a next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to poa grasses新一代测序方法克服多倍体系统发生学的多个基因复制问题,应用于poa草.pdfVIP

Griffin et al. BMC Biology 2011, 9:19 /1741-7007/9/19 METHODOLOGY ARTICLE Open Access A next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to Poa grasses Philippa C Griffin1*, Charles Robin1 and Ary A Hoffmann1,2 Abstract Background: Polyploidy is important from a phylogenetic perspective because of its immense past impact on evolution and its potential future impact on diversification, survival and adaptation, especially in plants. Molecular population genetics studies of polyploid organisms have been difficult because of problems in sequencing multiple-copy nuclear genes using Sanger sequencing. This paper describes a method for sequencing a barcoded mixture of targeted gene regions using next-generation sequencing methods to overcome these problems. Results: Using 64 3-bp barcodes, we successfully sequenced three chloroplast and two nuclear gene regions (each of which contained two gene copies with up to two alleles per individual) in a total of 60 individuals across 11 species of Australian Poa grasses. This method had high replicability, a low sequencing error rate (after appropriate quality control) and a low rate of missing data. Eighty-eight percent of the 320 gene/individual combinations produced sequence reads, and 80% of individuals produced sufficient reads to detect all four possible nuclear alleles of the homeologous nuclear loci with 95% probability. We applied this method to a group of sympatric Australian alpine Poa species, which we discovered to share an allopolyploid ancestor with a group of American Poa species. All markers revealed extensive allele sharing among the Australian species and so we recommend that the current taxonomy be re-examined. We also detected hypermutation in the trnH-psbA marker, suggesting it should not be use