4.4. Target Resequencing

About 184 products previously amplified for HRM analysis were selected for subsequent resequencing, one for each cluster within each fragment preferentially choosing the one with the maximum confidence percentage of cluster attribution (generally 100%). The selected amplified products were purified by adding 15 µmoles of ammonium acetate and 2.4 volumes of ethanol and then rinsing once with 70% ethanol. Sanger sequencing of purified products in forward and reverse were performed by an external service in 96-plate format. A total number of 4 to 10 FASTA sequences per target were obtained. Data were then read and edited by SeqScape Software (Version: 1.0, Applied Biosystems). On SeqScape, a reference data group was built up using available sequences for C. crenata. Automatically detected polymorphisms were then manually edited and analyzed, evaluating the confidence percentages of base calling and peak quality. All the SNPs were coded, adding to the two letters code of the fragment, three numbers indicating the position of the SNP on the original 161 bp fragment used as reference for the resequencing project. Thirty-seven SNPs were chosen among the SNPs falling in the central part of the fragment, so that at least 50 flanking base pairs were available, and were used for subsequent KASP assay design.

The association of each sequenced fragment with an annotated gene was performed by blasting (e-value threshold 1E-10) the sequences against the transcriptome of C. sativa cv. ‘Bouche de Bétizac’ and cv. ‘Madonna’, both retrieved from Aquadro et al. [13]. When no match was found in those transcriptomes, the sequence was blasted against the non-redundant nucleotide collection in NCBI, getting the closest phylogenetic result (mostly Quercus and Fagus). A functional annotation of the genes was then performed using Blast2GO [49] and gene ontology (GO) terms were predicted by assigning functional classifications [50] as well as the potential properties of gene products. The BLAST cut-off e-value was set to 10⁻⁵. SNP classification/annotation was assigned according to Ensembl Variation guidelines [51].