Genome-wide SNP discovery from de novo assemblies of pepper (Capsicum annuum) transcriptomes

Molecular breeding of pepper (Capsicum spp.) has been hampered by the paucity of molecular markers. This is primarily due to lack of availability of the pepper genome sequence and limited available sequence resources. However, during the past few years we have developed a wealth of genetic information for pepper including two ultra-saturated maps of pepper using an Affymetrix pepper GeneChip®. In spite of being useful in genetic map construction and QTL mapping, using the Affymerix GeneChip® is not technically and economically feasible for many small laboratories for marker discovery and breeding. In recent years with the more cost effective sequencing technologies such as Illumina, sequencing of expressed genes (transcriptomes), gene discovery and allele mining is no longer insurmountable. In order to exploit the speed and scale of data from new sequencing technologies and in an effort to enrich the sequence resources of pepper, we sequenced transcriptome sequences (RNAseq) of three pepper lines: Maor, Early Jalapeno and CM334. We selected a wide range of tissues to represent as many expressed genes possible. The reference sequence was constructed from >200 million Illumina reads (80-120 nt) using a combination of Velvet, CLC and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper lines. The SNPs were filtered to be 100 bp apart from any intron-exon junction as well as adjacent SNPs. After filtering >22,000 high quality putative SNPs were identified and bioinformatically mapped to pepper genetic maps.