Identification, utilisation and mapping of novel transcriptome-based markers from blackcurrant (Ribes nigrum)

Abstract

BackgroundDeep-level second generation sequencing (2GS) technologies are now being applied to non-model species as a viable and favourable alternative to Sanger sequencing. Large-scale SNP discovery was undertaken in blackcurrant (Ribes nigrum L.) using transcriptome-based 2GS 454 sequencing on the parental genotypes of a reference mapping population, to generate large numbers of novel markers for the construction of a high-density linkage map.ResultsOver 700,000 reads were produced, from which a total of 7,000 SNPs were found. A subset of polymorphic SNPs was selected to develop a 384-SNP OPA assay using the Illumina BeadXpress platform. Additionally, the data enabled identification of 3,000 novel EST-SSRs. The selected SNPs and SSRs were validated across diverse Ribes germplasm, including mapping populations and other selected Ribes species.SNP-based maps were developed from two blackcurrant mapping populations, incorporating 48% and 27% of assayed SNPs respectively. A relatively high proportion of visually monomorphic SNPs were investigated further by quantitative trait mapping of theta score outputs from BeadStudio analysis, and this enabled additional SNPs to be placed on the two maps.ConclusionsThe use of 2GS technology for the development of markers is superior to previously described methods, in both numbers of markers and biological informativeness of those markers. Whilst the numbers of reads and assembled contigs were comparable to similar sized studies of other non-model species, here a high proportion of novel genes were discovered across a wide range of putative function and localisation. The potential utility of markers developed using the 2GS approach in downstream breeding applications is discussed.

SNP Markers and Their Impact on Plant Breeding

“…Transcriptome resequencing using NGS technologies allows rapid and inexpensive SNP discovery within genes and avoids highly repetitive regions of a genome [22]. This methodology was successfully applied in several plant genomes, including maize [23], canola [24], eucalyptus [25], sugarcane [26], tree species [27], wheat [28], avocado [29], and black currant [30]. Originally developed for human disease diagnostic research, the NimbleGen sequence capture technology (Roche Applied Science, IN) [31] brought the detection of gene-based SNPs in plants into higher throughput and coverage level [32].…”

Transcriptome characterisation of Pinus tabuliformis and evolution of genes in the Pinusphylogeny

“…It also facilitates the analysis of the transcribed part of the genome, which is not easy to predict from the entire genome [13]. Next-generation sequencing is a viable and favourable alternative to Sanger sequencing and provides researchers with a relatively rapid and affordable option for developing genomic resources in non-model organisms [14-16]. The Roche 454 massively parallel pyrosequencing platform, GS FLX Titanium, can generate one million reads with an average read length of 400 bases at 99.5% accuracy per run [17,18].…”

Characterization of ESTs from black locust for gene discovery and marker development

“…In many species, the main limitation to understanding and characterizing important traits is the lack of sufficient genetic sequences for the development of high-density genetic maps and marker-assisted breeding studies (Russell et al, 2011). ESTs have been identified as useful sources of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), both of which are useful tools for mapping and marker-assisted breeding in plants (Morgante et al, 2002).…”