UMB SSR markers

PCR recipe

A

B

C

dNTPs (1.25 mM each)

1.0

1.0

1.0

MgCl22 (25 mM)

1.0

1.0

0.5

10 X buffer *

1.0

1.0

1.0

Forward Primer (5μM)

0.6

0.6

1.0

Reverse Primer (5μM)

0.6

0.6

1.0

Betaine (5 M)

0

2.0

0

DMSO (100%)

0

0.5

0

Taq polymerase*

0.075

0.075

0.075

H2O

2.725

0.225

2.425

DNA (10 ng/μL)

3.0

3.0

3.0

Total

10.0

10.0

10.0

                         * 10 X buffer is provided with purchase of Taq polymerase

 

 

    Table 2.  PCR programs for UMB SSR markers

Stage

Temp. (°C)

Time (min.)

Cycles

Stage

Temp. (°C)

Time (min.)

Cycles

 

 

Program A*

Program F*

Initial Hold

95

9:00

1

Initial Hold

-

-

-

Thermal Cycles-1

94

1:00

 

18

 

Pre-PCR

95

9:00

 

1

 

64

0:30

58

1:00

72

1:00

72

1:00

Thermal Cycles-2

94

1:00

 

30

 

Thermal Cycles

94

0:30

 

30

 

55

1:00

58

0:30

72

1:00

72

0:30

Ending Hold

72

5:00

1

Ending Hold

72

5:00

1

Final Hold

4

∞

1

Final Hold

4

∞

1

 

 

 

 

 

 

 

 

Program J*

Program L58

Initial Hold

95

8:00

1

Initial Hold

95

9:00

1

Thermal Cycles

94

1:00

 

35

 

Thermal Cycles

94

0:10

 

30

 

60

1:00

58

0:20

72

2:00

72

0:45

Ending Hold

72

5:00

1

Ending Hold

72

2:30

1

Final Hold

4

∞

1

Final Hold

4

∞

1

    * Programs A, F and J are as in Ramsay et al., 2000. 


Table 3.  PCR and mapping information for UMB markers.

 

 

 

Population2

Quality

 

PCR

PCR

Name

Chr.

BIN1

Polymorphism

Mapped

Score3

Alleles4

Program

Recipe

UMB101

1 (7H)

11-12

A/M; F/S; H/O; H/L; S/M

A/M; F/S; S/M

2

3

L58

A

UMB102

1 (7H)

11-12

A/M; H/O; H/L; S/M

S/M

2.5

2

L58

A

UMB103

1 (7H)

13

A/M; F/S; H/O; H/L; S/M

A/M; F/S; S/M

2

4

L58

A

UMB104

1 (7H)

7-8

C/M; F/S; H/O; H/L; S/M

F/S; S/M

1.5

2

F

A

UMB105

1 (7H)

10-11

A/M; C/M; H/O

A/M; C/M

3

2

L58

A

UMB106

1 (7H)

4-5

C/M; H/L; S/M

C/M; S/M

2

2

L58

A

UMB107

1 (7H)

4-5

F/S; H/O

F/S

3

4

J

C

UMB108

1 (7H)

2

A/M; F/S; H/O; H/L; S/M

F/S; S/M

1

3

A

B

UMB201

2 (2H)

6-7

C/M; F/S; H/O; H/L; S/M

C/M; F/S; S/M

2.5

4

L58

A

UMB202

2 (2H)

6-7

A/M; C/M; F/S; H/O; S/M

A/M; C/M; F/S; S/M

2

2

F

A

UMB203

2 (2H)

4-5

C/M; H/L

C/M

2

2

J

C

UMB204

2 (2H)

7-8

F/S; H/O

F/S

2.5

2

L58