Genetic diversity of the zigzag ladybird beetle, Cheilomenes sexmaculata F. (Coleoptera: Coccinellidae) with its distribution in India and implications for biological control

The zigzag beetle, Cheilomenes sexmaculata, the most important and abundant of all ladybird beetles, feeds on a diverse range of prey. The study was conducted understand the population structure of C. sexmaculata, individuals collected from five different zones, consisting of 25 subpopulations that uniformly representing India. From the ddRAD sequence data we have identified contigs, SNPs, and INDELs were identified for all these 25 subpopulations collected across the India.

Methods
The adult C. sexmaculata were collected from five different localities belonging to various zones of India viz., Delhi (North), Nagpur (Central), Jorhat (East), Anand (West) and Bengaluru (South) from May to June 2019. The samples were stored in 100% ethanol and frozen at -70°C. Genomic DNA was isolated from each individual separately by grinding in liquid nitrogen, using the method described earlier (Kim et al., 2012). We employed, restriction-site associated DNA sequencing (RADseq) coupled with Illumina sequencing, produces high coverage of homologous SNP (Single Nucleotide Polymorphism) loci. Population genomic analyses from ddRADseq depend on denovoassembly of a set of reference contigs. Raw SNPs/INDELs calls are represented in a single Hap Map file. 

Methods for processing the data: For samples 1-25, 100-1000ng of high-quality genomic DNA was completely digested using Sphl and Mluc1 in a 20-50ul reaction volume (2.4 U per enzyme, New England Biolabs (NEB), Ipswich, MA, USA) and incubated at 37 °C for 90 min. Adapter ligation was done with Adapters A and B by changing their sticky ends for SphI and Mluc1, respectively. The dual-indexed primers designed were used for the reactions. After adding the indexed primers in PCR, the obtained libraries were pooled based on concentration (Qubit 2.0 fluorometer analysis) and concentrated in a SpeedVac (Eppendorf, Hamburg, Germany). A manual size selection was applied (a range between 450 and 550 bp, which corresponds to DNA fragment size of interest between 310 and 410bp) in low-melting 1.5% agarose gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). The final libraries were quantified by Qubit 2.0 fluorometer (dsDNA kit, Thermo Fisher Scientific) and their quality was checked on a Fragment Analyzer system (DNA High Sensitivity kit, Agilent). The sequencing quality of each sample was checked using FastqC. The dDocent version 2.2.10 was used for this project. After dDocent checks, it was recognized the proper number of samples in the current directory, later dDocent, Trim Galore! Program was used to proceed with the quality trimming of sequence data. Without reference material, population genomic analyses from ddRADseq depend on denovoassembly of a set of reference contigs . After all executions of FreeBayes are completed, raw SNPs/INDELs calls are concatenated into a single variant call file (VCF) using VCF tools.  VCF file that contains all SNPs, INDELs, MNPs and complex events that are called in 90% of all individuals with a minimum quality score of 30. This VCF file is converted to HapMap with TASSEL.