ESTs are a Rich Source of Polymorphic SSRs for Genomics and Molecular Breeding Applications in Peanut

Sameer Khanal1, Shunxue Tang1, Vadim Beilinson2, Phillip San Miguel3, Baozhu Guo4, Niels Nielsen2, Thomas Stalker2, Marie-Michele Cordonnier-Pratt5, Lee H. Pratt5, Virgil Ed Johnson5, Christopher A. Taylor1, and Steven J. Knapp1

1Center for Applied Genetic Technologies, The University of Georgia, Athens, Georgia, 306022Crop Science Department, North Carolina State University, Raleigh, North Carolina, 276953Genomics Center, Purdue University, West Lafayette, Indiana, 479074USDA-ARS, Tifton, Georgia, 317935Laboratory for Genomics and Bioinformatics, The University of Georgia, Athens, Georgia, 30602

Narrow genetic diversity and a deficiency of polymorphic DNA markers have hindered genetic mapping and the application of genomics and molecular breeding approaches in cultivated peanut (Arachis hypogaea L.). Simple Sequence Repeat (SSR) markers have become the marker class of choice for molecular mapping and breeding of many plant species (Eujayl et al. 2003). Therefore, a complete collection of 556 polymorphic SSR markers was screened and found to be inadequate for developing a saturated linkage map for Arachis species (unpublished data). However, the development of a critical mass of SSR markers by mining Expressed Sequence Tag (EST) databases has emerged as a fast, efficient and low-cost option for many plant species including peanut (Eujayl et al. 2003, Moretzsohn et al. 2005). Also, the rate of detecting polymorphism among peanut lines is higher using EST derived SSR markers than those derived from the genomic sequences (Luo et al. 2005). Therefore, we developed and mined a peanut EST database for simple sequence repeats (SSRs), assessed the frequency of polymorphic SSRs in ESTs, and initiated the development of several hundred EST-SSR markers with the goal of breaking the DNA marker bottleneck in cultivated peanut. Our peanut EST database contains 84,229 ESTs assembled into 26,809 unigenes (unpublished).Our objectives of this study were to:1.Assess polymorphisms offered by different SSR repeat motifs, SSR repeat lengths, and repeat locations of SSRs.2.Assess the frequency of polymorphic EST-SSRs for developing a critical mass of DNA markers for genomics and molecular breeding applications in cultivated peanut.

Eujayl, I., M.K. Sledge, L. Wang, G.D. May, K. Chekhovskiy, J.C. Zwonitzer, and M.A.R. Mian. Medicago trunculata EST-SSRs reveal cross-species genetic markers for Medicago spp. Theor. Appl. Genet. 108(3):414-422.Liu, K. and S.V. Muse. 2005. PowerMarker: an integrated analysis environment for gentic marker analysis. Bioinform. Appl. 21(9):2128-2129.Luo, M., P. Dang, B.Z. Guo, G. He, C.C. Holbrook, M.G. Bausher, and R.D. Lee. 2005. Generation of expressed sequence tags (ESTs) for gene discovery and marker development in cultivated peanut. Crop Sci. 45:346-353.Moretzsohn, M.C., L. Leoi, K. Proite, Guimeraes P.M., Leal-Bertioli S.C.M., M.A. Gimenes, W.S. Martins, J.F.M. Valls, D. Grattapaglia, and D.J. Bertioli. 2005. A microsatellite-based, gene-rich linkage map for the AA genome of Arachis (Fabaceae). Theor. Appl. Genet.

111(6):1060-1071.Rozen, S. and H.J. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz, S. and S. Misener (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386.Temnykh, S., G. DeClerck, A. Lipovich, S. Cartinhour, and S. McCouch. 2001. Computational and experimental analysis of microsatellites in rice (Oryza sativa L): frequency,length, variation, transposon associations, and genetic marker potential. Genet. Res. 11:1441-1452.

SSRIT (Temnykh et al. 2001) was used for mining EST-SSRs obtained from the peanut EST database (unpublished). 80 EST-SSRs representing a broad spectrum of repeat motifs, repeat lengths and repeat locations were selected and Primer3 (Rozen and Skaletsky 2000), was used for designing primers. Primers were labeled with 6-FAM, HEX, or Tamra fluorescent dyes and were screened for polymorphisms against 28 tetraploid and 4 diploid germplasm accessions. Genotypes were determined using the ABI3730 DNA analyzer and GeneMapper Software Version 4 (Applied Biosystems, Foster City, CA).

Results and Conclusions1. SSRs are abundant in the ESTs.4,470 perfect SSRs were found interspersed in 3,716 unigenes. 13.86% of the unigenes contained SSRs.

Introduction

2. Dinucleotides are the most frequent repeat motifs (Figure 1).

7. ESTs are a rich source of polymorphic SSRs (Figure 5).Of 58 markers, 55 (94.8%) were polymorphic, 32 (55.2%) were polymorphic in tetraploid peanut (mean heterozygosity was 0.18), 27 (46.6%) were polymorphic in four cultivated peanut mapping populations, and 48 (82.8%) were polymorphic in two diploid mapping populations. The frequency of polymorphic EST-SSRs seems to be more than sufficient for developing a critical mass of DNA markers for genomics and molecular breeding applications in cultivated peanut.

Frequencies of SSR Motifs

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Figure 1. Frequencies of different repeat motifs out of a total of 4,470 SSRs mined from 26,809 unigenes.

Figure 2. Scatter plot of different SSR lengths plotted against heterozygosity observations.

SSR Length vs. Polymorphism

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Corr. (r) = 0.45

5. SSRs in exons and UTRs are equally polymorphic (Figure 3).Polymorphism Against SSR Locations

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Figure 3. Heterozygosity observations for SSRs in the exonic regions and in the untranslated regions.

Figure 5. Number of polymorphic markers in 4 tetraploid mapping populations and two diploid mapping populations

Polymorphisms in the Mapping Populations

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Tifrunner x GTC20NemaTAM x WS 14Chico x SSD-6NC12C x A. monticola (GKBSPSc 30062)A. duranensis (DUR25) x A. duranensis (DUR35)A. batizocoi (BAT3) x A. batizocoi (BAT8)

Materials and Methods

References

(57.7%)

(38.7%)

(2.37%) (0.6 %) (0.58 %)

3. Observations on Allele Frequencies and HeterozygositiesApproximately 5 alleles per marker for the panel and 3 alleles per marker for the tetraploid subset were scored. Average heterozygosity observed for 58 markers across the panel was 0.33 while that from the same set of markers on tetraploid subset was 0.18. 4. Longer SSRs are more polymorphic than the shorter ones.Although there was no strong correlelation between SSR length and heterozygosity (Figure 2), SSRs longer than 26 bp were two fold more polymorphic than SSRs shorter than 26 bp.

6. SSR markers can discriminate the botanical varieties of cultivated peanuts (Figure 4).

Figure 4. An unrooted cladogram generated by PowerMarker (Liu and Muse 2005) with 33 polymorphic SSRs. Accessions from four botanical varieties viz. Runner, Virginia, Valancia and Spanish are shown to cluster together.