PS4-3. Development of a Targeted TYRP1 Exon Sequencing Approach to Investigate Moorit and Muddy Moorit Wool Color in Wensleydale Sheep.

Abstract The moorit (brown) wool color is highly valued in heritage fiber markets and was presumed lost in Wensleydale sheep since 1924. However, this phenotype re-emerged in 2013 within a Wensleydale flock located in Idaho. The moorit phenotype has been associated with a single-nucleotide polymorphism in the tyrosinase-related protein 1 (TYRP1) gene, which is a key regulator of eumelanin synthesis in mammals. Furthermore, as this flock was selectively bred to increase the frequency of the moorit phenotype, some heterozygous individuals exhibited a previously undescribed dark blue-grey wool color, termed “muddy moorit”. The unexpected phenotype suggests additional variation at the TYRP1 locus. Therefore, the objective of this study was to develop a method to sequence exon regions of the TYRP1 gene to validate the association with the moorit wool color and investigate new genetic associations with the muddy moorit wool color in Wensleydale sheep. DNA was extracted from blood samples obtained from a single flock under artificial selection for the moorit phenotype (n = 84). Primers were designed to amplify all eight exons of TYRP1 for polymerase chain reactions (PCR) and sequencing, with n = 672 total PCR reactions performed during primer testing and validation. Sequencing was then performed using an in-house MinION platform (Oxford Nanopore Technologies) and base-called with Dorado. Sequencing data were quality filtered and mapped to the reference genome (ARS-UI_Ramb_v3.0) using minimap2. Identification of exonic variants including SNPs, insertions, and deletions was performed with Clair3. Previous research has already shown that moorit coloration has been associated with variation in TYRP1 gene; however, with this study, a method was designed to also examine a relationship between genetic variation in TYRP1 and the muddy moorit phenotype. Developing methods to study genetic variation in genes such as TYRP1 advances the understanding of pigment genetics and allelic effects in livestock. The results have direct implications for identification of new genetic variants to inform breeding decisions, breed registry discussions, and conservation strategies aimed at maintaining genetic diversity within at-risk heritage sheep breeds.