A tailed PCR procedure for cost-effective, two-order multiplex sequencing of candidate genes in polyploid plants.

Complex polyploid crop genomes can be recalcitrant towards conventional DNA sequencing approaches for allele mining in candidate genes for valuable traits. In the past, this has greatly complicated the transfer of knowledge on promising candidate genes from model plants to even closely related polyploid crops. Next-generation sequencing offers diverse solutions to overcome such difficulties. Here, we present a method for multiplexed 454 sequencing in gene-specific PCR amplicons that can simultaneously address multiple homologues of given target genes. We devised a simple two-step PCR procedure employing a set of barcoded M13/T7 universal fusion primers that enable a cost-effective and efficient amplification of large numbers of target gene amplicons. Sequencing-ready amplicons are generated that can be simultaneously sequenced in pools comprising multiple amplicons from multiple genotypes. High-depth sequencing allows resolution of the resulting sequence reads into contigs representing multiple homologous loci, with only insignificant off-target capture of paralogues or PCR artefacts. In a case study, the procedure was tested in the complex polyploid genome of Brassica napus for a set of nine genes identified in Arabidopsis as candidates for regulation of seed development and oil content. Up to six copies of these genes were expected in B. napus. SNP discovery was performed by pooled multiplex sequencing of 30 amplicons in 20 diverse B. napus accessions with interesting trait variation for oil content, providing a basis for comparative mapping to relevant quantitative trait loci and for subsequent marker-assisted breeding.

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.).

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.