Abstract
BackgroundPepper, Capsicum annuum L., Solanaceae, is a major staple economically important vegetable crop worldwide. Limited functional genomics resources and whole genome association studies could be substantially improved through the application of molecular approach for the characterization of gene content and identification of molecular markers. The massive parallel pyrosequencing of two pepper varieties, the highly pungent, Saengryeg 211, and the non-pungent, Saengryeg 213, including de novo transcriptome assembly, functional annotation, and in silico discovery of potential molecular markers is described. We performed 454 GS-FLX Titanium sequencing of polyA-selected and normalized cDNA libraries generated from a single pool of transcripts obtained from mature fruits of two pepper varieties.ResultsA single 454 pyrosequencing run generated 361,671 and 274,269 reads totaling 164.49 and 124.60 Mb of sequence data (average read length of 454 nucleotides), which assembled into 23,821 and 17,813 isotigs and 18,147 and 15,129 singletons for both varieties, respectively. These reads were organized into 20,352 and 15,781 'isogroups’ for both varieties. Assembled sequences were functionally annotated based on homology to genes in multiple public databases and assigned with Gene Ontology (GO) terms. Sequence variants analyses identified a total of 3,766 and 2,431 potential (Simple Sequence Repeat) SSR motifs for microsatellite analysis for both varieties, where trinucleotide was the most common repeat unit (84%), followed by di (9.9%), hexa (4.1%) and pentanucleotide repeats (2.1%). GAA repeat (8.6%) was the most frequent repeat motif, followed by TGG (7.2%), TTC (6.5%), and CAG (6.2%).ConclusionsHigh-throughput transcriptome assembly, annotation and large scale of SSR marker discovery has been achieved using next generation sequencing (NGS) of two pepper varieties. These valuable informations for functional genomics resource shall help to further improve the pepper breeding efforts with respect to genetic linkage maps, QTL mapping and marker-assisted trait selection.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-58) contains supplementary material, which is available to authorized users.
Highlights
Pepper, Capsicum annuum L., Solanaceae, is a major staple economically important vegetable crop worldwide
Results single stranded template DNA (sstDNA) Library construction The sstDNA libraries were constructed through total RNA isolation, mRNA purification, cDNA synthesis, cDNA fragmentation and adaptor ligation based on the standard protocols from commercial kits. cDNA was fragmented for the production of transcripts fragments with the standard length necessary for 454 pyrosequencing
For Saengryeg 211; 78.17% of the screened reads (282,705) were incorporated into assembled sequences, with remaining 5.01% as singletons (18,147). 60,789 reads (16.80% of reads subjected to assembly) were excluded because of being partially assembled (50,330; 13.91%), from repeat regions (111; 0.03%), outliers (5,075; 1.40%), or too short (5,273; 1.45%)
Summary
Capsicum annuum L., Solanaceae, is a major staple economically important vegetable crop worldwide. Despite the numerous available markers, the whole genome association studies in pepper are not possible as the marker density is not enough to target candidate genes underlying a QTL region and conduct association mapping for complex traits (Livingstone et al 1999; Minamiyama et al 2006; Barchi et al 2007; Wu et al 2009; Truong et al 2010; Kong et al 2012) These studies require a large number of markers and cost effective genotyping technology, where lack of markers covering the whole genome stands as the major limitation to the development of high throughput genotyping assays. The rapid identification of these markers associated with complex, economically important traits in crops has been hindered by the lack of whole genomic sequence, high-resolution maps and cost-effective platforms for high density genotyping This eventually restricts the commercial application of these genomic resources for gene discovery and molecular breeding
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