Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

Abstract

BackgroundBAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library.ResultsThis pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight.ConclusionsOur results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Cocoa Agronomy, Quality, Nutritional, and Health Aspects

“…and other diseases in cacao. Kuhn et al (2011) identified 19 COSII SNP markers that co-locate with existing QTLs in cacao, and that can be used for genotyping and off-typing in cacao breeding programs and for genetic mapping and studies to trace co-location of genes regulating important traits in cacao and other species. Feltus et al (2011) used a BAC-based physical map to identify candidate genes for bean shape, pod weight, and Black Pod resistance contained within QTL intervals.…”

“…Nevertheless, the future outlook for improved cacao planting material was deemed promising based on the ongoing research; conventional breeding and that involving genomic tools (Wilkinson, 2001;Figueira and Alemanno, 2005;Guiltinan, 2007;Guiltinan et al, 2008;Kuhn et al, 2011).…”

Development of chloroplast genomic resources for Cynara

“…This approach is the most straightforward for searching molecular resources functional in agriculture and ecology (Feltus et al . ; Henry ; Pootakham et al . ).…”

Sequencing Technologies and Their Use in Plant Biotechnology and Breeding

“…Rounsley et al (2009) sequenced and assembled a 19Mbp region of chromosome 3 in rice, using the 454 reads and was able to generate scaffolds with size ranging from 243 Kbp to 518 Kbp. Similar approaches have been used in cacao (Feltus et al, 2011) and Barley (Steuernagel et al, 2009). …”