Development of a panel of unigene-derived polymorphic EST–SSR markers in lentil using public database information

Lentil(Lens culinaris Medik.), a diploid(2n = 14) with a genome size greater than 4000 Mbp, is an important cool season food legume grown worldwide. The availability of genomic resources is limited in this crop species. The objective of this study was to develop polymorphic markers in lentil using publicly available curated expressed sequence tag information(ESTs). In this study, 9513 ESTs were downloaded from the National Center for Biotechnology Information(NCBI) database to develop unigene-based simple sequence repeat(SSR) markers. The ESTs were assembled into 4053 unigenes and then analyzed to identify 374 SSRs using the MISA microsatellite identification tool. Among the 374 SSRs, 26 compound SSRs were observed.Primer pairs for these SSRs were designed using Primer3 version 1.14. To classify the functional annotation of ESTs and EST–SSRs, BLASTx searches(using E-value 1 × 10-5) against the public UniP rot(http://www.uniprot.org/) and NCBI(http://www.ncbi.nlh.nih.gov/) databases were performed. Further functional annotation was performed using PLAZA(version3.0) comparative genomics and GO annotation was summarized using the Plant GO slim category. Among the synthesized 312 primers, 219 successfully amplified Lens DNA. A diverse panel of 24 Lens genotypes was used to identify polymorphic markers. A polymorphic set of 57 markers successfully discriminated the test genotypes. This set of polymorphic markers with functional annotation data could be used as molecular tools in lentil breeding.

Development of amplified consensus genetic markers in Taxodiaceae based on Cryptomeria japonica ESTs data

Amplified consensus genetic marker （ACGM） is a PCR-based marker technique that uses primers designed within conserved regions of coding sequences. After a comparison of Cryptomeria japonica and Arabidopsis ESTs to search for conserved sequences, 237 single e-PCR products were obtained. We randomly selected 110 candidate ACGM markers to test. Of the 110 candidate ACGM markers tested, 106 yielded stable and clear PCR products in C. japonica. We then tested the utility of these 106 primer pairs in 10 species, representing 7 genera of Taxodi- aceae. The number of specific amplification primer pairs among those 10 species varied from 49 to 103 （or 46.2±97.2%）. The 106 primer pairs （ACGM loci） were high transferable to Cryptomeria fortunei Hooibrenk （97.2%） but were low in Metasequoia glyptostroboides （46.2%）. The number of PCR bands per primer pair ranged from 1.06 to 1.15, which means that most of the ACGM primers can obtain a single band within these 10 Taxodiaceae species. In summary, our study shows that ACGM is a technique applicable for marker development even in species with limited sequence data.

Identification of SSR loci in Betula luminifera using birch EST data

Expressed sequence tags（ESTs） are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat（SSR） markers or microsatellites.However,EST databases have been developed for only a small number of species.This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera.Based on birch EST data,primers for 80 EST-SSR candidate loci were developed and tested in birch.Of these,59 EST-SSR loci yielded single,stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera.The results showed 28（47.6%） yielded stable and clear PCR products for at least one B.luminifera genotype.In addition,this study describes a rapid and inexpensive alternative for the development of SSRs in species with scarce available sequence data.