**3. Results**

#### *3.1. Genetic Data*

3.1.1. Population Diversity Metrics

From the 29 WCR genotyped, 25,304 SNPs were detected. The 90% call rate filter then removed 13,852 SNPs from the data set. Following this, the minor allele frequency filter, SNPs with frequencies <1%, hence removed another 3555 SNPs. Filtering for monomorphs, secondaries, and reproducibility set at 95% removed 772 SNPs. For final analyses, 7125 SNPs were used.

The overall population estimate was applied, and moderate observed heterozygosity (HO) was observed across all loci, with an estimated value of HO = 0.325. Moderate genetic diversity, estimated by expected heterozygosity (HE), was observed with an estimated value of HE = 0.302. Moderate inbreeding was observed (FIS = 0.121). There were no significant deviations from HWE for all loci. The low overall value of the genetic structure (FST = 0.0181) estimated for the five populations suggested a lack of genetic differentiation amongs<sup>t</sup> them as a whole.

Heterozygosity estimates (HO and HE) over all loci and populations were very similar. The average HO per population ranged from 0.315 (non-resistant) to 0.338 (Cry3Bb1\_Cry34/ 35Ab1), while average HE ranged from 0.315 (Cry34/35Ab1) to 0.349 (Cry3Bb1\_Cry34/35Ab1) (Table 2). Moderate levels of genetic diversity across all populations were therefore suggested.

**Table 2.** Expected heterozygosity (He) and observed heterozygosity (Ho) values for western corn rootworm populations over all loci.


Distribution of heterozygous WCR genotypes and SNP markers revealed moderate values of heterozygosity in 25 individuals out of 28, with heterozygosity <0.35 (Figure 2).

**Figure 2.** Frequency of heterozygous genotypes and heterozygosity of 7125 SNP markers.

In contrast, pairwise genetic structure does however show differentiation between pairwise population comparisons (Table 3). Pairwise FST θ estimates ranged from 0.0021 (non-resistant population versus Cry3Bb1 resistant population) to 0.0531 (Cry34/35Ab1 resistant population versus Cry3Bb1\_Cry34/35Ab1 resistant population). Cry34/35Ab1 and Cry3Bb1\_Cry34/35Ab1 populations showed the greatest genetic differentiation with respect to all other populations.


**Table 3.** Population pairwise estimates of fixation index (FST).

#### 3.1.2. Genetic Structure

STRUCTURE analysis revealed Δ *K* = 3 was the most likely number of clusters or populations present within the sampled US WCR individuals (Figure 3). Beetles were assigned to three clusters in consultation with results from STRUCTURE (Figure 4). Along with the results of the kinship analysis with the genetic clustering, a heat map of kinship matrix for evaluating the genetic differences among WCR genotypes was generated. Kinship coefficients between pairs of WCR genotypes varied very little on a scale of −1 to 1. However, the kinship matrix obtained from DArTseq SNP markers resulted in three distinct groups (Figure 5).

**Figure 3.** Results from Structure Harvester analysis to reveal the most likely value of *K* based on STRUCTURE results.

**Figure 4.** Determination of the optimal value of *K* = 3 and population structure of 29 WCR genotypes using DArTseq SNP markers.

**Figure 5.** Heat map plot of kinship matrix using average linkage clustering based on SNP markers depicts the existence of three different groups among WCR genotype.

Further analysis of genetic structure using neighbor-joining (NJ) cluster analysis differentiated WCR genotypes into tree clusters (Figure 6). Cluster I was the largest, and it comprised 18 genotypes that included non-resistant individuals, Cry34/35 and Cry3Bb1 resistant. Cluster II contained individuals with combined *Bt* toxins Cry3Bb1 and Cry34/35 toxin, and Cluster III contained individuals adapted to crop rotation.

**Figure 6.** The neighbor-joining cluster analysis using DArTseq SNP markers for grouping 29 WCR genotypes.
