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High-throughput SNP discovery and genotyping for constructing a saturated linkage map of chickpea (Cicer arietinum L.).

Gaur R, Azam S, Jeena G, Khan AW, Choudhary S, Jain M, Yadav G, Tyagi AK, Chattopadhyay D, Bhatia S - DNA Res. (2012)

Bottom Line: Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%.The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean.The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, PO Box 10531, New Delhi 110067, India.

ABSTRACT
The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina's GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.

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Representative clustering patterns generated by the Illumina GoldenGate SNP Genotyping assay. For a given SNP marker, genotypes are called for each sample (dots) by their normalized signal intensity (Norm R, y-axis), i.e. sum of intensities of two fluorescent signals, and allele frequency (Norm theta, x-axis) relative to a cluster position (shaded area). The data point colour codes represent: red, AA (homozygous); blue, BB (homozygous); purple, AB (heterozygous); black, no call (missing data). (A) High-quality SNPs (e.g. CaSNP116 and CaSNP290) showing well-separated clusters of homozygous alleles (red and blue) and heterozygotes (purple). Some data points located between or in the border of these clusters (marked by an arrow) are unsuccessfully genotyped samples for which no calls were generated and considered as missing data. (B) SNPs which were considered as false or monomorphic (failed to detect an SNP in the parents and the mapping population) that grouped into a single cluster (e.g. CaSNP917 and ESNP46). (C) Technically unsatisfactory SNPs (e.g. CaSNP61 and CaSNP226) represented by insufficient allele cluster separation.
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DSS018F1: Representative clustering patterns generated by the Illumina GoldenGate SNP Genotyping assay. For a given SNP marker, genotypes are called for each sample (dots) by their normalized signal intensity (Norm R, y-axis), i.e. sum of intensities of two fluorescent signals, and allele frequency (Norm theta, x-axis) relative to a cluster position (shaded area). The data point colour codes represent: red, AA (homozygous); blue, BB (homozygous); purple, AB (heterozygous); black, no call (missing data). (A) High-quality SNPs (e.g. CaSNP116 and CaSNP290) showing well-separated clusters of homozygous alleles (red and blue) and heterozygotes (purple). Some data points located between or in the border of these clusters (marked by an arrow) are unsuccessfully genotyped samples for which no calls were generated and considered as missing data. (B) SNPs which were considered as false or monomorphic (failed to detect an SNP in the parents and the mapping population) that grouped into a single cluster (e.g. CaSNP917 and ESNP46). (C) Technically unsatisfactory SNPs (e.g. CaSNP61 and CaSNP226) represented by insufficient allele cluster separation.

Mentions: The genotyping data of the 768 SNPs across the 129 RILs were analysed using the GenomeStudio software (Illumina), which clusters and calls the data automatically, allowing visualization of the data directly for downstream analysis. For each SNP, the genotyping data representation included three main clusters corresponding to AA homozygote, AB heterozygote and BB homozygote. As in the present study, an F9-F10 RIL mapping population was used, which is expected to contribute very few heterozygotes, most of the SNP markers produced two main clusters representing the two homozygous genotypes, with sometimes a small additional cluster corresponding to heterozygous genotypes (Fig. 1A). A few data points were sometimes ambiguously located outside these clusters (indicated by arrows in Fig. 1A) and represented those for which no calls were generated and were therefore scored as missing data. In our genotyping data set, the average level of heterozygosity was 6.5%, expected for a RIL population, whereas the level of missing data per marker averaged at 5.6%.Figure 1.


High-throughput SNP discovery and genotyping for constructing a saturated linkage map of chickpea (Cicer arietinum L.).

Gaur R, Azam S, Jeena G, Khan AW, Choudhary S, Jain M, Yadav G, Tyagi AK, Chattopadhyay D, Bhatia S - DNA Res. (2012)

Representative clustering patterns generated by the Illumina GoldenGate SNP Genotyping assay. For a given SNP marker, genotypes are called for each sample (dots) by their normalized signal intensity (Norm R, y-axis), i.e. sum of intensities of two fluorescent signals, and allele frequency (Norm theta, x-axis) relative to a cluster position (shaded area). The data point colour codes represent: red, AA (homozygous); blue, BB (homozygous); purple, AB (heterozygous); black, no call (missing data). (A) High-quality SNPs (e.g. CaSNP116 and CaSNP290) showing well-separated clusters of homozygous alleles (red and blue) and heterozygotes (purple). Some data points located between or in the border of these clusters (marked by an arrow) are unsuccessfully genotyped samples for which no calls were generated and considered as missing data. (B) SNPs which were considered as false or monomorphic (failed to detect an SNP in the parents and the mapping population) that grouped into a single cluster (e.g. CaSNP917 and ESNP46). (C) Technically unsatisfactory SNPs (e.g. CaSNP61 and CaSNP226) represented by insufficient allele cluster separation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473369&req=5

DSS018F1: Representative clustering patterns generated by the Illumina GoldenGate SNP Genotyping assay. For a given SNP marker, genotypes are called for each sample (dots) by their normalized signal intensity (Norm R, y-axis), i.e. sum of intensities of two fluorescent signals, and allele frequency (Norm theta, x-axis) relative to a cluster position (shaded area). The data point colour codes represent: red, AA (homozygous); blue, BB (homozygous); purple, AB (heterozygous); black, no call (missing data). (A) High-quality SNPs (e.g. CaSNP116 and CaSNP290) showing well-separated clusters of homozygous alleles (red and blue) and heterozygotes (purple). Some data points located between or in the border of these clusters (marked by an arrow) are unsuccessfully genotyped samples for which no calls were generated and considered as missing data. (B) SNPs which were considered as false or monomorphic (failed to detect an SNP in the parents and the mapping population) that grouped into a single cluster (e.g. CaSNP917 and ESNP46). (C) Technically unsatisfactory SNPs (e.g. CaSNP61 and CaSNP226) represented by insufficient allele cluster separation.
Mentions: The genotyping data of the 768 SNPs across the 129 RILs were analysed using the GenomeStudio software (Illumina), which clusters and calls the data automatically, allowing visualization of the data directly for downstream analysis. For each SNP, the genotyping data representation included three main clusters corresponding to AA homozygote, AB heterozygote and BB homozygote. As in the present study, an F9-F10 RIL mapping population was used, which is expected to contribute very few heterozygotes, most of the SNP markers produced two main clusters representing the two homozygous genotypes, with sometimes a small additional cluster corresponding to heterozygous genotypes (Fig. 1A). A few data points were sometimes ambiguously located outside these clusters (indicated by arrows in Fig. 1A) and represented those for which no calls were generated and were therefore scored as missing data. In our genotyping data set, the average level of heterozygosity was 6.5%, expected for a RIL population, whereas the level of missing data per marker averaged at 5.6%.Figure 1.

Bottom Line: Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%.The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean.The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, PO Box 10531, New Delhi 110067, India.

ABSTRACT
The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina's GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.

Show MeSH
Related in: MedlinePlus