Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assaysReport as inadecuate




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BMC Genomics

, 14:603

Plant genomics

Abstract

BackgroundEfficient 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.

ResultsA 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.

ConclusionsIllumina’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.

KeywordsBAC library Brassica napus Genotyping Multidimensional pools PCR screening Polyploidy SNP Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-603 contains supplementary material, which is available to authorized users.

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Author: Hieu Xuan Cao - Renate Schmidt

Source: https://link.springer.com/







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