Computational protocol: Morphological and Genetic Analysis of Four Color Morphs of Bean Leaf Beetle

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[…] DNA was extracted from the thorax of 22 randomly selected BLB adults of each phenotype using the CTAB extraction protocol as modified by . The procedures for extraction included homogenization of the thorax, addition and incubation of Proteinase K and RNase A, and centrifugation followed by supernatant transfer and cold storage. Quantification of DNA from individuals for each sample location using a spectrophotometer and visualization on an agarose gel were the same as previously reported (). Diluted DNA samples were then kept at −20°C until they were used after which the samples were kept at −80°C as vouchers in the Genetics Laboratory of the Entomology Department at the University of Nebraska - Lincoln.A modified AFLP protocol () was used to assess the genetic variability within and among BLB subpopulations. DNA extracted from 15 to 20 individuals per color morph of BLB was used. All steps of AFLP-PCR were completed following the same procedure as in and included: 1) restriction of the DNA with MseI and EcoRI restriction enzymes; 2) adapter ligation with MseI and EcoRI adapters; 3) pre-amplification (); and 4) selective amplification with 3 combinations of MseI and EcoRI primers (). Afterwards, 1.5 µl of each sample with specific primer combinations was electrophoresed on KBplus 6.5% polyacrylamide gel in the GeneReadIR 4200 DNA analyzer (LI-COR, Lincoln, NE) for 2 h to separate the DNA markers. The gel image was saved for scoring (developing a binary matrix of 1’s = band present and 0’s = band absent) and further analysis.Each primer combination was used with subsamples of DNA from 24 randomly selected individuals for assessing the genotyping error as reported previously (). The error rate, calculated as the ratio of the total number of mismatches (presence or absence of a band) at a locus to the number of the replicated individuals, should never exceed 10% to better ensure reliability and reproducibility of results (, ).Bootstrap analysis was used as a way of testing the robustness of the dataset for further analysis by the creation of a pseudo-replicate which re-sampled the data 10,000 times using BOOD-P software ver. 3.1 (). DBOOT () was used to evaluate the correlation between the coefficient of variation and the number of molecular markers observed, thus providing an estimate of the robustness of the data (). Genetic similarity was estimated using the Jaccard index through the SIMQUAL procedure using NTSYSpc (). Dendrograms were constructed to illustrate genetic similarity, following the methodology described by .POPGENE version 1.32 () was used to determine the degree of genetic polymorphisms both within and between color morphs of BLB. Genetic differentiation between color morphs samples was assessed in POPGENE version 1.32 () using Nei’s gene diversity index (GST); gene flow (Nm) between color morphs was estimated from GST, expressed as (Nm) = (1−GST)/4GST (, , ). Fall armyworm larvae, Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae), were used as the out-group to test the robustness of the dendrogram developed in the POPGENE analysis. The potential that the samples exhibited population structure was investigated using the program STRUCTURE (). One to four potential populations (K = 1 to K = 4) were evaluated (burn-in = 10,000; replicates = 10,000); 15 iterations were done for each K value. Results from STRUCTURE were further evaluated using STRUCTURE HARVESTER (). Analysis of molecular variance (AMOVA) was used to evaluate genetic variability among groups and within groups (color morphs) using the software package Arlequin version 3.5 (). AMOVA were calculated with color morphs based on results from STRUCTURE (considered as three sub-populations of G+S, G-S, and R+S with R-S) and, for comparison, an AMOVA was calculated for all color morphs belonging to one population. ARLEQUIN was also used for pairwise comparisons to test genetic divergence (FST-Wright’s inbreeding coefficient) (, ). […]

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