Fig. 1
Outline of the initial crosses, backcrosses, in vivo passage and selection experiments. a. Ivermectin resistant populations MHco10(CAVR) or MHco4(WRS) (“resistant” haplotypes are depicted as red lines) were crossed with an ivermectin sensitive population MHco3(ISE) (“susceptible” haplotypes as blue lines) to generate heterozygous F1 progeny. F1 eggs were cultured in vitro to L3, before maturation in vivo to L4/immature adults, from which females were used to initiate the backcross. b. The first round of backcross was performed by crossing heterozygous females from the initial cross with susceptible MHco3(ISE) males in vivo, resulting in F2 progeny with reduced heterozygosity due to enrichment of MHco3(ISE) haplotypes. Resistance alleles were maintained in the backcross population by selection with ivermectin, before seeding the next round of the backcross with cross-derived heterozygous females and new susceptible MHco3(ISE) males. This process was repeated for four rounds of backcrossing, resulting in the backcrossed population becoming genetically similar to the MHco3(ISE) parental line in all regions of the genome not linked to ivermectin resistance. After four rounds of backcrossing, introgressed L3 progeny were used to infect a new recipient sheep, resulting in segregation of susceptible and resistant alleles in both haplotypes among the progeny (mixed red/blue haplotypes). Eggs were cultured to L3 before infecting two sheep, one exposed to ivermectin and one that did not receive drug. Post ivermectin treatment, L4/immature adults from both sheep were recovered on necropsy for sequencing. c. Post-backcross L3 were further passaged into a worm-naive sheep and treated with ivermectin, after which eggs were recovered and cultured to infective L3 for reinfection. This process was repeated for four generations of passage with selection (but without backcrossing). L4/immature adults were recovered after passage three and four for sequence analysis