Abstract
BackgroundHomoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.ResultsSequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina’s GoldenGate® Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.ConclusionsThis proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-560) contains supplementary material, which is available to authorized users.
Highlights
Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome
Identification of regions suitable for development of intergenomic Single nucleotide polymorphisms (SNPs) assays A region represented by 15 whole genome sequence (WGS) contigs and spanning approximately one Mbp on Brassica rapa chromosome A1 and its counterpart on Brassica oleracea chromosome C1 were chosen for this study
Intergenomic SNP assays should be placed in regions in which the 20–25 bp of homoeologous sequences flanking the SNP site are identical, they should be distinguished by multiple mismatches and/or indels very close to the assay site when compared to homologous sequences elsewhere in the Brassica napus genome [21]
Summary
Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. The. Studies in Brassica species greatly benefit from genomic resources that have been assembled. BAC contig maps for the Brassica rapa [12] and Brassica oleracea genomes were produced [13]. In both studies, highinformation-content fingerprinting data of BACs were exploited to establish overlaps between BACs. Integration of the resulting contigs with molecular marker maps was achieved by hybridising sequence-tagged probes to the gridded BAC libraries. Large collections of expressed sequence tags (ESTs) and BAC end sequences were put together for different Brassica species [17]. A draft genome sequence was released for Brassica rapa [18]
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