title: A high-throughput pipeline for detecting locus-specific polymorphism in hexaploid wheat (Triticum aestivum L.).
abstract: BACKGROUND
  Bread wheat (Triticum aestivum L., 2n = 6x = 42) is an allohexaploid with a huge genome. Due to the presence of extensive homoeologs and paralogs, generating locus-specific sequences can be challenging, especially when a large number of sequences are required. Traditional methods of generating locus-specific sequences are rather strenuous and time-consuming if large numbers of sequences are to be handled.
  
  
  RESULTS
  To improve the efficiency of isolating sequences for targeted loci, a time-saving and high-throughput pipeline integrating orthologous sequence alignment, genomic sequence retrieving, and multiple sequence alignment was developed. This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.
  
  
  CONCLUSIONS
  The high-throughput pipeline developed in this study makes it feasible to efficiently identify locus-specific sequences for large numbers of sequences. It could find applications in all research projects where locus-specific sequences are required. In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat. Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.
authors.affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China ; CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia.
authors.forename: Jian
authors.initials: J
authors.lastname: Ma
authors.affiliation: CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia.
authors.forename: Jiri
authors.initials: J
authors.lastname: Stiller
authors.affiliation: CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia ; School of Plant Biology, The University of Western Australia, Perth, WA 6009 Australia ; National Foxtail Millet Improvement Centre, Institute of Millet Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China.
authors.forename: Ya-Xi
authors.initials: YX
authors.lastname: Liu
authors.affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China.
authors.forename: You-Liang
authors.initials: YL
authors.lastname: Zheng
authors.affiliation: CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia ; School of Plant Biology, The University of Western Australia, Perth, WA 6009 Australia.
authors.forename: Chunji
authors.initials: C
authors.lastname: Liu
keywords: Allopolyploid; Genome-specificity; Multiple sequence alignment; Primer design; Sequence polymorphism; blastn