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Transcriptome and complexity-reduced, DNA-based identification of intraspecies single-nucleotide polymorphisms in the polyploid Gossypium hirsutum L.

Zhu QH, Spriggs A, Taylor JM, Llewellyn D, Wilson I - G3 (Bethesda) (2014)

Bottom Line: Using transcriptome data, we successfully identified 37,413 varietal SNPs and, of these, 22,121 did not have an additional varietal SNP within their 20-bp flanking regions so can be used in most SNP genotyping assays.Of the 1583 successful SNP assays achieved using different genotyping platforms, 1363 were verified.Many of the SNPs behaved as dominant markers because of coamplification from homeologous loci, but the number of SNPs acting as codominant markers increased when one or more subgenome-specific SNP(s) were incorporated in their assay primers, giving them greater utility for breeding applications.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Plant Industry, Canberra, ACT 2601, Australia Qianhao.Zhu@csiro.au Iain.Wilson@csiro.au.

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Verification of transcriptome-based predicted varietal SNPs by the CAPS method. The subgenome-specific SNPs are shown in pink and green, and the varietal SNPs are shown in red and blue. The restriction sites used for cleavage of the generated polymerase chain reaction fragments are underlined. The numbers in parentheses after the variety names represent the number of RNA-seq reads with identical sequences to that shown. Lanes 1 and 2 of the agarose gel represent DeltaOpal and Sicot 70, respectively. DNA size markers are indicated in bp. Sub-genome designations (At and Dt) are inferred by comparison to G. raimondii and G. arboreum sequences.
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fig2: Verification of transcriptome-based predicted varietal SNPs by the CAPS method. The subgenome-specific SNPs are shown in pink and green, and the varietal SNPs are shown in red and blue. The restriction sites used for cleavage of the generated polymerase chain reaction fragments are underlined. The numbers in parentheses after the variety names represent the number of RNA-seq reads with identical sequences to that shown. Lanes 1 and 2 of the agarose gel represent DeltaOpal and Sicot 70, respectively. DNA size markers are indicated in bp. Sub-genome designations (At and Dt) are inferred by comparison to G. raimondii and G. arboreum sequences.

Mentions: Using this rationale, we deployed an analytical approach to identify only the varietal SNPs among our RNA-seq data that were flanked by at least one subgenome-specific SNP. The approach was first tested using the short reads from four varieties (MCU-5, Siokra 1−4, DeltaOpal and Sicot 70), and this identified 4894 varietal SNPs. Ten of the predicted varietal SNPs and 10 equivocal SNPs without a supporting adjacent subgenome-specific SNP were selected for validation by converting them to CAPS markers. Of the 10 predicted varietal SNPs with flanking subgenome-specific SNP(s), 7 were confirmed to be polymorphic. Two such examples are shown in Figure 2. In contrast, of the 10 equivocal SNPs, a polymorphism was confirmed in only one case, supporting our hypothesis. We then extended the analysis to call varietal SNPs by parallel processing the RNA-seq data from all 18 varieties. In total, 37,413 nonredundant varietal SNPs were identified among these G. hirsutum varieties (File S1).


Transcriptome and complexity-reduced, DNA-based identification of intraspecies single-nucleotide polymorphisms in the polyploid Gossypium hirsutum L.

Zhu QH, Spriggs A, Taylor JM, Llewellyn D, Wilson I - G3 (Bethesda) (2014)

Verification of transcriptome-based predicted varietal SNPs by the CAPS method. The subgenome-specific SNPs are shown in pink and green, and the varietal SNPs are shown in red and blue. The restriction sites used for cleavage of the generated polymerase chain reaction fragments are underlined. The numbers in parentheses after the variety names represent the number of RNA-seq reads with identical sequences to that shown. Lanes 1 and 2 of the agarose gel represent DeltaOpal and Sicot 70, respectively. DNA size markers are indicated in bp. Sub-genome designations (At and Dt) are inferred by comparison to G. raimondii and G. arboreum sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199696&req=5

fig2: Verification of transcriptome-based predicted varietal SNPs by the CAPS method. The subgenome-specific SNPs are shown in pink and green, and the varietal SNPs are shown in red and blue. The restriction sites used for cleavage of the generated polymerase chain reaction fragments are underlined. The numbers in parentheses after the variety names represent the number of RNA-seq reads with identical sequences to that shown. Lanes 1 and 2 of the agarose gel represent DeltaOpal and Sicot 70, respectively. DNA size markers are indicated in bp. Sub-genome designations (At and Dt) are inferred by comparison to G. raimondii and G. arboreum sequences.
Mentions: Using this rationale, we deployed an analytical approach to identify only the varietal SNPs among our RNA-seq data that were flanked by at least one subgenome-specific SNP. The approach was first tested using the short reads from four varieties (MCU-5, Siokra 1−4, DeltaOpal and Sicot 70), and this identified 4894 varietal SNPs. Ten of the predicted varietal SNPs and 10 equivocal SNPs without a supporting adjacent subgenome-specific SNP were selected for validation by converting them to CAPS markers. Of the 10 predicted varietal SNPs with flanking subgenome-specific SNP(s), 7 were confirmed to be polymorphic. Two such examples are shown in Figure 2. In contrast, of the 10 equivocal SNPs, a polymorphism was confirmed in only one case, supporting our hypothesis. We then extended the analysis to call varietal SNPs by parallel processing the RNA-seq data from all 18 varieties. In total, 37,413 nonredundant varietal SNPs were identified among these G. hirsutum varieties (File S1).

Bottom Line: Using transcriptome data, we successfully identified 37,413 varietal SNPs and, of these, 22,121 did not have an additional varietal SNP within their 20-bp flanking regions so can be used in most SNP genotyping assays.Of the 1583 successful SNP assays achieved using different genotyping platforms, 1363 were verified.Many of the SNPs behaved as dominant markers because of coamplification from homeologous loci, but the number of SNPs acting as codominant markers increased when one or more subgenome-specific SNP(s) were incorporated in their assay primers, giving them greater utility for breeding applications.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Plant Industry, Canberra, ACT 2601, Australia Qianhao.Zhu@csiro.au Iain.Wilson@csiro.au.

Show MeSH
Related in: MedlinePlus