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Open Access Methodology article

A DArT platform for quantitative bulked segregant analysis

Peter Wenzl12, Harsh Raman3, Junping Wang34, Meixue Zhou4, Eric Huttner12 and Andrzej Kilian12*

Author Affiliations

1 Diversity Arrays Technology P/L, 1 Wilf Crane Cr., Yarralumla, Canberra ACT 2600, Australia

2 Triticarte P/L, 1 Wilf Crane Cr., Yarralumla, Canberra ACT 2600, Australia

3 NSW Agricultural Genomics Centre and NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, PMB, Wagga Wagga NSW 2650, Australia

4 Tasmanian Institute of Agricultural Research, University of Tasmania, PO Box 46, Kings Meadows TAS 7249, Australia

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BMC Genomics 2007, 8:196  doi:10.1186/1471-2164-8-196

Published: 28 June 2007

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.