Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers

Abstract

BackgroundField pea (Pisum sativum L.) and faba bean (Vicia faba L.) are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. To date, both species have been relative 'genomic orphans' due to limited availability of genetic and genomic information. A significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies.ResultscDNA samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using 454 Roche GS FLX Titanium technology. A total of 720,324 and 304,680 reads for field pea and faba bean, respectively, were de novo assembled to generate sets of 70,682 and 60,440 unigenes. Consensus sequences were compared against the genome of the model legume species Medicago truncatula Gaertn., as well as that of the more distantly related, but better-characterised genome of Arabidopsis thaliana L.. In comparison to M. truncatula coding sequences, 11,737 and 10,179 unique hits were obtained from field pea and faba bean. Totals of 22,057 field pea and 18,052 faba bean unigenes were subsequently annotated from GenBank. Comparison to the genome of soybean (Glycine max L.) resulted in 19,451 unique hits for field pea and 16,497 unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. Simple sequence repeat (SSR)-containing expressed sequence tags (ESTs) were identified from consensus sequences, and totals of 2,397 and 802 primer pairs were designed for field pea and faba bean. Subsets of 96 EST-SSR markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. For field pea, 86 primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between 6 genotypes. In the case of faba bean, 81 primer pairs displayed successful amplification, of which 48% detected polymorphism.ConclusionsThe generation of EST datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation. EST-SSR loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. The subsets from these primer pairs proved highly useful for polymorphism detection within Pisum and Vicia germplasm.

Short Communication Development and characterization of novel transcriptome-derived microsatellites for genetic analysis of persimmon

“…Except for the mononucleotide repeat unit, dinucleotide repeat units were predominant, followed by tri-, tetra-, penta-, and hexanucleotide repeat units, which was consistent with the theory reported by Kaur et al (2012). However, trinucleotide repeat units were predominant in peanut (Zhang et al, 2012b), red pepper (Lu et al, 2012), field pea and faba bean (Kaur et al, 2012). AG/CT motifs were abundant among the dinucleotide EST-SSRs, while AAG/CTT motifs were predominant in trinucleotide EST-SSRs.…”

“…Furthermore, it has been emphasized that the frequency of SSRs is correlated with many factors, such as SSR mining software, SSR finding criteria, dataset size, and different materials (Varshney et al, 2005). Except for the mononucleotide repeat unit, dinucleotide repeat units were predominant, followed by tri-, tetra-, penta-, and hexanucleotide repeat units, which was consistent with the theory reported by Kaur et al (2012). However, trinucleotide repeat units were predominant in peanut (Zhang et al, 2012b), red pepper (Lu et al, 2012), field pea and faba bean (Kaur et al, 2012).…”

“…The polymorphic rate of EST-SSR in the present study for persimmon was 63.23%, which was higher than that in sesame (11.59%) (Zhang et al, 2012a), pigeonpea (12.91%) (Dutta et al, 2011), faba bean (29.6%) (Kaur et al 2012), peanut (40.63%) (Zhang et al, 2012b), field pea (46.5%) (Kaur et al, 2012), and sweet potato (51.09%) (Wang et al, 2010).…”

“…SSR markers have been explored from NGS data for many plant species (Wang et al, 2010;Dutta et al, 2011;Zhu et al, 2012;Kaur et al, 2012). In the present study, Roche 454 sequencing technology was applied to sequence the persimmon transcriptome.…”

Identification of novel and useful EST-SSR markers from de novo transcriptome sequence of wheat (Triticum aestivum L.)

“…Such SSRs are referred to as EST-SSRs. In recent years, EST-SSR mining from coding sequences determined by RNA-seq technology has become increasingly popular in plant research (Kaur et al, 2012;Li et al, 2012).…”