Jivanji et al. Genet Sel Evol (2019) 51:62 https://doi.org/10.1186/s12711-019-0506-2 Genetics Selection Evolution RESEARCH ARTICLE Open Access Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle Swati Jivanji1* , Gemma Worth2, Thomas J. Lopdell2, Anna Yeates2, Christine Couldrey2, Edwardo Reynolds1, Kathryn Tiplady2, Lorna McNaughton2, Thomas J. J. Johnson2, Stephen R. Davis2, Bevin Harris2, Richard Spelman2, Russell G. Snell3, Dorian Garrick1 and Mathew D. Littlejohn2 Abstract Background: White spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein–Friesian cattle, and several previous studies have detected genetic loci with major efects for white spotting in animals with Holstein–Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date. Results: Using imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly signifcant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these efects. These results are in broad agreement with previous studies, although we also report a third signifcant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcrip- tion factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed exami- nation of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region. Conclusions: Our fndings extend those of previous studies that reported KIT as a likely causal gene for white spot- ting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the efects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics. Background several genes with major efects have been described and Coat patterning traits provide visual characteristics are relevant across species, as well as many other loci that allow diferentiation between domesticated animal with small efects that account for the remaining genetic breeds and between strains within breeds. White spotting variance [1]. Tis oligogenic architecture derives from the is one of these phenotypes, and is a feature of a variety of multifaceted biology that contributes to white spotting of mammals including cattle, horses, dogs, cats and mice. the coat, which is hypothesised to arise from abnormal White spotting is a complex quantitative trait, for which melanocyte precursor migration and/or development. Mouse models have demonstrated that pigment cells *Correspondence: [email protected] originate from the neural crest cells via the SOX10 posi- 1 Massey University Manawatu, Private Bag 11 222, Palmerston tive glial bipotent progenitor cells during embryogenesis, North 4442, New Zealand and migrate dorsally via the neural tube [2]. Tese cells Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jivanji et al. Genet Sel Evol (2019) 51:62 Page 2 of 18 proceed to diferentiate into melanoblasts by acquiring trait [1]. Together these studies converge on the involve- expression of the genes micropthalmia-associated tran- ment of KIT and MITF gene expression in white spot- scription factor (MITF), proto-oncogene receptor tyrosine ting in dairy cattle, however the causal variants that drive kinase (KIT) and dopachrome tautomerase (DCT), and these efects have yet to be defnitively identifed and may migrate down the ventral axis of the body. When the cells be breed-specifc. reach their destination, they migrate into the epidermis Here, our aim was to investigate white spotting in New where some melanoblasts localise to the hair follicle and Zealand dairy cattle, by using whole-genome sequence diferentiate into melanocytes. A subset of melanoblasts genotype data to conduct the largest GWAS of white dediferentiate, losing MITF and KIT gene expression, spotting to date. We report three genome-wide signif- and colonise the hair follicle bulge where they act as mel- cant QTL for white spotting. Efects on chromosomes 6 anocyte stem cells and replenish diferentiated melano- and 22 extend on previous associations at these loci, and cytes during subsequent hair cycles [2]. Disruption of any further implicate the KIT and MITF genes as responsible of the above processes is expected to result in parts of the for these efects. For the frst time, we also report a QTL body lacking mature melanocytes, and thus regions of on chromosome 2 that implicates the PAX3 gene in white abnormal pigmentation in the hair coat. spotting of dairy cattle and highlight an amino acid sub- Quantitative trait loci (QTL) and mutations that cause stitution that may underlie this efect. white spotting have been described for a variety of spe- cies. Genetic studies in the horse revealed an inversion in Methods the KIT gene associated with the Tobiano white-spotting Study population [3], and a mutation in the PAX3 gene associated with a White spotting data were derived from several cohorts of splashed white pattern [4, 5]. Several mutations in the animals that included: 885 outbred dairy bulls (223 J, 327 KIT gene have also been associated with complete white HF, and 335 HF × J), 1389 outbred dairy cows (51 J, 265 [6] or roan coat phenotypes [7]. Studies on white spotting HF, and 1073 HF × J), and 699 HF × J F2 cross cows from in dogs have revealed associations with the MITF gene an experimental pedigree. Breed defnitions, in these [8], and in mice more than 10 genes have been reported cases, defne animals from a 4-generation pedigree that 16 15 to be associated with white spotting traits, including the were /16 J or HF as purebreds, with /16 animals defned KIT and MITF genes [9]. Comparatively few studies have as crossbreeds. Te F2 animals were ½ HF × ½ J, repre- investigated the genetics of white spotting in cattle. Liu senting a study population that was previously described et al. [10] found signifcant QTL on chromosomes 6, 18 in several publications [10, 13–15]. Genotyping data were and 22 using linkage analysis within Holstein–Friesian available for 2973 animals, with genotype and pheno- (HF) × Jersey (J) crossbred cows. It has been suggested type information derived as described in the following that the QTL on chromosomes 6 and 22 might be under- sections. pinned by the KIT and MITF genes, respectively [10]. Fontanesi et al. [11] compared the sequences of the MITF Measurements of white spotting in our study population gene in white spotted Italian Holstein and Simmental cat- For animals in the F2 population, proportion of white tle, and solid coloured Italian Brown and Reggiana cat- spotting values that had been derived for a previous study tle, and found a haplotype (carrying allele g.31831615T) [10] were used directly in the current study. Video foot- that is associated with white spotting. Tis haplotype age was recorded on 1389 cows walking single fle either accounts for some, but not all of the variation observed into or out of the milking shed using a GoPro HERO4 in the white spotting phenotype [11]. More recently, camera, at a 4000 pixel horizontal resolution. Still images Hofstetter et al. [12] investigated atypical white spotting that provide a clear side-on view of each animal were cap- in Brown Swiss cattle. Tey identifed two completely tured from the video footage using VideoPad Video Edi- linked single nucleotide variants within the 5′ regula- tor (v5.3). Additional side-on images representing either tory region of the MITF gene associated with white spot- the right or left profle of 885 bulls were made available ting, and although these variants largely account for the by LIC and incorporated into the dataset. First, cows and manifestation of white spotting, they do not account for bulls were scored for the presence or absence of white the variability between individuals, which provides fur- on their coat and, then, the proportion of white spotting ther evidence for a polygenic trait [12]. Hayes et al. [1] was quantifed. Quantifcation was carried out manually detected the MITF and KIT genes in a genome-wide using the image processing software, GNU Image Manip- association study (GWAS) that investigated the pro- ulation Program (GIMP, v2.9.8), to generate an objective portion of black in black and white Holstein cows, and measurement of the proportion of white color. Te free- reported an additional signal on chromosome 8, which hand tool was used to trace each animal and remove the carries PAX5 i.e. another potential candidate gene for this background. Te pixel count from the remaining image, Jivanji et al.