- Email: [email protected]
Genome-wide association study of first lambing age and lambing interval in sheep
Small Ruminant Research 178 (2019) 43–45
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Genome-wide association study of first lambing age and lambing interval in sheep
T
⁎
R. Abdolia, S.Z. Mirhoseinia, , N. Ghavi Hossein-Zadeha, P. Zamanib, M.H. Moradic, M.H. Ferdosid, C. Gondroe a
Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, 41996-13776, Iran Department of Animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, 65178-33131, Iran c Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arāk, 38156-8-8349, Iran d Animal Genetics and Breeding Unit (AGBU), University of New England, Armidale, NSW, 2351, Australia e Department of Animal Science, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, 48824, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Reproductive traits Genomic scan Single nucleotide polymorphism GWAS
First lambing age and lambing interval are among the most important reproductive traits affecting profitability of sheep breeding systems. Genome-wide association studies (GWAS) were performed on estimated breeding values (EBVs) for age at first lambing and the lambing interval between first and second lambing in LoriBakhtiari ewes. Animals were genotyped using the Illumina Ovine SNP50 K genotyping array. Two single nucleotide polymorphisms (SNPs) associated with lambing interval were identified on chromosomes 1 and 2 (Bonferroni adjusted P-value < 0.05). No SNP associated with first lambing age was detected. SNP s50067.1 on sheep chromosome 2: 86798921 is within a candidate gene; ADAMTS like 1 (ADAMTSL1), known to be involved in proteolysis, peptidase activity, biological process and reproductive pathways in mammalian species. No candidate gene was detected for SNP OAR1_57568179.1 on chromosome 1. The results of the present study may provide new insights into the genomic regions involved in reproductive traits of sheep.
1. Introduction Lori-Bakhtiari sheep are an indigenous fat-tailed meat breed mainly raised in the southwestern part of Iran. About 1,700,000 head of this breed are concentrated in the Charmahal and Bakhtiari province and in some neighboring provinces. Lori-Bakhtiari is a major source of red meat for the region and has good maternal characteristics (Vatankhah et al., 2008). Reproductive ability plays an important role on the profitability of sheep breeding systems. Reproduction is an intricate composite process, which in addition to environmental conditions, is influenced by genetic factors (Snowder and Fogarty, 2009). First lambing age and lambing interval are two important economic reproductive traits which are directly related to the reproductive efficiency and profitability of a sheep breeding enterprise (Mohammadi et al., 2011). Alongside the length of the natural breeding season and longevity, they also have an impact on ewe lifetime production (Schoeman et al., 1991). A number of studies have investigated the association of candidate genes, e.g. haemoglobin, teransferin, melatonin receptor 1A (MTNR1A) and aromatase (CYP 19) with first lambing age and lambing interval in
⁎
sheep (Kumar et al., 2011; Yadav et al., 2013; Davari Varanlou et al., 2017). However, no genome-wide scans have been reported for these reproductive traits. Herein, we report on the results for a genome-wide association study (GWAS) for age at first lambing and lambing interval in Lori-Bakhtiari ewes. 2. Methods The phenotypic data used in the present study were collected from 1989 to 2017 (28 years) in the Sholi Sheep Breeding Station, located in Sharekord city, Charmahal and Bakhtiari province, Iran. The flock was managed under a semi-migratory or village system. The animals were kept on the range feeding on cereal pastures from mid-spring to lateautumn and kept indoors from December to May at the experimental station and fed a ration composed of alfalfa, barley and wheat stubble. The breeding period extends from late August to late October (ewes were randomly assigned to the rams). Lambing started in late January. All ewes were bred to rams for the first time at an average age of 18 months. Lambs were ear-tagged and weighed immediately after lambing, and their birth dates were recorded. From 15 days of age
Corresponding author. E-mail address: [email protected] (S.Z. Mirhoseini).
https://doi.org/10.1016/j.smallrumres.2019.07.014 Received 19 October 2018; Received in revised form 30 May 2019; Accepted 25 July 2019 Available online 26 July 2019 0921-4488/ © 2019 Published by Elsevier B.V.
Small Ruminant Research 178 (2019) 43–45
R. Abdoli, et al.
effects had a multivariate normal distribution with mean 0 and variance In σ 2e , where σ 2e is the residual variance and In is an identity matrix with an order equal to the number of single records. Marginal posterior distributions for genetic parameters and (co)variance components were estimated using the TM program (Legarra et al., 2011). An inverted Wishart distribution was assumed for the priors of genetic and residual (co)variance matrices. The Gibbs sampler was run 200,000 rounds, and the first 40,000 rounds were discarded as a burn-in period (Legarra et al., 2011). A thinning interval of 100 rounds was used to retain sampled values that decreased lag correlation among thinned samples. A number of 124 ewes were selected from different half-sib families for genotyping. The samples were genotyped on the Ovine SNP50 genotyping array (Illumina Inc., San Diego, CA, USA), which includes 51,135 SNPs. Quality control was performed using Plink ver. 1.9 beta (www.coggenomics.org/plink/1.9/). Samples with call rates < 99% were excluded before performing manual re-clustering. SNPs with no assigned genomic location, GenCall (GC) scores < 0.6, call rates < 95%, large Hardy-Weinberg equilibrium deviations (P-value < 1E−6) and a minor allele frequency lower than 0.01 were also removed from the final analysis. Plink was used for the genome-wide association study and the estimated breeding values (EBVs) for age at first lambing and lambing intervals, calculated as described above, were used as phenotypes. SNPs were considered significantly associated to the trait for Bonferroni adjusted p-values < 0.05. The QQ-plot and Manhattan Plot were drawn up using the “qqman” package in R version 3.5.1 (R core team, 2018). To avoid biases due to the existence of any population stratification, first two principal components were included in the model as covariates to consider population structure. Search on BioMart-Ensembl (www.ensembl.org/biomart) and NCBI (https://www.ncbi.nlm.nih.gov/) databases were performed to identify putative candidate genes mapped to the sheep genome within a window of 100 kb upstream and downstream of the significant SNPs.
Table 1 Characteristics of data set used in this study. Trait
Number
Mean (day)
SD (day)
CV (%)
Age at first lambing Interval from first to second lambing
2425 1887
767.65 396.86
161.23 121.73
21.00 30.67
SD: standard deviation; CV: coefficient of variation.
onwards, lambs had access to creep feed ad libitum and were weaned at an average age of 90 ± 5 days. After weaning, male and female lambs were separated. Surplus male lambs chosen for fattening were separated from the rest of the animals. Female lambs were kept in the pasture of cultivated alfalfa, while the rest of the males were kept indoors and fed a maintenance and growth ration up to 12 months of age. (Vatankhah et al., 2008). Phenotypic data consisted of 2425 first lambing age and 1887 records of lambing interval between first and second lambing (Table 1). Pedigree information was available for 10,645 animals, of which there were 378 sires and 2805 dams. The founder population consisted of 473 sheep (and 10,172 non-founders), 5066 animals had some level of inbreeding. The average inbreeding coefficient was 1.23% in the Lori-Bakhtiari sheep. These data were used to calculate the estimated breeding values (EBVs) of the sheep. Fixed effects to be included in the model were chosen after testing whether the effects were statistically significant with a linear fixed effects model analysed with the GLM procedure of SAS version 9.4 (SAS Institute, Cary, NC). The significance threshold for inclusion of effects into the model was P < 0.05. The fixed class effects included were lambing year and month. Genetic analysis of the traits was conducted using a univariate animal model as follows:
y = Xb + Za a + e Where, y is a N × 1 vector of records, b indicates the fixed effect variables in the model with incidence matrix X, a is the vector of random direct genetic effects with the association matrix Za and e indicates the vector of residual (temporary environmental) effects. From a Bayesian perspective, it was assumed that the prior distribution of the direct additive effects followed a multivariate normal with mean 0 and variance A σ 2a , where A is the additive numerator relationship matrix and σ 2a is direct additive variance. Also, it was assumed that residual
3. Results and discussion Lambing year and month were associated with the studied traits (P < 0.0001) and were thus included as fixed effects in the model used for estimation of variance components and breeding values. Average
Fig. 1. (a) Manhattan plot of genome-wide association study (-log10 P-values) for lambing interval. (b) Q-Q plot for the association analysis of lambing interval (λ=1.20). (c) Manhattan plot of genome-wide association study (-log10 P-values) for age at first lambing. (d) Q-Q plot for the association analysis of age at first lambing (λ=1.03). 44
Small Ruminant Research 178 (2019) 43–45
R. Abdoli, et al.
Table 2 SNPs significantly associated with lambing interval in Lori-Bakhtiari sheep. Unadjusted
SNP*
Chr
Position (bp)
P-value
s50067.1 OAR1_57568179.1
2 1
86798921 54660528
2.182E-09 1.259E-06
Bonferroni
Gene
9.776E-05 5.641E-02
ADAMTSL1 ——————————
Chr: chromosome number.
heritabilities (h2) for age at first lambing and lambing interval were expectedly low (0.07 and 0.02, respectively). After quality control, two samples were removed from the initial dataset due to high numbers of missing genotypes. Excluded SNPs consisted of 1432 with missing genotype data, 3081 that had average GC scores below 0.6, 1820 variants with minor allele frequencies < 0.01 and 1 SNP out of Hardy-Weinberg equilibrium (P < 1E−6). After filtering, 122 sheep and 44,801 SNPs were used for the GWAS; total genotyping rate in the remaining samples was 0.99. The traits have low heritabilities and the number of genotypes available for this study was small. Both factors contributed to the low power of the association study, but the use of EBVs can increase power to some extent as we have a better estimate of the actual genetic variance and don’t need as many animals to get reasonable estimates. After Bonferroni correction (adjusted p < 0.05), two SNPs on chromosomes 2 (s50067.1) and 1 (OAR1_57568179.1) were identified as being associated with lambing interval (Manhattan plot, Fig. 1a and Q-Q plot, Fig. 1b). There were no SNPs associated with first lambing age (Manhattan plot, Fig. 1c and Q-Q plot, Fig. 1d). No candidate genes were found neighboring the SNP on chromosome 1 (OAR1_57568179.1). However, the SNP on chromosome 2 (s50067.1) associated with lambing interval was located within the candidate gene; ADAMTS like 1 (ADAMTSL1), known to be involved in proteolysis, peptidase activity and biological process in sheep (Table 2). The ADAMTSL1 gene is located on sheep chromosome 2: 86,407,235-86,900,674 with 31 exons (http://www.ensembl.org). ADAMTSL1 is a protein coding gene with related pathways involved in metabolism of proteins and O-linked glycosylation. Gene ontology clarifications related to ADAMTSL1 include peptidase and metallopeptidase activities (https://www.genecards.org). The ADAMTS superfamily consists of 19 genes and seven ADAMTS-like proteins in human, mice and other mammals (Apte, 2009). In a previous study to elucidate the biological role of the ADAMST-1 in mouse, it was suggested that ADAMST-1 gene is necessary for normal growth, fertility, and organ morphology and function (Shindo et al., 2000). By inducing luteinizing hormone surges through a progesterone receptor-dependent mechanism, ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles in mice, and observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded cumulus oocyte complex (COC) during the process of matrix expansion. The anovulatory phenotype of female progesterone receptor knockout (PRKO) mice is at least partially due to loss of this function (Russell et al., 2003). To date, there have been no reports of an association between the ADAMTSL1 gene and reproductive traits in sheep. It is, however, a biologically sensible candidate gene that is known to be involved in
reproductive pathways in other mammalian species. Overall, our GWAS identified two SNPs on chromosomes 1 and 2 associated with lambing interval in Iranian fat-tailed Lori-Bakhtari sheep. The SNP on chromosome 2 (s50067.1) was located within a putative candidate gene (ADAMTSL1) with important known functions in reproductive pathways of mammals. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgment The authors are grateful to Guilan and Bu-Ali Sina Universities for their financial support of the present study. References Apte, S.S., 2009. A disintegrin-like and metalloprotease (reprolysin- type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J. Biol. Chem. 284, 31493–31497. https://doi.org/10.1074/jbc.R109.052340. Davari Varanlou, Z., Hassani, S., Ahani Azari, M., Samadi, F., Zakizadeh, S., Khan Ahmadi, A.R., 2017. Association between MTNR1A and CYP19 genes polymorphisms and economic traits in Kurdi sheep. Iran. J. Appl. Anim. Sci. 7, 69–74. Kumar, B., Taraphder, S., Sahoo, A.K., Dhara, K.C., Samanta, I., Misra, S.S., 2011. Haemoglobin polymorphism and its effect on different economic traits of Garole sheep. Indian J. Anim. Sci. 81, 417–419. Legarra, A., Varona, L., Lopez de Maturana, E., 2011. TM User’s Guide. Mohammadi, A.R., Abbasi, M.A., Moghaddam, A.A., Zare Shahneh, A., 2011. Determination of some reproductive traits in Iranian Afshari sheep breed. Aust. J. Basic Appl. Sci. 5, 2742–2751. R Core Team, 2018. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Russell, D.L., Doyle, K.M., Ochsner, A.A., Sandy, J.D., Richards, J.S., 2003. Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. J. Biol. Chem. 278, 42330–42339. https://doi.org/10. 1074/jbc.M300519200. Schoeman, S.J., Albertyn, J.R., Groeneveld, H.T., 1991. Lifetime reproduction of Karukul ewes as influenced by season of birth, age at first lambing and lambing interval. S. Afr. J. Anim. Sci. 21, 169–172. Shindo, T., Kurihara, H., Kuno, K., Yokoyama, H., Wada, T., Kurihara, Y., Imai, T., Wang, Y., Ogata, M., Nishimatsu, H., Moriyama, N., Oh-hashi, Y., Morita, H., Ishikawa, T., Nagai, R., Yazaki, Y., Matsushima, K., 2000. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J. Clin. Invest. 105, 1345–1352. https://doi.org/10.1172/JCI8635. Snowder, G.D., Fogarty, N.M., 2009. Composite trait selection to improve reproduction and ewe productivity: a review. Anim. Prod. Sci. 48, 9–16. https://doi.org/10.1071/ EA08184. Vatankhah, M., Talebi, M.A., Edriss, M.A., 2008. Estimation of genetic parameters for reproductive traits in Lori-Bakhtiari sheep. Small. Rum. Res. 74, 216–220. https:// doi.org/10.1016/j.smallrumres.2007.02.008. Yadav, D.K., Taraphder, S., Dhara, K.C., Batabyal, S., Samanta, I., Mitra, M., 2013. Association of transferrin polymorphism with different economic traits of Garole sheep. Int. J. Livest. Prod. 3, 6–11. https://doi.org/10.5897/IJLP11.013.
45
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Genome-wide association study of first lambing age and lambing interval in sheep
T
⁎
R. Abdolia, S.Z. Mirhoseinia, , N. Ghavi Hossein-Zadeha, P. Zamanib, M.H. Moradic, M.H. Ferdosid, C. Gondroe a
Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, 41996-13776, Iran Department of Animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, 65178-33131, Iran c Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arāk, 38156-8-8349, Iran d Animal Genetics and Breeding Unit (AGBU), University of New England, Armidale, NSW, 2351, Australia e Department of Animal Science, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, 48824, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Reproductive traits Genomic scan Single nucleotide polymorphism GWAS
First lambing age and lambing interval are among the most important reproductive traits affecting profitability of sheep breeding systems. Genome-wide association studies (GWAS) were performed on estimated breeding values (EBVs) for age at first lambing and the lambing interval between first and second lambing in LoriBakhtiari ewes. Animals were genotyped using the Illumina Ovine SNP50 K genotyping array. Two single nucleotide polymorphisms (SNPs) associated with lambing interval were identified on chromosomes 1 and 2 (Bonferroni adjusted P-value < 0.05). No SNP associated with first lambing age was detected. SNP s50067.1 on sheep chromosome 2: 86798921 is within a candidate gene; ADAMTS like 1 (ADAMTSL1), known to be involved in proteolysis, peptidase activity, biological process and reproductive pathways in mammalian species. No candidate gene was detected for SNP OAR1_57568179.1 on chromosome 1. The results of the present study may provide new insights into the genomic regions involved in reproductive traits of sheep.
1. Introduction Lori-Bakhtiari sheep are an indigenous fat-tailed meat breed mainly raised in the southwestern part of Iran. About 1,700,000 head of this breed are concentrated in the Charmahal and Bakhtiari province and in some neighboring provinces. Lori-Bakhtiari is a major source of red meat for the region and has good maternal characteristics (Vatankhah et al., 2008). Reproductive ability plays an important role on the profitability of sheep breeding systems. Reproduction is an intricate composite process, which in addition to environmental conditions, is influenced by genetic factors (Snowder and Fogarty, 2009). First lambing age and lambing interval are two important economic reproductive traits which are directly related to the reproductive efficiency and profitability of a sheep breeding enterprise (Mohammadi et al., 2011). Alongside the length of the natural breeding season and longevity, they also have an impact on ewe lifetime production (Schoeman et al., 1991). A number of studies have investigated the association of candidate genes, e.g. haemoglobin, teransferin, melatonin receptor 1A (MTNR1A) and aromatase (CYP 19) with first lambing age and lambing interval in
⁎
sheep (Kumar et al., 2011; Yadav et al., 2013; Davari Varanlou et al., 2017). However, no genome-wide scans have been reported for these reproductive traits. Herein, we report on the results for a genome-wide association study (GWAS) for age at first lambing and lambing interval in Lori-Bakhtiari ewes. 2. Methods The phenotypic data used in the present study were collected from 1989 to 2017 (28 years) in the Sholi Sheep Breeding Station, located in Sharekord city, Charmahal and Bakhtiari province, Iran. The flock was managed under a semi-migratory or village system. The animals were kept on the range feeding on cereal pastures from mid-spring to lateautumn and kept indoors from December to May at the experimental station and fed a ration composed of alfalfa, barley and wheat stubble. The breeding period extends from late August to late October (ewes were randomly assigned to the rams). Lambing started in late January. All ewes were bred to rams for the first time at an average age of 18 months. Lambs were ear-tagged and weighed immediately after lambing, and their birth dates were recorded. From 15 days of age
Corresponding author. E-mail address: [email protected] (S.Z. Mirhoseini).
https://doi.org/10.1016/j.smallrumres.2019.07.014 Received 19 October 2018; Received in revised form 30 May 2019; Accepted 25 July 2019 Available online 26 July 2019 0921-4488/ © 2019 Published by Elsevier B.V.
Small Ruminant Research 178 (2019) 43–45
R. Abdoli, et al.
effects had a multivariate normal distribution with mean 0 and variance In σ 2e , where σ 2e is the residual variance and In is an identity matrix with an order equal to the number of single records. Marginal posterior distributions for genetic parameters and (co)variance components were estimated using the TM program (Legarra et al., 2011). An inverted Wishart distribution was assumed for the priors of genetic and residual (co)variance matrices. The Gibbs sampler was run 200,000 rounds, and the first 40,000 rounds were discarded as a burn-in period (Legarra et al., 2011). A thinning interval of 100 rounds was used to retain sampled values that decreased lag correlation among thinned samples. A number of 124 ewes were selected from different half-sib families for genotyping. The samples were genotyped on the Ovine SNP50 genotyping array (Illumina Inc., San Diego, CA, USA), which includes 51,135 SNPs. Quality control was performed using Plink ver. 1.9 beta (www.coggenomics.org/plink/1.9/). Samples with call rates < 99% were excluded before performing manual re-clustering. SNPs with no assigned genomic location, GenCall (GC) scores < 0.6, call rates < 95%, large Hardy-Weinberg equilibrium deviations (P-value < 1E−6) and a minor allele frequency lower than 0.01 were also removed from the final analysis. Plink was used for the genome-wide association study and the estimated breeding values (EBVs) for age at first lambing and lambing intervals, calculated as described above, were used as phenotypes. SNPs were considered significantly associated to the trait for Bonferroni adjusted p-values < 0.05. The QQ-plot and Manhattan Plot were drawn up using the “qqman” package in R version 3.5.1 (R core team, 2018). To avoid biases due to the existence of any population stratification, first two principal components were included in the model as covariates to consider population structure. Search on BioMart-Ensembl (www.ensembl.org/biomart) and NCBI (https://www.ncbi.nlm.nih.gov/) databases were performed to identify putative candidate genes mapped to the sheep genome within a window of 100 kb upstream and downstream of the significant SNPs.
Table 1 Characteristics of data set used in this study. Trait
Number
Mean (day)
SD (day)
CV (%)
Age at first lambing Interval from first to second lambing
2425 1887
767.65 396.86
161.23 121.73
21.00 30.67
SD: standard deviation; CV: coefficient of variation.
onwards, lambs had access to creep feed ad libitum and were weaned at an average age of 90 ± 5 days. After weaning, male and female lambs were separated. Surplus male lambs chosen for fattening were separated from the rest of the animals. Female lambs were kept in the pasture of cultivated alfalfa, while the rest of the males were kept indoors and fed a maintenance and growth ration up to 12 months of age. (Vatankhah et al., 2008). Phenotypic data consisted of 2425 first lambing age and 1887 records of lambing interval between first and second lambing (Table 1). Pedigree information was available for 10,645 animals, of which there were 378 sires and 2805 dams. The founder population consisted of 473 sheep (and 10,172 non-founders), 5066 animals had some level of inbreeding. The average inbreeding coefficient was 1.23% in the Lori-Bakhtiari sheep. These data were used to calculate the estimated breeding values (EBVs) of the sheep. Fixed effects to be included in the model were chosen after testing whether the effects were statistically significant with a linear fixed effects model analysed with the GLM procedure of SAS version 9.4 (SAS Institute, Cary, NC). The significance threshold for inclusion of effects into the model was P < 0.05. The fixed class effects included were lambing year and month. Genetic analysis of the traits was conducted using a univariate animal model as follows:
y = Xb + Za a + e Where, y is a N × 1 vector of records, b indicates the fixed effect variables in the model with incidence matrix X, a is the vector of random direct genetic effects with the association matrix Za and e indicates the vector of residual (temporary environmental) effects. From a Bayesian perspective, it was assumed that the prior distribution of the direct additive effects followed a multivariate normal with mean 0 and variance A σ 2a , where A is the additive numerator relationship matrix and σ 2a is direct additive variance. Also, it was assumed that residual
3. Results and discussion Lambing year and month were associated with the studied traits (P < 0.0001) and were thus included as fixed effects in the model used for estimation of variance components and breeding values. Average
Fig. 1. (a) Manhattan plot of genome-wide association study (-log10 P-values) for lambing interval. (b) Q-Q plot for the association analysis of lambing interval (λ=1.20). (c) Manhattan plot of genome-wide association study (-log10 P-values) for age at first lambing. (d) Q-Q plot for the association analysis of age at first lambing (λ=1.03). 44
Small Ruminant Research 178 (2019) 43–45
R. Abdoli, et al.
Table 2 SNPs significantly associated with lambing interval in Lori-Bakhtiari sheep. Unadjusted
SNP*
Chr
Position (bp)
P-value
s50067.1 OAR1_57568179.1
2 1
86798921 54660528
2.182E-09 1.259E-06
Bonferroni
Gene
9.776E-05 5.641E-02
ADAMTSL1 ——————————
Chr: chromosome number.
heritabilities (h2) for age at first lambing and lambing interval were expectedly low (0.07 and 0.02, respectively). After quality control, two samples were removed from the initial dataset due to high numbers of missing genotypes. Excluded SNPs consisted of 1432 with missing genotype data, 3081 that had average GC scores below 0.6, 1820 variants with minor allele frequencies < 0.01 and 1 SNP out of Hardy-Weinberg equilibrium (P < 1E−6). After filtering, 122 sheep and 44,801 SNPs were used for the GWAS; total genotyping rate in the remaining samples was 0.99. The traits have low heritabilities and the number of genotypes available for this study was small. Both factors contributed to the low power of the association study, but the use of EBVs can increase power to some extent as we have a better estimate of the actual genetic variance and don’t need as many animals to get reasonable estimates. After Bonferroni correction (adjusted p < 0.05), two SNPs on chromosomes 2 (s50067.1) and 1 (OAR1_57568179.1) were identified as being associated with lambing interval (Manhattan plot, Fig. 1a and Q-Q plot, Fig. 1b). There were no SNPs associated with first lambing age (Manhattan plot, Fig. 1c and Q-Q plot, Fig. 1d). No candidate genes were found neighboring the SNP on chromosome 1 (OAR1_57568179.1). However, the SNP on chromosome 2 (s50067.1) associated with lambing interval was located within the candidate gene; ADAMTS like 1 (ADAMTSL1), known to be involved in proteolysis, peptidase activity and biological process in sheep (Table 2). The ADAMTSL1 gene is located on sheep chromosome 2: 86,407,235-86,900,674 with 31 exons (http://www.ensembl.org). ADAMTSL1 is a protein coding gene with related pathways involved in metabolism of proteins and O-linked glycosylation. Gene ontology clarifications related to ADAMTSL1 include peptidase and metallopeptidase activities (https://www.genecards.org). The ADAMTS superfamily consists of 19 genes and seven ADAMTS-like proteins in human, mice and other mammals (Apte, 2009). In a previous study to elucidate the biological role of the ADAMST-1 in mouse, it was suggested that ADAMST-1 gene is necessary for normal growth, fertility, and organ morphology and function (Shindo et al., 2000). By inducing luteinizing hormone surges through a progesterone receptor-dependent mechanism, ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles in mice, and observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded cumulus oocyte complex (COC) during the process of matrix expansion. The anovulatory phenotype of female progesterone receptor knockout (PRKO) mice is at least partially due to loss of this function (Russell et al., 2003). To date, there have been no reports of an association between the ADAMTSL1 gene and reproductive traits in sheep. It is, however, a biologically sensible candidate gene that is known to be involved in
reproductive pathways in other mammalian species. Overall, our GWAS identified two SNPs on chromosomes 1 and 2 associated with lambing interval in Iranian fat-tailed Lori-Bakhtari sheep. The SNP on chromosome 2 (s50067.1) was located within a putative candidate gene (ADAMTSL1) with important known functions in reproductive pathways of mammals. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgment The authors are grateful to Guilan and Bu-Ali Sina Universities for their financial support of the present study. References Apte, S.S., 2009. A disintegrin-like and metalloprotease (reprolysin- type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J. Biol. Chem. 284, 31493–31497. https://doi.org/10.1074/jbc.R109.052340. Davari Varanlou, Z., Hassani, S., Ahani Azari, M., Samadi, F., Zakizadeh, S., Khan Ahmadi, A.R., 2017. Association between MTNR1A and CYP19 genes polymorphisms and economic traits in Kurdi sheep. Iran. J. Appl. Anim. Sci. 7, 69–74. Kumar, B., Taraphder, S., Sahoo, A.K., Dhara, K.C., Samanta, I., Misra, S.S., 2011. Haemoglobin polymorphism and its effect on different economic traits of Garole sheep. Indian J. Anim. Sci. 81, 417–419. Legarra, A., Varona, L., Lopez de Maturana, E., 2011. TM User’s Guide. Mohammadi, A.R., Abbasi, M.A., Moghaddam, A.A., Zare Shahneh, A., 2011. Determination of some reproductive traits in Iranian Afshari sheep breed. Aust. J. Basic Appl. Sci. 5, 2742–2751. R Core Team, 2018. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Russell, D.L., Doyle, K.M., Ochsner, A.A., Sandy, J.D., Richards, J.S., 2003. Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. J. Biol. Chem. 278, 42330–42339. https://doi.org/10. 1074/jbc.M300519200. Schoeman, S.J., Albertyn, J.R., Groeneveld, H.T., 1991. Lifetime reproduction of Karukul ewes as influenced by season of birth, age at first lambing and lambing interval. S. Afr. J. Anim. Sci. 21, 169–172. Shindo, T., Kurihara, H., Kuno, K., Yokoyama, H., Wada, T., Kurihara, Y., Imai, T., Wang, Y., Ogata, M., Nishimatsu, H., Moriyama, N., Oh-hashi, Y., Morita, H., Ishikawa, T., Nagai, R., Yazaki, Y., Matsushima, K., 2000. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J. Clin. Invest. 105, 1345–1352. https://doi.org/10.1172/JCI8635. Snowder, G.D., Fogarty, N.M., 2009. Composite trait selection to improve reproduction and ewe productivity: a review. Anim. Prod. Sci. 48, 9–16. https://doi.org/10.1071/ EA08184. Vatankhah, M., Talebi, M.A., Edriss, M.A., 2008. Estimation of genetic parameters for reproductive traits in Lori-Bakhtiari sheep. Small. Rum. Res. 74, 216–220. https:// doi.org/10.1016/j.smallrumres.2007.02.008. Yadav, D.K., Taraphder, S., Dhara, K.C., Batabyal, S., Samanta, I., Mitra, M., 2013. Association of transferrin polymorphism with different economic traits of Garole sheep. Int. J. Livest. Prod. 3, 6–11. https://doi.org/10.5897/IJLP11.013.
45