A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies

Sequencing of BAC pools by different next generation sequencing platforms and strategies

<p>Abstract</p> <p>Background</p> <p>Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs) improve the assemblies by scaffolding and whether barcoding of BACs is dispensable.</p> <p>Results</p> <p>Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library.</p> <p>Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%.</p> <p>Conclusion</p> <p>Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.</p

Discovery of single nucleotide polymorphism in Capsicum and SNP markers for cultivar identification

Molecular markers based on single nucleotide polymorphisms (SNPs) are abundant and evenly distributed in a whole genome enough to distinguish individuals in a population. In recent years, sets of SNP markers have been designed and applied for cultivar identification in various crop species. This paper is the first to report the development of a panel of SNP markers for variety identification in peppers. We used conserved ortholog set II (COSII) markers developed from conserved unigenes between tomato and Arabidopsis to identify SNPs in peppers. We tested 438 COSII primer sets amplified as single PCR products out of a total 600 COSII primer sets. Among the 438 COSII primers, 170 primer sets (38.8%) showed polymorphisms between Capsicum annuum 'RNaky (RN)'and C. chinense 'PI 159234 (234)'. In contrast, only 48 primer sets (11.0%) out of 438 primers sets were polymorphic between C. annuum 'Perennial (PER), and 'Dempsey (DEMP)'. The average frequency of SNPs plus InDels between C. annuum and C. chinense was 1/189 bp and between C. annuum spp. was 1/948 bp. Primer sets showing SNP between C. annuum PER and DEMP were re-designed to Allele Specific PCR (AS-PCR) primers and we finally selected a total of 40 SNP markers for cultivar identification. As the result, we were able to discriminate 97.5% of the 81 commercial hot cultivars and 100% of the 17 sweet pepper cultivars. We conclude the paper by discussing the use of the SNP marker set for cultivar identification and other applications. © 2010 Springer Science+Business Media B.V