Genes associated with variation in longevity and fecundity in sheep

Abstract

Longevity is an important trait in animal production systems because long-lived animals reduce the requirement to carry non-productive replacement livestock. Many factors affect animal longevity including animal fecundity and performance for whichever trait, or traits, is valuable in that production system. Longevity is therefore an economically important trait and should be a consideration in any breeding programme. Little research has been undertaken on longevity in sheep. So-called “longevity genes” in other animal species have not been identified in sheep and/or effort has been primarily put into improving other sheep production traits. In this study, the aim was to identify sheep homologues for some putative longevity genes, including IGF1R, FOXO3, CAST and TLR4, to assess the extent of variation in these genes, and to ascertain whether variation in these genes is associated with longevity. IGF1R is structurally conserved across species and its product is an evolutionarily conserved regulator of the insulin-like growth factor (IGF) signalling pathway from lower animals to mammals. Variation in IGF1R has been described in many species and associated with variation in longevity. In this study, a fragment of intron 2 and exon 3 of ovine IGF1R that encompasses the α subunits of the ligand-binding region, was screened for genetic variation. Three novel sequences (named A, B and C) were identified in this region of the gene. The three sequences were subsequently used to investigate associations with longevity. FOXO3, a subgroup member of the Forkhead transcription factor family, plays an important role in mediating the effects of insulin and other growth factors on metabolism. Genetic variation in FOXO3 has been associated with longevity in a number of species. In this study, the coding regions (two exons) of ovine FOXO3 were firstly sequenced and identified. The entire exon 2 of ovine FOXO3 was 1422 bp long and encompasses the C-terminus of the DNA-binding domain and a transcription activation domain. These are key regulators of transcription activity in the target genes that FOXO3 binds. Seven haplotypes with 10 nucleotide substitutions were identified across the 1422-bp exon 2 fragment. One of these substitutions was predicted to produce an amino acid change (p.M201V). The effect of the seven haplotypes (A to G) on sheep longevity was investigated. CAST plays a role in protein-turnover and appears to be implicated in a range of phenotypes such as exercise-induced muscle injury, diabetes and degenerative neural diseases in humans. It has post-mortem effects on meat quality traits in livestock species and body weight traits in sheep. Recently, genetic variation in a non-coding region of CAST has been linked to longevity and fertility in dairy cattle, suggesting that this gene might play the same role in sheep. In this study, two highly polymorphic regions of ovine CAST were investigated revealing five sequences for exon 6 (A to E) and four sequences (A to D) for part of intron 12, respectively. Extended haplotype variation across the two regions was also investigated to better understand genetic diversity in ovine CAST. Nine haplotypes were defined across this extended region and four haplotypes were identified that suggested historical recombination events occurred within this gene. This intragenic recombination may make the discovery of phenotypic associations more challenging. Therefore, the five sequences (A to E) defined in the exon 6 of ovine CAST were further investigated in the context of sheep longevity. TLR4 is a receptor for lipopolysaccharide (LPS) found on Gram-negative bacteria as well as a number of other endogenous ligands that drive immune responses to pathogens. In mammals, an optimized immune system plays an important role in good health and longevity. Variation in TLR4 has been shown to be associated with various infectious and age-related diseases. In this study, seven sequences (A to G) were identified for exon 3 of ovine TLR4 which encodes an important ligand-binding site, and these sequences were investigated and assessed for their effect on longevity. Using the PCR-SSCP method, 1826 sheep, from six different breeds and 36 different stud breeding flocks, were genotyped for the variation described above in IGF1R, FOXO3, CAST and TLR4. The age of these sheep varied from 2 to 16 years old and fecundity data (the number of lambs raised per each year of their productive life) was collected for many of the ewes to investigate the relationship between longevity and fecundity. A general linear mixed model (GLMM) in SPSS version 17 (SPSS Science Inc., Chicago, IL, USA) was used to assess the effects of the variation in IGF1R, FOXO3, CAST and TLR4. Genetic association study has revealed that genetic variation in both IGF1R and FOXO3 had a significant association with sheep longevity, but genetic variation in CAST and TLR4 had no significant effect on sheep longevity across breeds and flocks. Ovine IGF1R C was a significantly associated with an increase in age of about half a year (breed correction, P = 0.009 and flock correction, P = 0.024), while the D haplotype of FOXO3, which carries the amino acid substitution p.M201V, was associated with a decrease in age of about five months (breed correction, P = 0.006 and flock correction, P = 0.034). The presence of TLR4 A and TLR4 C was associated or was trending to an association with fecundity when the model was corrected for ‘breed’ (1.64 ± 0.03, P = 0.004) and ‘flock’ (1.75 ± 0.02, P = 0.066), respectively. The Spearman’s correlation coefficient between longevity and fecundity in the sheep studied was -0.248 (P<0.001), suggesting a weak negative interaction between these traits.