4.5. Resequencing and InDel Detection

Genomic DNA of ‘Greensis’ and ‘Whasan’ was isolated from young leaves using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA libraries with approximately 550 bp of short inserts were constructed using the Truseq DNA PCR-Free kit (Illumina, San Diego, CA, USA) and sequenced with paired-ends using the Illumina Hiseq 2500 platform. Base calling of 101 bp at each end was performed using CASAVA v1.8.2 and the reads were trimmed using SolexQA [31] and Cutadapt [32]. The genome assembly data of P. bretschneideri cv. Dangshansuli v1.1 [33] were used as a reference for the alignment of the trimmed reads. The Burrows-Wheeler Aligner was employed to align the reads [34] using the default option. BAM files were produced using SAMtools [35] and Picard (http://broadinstitute.github.io/picard, Accessed 16 October 2017) [36] to remove PCR duplicates. The BAM files were loaded into the Integrative Genomics Viewer (IGV, [37]) to detect sequence variations between ‘Greensis’ and ‘Whasan’.

Sequences of InDels, which existed in ‘Greensis’ and ‘Whasan’ genomes, were extracted in IGV, and homology with public genes was analyzed using the blastn function in the National Center for Biotechnology Information (NCBI).