Association of SMAD3 and B4GALNT2 sequence variation with fertility in sheep : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

This research explored the presence of variaition in two genes that may be associated with fertility in sheep. Polymerase Chain Reaction – Single Strand Conformational Polymorphism (PCR-SSCP) was used to identifiy variation in exon 8 and exon 6 of Small Mothers Against Decapentaplegic Family Member (SMAD3) and exon 4 and exon 8 of Beta-1,4-N-Acetyl-Galactosaminyltransferase 2 (B4GALNT2). Nucleotide sequence variation identified in these genes was identified using Sanger DNA sequencing technology. Once sequence variation was confirmed, statistical analyses using the animal model in R Studio was employed to ascertain whether the variation identified in exon 8 of SMAD3 was associated with fertility in Finnish-Landrace, Finnish-Landrace X Texel-cross, composite and Romney sheep breeds and between exon 4 of B4GALNT2 and Finnish-Landrace, Finnish-Landrace X Texel-cross and composite sheep. No variation in exon 6 of SMAD3 or exon 8 of B4GALNT2 was identified, and so no association studies were carried out for those two regions. For SMAD3, 439 ewes from the Finnish-Landrace, Finnish-Landrace X Texel-cross, composite and Romney sheep breeds were used for association analyses. For B4GALNT2, 143 ewes from Finnish-Landrace, Finnish-Landrace X Texel-cross and composite sheep were used for the association analyses. Lambing records for the Romney ewes were from the year 2015 and lambing records for the Finnish-Landrace, Finnish-Landrace X Texel-cross and composite sheep were from the year 2016. Four variant sequences (A, B, C and D) of exon 8 of SMAD3 were identified. The only variant associated with litter-size was Variant B (OL434430) in the Finnish-Landrace and Finnish-Landrace X Texel-cross and Romney sheep. This variant was associated with litter-size in a presence/ absence model in the Finnish-Landrace and Finnish-Landrace x Texel-cross (p < 0.001) and the Romney sheep (P = 0.011). Two variants (A and B) of exon 4 of B4GALNT2 were identified, and variant B was associated with litter-size in the sheep studied (P = 0.030) in a presence/absence model. The results in this study suggest that the narrative being built around B4GALNT2 and SMAD3 as possible new candidates for fertility in sheep may be warranted for all sheep breeds. This study adds to the growing narrative that the classic fecundity genes and the their pathways do not capture the entire story surrounding what we know about sheep fertlity. In addition to their relevence in sheep reprodution, studying SMAD3 and B4GALNT2 may offer a better understanding into other fields such as human fertility.