Dataset Information

Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L.

ABSTRACT: Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci -QTL- analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.

SUBMITTER: Clarke WE

PROVIDER: S-EPMC3849492 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L.

Clarke Wayne E WE Parkin Isobel A IA Gajardo Humberto A HA Gerhardt Daniel J DJ Higgins Erin E Sidebottom Christine C Sharpe Andrew G AG Snowdon Rod J RJ Federico Maria L ML Iniguez-Luy Federico L FL

PloS one 20131203 12

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait lo ...[more]

PMID: 24312619

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Project description:BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay. RESULTS: A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%. CONCLUSIONS: Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.

Project description:BackgroundStudies in resynthesized Brassica napus allopolyploids indicate that homoeologous chromosome exchanges in advanced generations (S(5ratio6)) alter gene expression through the loss and doubling of homoeologous genes within the rearrangements. Rearrangements may also indirectly affect global gene expression if homoeologous copies of gene regulators within rearrangements have differential affects on the transcription of genes in networks.Methodology/principal findingsWe utilized Arabidopsis 70mer oligonucleotide microarrays for exploring gene expression in three resynthesized B. napus lineages at the S(0ratio1) and S(5ratio6) generations as well as their diploid progenitors B. rapa and B. oleracea. Differential gene expression between the progenitors and additive (midparent) expression in the allopolyploids were tested. The S(5ratio6) lines differed in the number of genetic rearrangements, allowing us to test if the number of genes displaying nonadditive expression was related to the number of rearrangements. Estimates using per-gene and common variance ANOVA models indicated that 6-15% of 26,107 genes were differentially expressed between the progenitors. Individual allopolyploids showed nonadditive expression for 1.6-32% of all genes. Less than 0.3% of genes displayed nonadditive expression in all S(0ratio1) lines and 0.1-0.2% were nonadditive among all S(5ratio6) lines. Differentially expressed genes in the polyploids were over-represented by genes differential between the progenitors. The total number of differentially expressed genes was correlated with the number of genetic changes in S(5ratio6) lines under the common variance model; however, there was no relationship using a per-gene variance model, and many genes showed nonadditive expression in S(0ratio1) lines.Conclusions/significanceFew genes reproducibly demonstrated nonadditive expression among lineages, suggesting few changes resulted from a general response to polyploidization. Furthermore, our microarray analysis did not provide strong evidence that homoeologous rearrangements were a determinant of genome-wide nonadditive gene expression. In light of the inherent limitations of the Arabidopsis microarray to measure gene expression in polyploid Brassicas, further studies are warranted.

Dataset Information

Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L.

Publications

Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP) in Brassica napus L.

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