1) Researchers developed a pipeline to build and annotate a Sanger-454 transcript assembly database for groundnut, containing over 100,000 unigenes. 2) They mined this EST database to identify over 7,000 perfect simple sequence repeats (SSRs), selecting around 2,100 to develop into EST-SSR markers. 3) A pilot study screening 58 EST-SSR markers on elite cultivars and wild relatives found around half were polymorphic among cultivars but diversity was low, while 80% were polymorphic between wild relatives, demonstrating their greater utility for genetic mapping.

1. • 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 Groundnut
Sameer 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, USA
2USDA-ARS, Tifton, Georgia, 31793, USA
3Crop Science Department, North Carolina State University, Raleigh, North Carolina, 27695, USA
4Genomics Center, Purdue University, West Lafayette, Indiana, 47907, USA
5Laboratory for Genomics and Bioinformatics, The University of Georgia, Athens, Georgia, 30602, USA
6Advanced 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 Statistics
ESTs Unigenes
Sanger 454 Total Singletons Contigs Total
71,448 304,215 375,663 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.