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


RESULTS
Over 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.


CONCLUSIONS
The 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.
Cell & Molecular Sciences, James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK. joanne.russell@hutton.ac.uk
Joanne R
JR
Russell
Micha
M
Bayer
Clare
C
Booth
Linda
L
Cardle
Christine A
CA
Hackett
Pete E
PE
Hedley
Linzi
L
Jorgensen
Jenny A
JA
Morris
Rex M
RM
Brennan
D002874:Chromosome Mapping / Q000379:methods*; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D008040:Genetic Linkage; D005819:Genetic Markers; D005838:Genotype; D060005:Genotyping Techniques; D018895:Microsatellite Repeats; D020641:Polymorphism, Single Nucleotide; D040641:Quantitative Trait Loci; D031965:Ribes / Q000235:genetics*; D017422:Sequence Analysis, DNA; D059467:Transcriptome
D018744:DNA, Plant; D005819:Genetic Markers