Animal Reproduction Science 151 (2014) 71–77
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits A. Maitra a , Rekha Sharma a,∗ , Sonika Ahlawat a , M.S. Tantia a , Manoranjan Roy b , Ved Prakash c a b c
National Bureau of Animal Genetic Resources, Karnal, India West Bengal University of Animal and Fishery Sciences, Kolkata, India Central Sheep and Wool Research Institute, Avikanagar, India
a r t i c l e
i n f o
Article history: Received 1 May 2014 Received in revised form 1 September 2014 Accepted 16 September 2014 Available online 28 September 2014 Keywords: Goats KiSS1 GPR54 Precocity Prolificacy
a b s t r a c t KiSS1 is considered to be a key mediator of molecular mechanism of reproduction (puberty and prolificacy) in mammals. Kisspeptins are a family of structurally related peptides, encoded by KiSS1 gene, with ability to regulate gonadotropin-releasing hormone and hence hypothalamic–pituitary–gonadal axis. The present study investigated the polymorphism of caprine KiSS1 gene in 9 Indian goat breeds differing in sexual precocity and prolificacy. Comparison of KiSS1 amplified sequences of indigenous goats resulted in identification of nine SNPs (intron (1) G296C, T455G, T505A, T693C, T950C and intron (2) T1125C, A2510G, C2540T, A2803G) of which four are novel. These loci were not segregating together (r2 < 0.33). Mutations existed in both, sexually precocious and late-maturing goat breeds as well as low and high prolificacy goat breeds. Three loci reported to be associated with goat litter size (G296C, G2510A and C2540T) were identified in Indian goats as well. Association between loci of KiSS1 gene and age of puberty as well as litter size was explored in Black Bengal (N = 158), a sexually precocious and prolific goat breed of India by designing PCR–RFLP. None of the mutations were found to be associated with reproductive traits however, difference in litter size as well age of sexual maturity for different genotypes indicates that the study on additional data based on more number of breeds and animals would be interesting to perform. Considering the importance of the reproductive trait in small ruminants, the results extend the limited information on genetic variation of the caprine KiSS1, which might contribute toward molecular breeding to enhance productivity of goat. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The Kisspeptin/GPR54 pathway is considered as a key gatekeeper of pubertal development and reproductive function. So far, there have been some studies of KiSS1 gene as a candidate gene for reproductive traits in animals,
∗ Corresponding author. Tel.: +91 9255482422; fax: +91 184 2267654. E-mail address: [email protected] (R. Sharma). http://dx.doi.org/10.1016/j.anireprosci.2014.09.013 0378-4320/© 2014 Elsevier B.V. All rights reserved.
which revealed that this gene plays an important role in animal reproduction (Ardlie et al., 2002; Huijbregts et al., 2012; Tomikawa et al., 2010). Kisspeptins are the peptide products of KiSS1 gene, which operate via their cognate receptors, G-protein-coupled receptor (GPR54). These neuropeptides have emerged as essential upstream regulators of neurons that reside in the basal forebrain and produce Gonadotropin Releasing Hormone (GnRH) (Popa et al., 2005; Knoll et al., 2013). Human KiSS1 gene maps to chromosome 1q32 and consists of three exons
72
A. Maitra et al. / Animal Reproduction Science 151 (2014) 71–77
(West et al., 1998; Luan et al., 2007). The latter two exons of KiSS1 gene are finally translated into a 145 amino acid protein along with a putative signal sequence, from which different forms of kisspeptin consisting 54, 14, 13 and 10 amino acid are derived (Luan et al., 2007). KiSS1 neurons in the hypothalamus participate in crucial features of reproductive maturation and function, such as brain-level sex differentiation, puberty onset and neuroendocrine regulation of gonadotropin secretion and ovulation (Caraty et al., 2010). Mutations of GPR54 (KiSS1R) are associated with hypogonadotrophic hypogonadism in humans (de Roux et al., 2003; Seminara et al., 2003), a phenotype which is also observed in mice carrying inactivating mutations of KiSS1 gene (Tena-Sempere, 2010). Activated mutations of KiSS1 gene (Ko et al., 2010; Luan et al., 2007) have been shown to cause central precocious puberty (CPP) in humans with maturation of the hypothalamic–pituitary–gonadal (ovarian) axis (HPGA) by a gonadotropin dependent manner. Up to now, the literature concerning KiSS1 and goat reproduction is limited. Due to the importance of KiSS1 as a regulator of puberty onset, it is probable that the polymorphisms in this gene have some relationship with reproductive traits such as high prolificacy, sexual precocity and year-round estrus phenotypes of goats. Indian goat breeds differ pertaining to these reproductive traits. Goat genetic resource comprises of 23 recognized breeds in India (Sharma et al., 2013) which is valuable gene pool for adaptive and economic traits, providing diversified genetic material that can help to meet future challenges. Black Bengal goat is unique among all Indian goat breeds as these show significant characteristics of sexual precocity and high prolificacy. The objectives of the present study were firstly, to sequence characterize Indian goat KiSS1 gene through polymerase chain reaction (PCR) and sequence assembly; secondly, to identify single nucleotide polymorphisms (SNPs) in the gene by sequence alignment between goat breeds (Black-Bengal, Malabari, Beetal, Barbari, Osmanabadi, Sangamneri, Jakhrana, Ganjam and Sirohi) differing in fecundity and age at puberty and finally, to study the relationship of the identified mutations with age at sexual maturity as well as litter size within Black Bengal goat. 2. Materials and methods 2.1. Animal selection, sample collection and DNA isolation Nine well-recognized breeds of Indian goats from different geographic and agro-climatic parts across India (Fig. 1) were selected which differ in prolificacy (number of kids per kidding) and age of sexual maturity (Table 1). Each breed was represented by five animals for polymorphism scanning. Unrelated animals were selected at random from their breeding tracts by picking up only two samples per herd and only two herds per village. One hundred and fifty eight Black Bengal goats were utilized (Kotulpur goat cum fodder form, West Bengal, India) for association between identified genotypes in KiSS1 gene with reproductive traits (age of puberty and litter size at first, second, third and
Fig. 1. Geographical distribution of selected goat breeds.
fourth parity). Five milliliter blood per goat was collected aseptically from the jugular vein in a vacutainer tube (B.D. Bioscience, Germany) containing EDTA as anticoagulant. All samples were delivered back to the laboratory in an ice box. Genomic DNA was extracted from white blood cells using standard phenol-chloroform extraction protocol (Sambrook et al., 1989).
2.2. Primers and PCR amplification Three pairs of primers were utilized for amplification of 5 UTR (includes exon 1 and intron 1), exon 2 and intron 2 of KiSS1 gene (Table 2). Two pair of primers was designed according to the genomic DNA sequence of goat KiSS1 gene (GU142847) and a primer pair was taken from literature. The primers were synthesized by IDT (Integrated DNA Technologies, Inc.). Polymerase chain reaction was carried out in 25 L reaction volumes with about 50–100 ng genomic DNA using i-cycler (BioRAD, USA). The reaction mixture consisted of 200 M each of dATP, dCTP, dGTP, dTTP, 1.5 mM MgCl2 , 50 pmol primer, 0.5 U Taq polymerase (Sigma–Aldrich® ) and corresponding Taq buffer. Amplification conditions were as follows: initial denaturation for 1 min at 94 ◦ C; followed by 30 cycles of denaturation at 94 ◦ C for 1 min, annealing at precise temperature (Table 2) for 1 min, extension at 72 ◦ C for 1 min; and finally extension at 72 ◦ C for 5 min. The PCR products were separated by electrophoresis on 1.8% agarose gels in parallel with a 100 bp DNA ladder, enzymatically purified and sequenced from both directions using ABI-3100 Automated DNA Sequencer (Applied Biosystem, USA).
A. Maitra et al. / Animal Reproduction Science 151 (2014) 71–77
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Table 1 Geographical distribution and characteristics of selected Indian goat breeds. Breed
Geographical distribution
Prolificacy
Twinning percentage (%)
Sexual maturity (age at puberty)
Beetal Barbari Black-Bengal Malabari Osmanabadi Sangamneri Jakhrana Ganjam Sirohi
Punjab Uttar Pradesh West Bengal, Bihar, Jharkhand Kerala Maharashtra Maharashtra Rajasthan Orissa Rajasthan
High High High High Medium Medium Medium Low Low
>50 >50 >50 >50 25–50 25–50 25–50
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits A. Maitra a , Rekha Sharma a,∗ , Sonika Ahlawat a , M.S. Tantia a , Manoranjan Roy b , Ved Prakash c a b c
National Bureau of Animal Genetic Resources, Karnal, India West Bengal University of Animal and Fishery Sciences, Kolkata, India Central Sheep and Wool Research Institute, Avikanagar, India
a r t i c l e
i n f o
Article history: Received 1 May 2014 Received in revised form 1 September 2014 Accepted 16 September 2014 Available online 28 September 2014 Keywords: Goats KiSS1 GPR54 Precocity Prolificacy
a b s t r a c t KiSS1 is considered to be a key mediator of molecular mechanism of reproduction (puberty and prolificacy) in mammals. Kisspeptins are a family of structurally related peptides, encoded by KiSS1 gene, with ability to regulate gonadotropin-releasing hormone and hence hypothalamic–pituitary–gonadal axis. The present study investigated the polymorphism of caprine KiSS1 gene in 9 Indian goat breeds differing in sexual precocity and prolificacy. Comparison of KiSS1 amplified sequences of indigenous goats resulted in identification of nine SNPs (intron (1) G296C, T455G, T505A, T693C, T950C and intron (2) T1125C, A2510G, C2540T, A2803G) of which four are novel. These loci were not segregating together (r2 < 0.33). Mutations existed in both, sexually precocious and late-maturing goat breeds as well as low and high prolificacy goat breeds. Three loci reported to be associated with goat litter size (G296C, G2510A and C2540T) were identified in Indian goats as well. Association between loci of KiSS1 gene and age of puberty as well as litter size was explored in Black Bengal (N = 158), a sexually precocious and prolific goat breed of India by designing PCR–RFLP. None of the mutations were found to be associated with reproductive traits however, difference in litter size as well age of sexual maturity for different genotypes indicates that the study on additional data based on more number of breeds and animals would be interesting to perform. Considering the importance of the reproductive trait in small ruminants, the results extend the limited information on genetic variation of the caprine KiSS1, which might contribute toward molecular breeding to enhance productivity of goat. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The Kisspeptin/GPR54 pathway is considered as a key gatekeeper of pubertal development and reproductive function. So far, there have been some studies of KiSS1 gene as a candidate gene for reproductive traits in animals,
∗ Corresponding author. Tel.: +91 9255482422; fax: +91 184 2267654. E-mail address: [email protected] (R. Sharma). http://dx.doi.org/10.1016/j.anireprosci.2014.09.013 0378-4320/© 2014 Elsevier B.V. All rights reserved.
which revealed that this gene plays an important role in animal reproduction (Ardlie et al., 2002; Huijbregts et al., 2012; Tomikawa et al., 2010). Kisspeptins are the peptide products of KiSS1 gene, which operate via their cognate receptors, G-protein-coupled receptor (GPR54). These neuropeptides have emerged as essential upstream regulators of neurons that reside in the basal forebrain and produce Gonadotropin Releasing Hormone (GnRH) (Popa et al., 2005; Knoll et al., 2013). Human KiSS1 gene maps to chromosome 1q32 and consists of three exons
72
A. Maitra et al. / Animal Reproduction Science 151 (2014) 71–77
(West et al., 1998; Luan et al., 2007). The latter two exons of KiSS1 gene are finally translated into a 145 amino acid protein along with a putative signal sequence, from which different forms of kisspeptin consisting 54, 14, 13 and 10 amino acid are derived (Luan et al., 2007). KiSS1 neurons in the hypothalamus participate in crucial features of reproductive maturation and function, such as brain-level sex differentiation, puberty onset and neuroendocrine regulation of gonadotropin secretion and ovulation (Caraty et al., 2010). Mutations of GPR54 (KiSS1R) are associated with hypogonadotrophic hypogonadism in humans (de Roux et al., 2003; Seminara et al., 2003), a phenotype which is also observed in mice carrying inactivating mutations of KiSS1 gene (Tena-Sempere, 2010). Activated mutations of KiSS1 gene (Ko et al., 2010; Luan et al., 2007) have been shown to cause central precocious puberty (CPP) in humans with maturation of the hypothalamic–pituitary–gonadal (ovarian) axis (HPGA) by a gonadotropin dependent manner. Up to now, the literature concerning KiSS1 and goat reproduction is limited. Due to the importance of KiSS1 as a regulator of puberty onset, it is probable that the polymorphisms in this gene have some relationship with reproductive traits such as high prolificacy, sexual precocity and year-round estrus phenotypes of goats. Indian goat breeds differ pertaining to these reproductive traits. Goat genetic resource comprises of 23 recognized breeds in India (Sharma et al., 2013) which is valuable gene pool for adaptive and economic traits, providing diversified genetic material that can help to meet future challenges. Black Bengal goat is unique among all Indian goat breeds as these show significant characteristics of sexual precocity and high prolificacy. The objectives of the present study were firstly, to sequence characterize Indian goat KiSS1 gene through polymerase chain reaction (PCR) and sequence assembly; secondly, to identify single nucleotide polymorphisms (SNPs) in the gene by sequence alignment between goat breeds (Black-Bengal, Malabari, Beetal, Barbari, Osmanabadi, Sangamneri, Jakhrana, Ganjam and Sirohi) differing in fecundity and age at puberty and finally, to study the relationship of the identified mutations with age at sexual maturity as well as litter size within Black Bengal goat. 2. Materials and methods 2.1. Animal selection, sample collection and DNA isolation Nine well-recognized breeds of Indian goats from different geographic and agro-climatic parts across India (Fig. 1) were selected which differ in prolificacy (number of kids per kidding) and age of sexual maturity (Table 1). Each breed was represented by five animals for polymorphism scanning. Unrelated animals were selected at random from their breeding tracts by picking up only two samples per herd and only two herds per village. One hundred and fifty eight Black Bengal goats were utilized (Kotulpur goat cum fodder form, West Bengal, India) for association between identified genotypes in KiSS1 gene with reproductive traits (age of puberty and litter size at first, second, third and
Fig. 1. Geographical distribution of selected goat breeds.
fourth parity). Five milliliter blood per goat was collected aseptically from the jugular vein in a vacutainer tube (B.D. Bioscience, Germany) containing EDTA as anticoagulant. All samples were delivered back to the laboratory in an ice box. Genomic DNA was extracted from white blood cells using standard phenol-chloroform extraction protocol (Sambrook et al., 1989).
2.2. Primers and PCR amplification Three pairs of primers were utilized for amplification of 5 UTR (includes exon 1 and intron 1), exon 2 and intron 2 of KiSS1 gene (Table 2). Two pair of primers was designed according to the genomic DNA sequence of goat KiSS1 gene (GU142847) and a primer pair was taken from literature. The primers were synthesized by IDT (Integrated DNA Technologies, Inc.). Polymerase chain reaction was carried out in 25 L reaction volumes with about 50–100 ng genomic DNA using i-cycler (BioRAD, USA). The reaction mixture consisted of 200 M each of dATP, dCTP, dGTP, dTTP, 1.5 mM MgCl2 , 50 pmol primer, 0.5 U Taq polymerase (Sigma–Aldrich® ) and corresponding Taq buffer. Amplification conditions were as follows: initial denaturation for 1 min at 94 ◦ C; followed by 30 cycles of denaturation at 94 ◦ C for 1 min, annealing at precise temperature (Table 2) for 1 min, extension at 72 ◦ C for 1 min; and finally extension at 72 ◦ C for 5 min. The PCR products were separated by electrophoresis on 1.8% agarose gels in parallel with a 100 bp DNA ladder, enzymatically purified and sequenced from both directions using ABI-3100 Automated DNA Sequencer (Applied Biosystem, USA).
A. Maitra et al. / Animal Reproduction Science 151 (2014) 71–77
73
Table 1 Geographical distribution and characteristics of selected Indian goat breeds. Breed
Geographical distribution
Prolificacy
Twinning percentage (%)
Sexual maturity (age at puberty)
Beetal Barbari Black-Bengal Malabari Osmanabadi Sangamneri Jakhrana Ganjam Sirohi
Punjab Uttar Pradesh West Bengal, Bihar, Jharkhand Kerala Maharashtra Maharashtra Rajasthan Orissa Rajasthan
High High High High Medium Medium Medium Low Low
>50 >50 >50 >50 25–50 25–50 25–50
