Pooled whole genome sequencing method (Pool-Seq) to identify genomic regions associated to Dryocosmus kuriphilus Yasumatsu resistance in European Castanea sativa Mill.

The rapid advances of next generation sequencing (NGS), its decreasing cost and the development of efficient technologies for big data analysis allow a wide range of NGS applications in non-model plants. An important advantage of NGS studies is the identification of many markers with known genome position and the direct identification of causal mutations when they are associated to trait variation. The pool-seq method is a cost-effective approach to perform genome-wide association studies (GWAS), consisting in sequencing pool of individuals with extreme phenotypes. This approach was used to study the genetic basis of resistance to the insect Dryocosmus kuriphilus (Asian chestnut galls wasp, ACGW), which was introduced from China to Italy in 2002 and has become one the most damaging pests of Castanea sativa (C. sativa). A comparative field trial at IRET-CNR, including provenances from Spain, Italy, and Greece, was screened for severity of infestation by ACGW. A great diversity among plants and a significant heterogeneity among the geographical provenances were found. The phenotypic data suggest that the Greek provenance Hortiatis could bring specific genetic factors of resistance to ACGW. A suitable set of plant material was selected for a Pool-Seq GWAS experiment, by comparing susceptible and resistant plants and reducing risk of spurious association due to lack of population structure. DNA pools of the two phenotypic groups were sequenced and reads were aligned to the reference genome of C. mollissima. The two pools will be compared to identify SNPs associated to resistance. The application of PoPoolation2 software, allows to identify significant differences in SNP allele frequencies between our DNA pools. Genomic regions containing significant SNP differences will be associated to plant resistance and will be screened to identify candidate genes and clarify the mechanisms of resistance.