• We developed a pipeline for building and annotating Sanger-454 transcript assemblies and developed a groundnut EST database (Figure 1).

EST-SSR Marker Resources for GroundnutSameer Khanal1, Shunxue Tang1, Yufang Guo1, Yan Li2, Vadim Beilinson3, Phillip San Miguel4, Baozhu Guo2,

Niels Nielsen3, Thomas Stalker3, Marie-Michele Cordonnier-Pratt5, Lee H. Pratt5, Virgil Ed Johnson5, Christopher A. Taylor1, Bruce Roe6, David Hoisington7, Rajeev Varshney7, and Steven J. Knapp1

1Institute of Plant Breeding, Genetics, and Genomics, The University of Georgia, Athens, Georgia, 30602, USA2USDA-ARS, Tifton, Georgia, 31793, USA

3Crop Science Department, North Carolina State University, Raleigh, North Carolina, 27695, USA4Genomics Center, Purdue University, West Lafayette, Indiana, 47907, USA

5Laboratory for Genomics and Bioinformatics, The University of Georgia, Athens, Georgia, 30602, USA6Advanced Center for Genome Technology (ACGT), University of Oklahoma, Norman, OK 73019, USA

7International Crops Research Institute for Semi Arid Tropics (ICRISAT), Patancheru, 502 324, AP, INDIA

Background

Narrow genetic diversity and a deficiency of polymorphic DNA markers have hindered genetic mapping, forward genetics, and the application of genomics and molecular breeding approaches in groundnut (Arachis hypogaea). To further break the DNA marker bottleneck, we mined an EST database for simple sequence repeats (SSRs) and developed 2,100 EST-SSR markers for groundnut. The EST database was developed by Sanger and next-generation (454-FLX) DNA sequencing technologies. Here, we describe a pipeline for assembling and annotating Sanger-454 ESTs, the development of the groundnut EST database (ESTdb), SSR markers developed from ESTs, and polymorphisms of EST-SSRs among 28 elite tetraploid and four wild diploid inbred lines.

Mining a Groundnut EST Database for SSRs

• Sanger and 454 ESTs were produced from normalized and non-normalized leaf and developing-seed cDNA libraries of Tifrunner, GTC20, NC12C, A13, and New Mexico Valencia A (Table 1).

Table 1. Groundnut ESTs and Transcript Assembly StatisticsESTs Unigenes

Sanger 454 Total Singletons Contigs Total71,448 304,215 375,663 63,218 37,914 101,132

Figure 1. Uniscript Alignment in Groundnut ESTdb for a Sanger-454 Contig Harboring an Trinculeotide (CAA5) Simple Sequence Repeat.

Figure 2. Simple Sequence Repeats Identified by Mining the Groundnut EST Database (101,132 Uniscripts).

Pilot Study: EST-SSR Polymorphisms in Groundnut

• To gain an understanding of the potential utility of EST-SSRs for genotyping applications in groundnut, particularly in modern cultivars, 58 EST-SSR markers were developed by targeting a broad range of repeat motifs and repeat lengths identified in the database.

• The 58 EST-SSR markers were screened for allele length polymorphisms among 28 elite tetraploid, two A. duranensis, and two A. batizocoi lines. The diploid lines are the parents of intraspecific A- and B-genome diploid mapping populations. The tetraploid lines are the parents of elite x elite mapping populations and important founders of modern cultivars (Figure 3).

• We identified 7,314 perfect SSRs in 6,371 unigenes and selected 2,134 for EST-SSR marker development (Figure 2), 96% of which were dinucleotide and trinucleotide repeats. These are currently being screened for polymorphisms and genetically mapped in several elite x elite tetraploid and wild diploid mapping populations.

Figure 3. Genetic Distance-Based Dendrograms Estimated from Genotypes for 58 EST-SSR Markers.

• 80% of the EST-SSR markers were polymorphic in one or both of the diploid mapping populations (DUR25 x DUR35 and BAT3 x BAT9) (Figure 3).

• Half of the EST-SSR markers were polymorphic among tetraploid germplasm accessions; however, the mean heterozygosity (H) or polymorphic information content was low (H = 0.17). Trinucleotide repeats (H = 0.20) were more polymorphic than dinucleotide repeats (H = 0.10).

• The well-known diversity bottleneck in modern cultivars is illustrated by the tight cluster of tetraploid nodes in the diploid-tetraploid dendrogram (Figure 3).

• SSR length was positively correlated with allelic diversity (r = 0.45; p < 0.0001) (Figure 4). SSRs longer than 26 bp (H = 0.26) were significantly more polymorphic than SSRs shorter than 26 bp (H = 0.10).

Conclusions

• Twenty to 30% of the 2,134 EST-SSR markers developed in the present study are predicted to be polymorphic and have utility in modern cultivars, whereas 80-90% are predicted to be polymorphic in two diploid mapping populations.

• Large-scale DNA resequencing approaches are needed to identify DNA sequence variants in modern cultivars of groundnut to further increase the supply of polymorphic DNA markers for molecular breeding and other genotyping applications.