Genetic × environment variation in sheep lines bred for divergent resistance to strongyle infection

Abstract Drug‐resistant parasites threaten livestock production. Breeding more resistant hosts could be a sustainable control strategy. Environmental variation linked to animal management practices or to parasite species turnover across farms may however alter the expression of genetic potential. We created sheep lines with high or low resistance to Haemonchus contortus and achieved significant divergence on both phenotypic and genetic scales. We exposed both lines to chronic stress or to the infection by another parasite Trichostrongylus colubriformis, to test for genotype‐by‐environment and genotype‐by‐parasite species interactions respectively. Between‐line divergence remained significant following chronic stress exposure although between‐family variation was found. Significant genotype‐by‐parasite interaction was found although H. contortus‐resistant lambs remained more resistant against T. colubriformis. Growth curves were not altered by the selection process although resistant lambs were lighter after the second round of divergence, before any infection took place. Breeding for resistance is a sustainable strategy but allowance needs to be made for environmental perturbations and worm species.

Genotyping was performed for 274 of these lambs with a set of 1,000 SNPs, including markers in 8 QTL regions previously detected in a backcross between Romane and Black Belly breeds (Sallé et al., 2012) and in two other European sheep populations (Riggio et al., 2014). Sires (n = 37) were also genotyped for the 1,000 SNPs to assign missing sire-offspring kinship in 216 lambs. The total pedigree considered in the following analyses included 3 generations and 2,572 individuals. The founder individuals mated to create the first generation of divergent individuals (G1) were selected among 274 animals with phenotype and genotype information. Their breeding value was estimated following a single-step Best Linear Unbiased Prediction (BLUP) approach that uses both the genomic and pedigree information (with equal weights).  A second generation of lambs was subsequently created (G2). The most extreme G1 lambs were chosen to retain 3 sires within each line (Table 4). To increase the genetic gain in females, 82 females were selected from both G0 and G1 ewes, ensuring 2 standard deviations between the average expected breeding values of the two lines. Controlled matings were performed between individuals with limited kinship ensuring an expected inbreeding coefficient in the G2 lambs lower than 0.04. Lambing took place in 55 out of the 82 selected ewes and generated 111 offsprings, 80 of which were selected according to the expected breeding values (average of their parents genetic merits ; Table 5).  Diagram of the selection procedure

Supplementary Figure 1. QQ-plot of the association between genotyped SNPs and faecal egg count at first and second infection
The figure represents the relationship between expected and observed P-values (represented as -log10(P)) following association between faecal egg count at first (FEC1) or second infection (FEC2) and 898 filtered SNPs located within 8 QTL regions in the G0 population. Associated genomic inflation factors were 1.19 and 1.14 for FEC1 and FEC2 respectively.

Supplementary technical note 2 : Changes in behavioural reactivity
and standing-lying behaviour following chronic stress exposure

Measures of behavioural reactivity and standing-lying behaviour
Measurements focused on behavioural reactivity and standing-lying behaviour that were recorded before the onset of behavioural treatments, five weeks later, and 14 days after the first and second infections. Additional recordings of standing-lying behaviour were performed on the same day and just before experimental infection took place.
Behavioural reactivity was measured by an arena test (2 m x 7 m dirt floor with 2 m high solid walls and 7 equal-sized areas delimited by lines on the floor) that evaluate social attractiveness of two lambs for their flock-mates (n = 3, kept behind a wire mesh) or for a standing human being. The first phase of the test (1 min) evaluated reaction to novelty and social isolation (measured by locomotion, i.e. number of squares entered, and vigilance, i.e. head in upright position and ears perpendicular to the head). Tested lambs were first isolated from their flock-mates (hidden behind a curtain) for 1 min before the curtain was lifted to allow social proximity between tested lambs and their mates. The second phase consisted in an operator entering the arena and standing in front of the wire mesh for 1 min to measure lamb social attraction and reaction to a stationary person (locomotion, vigilance, bleat, physical contact with their flock-mates and with the operator). Lamb behaviour was recorded with a camera (Sony SPT-MC128CE, Sony Corp., Tokyo, Japan) and video recorder (Sony SVT-1000P, Sony Corp., Tokyo, Japan). Vocalisations were recorded directly by an observer hidden from lambs.
Standing-lying behaviour was recorded on females with an accelerometer (HOBO ® Pendant ® ) attached with a cohesive bandage to the lateral side of the hind leg (left or right in half the lambs each). The accelerometer was positioned so that the x-axis was vertical and towards the ground, and the y-axis parallel to the ground and towards the rear of the animal.

Results
Average values recorded in resistant and susceptible lines either before or after chronic stress are presented in supplementary Figure 3 Table 2). This variable was hence not considered further. No significant difference was found in other behavioural data between both treatment groups at that time (supplementary Table 2).
Following exposure to chronic stress for five weeks and before any infection took place, lambs displayed altered behaviour (supplementary Figure 3, supplementary

Supplementary Figure 4. Environmental effect on resistance potential varies across sire families
This figure represents the within-sire family variation in Faecal Egg Count (FEC, given in phenotypic standard deviation p) across environmental conditions. FEC were normalized (fourth-root transformation) and then mean centered and scaled to unit standard deviation within each experimental block for the sake of comparison across conditions. First sire name letter indicates the divergent line it belongs to (R, resistant or S, susceptible). The picture indicates that the magnitude of G x E interaction varies among sires across stress conditions (top panel): sires S-132361 and S-132550 had their progenies showing higher or lower susceptibility under chronic stress respectively. The trend in resistance was conserved across GIN species in every sire family (bottom panel).