Expression Levels of LCORL Are Associated with Body Size in Horses Julia Metzger, Rahel Schrimpf, Ute Philipp, Ottmar Distl* Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover, Germany Abstract Body size is an important characteristic for horses of various breeds and essential for the classification of ponies concerning the limit value of 148 cm (58.27 inches) height at the withers. Genome-wide association analyses revealed the highest associated quantitative trait locus for height at the withers on horse chromosome (ECA) 3 upstream of the candidate gene LCORL. Using 214 Hanoverian horses genotyped on the Illumina equine SNP50 BeadChip and 42 different horse breeds across all size ranges, we confirmed the highly associated single nucleotide polymorphism BIEC2-808543 (2log10P = 8.3) and the adjacent gene LCORL as the most promising candidate for body size. We investigated the relative expression levels of LCORL and its two neighbouring genes NCAPG and DCAF16 using quantitative real-time PCR (RT-qPCR). We could demonstrate a significant association of the relative LCORL expression levels with the size of the horses and the BIEC2- 808543 genotypes within and across horse breeds. In heterozygous C/T-horses expression levels of LCORL were significantly decreased by 40% and in homozygous C/C-horses by 56% relative to the smaller T/T-horses. Bioinformatic analyses indicated that this SNP T.C mutation is disrupting a putative binding site of the transcription factor TFIID which is important for the transcription process of genes involved in skeletal bone development. Thus, our findings suggest that expression levels of LCORL play a key role for body size within and across horse breeds and regulation of the expression of LCORL is associated with genetic variants of BIEC2-808543. This is the first functional study for a body size regulating polymorphism in horses and a further step to unravel the mechanisms for understanding the genetic regulation of body size in horses. Citation: Metzger J, Schrimpf R, Philipp U, Distl O (2013) Expression Levels of LCORL Are Associated with Body Size in Horses. PLoS ONE 8(2): e56497. doi:10.1371/ journal.pone.0056497 Editor: Elissa Z. Cameron, University of Tasmania, Australia Received August 26, 2012; Accepted January 10, 2013; Published February 13, 2013 Copyright: ß 2013 Metzger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Mehl-Mu¨lhens Stiftung, Ko¨ln (DI-MM/1-1). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction advantage and performances are evaluated correspondingly [13,14]. Due to the selection for specific functions, the domestic Body size is an important model trait for studying genetic horse has been modified within breeds into diverse skeletal influences on quantitative traits and has been intensely investigat- morphologic types. The heritability of height at the withers was ed in human and also in domestic animals [1–17]. In human, adult estimated to be medium to high in pony breeds. Particularly, in height is described to be a complex trait influenced by many genes Haflinger and Shetland ponies high heritabilities at 0.79–0.89 and environmental factors [5]. Several genetic variants affecting were found [8–10] while Icelandic and Hanoverian warmblood the adult height have been identified using association analyses showed medium values at 0.5–0.6 [11,12]. The first attempt to [5,6]. identify patterns of skeletal size and shape variation among In horses, body size is an important criterion for the evaluation domestic horses has been made by principal component analyses of different breeds concerning appearance and function and is [15]. Overall body size was used as a principal component crucial for the classification of horses. According to the Fe´de´ration including thirty measurements all over horse’s body like head Equestre Internationale (FEI) veterinary regulations ponies taking length, height at withers, height at croup, chest width and neck part in any FEI competition have to be measured at the highest length. It grouped small ponies together with low scores and large point of the withers. The limit height is in accordance with the draft breeds with high median scores. Light horses showed mid- definition of a pony 148 cm (centimetres) (58.27 inches) without values [15]. Several genome-wide association studies (GWAS) shoes or with competition shoes 149 cm (58.66 inches). If this have been performed for height at withers in horses [1,2,16,17]. height is exceeded the animal is then classified as a horse [7]. In The involvement of LCORL (ligand-dependent nuclear receptor compres- some breeds, the limit values are even lower. The American sor-like protein) in height at withers has been primarily shown in a Miniature Horse Association requires a limit height at the last GWAS in Hanoverian stallions [16]. A highly significant QTL on hairs of the mane of 86.4 cm (34 inches). For breeders, body size horse chromosome (ECA) 3 in the region of LCORL was detected of horses is an essential parameter to improve marketability, for conformation traits like head, neck, frame and development function and performance. The important effect of size for [17]. In Franches-Montagnes horses, quantitative trait loci (QTL) competitive jumping ability in ponies was suggested [8]. Generally, on ECA3 and 9 were significantly associated with withers height. larger animals within each height class possess competitive Both associated SNPs were located in large intergenic regions [1]. PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e56497 LCORL Expression Associated with Equine Body Size Figure 1. Genome-wide association analysis shows a highly significant peak on equine chromosome (ECA) 3 in Hanoverian. (A) Manhattan-plot of the 2log10P-values from genome-wide association analysis (MLM) of body size in Hanoverian warmblood horses. The highest peak is located at 105 Mb on ECA3. (B) Q-Q plot of observed versus expected 2log10P-values from a genome- wide association study (GWAS) in Hanoverian warmblood horses. The expected distribution (solid line) and the observed 2log10P-values plotted against the expected 2log10P-values (black dots) are shown. The peak value (BIEC2-808543) is located on horse chromosome 3 at 105.55 Mb. doi:10.1371/journal.pone.0056497.g001 In thoroughbred horses, the same locus on ECA3 was found as strongest statistical effect was detected for the SNP ADI- highly associated with body size. A scan of 48 horses from 16 POQ:g.1596G.A, possibly influencing the anchoring of the different breeds revealed that four loci on ECA3, 6, 9 and 11 transcription pre-initiation transcription factor IID (TFIID) complex explain 83% of the variance for size. The highest associated SNP and therefore affecting the stability of the initiation complex [18]. was located near the candidate gene LCORL and this finding was Analyses of TFIID in mice resulted in dwarf phenotypes with an in line with the other analyses for withers height in horses about 50% reduced body weight. According to its multiple [2,16,17]. In human, the candidate gene LCORL has been molecular functions, TFIID is considered as a central component discussed to be involved in trunk and hip axis length [6]. GWAS of the transcription apparatus [19]. Furthermore, analyses showed in cattle for growth traits such as birth weight, body length, carcass a correlation between body weight, withers height and further size weight and longissimus muscle area, revealed an association in the measurements [20]. region of NCAPG (non-SMC condensing I complex subunit) and LCORL. In thoroughbred horses, a mass to height at the withers ratio Coding regions of the candidate genes were sequenced and was used to test the influence of the MSTN (myostatin) gene revealed further associated markers [4]. In Angus cattle, g.66493737C.T genotypes on body mass. Sprinters are proposed polymorphisms in the candidate genes adiponectin (ADIPOQ) and to be generally shorter animals with greater muscle mass [21,22]. somatostatin (SST) were tested for growth and carcass traits. The PLOS ONE | www.plosone.org 2 February 2013 | Volume 8 | Issue 2 | e56497 LCORL Expression Associated with Equine Body Size The objective of our study was to investigate the role of the heavier horses predominantly showed the genotype C/C (Table candidate gene LCORL and its flanking polymorphisms with the S1). Heterozygous horses in pony breeds showed relatively high development of body size in horses. First, we validated the withers height values and those stallions served breeders to pass on association of the genomic region around LCORL in Hanoverian larger height at the wither to the offspring. The results show that warmblood horses as well as substantiated the association of this this SNP proved to be a highly predictive marker of genetic locus with body size in a large number of horse breeds with potential for body size. extreme size and then we sequenced genomic and copy DNA of LCORL for polymorphism detection. In order to show a functional Candidate region analysis relationship of LCORL with body size and the associated SNP Bioinformatic sequence analysis of the genomic region of the upstream of LCORL, we employed expression analyses for LCORL highly associated SNP (BIEC2-808543) using Patch 1.0 and and its two adjacent genes for horses with differing height at SIGNAL SCAN showed that the polymorphism was located in a withers within and across breeds. putative DNA consensus sequence element, the transcription factor binding site of TFIID, which influences the transcription by Results RNA polymerase II [23,24].