Snipping polymorphisms from large EST collections in barley (Hordeum vulgare L.)

Abstract

The public EST (expressed sequence tag) databases represent an enormous but heterogeneous repository of sequences, including many from a broad selection of plant species and a wide range of distinct varieties. The significant redundancy within large EST collections makes them an attractive resource for rapid pre-selection of candidate sequence polymorphisms. Here we present a strategy that allows rapid identification of candidate SNPs in barley (Hordeum vulgare L.) using publicly available EST databases. Analysis of 271,630 EST sequences from different cDNA libraries, representing 23 different barley varieties, resulted in the generation of 56,302 tentative consensus sequences. In all, 8171 of these unigene sequences are members of clusters with six or more ESTs. By applying a novel SNP detection algorithm (SNiPpER) to these sequences, we identified 3069 candidate inter-varietal SNPs. In order to verify these candidate SNPs, we selected a small subset of 63 present in 36 ESTs. Of the 63 SNPs selected, we were able to validate 54 (86%) using a direct sequencing approach. For further verification, 28 ESTs were mapped to distinct loci within the barley genome. The polymorphism information content (PIC) and nucleotide diversity (pi) values of the SNPs identified by the SNiPpER algorithm are significantly higher than those that were obtained by random sequencing. This demonstrates the efficiency of our strategy for SNP identification and the cost-efficient development of EST-based SNP-markers.

Single nucleotide polymorphisms in rye (Secale cereale L.): discovery, frequency, and applications for genome mapping and diversity studies

“…The barley amplicons were of mean size 300-450 bp. All PCR procedures followed those described elsewhere (Kota et al 2001(Kota et al , 2003.…”

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches’ broom disease in cacao

“…For example, in barley, a total of 3069 candidate SNPs were obtained from the analysis of detection algorithm (SNiPpER) in 8171 EST sequences (0.37 SNP per sequence; Kota et al, 2003). In Lycopersicon esculentum, it was possible to obtain 101 potential SNPs from 1245 contigs, using in silico detection (0.08 SNP per sequence; Yang et al, 2004).…”

Development and characterization of 35 single nucleotide polymorphism markers for the brown algaFucus vesiculosus

“…Rapid identification and verification of SNP markers using EST data sources is advantageous in leading to high-volume, cost-effective SNP discovery in plants ( Kota et al, 2003;Pavy et al, 2006;Varshney et al, 2007). One of the main advantages is that markers within or closely associated with coding regions can be identified (Picoult-Newberg et al, 1999).…”