Extreme-QTL mapping of monepantel resistance in Haemonchus contortus.

BACKGROUND: Haemonchus contortus, a gastrointestinal nematode parasite of sheep, is mainly controlled by anthelmintics; the occurrence of anthelmintic resistance leads to treatment failures and increases economic burden. Because molecular mechanisms involved in drug resistance can be elucidated by genomic studies, an extreme quantitative trait locus (X-QTL) mapping approach was used to identify co-segregation of the resistance phenotype with genetic markers to detect the genome-wide variants associated with monepantel resistance in H. contortus. METHODS: A cross between H. contortus isolates using parental susceptible (Par-S) males and monepantel resistant (Par-R) females resulted in SR progeny, while reciprocal cross resulted in RS progeny. Pools (n = 30,000) of infective larvae (L3) recovered from Par-R, and from SR and RS populations in the F3 generation, collected both before (unselected group) and 7 days after (selected group) selection with monepantel treatment in sheep hosts, were subjected to genome sequencing (Pool-Seq). Pairwise comparisons of allele frequencies between unselected and selected groups were performed for each population by Fisher's exact test (FET) and for both populations combined by a Cochran-Mantel-Haenszel (CMH) test. RESULTS: Mapping rates varied from 80.29 to 81.77% at a 90.4X mean coverage of aligned reads. After correction for multiple testing, significant (P < 0.05) changes in allele frequencies were detected by FET for 6 and 57 single nucleotide polymorphisms (SNPs) in the SR and RS populations, respectively, and by the CMH test for 124 SNPs in both populations. The significant variants located on chromosome 2 generated a selection signal in a genomic region harboring the mptl-1, deg-3 and des-2 genes, previously reported as candidates for monepantel resistance. In addition, three new variants were identified in the mptl-1 gene. CONCLUSIONS: This study expands knowledge on genome-wide molecular events underlying H. contortus resistance to monepantel. The identification of a genome region harboring major genes previously associated with monepantel resistance supports the results of the employed X-QTL approach. In addition, a deletion in exon 11 of the mptl-1 gene should be further investigated as the putative causal mutation leading to monepantel resistance.

PCR-based genotyping of SNP markers in sheep.

Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl2 and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.