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. 2009 Aug;119(3):507-17.
doi: 10.1007/s00122-009-1059-5. Epub 2009 May 18.

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Eduard Akhunov  1 , Charles Nicolet, Jan Dvorak
Affiliations

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Eduard Akhunov et al. Theor Appl Genet. 2009 Aug.

Abstract

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach.

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Figures

Fig. 1
Fig. 1
GoldenGate SNP genotyping in polyploid wheat. a Principles of GoldenGate SNP genotyping in tetraploid wheat. The toppanel shows two genotypes of T. dicoccoides polymorphic for A/T SNP in the A-genome and monomorphic for the T base at the orthologous site in the B-genome. The bottompanel shows the same two genotypes except for the LSO annealing site in the B-genome showing a secondary mutation resulting in imperfect match. Upstream ASOs are blue and red and downstream LSO is black. b The expected Cy3/Cy5 fluorescence ratios for homozygous (AA, TT) and heterozygous (AT) diploid, tetraploid and hexaploid plants. The genotypes of the polymorphic sites are shown in parentheses
Fig. 2
Fig. 2
SNP sites failing to produce a two-cluster pattern. Genotyping plots were generated by graphing normalized Cy3 and Cy5 fluorescence intensities
Fig. 3
Fig. 3
Genotype calls in a sample of 53 accessions of tetraploid T. dicoccoides (a) and 38 accessions of hexaploid T. aestivum (b). The accession numbers of mapped EST unigenes are shown on the top of each panel. Genotyping plots were generated by plotting Cy3 and Cy5 normalized fluorescence intensities. a SNP sites polymorphic in the A-genome of T. dicoccoides. b SNP sites that were polymorphic in the hexaploid wheat A- or D-genomes
Fig. 4
Fig. 4
Distribution of minor allele frequencies of SNPs in the A- and D-genomes of T. aestivum and the A-genome of T. dicoccoides detected with the GoldenGate assay and sequencing in the SNP discovery panel
See this image and copyright information in PMC

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