SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napusThis article is one of a selection of papers from the conference “Exploiting Genome-wide Association in Oilseed Brassicas:  a model for genetic improvement of major OECD crops for sustainable farming”.

Durstewitz, Gregor; Polley, Andreas; Plieske, Joerg; Luerssen, Hartmut; Graner, Eva-Maria; Wieseke, Ralf; Ganal, Martin W.

doi:10.1139/g10-079

Cited by 55 publications

(35 citation statements)

References 18 publications

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“…The SNPs were chosen to cover a range of the 76 supercontigs on which SNPs were predicted, from the list of 21,814 SNPs described in Zander et al (2013). A designability assessment conducted using the Illumina Assay Design Tool (ADT) scored the 384 SNPs at 0.4 or above, which is deemed a good score for the Illumina GoldenGate assay (Durstewitz et al, 2010). Sample preparation for the Illumina GoldenGate assay was performed according to the Illumina GoldenGate Genotyping Assay guide (According to manufacturer's instructions).…”

Section: Illumina Goldengate Assaymentioning

confidence: 99%

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Population Diversity of Leptosphaeria maculans in Australia

Patel¹,

Zander²,

Wouw³

et al. 2015

IJB

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The fungal pathogen Leptosphaeria maculans, causal agent of blackleg disease, is a primary cause of canola (Brassica napus) crop loss in Australia. Expanding our knowledge of the occurrence of this pathogen in Australia will provide valuable insights into developing methods of resistance against it. In this study, we examine the population diversity of L. maculans in Australia using single nucleotide polymorphisms (SNPs). An Illumina GoldenGate 384 SNP assay was developed and used to genotype 59 blackleg isolates collected from across Australia, in different years and from different stubble sources. Limited linkage disequilibrium, absence of significant clustering in the principal component analysis and a mixed dendrogram suggest that the Australian L. maculans population as a whole is panmictic. Some evidence of clonality concentrated in each state was also observed. There was a lack of correlation between SNP haplotypes, stubble cultivar and year of collection. These results suggest a high rate of sexual reproduction and evolutionary diversification in the pathogen. These features could enable the pathogen to overcome resistance and continue to cause disease in Brassica crops. Analysis of these fungal population isolates will help shed some light on evolution and pathogenicity questions in this important crop pathogen.

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Section: Illumina Goldengate Assaymentioning

confidence: 99%

“…Previous studies using the Illumina GoldenGate assay have shown that it can be used to reliably score SNPs for genetic analysis (Durstewitz et al, 2010). Furthermore, it is cost-effective and flexible for analysing large numbers of SNPs (Appleby et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Population Diversity of Leptosphaeria maculans in Australia

Patel¹,

Zander²,

Wouw³

et al. 2015

IJB

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“…In recent years, many SNPs have been discovered in B. napus [22, 23] and B. oleracea [24]. However, these SNPs are inadequate for large-scale applications [23].…”

Section: Introductionmentioning

confidence: 99%

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

et al. 2013

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BackgroundSingle 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.ResultsA 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%.ConclusionsDespite 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.

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“…Due to polyploidy, two classes of polymorphisms need to be considered. Sites that are polymorphic between accessions represent the so-called intragenomic SNPs [23] that are especially versatile for genetic mapping and discrimination of accessions. Large SNP collections have been put together for Brassica napus [24–26].…”

Section: Introductionmentioning

confidence: 99%

“…Large SNP collections have been put together for Brassica napus [24–26]. SNPs that differentiate between the homoeologous A and C genomes were termed interhomoeolog polymorphism [24] or intergenomic SNP [23]. This SNP class proved to be valuable for the screening of BAC libraries, since intergenomic SNPs identify and at the same time differentiate homoeologous sequences [21].…”

Section: Introductionmentioning

confidence: 99%

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

Cao

Schmidt

2014

BMC Genomics

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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.

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Cited by 55 publications

References 18 publications

Population Diversity of Leptosphaeria maculans in Australia

Population Diversity of Leptosphaeria maculans in Australia

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

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

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