Development of Linked DNA Marker to Isolation of Gene Controlling Disease Susceptibility to Black Spot in Strawberry Based on NGS Analysis.

为了开发分离疾病特异性基因的DNA maker，我们将AFLP（扩增片段长度多态性）批量标记法、NGS（下一代测序法）和CD-HIT-EST序列聚类技术相结合。采用AFLP法制备了易感基因型和抗病基因型的基因组DNA。通过CD-HIT-EST将确定的序列聚类为89,531。通过读取计数偏差（读取计数从敏感体/抗性体中得出的比率）过滤了总共3,679个序列簇。为了验证这些序列簇作为连锁标记的有效性，我们设计了PCR引物，并对花菇基因组的序列进行了定位。目前，在测试的231个引物组合中，126个引物组存在多态性扩增。这些结果表明，随着我们的方法被证明是有用的DNA制造者的发展；我们已经成功地开发了至少126个与疾病特异性位点（loci）相关的基因。

To develop DNA maker for isolation of disease specificity gene, we have combined AFLP (amplified fragment length polymorphism) bulk tagging method, NGS (next generation sequencing) and sequence clustering by CD-HIT-EST. Both bulks of genomic DNA derived from disease susceptible and resistant genotype were prepared according to AFLP method. Determined sequences were clustered into 89,531 by CD-HIT-EST. Total of 3,679 sequence clusters was filtered by read count bias (a ratio of read count derived from susceptible bulk / resistant bulk). To check availability of these sequence clusters as linked makers, PCR primers were designed through mapping of sequences on Fragaria vesca genome. Currently, of tested 231 primer combinations, 126 primer sets showed polymorphic amplification. These results reveal that along with our methods was proven to be useful for development of DNA makers; we have succeeded to develop at least 126 linked makers to disease specificity locus (loci).