Dataset Information

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions.

ABSTRACT:

Background

Homoeologous 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.

Results

Sequence 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.

Conclusions

This 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.

SUBMITTER: Cao HX

PROVIDER: S-EPMC4102721 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Publications

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions.

Cao Hieu Xuan HX Schmidt Renate R

BMC genomics 20140704

<h4>Background</h4>Homoeologous 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.<h4>Results</h4>Sequence alignments between Brassica rapa ...[more]

PMID: 24996518

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Project description:BACKGROUND: Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. RESULTS: A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina's GoldenGate Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. CONCLUSIONS: Illumina's GoldenGate Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome.

Project description:All crop species are current or ancient polyploids. Following whole genome duplication, structural and functional modifications result in differential gene content or regulation in the duplicated regions, which can play a fundamental role in the diversification of genes underlying complex traits. We have investigated this issue in Brassica napus, a species with a highly duplicated genome, with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker, a disease caused by the fungal pathogen Leptosphaeria maculans. Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E, J, R, U, and W were involved in resistance to stem canker. The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions, as overall in the whole B. napus genome. The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B. napus progenitors. About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block. Genes retained in several copies were mainly involved in response to stress, signaling, or transcription regulation. Genes with resistance-associated markers were mainly retained in more than two copies. These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes. Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions. Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype.

Dataset Information

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions.

Background

Results

Conclusions

Publications

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions.

Similar Datasets

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