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

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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).