Dataset Information

A DArT platform for quantitative bulked segregant analysis.

ABSTRACT:

Background

Bulked segregant analysis (BSA) identifies molecular markers associated with a phenotype by screening two DNA pools of phenotypically distinct plants for markers with skewed allele frequencies. In contrast to gel-based markers, hybridization-based markers such as SFP, DArT or SNP generate quantitative allele-frequency estimates. Only DArT, however, combines this advantage with low development and assay costs and the ability to be deployed for any plant species irrespective of its ploidy level. Here we investigate the suitability of DArT for BSA applications using a barley array as an example.

Results

In a first test experiment, we compared two bulks of 40 Steptoe/Morex DH plants with contrasting pubescent leaves (mPub) alleles on chromosome 3H. At optimized levels of experimental replication and marker-selection threshold, the BSA scan identified 433 polymorphic markers. The relative hybridization contrast between bulks accurately reflected the between-bulk difference in the frequency of the mPub allele (r = 0.96). The 'platform noise' of DArT assays, estimated by comparing two identical aliquots of a DNA mixture, was significantly lower than the 'pooling noise' reflecting the binomial sampling variance of the bulking process. The allele-frequency difference on chromosome 3H increased in the vicinity of mPub and peaked at the marker with the smallest distance from mPub (4.6 cM). In a validation experiment with only 20 plants per bulk we identified an aluminum (Al) tolerance locus in a Dayton/Zhepi2 DH population on chromosome 4H with < 0.8 cM precision, the same Al-tolerance locus that had been mapped before in other barley populations.

Conclusion

DArT-BSA identifies genetic loci that influence phenotypic characters in barley with at least 5 cM accuracy and should prove useful as a generic tool for high-throughput, quantitative BSA in plants irrespective of their ploidy level.

SUBMITTER: Wenzl P

PROVIDER: S-EPMC1920522 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Publications

A DArT platform for quantitative bulked segregant analysis.

Wenzl Peter P Raman Harsh H Wang Junping J Zhou Meixue M Huttner Eric E Kilian Andrzej A

BMC genomics 20070628

<h4>Background</h4>Bulked segregant analysis (BSA) identifies molecular markers associated with a phenotype by screening two DNA pools of phenotypically distinct plants for markers with skewed allele frequencies. In contrast to gel-based markers, hybridization-based markers such as SFP, DArT or SNP generate quantitative allele-frequency estimates. Only DArT, however, combines this advantage with low development and assay costs and the ability to be deployed for any plant species irrespective of ...[more]

PMID: 17597543

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Project description:Positional gene isolation in unsequenced species generally requires either a reference genome sequence or an inference of gene content based on conservation of synteny with a genomic model. In the large unsequenced genomes of the Triticeae cereals the latter, i.e. conservation of synteny with the rice and Brachypodium genomes, provides a powerful proxy for establishing local gene content and order. However, efficient exploitation of conservation of synteny requires 'homology bridges' between the model genome and the target region that contains a gene of interest. As effective homology bridges are generally the sequences of genetically mapped genes, increasing the density of these genes around a target locus is an important step in the process. We used bulked segregant analysis (BSA) of transcript abundance data to identify genes located in a specific region of the barley genome. The approach is valuable because only a relatively small proportion of barley genes are currently placed on a genetic map. We analyzed eQTL datasets from the reference Steptoe × Morex doubled haploid population and showed a strong association between differential gene expression and cis-regulation, with 83% of differentially expressed genes co-locating with their eQTL. We then performed BSA by assembling allele-specific pools based on the genotypes of individuals at the partial resistance QTL Rphq11. BSA identified a total of 411 genes as differentially expressed, including HvPHGPx, a gene previously identified as a promising candidate for Rphq11. The genetic location of 276 of these genes could be determined from both eQTL datasets and conservation of synteny, and 254 (92%) of these were located on the target chromosome. We conclude that the identification of differential expression by BSA constitutes a novel method to identify genes located in specific regions of interest. The datasets obtained from such studies provide a robust set of candidate genes for the analysis and serve as valuable resources for targeted marker development and comparative mapping with other grass species.

Dataset Information

A DArT platform for quantitative bulked segregant analysis.

Background

Results

Conclusion

Publications

A DArT platform for quantitative bulked segregant analysis.

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