A Genome Scan for Quantitative Trait Loci Affecting Average Daily Gain and Kleiber Ratio in Baluchi Sheep Running Title: GWAS for Growth rate Majid Pasandideh*, Ghodrat Rahimi-Mianji, Mohsen Gholizadeh Laboratory for Molecular Genetics and Animal Biotechnology, Faculty of Animal and Aquatic Science, Sari Agricultural Sciences and Natural Resources University, Sari, P.O. Box -578, Iran Postal address: Majid Pasandideh, No 1, Valiasr 8 Alley, Valiasr (Farhangian) Town, Bojnord City, North Khorasan Province, Iran. Postal Code: 94178-94815 Corresponding Author Email: [email protected] [email protected] [email protected] Keywords: Baluchi sheep, Genome-wide association study, Growth rate, Single nucleotide polymorphism Abstract 1 Genome-wide association study (GWAS) is an efficient tool for detection SNPs and candidate genes in quantitative traits. Growth rate is an important trait for increasing of meat production in sheep. A total of 96 Baluchi sheep were genotyped using Illumina OvineSNP50 BeadChip to run a GWAS for average daily gain (ADG) and Kleiber ratio (KR) traits in different periods of age in sheep. Traits included were average daily gain from birth to 3 months (ADG0-3), from 3 months to 6 months (ADG3- 6), from 6 months to 9 months (ADG6-9), from 9 months to yearling (ADG9-12), from birth to 6 months (ADG0-6), from 3 months to 9 months (ADG3-9), from 3 months to yearling (ADG3-12) and corresponding Kleiber ratios (KR0-3, KR3-6, KR6-9, KR9-12, KR0-6, KR3-9 and KR3-12, respectively). A total of 42,243 SNPs passed the quality-control filters and were analyzed by PLINK software in a linear mixed model. Two SNPs were identified on two chromosomes at the 5% genome-wide significance level for KR(3-9) and KR(6-9). Two candidate genes namely MAGI1 and ZNF770 were identified correspondingly harboring and close to these QTL. Also, a total of 21 SNPs were found on chromosomes 2, 3, 5, 6, 7, 10, 17, 19, 20 and 25 at the 5% chromosome-wide significance level for ADG and KR traits. Thus, we suggest more studies to discovery of causative variants for growth traits in sheep. Keywords: Baluchi sheep, Genome-wide association study, Growth rate, Single nucleotide polymorphism Introduction In Iran, the consumption of lamb and mutton as protein sources are more prevalent than meat from beef cattle and goats. Due to the fact that meat production from sheep is not enough, it is necessary to increase the efficiency of sheep production by improving litter size, lamb weight and growth rate. The Baluchi sheep is one of the most important native breeds in Iran, constituting about 30% of the total sheep population (Yazdi et al. 1997). This breed has medium size, fat-tailed with the 2 predominant white coat color, whereas the muzzle is black. These animals are mostly kept on pasture and adapted to the harsh environment and dry and hot climate in the eastern part of Iran with low quality pasture. Increasing in size and number of cells is applied as primarily definition of growth. However, this does not comprise the phenomenology and etiology of growth. Because, growth is a continuous function during life of animal, it is better to use growth rate or increasing in weight and size during different stages of life for measuring growth (Nkrumah et al. 2007). Therefore, the average daily gain (ADG) in weight has been one of the most valuable traits and plays an essential role in sheep breeding programs. A portion of feed intake is served for animal maintenance requirements and the rest can be used for production (milk, wool and meat). Due to maintenance need is higher for larger animals, they must obtain more feed intake to have the same body mass gain as the smaller animal (Kleiber 1936). Therefore, a method must be used for comparison of larger and smaller animals. Metabolic mass (W0.75) in kleiber ratio provides the possibility of comparison the large and the small animals without to have an advantage to either (Kleiber 1947). Selection for mass or growth rate can result in undesirable correlated responses such as increasing of fat deposit in the animal body (Badenhorst 1990). The fat deposit can disrupt the homeostatic balance leading to lower fertility (Badenhorst 2011). Selection for efficiency will not decrease fertility as a result of increasing fat deposits in the body. Therefore, efficiency of feed conversion is a more acceptable selection criterion than mass gain for evaluation growth rate (Badenhorst 2011). However, it is difficult to determine the efficiency of feed conversion for each animal individually in field condition. The Kleiber ratio is based on the fact that there is a direct relationship between the animal weight and the maintenance requirements and production. Therefore, Kleiber ratio is used as an alternative selection criterion for efficiency (Kleiber 1947). 3 Mapping of quantitative trait loci (QTL) is a set of procedures to detect the genetic variation using linkage mapping and traditional statistical and quantitative genetic approaches. There have been two approaches for QTL mapping since many years ago: candidate gene approach and linkage mapping analyses by microsatellite markers (Hirschhorn and Daly 2005). It is difficult to use candidate gene approach for quantitative traits, because it is limited to how much of the biology of the investigated trait is recognized (Andersson and Georges 2004; Al-Mamun et al. 2015). The limitations of QTL mapping by microsatellite markers are the low map resolution and the large confidence interval (CI). Therefore, it is difficult to identify the important genes and the underlying mutations for the interested traits using the marker information (Goddard and Hayes 2009). Recently, due to the accessibility of a high-density SNP chip, the genome wide association study (GWAS) has become a useful technique for the identification of causal genes for the economical traits in livestock. The GWAS with the applying of large scales of SNPs in whole genome investigates the correlation between genetic variations and phenotypic variations in trait. The goal of GWAS is using of linkage disequilibrium (LD) information between markers and causative mutations that tend to be inherited together to next generation (Bush and Moore 2012). GWAS has been widely applied to detect of causative mutation in human in order to improve treatment or produce useful diagnostic for diseases (Scott et al. 2007; Lasky-Su et al. 2008; Lee et al. 2008). In animal, especially cattle, this method has emphasized on economically important traits to expedite the genetic improvement (Cole et al. 2011; Zanella et al. 2011). A GWAS analysis in Australian Merino sheep has detected 39 SNPs associated with body weight and a major QTL region (included 13 SNPs) on OAR6 (Al-Mamun et al. 2015). Gholizadeh et al. (2015) identified one SNP with genome wide significance effect within SYNE1 gene on chromosome 8 to be associated with yearling weight in Baluchi sheep. A GWA study in purebred sheep reported 36 significant SNPs for growth and meat production traits and 5 most crucial candidate genes associated with post-weaning gain (Zhang et al. 2013). Our objectives in this study were to 4 perform a GWAS in Baluchi sheep to identify the significant SNPs associated with ADG and KR traits in different periods of age using the ovine SNP50 BeadChip (Illumina, San Diego, USA) and to explore the candidate genes around these SNPs. Materials and methods Ethical statement This study has been performed with the approval of Abbasabad Sheep Breeding Station of Mashhad, Iran and Iran national science foundation (INSF) (number 89,002,382). All institutional and national guidelines for the care and use of laboratory animals were followed. Animal resources and measurement of traits The sheep population used in this study consisted of 96 Baluchi sheep (93 females and 3 males) collected from Abbasabad Sheep Breeding Station, Mashhad, Iran. The animals were kept with conventional industry practices. The mating period was between late summer (August) and early autumn (September) and included at most three estrous cycles (51 days). Lambing started in early February and ended in late March. The lambs were kept with their mothers until the mean weaning age of all lambs at about 3 months. During spring and summer, the animals were kept on pasture and kept indoors in winter. This study focused on growth rate traits included: average daily gain from birth to 3 months (ADG0-3), from 3 months to 6 months (ADG3-6), from 6 months to 9 months (ADG6- 9), from 9 months to yearling (ADG9-12), from birth to 6 months (ADG0-6), from 3 months to 9 months (ADG3-9), from 3 months to yearling (ADG3-12) and corresponding Kleiber ratios (KR0-3, KR3-6, KR6- 9, KR9-12 ,KR0-6, KR3-9 and KR3-12, respectively). Average daily gain and Kleiber ratios for each period were calculated as follow: ADG = (end weight – first weight) / number of days in period KR = ADG for each period / (end weight) 0.75 Sampling, genotyping and data quality control 5 Venous jugular blood samples were collected using vacuum tubes treated with 0.25% Ethylene Diamine Tetracetic Acid (EDTA) as an anticoagulant. Genomic DNA was extracted from blood samples using a modified salting out protocol following Miller et al. (1988). DNA was genotyped using the Illumina OvineSNP50 BeadChip containing 54,241 SNPs at the Sci-Life Lab in Uppsala, Sweden. PLINK software version 1.07 (Purcell et al. 2007) was used for data quality control. SNPs that passed the quality control criteria (genotyping frequency >95%, minor allele frequency >0.05 and Hardy–Weinberg equilibrium P>0.001) were included for further analysis. Population stratification was assessed by a quantile-quantile (Q-Q) plot using the ggplot package in R software and genomic inflation factor (based on median chi-squared) in PLINK software.