SNP allele frequency estimation in pooled DNA of Frankliniella occidentalis (Pergande, 1895) (Thysanoptera: Thripidae) by next-generation sequencing

Next-generation sequencing combined with bioinformatic analysis has become an instrumental tool for quick and reliable SNP discovery in genomes. Here, we utilized a genomic DNA library prepared from 200 Frankliniella occidentalis adults, collected from multiple weed and cultivated plant species in Konya, Karaman and Mersin (2016-2017), Turkey. Specimens were morphologically identified and preserved in ethyl alcohol. High molecular weight genomic DNA was extracted using the CTAB method. For NGS-seq library preparation, 40 mu l of DNA was fragmented in a Monorex ultrasonic bath. The fragmented DNA was cleaned and size selected using AMPure XP beads and subjected to the Illumina TruSeq Nano library preparation protocol. The resulting library was bidirectionally sequenced on the HiSeq-X platform. Raw sequencing reads were trimmed and filtered by the "fastp" and "trim galore" bioinformatic programs. After data filtering, a total of 645,144 NGS reads were "bowtie2" aligned with the reference mitochondrial genome (NC_018370.1). The aligned reads were subjected to the SNP calling pipeline using "freebayes," resulting in 61 SNPs between the sample and reference mitochondria. Of those SNPs, 46 were found to be in protein-coding genes (PCGs), whereas 15 SNPs were located in tRNA regions. All 13 PCGs on the mitochondria were found to have at least one SNP site. Among the PCGs, cox1 was the most polymorphic gene, bearing 8 SNPs along its 1553 bp length. On the other hand, the nad5 gene (1598 bp length) had only a single SNP location. Despite the results suggesting that the cox1 gene may be the most useful gene for population and phylogenetic studies, other PCGs, such as cox3 and cob, could also be utilized, as their polymorphism rates were comparable to that of the cox1 gene.