Association analysis between variants in KISS1 gene and litter size in goats

Background Kisspeptins are the peptide products of KISS1 gene, which operate via the G - protein-coupled receptor GPR54. These peptides have emerged as essential upstream regulators of neurons secreting gonadotropin-releasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the hypothalamic–pituitary– gonadal (HPG) axis. The present study detected the polymorphisms of caprine KISS1 gene in three goat breeds and investigated the associations between these genetic markers and litter size. Results Three goat breeds (n = 680) were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron–exon boundaries and the proximal flanking regions of KISS1 gene by DNA sequencing and PCR–RFLP. Eleven novel SNPs (g.384G>A, g.1147T>C, g.1417G>A, g.1428_1429delG, g.2124C>T, g.2270C>T, g.2489T>C, g.2510G>A, g.2540C>T, g.3864_3865delCA and g.3885_3886insACCCC) were identified. It was shown that Xinong Saanen and Guanzhong goat breeds were in Hardy-Weinberg disequilibrium at g.384G>A locus (P < 0.05). Both g.2510G>A and g.2540C>T loci were closely linked in Xinong Saanen (SN), Guanzhong (GZ) and Boer (BG) goat breeds (r2 > 0.33). The g.384G>A, g.2489T>C, g.2510G>A and g.2540C>T SNPs were associated with litter size (P<0.05). Individuals with AATTAATT combinative genotype of SN breed (SC) and TTAATT combinative genotype of BG breed (BC) had higher litter size than those with other combinative genotypes in average parity. The results extend the spectrum of genetic variation of the caprine KISS1 gene, which might contribute to goat genetic resources and breeding. Conclusions This study explored the genetic polymorphism of KISS1 gene, and indicated that four SNPs may play an important role in litter size. Their genetic mechanism of reproduction in goat breeds should be further investigated. The female goats with SC1 (AATTAATT) and BC7 (TTAATT) had higher litter size than those with other combinative genotypes in average parity and could be used for the development of new breeds of prolific goats. Further research on a large number of animals is required to confirm the link with increased prolificacy in goats.


Background
Kisspeptins are the peptide products of KISS1 gene, which operate via the G -protein-coupled receptor GPR54 (also known as KISS1R). These peptides have emerged as essential upstream regulators of neurons secreting gonadotropinreleasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the hypothalamic-pituitarygonadal (HPG) axis [1]. They are potent elicitors of gonadotropin secretion in various species and physiological settings. Moreover, KISS1 neurons in the hypothalamus participate in crucial features of reproductive maturation and function, such as brain-level sex differentiation, puberty onset and the neuroendocrine regulation of gonadotropin secretion and ovulation [2]. Irwig et al. (2004) and Navarro et al. (2004) have provided evidences in rats that kisspeptin-expressing neurons are targets for regulation by sex steroids [3,4], furthermore, these neurons are directly regulated by the negative and positive feedback actions of sex steroids in distinct regions of the forebrain [5].
Mutations of KISS1R are associated with hypogonadotrophic hypogonadism in humans [6,7], a phenotype which is also observed in mice carrying inactivating mutations of KISS1 or KISS1R genes [8]. In addition, to their prominent expression at hypothalamic levels, fragmentary evidences suggest that KISS1 and/or KISS1R mRNAs or proteins are also present in several peripheral reproductive tissues including the ovary [9,10], oviduct [11] and testes [12]. In humans, Pinto et al. (2012) reported kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation [13]. One novel nonsynonymous single nucleotide polymorphism (G54650055T) substituting one amino acid in kisspeptin (P110T) was found to be statistically related to central precocious puberty (P<0.025) in humans [14]. In sheep, KISS1 mRNAexpressing cells are found in the arcuate nucleus (ARC) and dorsallateral preoptic area and both appear to mediate the positive feedback effect of estradiol to generate the preovulatory GnRH/LH surge [15]. The LH surge has been associated with an increase in the LH response to kisspeptin in humans and sheep [16,17], indicating the surge may be generated by increased kisspeptin output and sensitivity. These findings indicate that KISS1 gene is an excellent candidate gene for reproductive traits in human and livestock.
Based on above considerations, here we detected the polymorphisms of caprine KISS1 gene in three goat breeds and investigated the associations between these genetic markers and litter size. This study provides some useful information on goat genetic resources and breeding.

SNPs identification and genotypes
In the current study, sequencing of the amplicons of different primer pairs identified eleven polymorphic nucleotide sites in caprine KISS1 gene. The g.384G>A mutation was in the 5′UTR (Additional file 1: Table S1), which was not found in BG breed. The g.3864_3865delCA and g.3885_3886insACCCC mutations were in the 3′UTR. Other mutations were in the intron 1 (g.1147T>C, g.141 7G>A, g.1428_1429delG, g.2124C>T, g.2270C>T, g.2489 T>C, g.2510G>A and g.2540C>T). SNP accession number is showed in Additional file 1: Table S1. Four SNPs (g.384G>A, g.2489T>C, g.2510G>A and g.2540C>T) were genotyped in three goat breeds (Figures 1, 2, 3, and 4). At g.384G>A locus, the PIC was 0.37 in SN and GZ breeds (Additional file 1: Table S2). At g.2489T>C locus, the PIC was 0.24-0.29 in three goat breeds. At other two loci, the PIC was 0.36-0.38 in three goat breeds. Genotypic distribution and allelic frequencies of four SNPs are shown in Additional file 1: Table S2. It was shown that SN and GZ breeds were in Hardy-Weinberg disequilibrium at g.384G>A locus (P < 0.05) (Additional file 1: Table S2). To reveal the linkage relationships between the four SNPs, the linkage disequilibrium was estimated in these breeds (Additional file 1: Table S3). If r 2 > 0.33, the linkage disequilibrium was considered strong [18]. Following the result, both g.2510G>A and g.2540C>T loci were closely linked in three goat breeds (Additional file 1: Table S3).

Association analysis of SNPs with litter size
At g.384G>A locus in SN breed (Table 1), individuals with AA genotype had greater litter size than those with GG genotype in the second, third and average parity (P < 0.05); in GZ breed, individuals with AA genotype had greater litter size than those with GG genotype in the third and average parity (P < 0.05), and individuals with GA genotype had greater litter size than those with GG genotype in the first and average parity (P < 0.05). At g.2510G>A locus in SN breed (Table 2), individuals with AA genotype had greater litter size than those with GA genotype in the third parity (P < 0.05); in BG breed, individuals with AA genotype had greater litter size than those with GG genotype in the fourth and average parity (P < 0.05). At Figure 1 The electrophoresis patterns obtained after digestion with MwoI endonuclease at g.384G>A locus. Note: Fragments including 19 bp of GG and GA genotypes were invisible.

Figure 2
The electrophoresis patterns obtained after digestion with SfaNI endonuclease at g.2489T>C locus.
g.2540C>T locus in GZ breed (Table 2), individuals with TT genotype had greater litter size than those with CC genotype in the fourth parity (P < 0.05); in BG breed, individuals with TT genotype had greater litter size than those with CC and CT genotypes in average parity (P < 0.05).

Effects of combinative genotypes on litter size
Association analysis of combinative genotypes of g.38 4G>A, g.2489T>C, g.2510G>A and g.2540C>T loci with litter size was done in SN and GZ breeds. In SN breed (Additional file 1: Table S4), individuals with SC3 (AATCGACT) had the greatest litter size in comparison with other combinative genotypes in the first parity. Individuals with SC1 (AATTAATT) and SC3 (AATCGACT) had greater litter size than those with SC4 (AATTGACT) and SC10 (GGTTAATT) in the second parity (P < 0.05). Individuals with SC1 (AATTAATT) had greater litter size than those with SC2 (AATTGGCC) and SC4 (AATTGACT) in the fourth parity (P < 0.05). Individuals with SC3 (AATCGACT) had greater litter size than those with SC4 (AATTGACT), SC5 (GATCGGCC) and SC13 (GATTGGCC) in average parity (P < 0.05). In GZ breed (Additional file 1: Table S5), Individuals with GC2 (GATCGGCC) had greater litter size than those with GC12 (GGTCGACT) and GC14 (GGTCGATT) in the first parity (P < 0.05). Individuals with GC14 (GGTCGATT) had the lowest litter size in comparison with other combinative genotypes in the third parity. Association analysis of combinative genotypes of g.2489T>C, g.2510G>A and g.2540C>T loci with litter size was done in BG breeds. In BG breed (Additional file 1: Table S6), individuals with BC7(TTAATT) had greater litter size than those with BC6 (TTGACT) in the third, fourth and average parity (P < 0.05). Individuals with BC6 (TTGACT) had the lowest litter size in comparison with other combinative genotypes in average parity.

Discussion
According to the classification of PIC (low polymorphism if PIC value < 0.25, moderate polymorphism if 0.25 < PIC value < 0.50, and high polymorphism if PIC > 0.50), SN and GZ breeds at g.384G>A locus had moderate genetic diversity, and SN, GZ and BG breeds had also moderate genetic diversity at g.2510G>A and g.2540C>T loci. The g.384G>A locus was in Hardy-Weinberg disequilibrium in SN and GZ breeds (P<0.05), which showed the genotypic frequencies were affected by selection, mutation or migration.
The reproductive traits are complex quantitative traits involving multiple genes, loci and interactions, so it is important to analyze the combined effect of multiple genes or loci on reproductive traits. In the study, association between multiple locus and litter size from the first to the fourth parity was analyzed. Mean litter size of goat tended to increase in later parity. Individuals with SC1 (AATTAATT) had higher litter size than those with SC4 (AATTGACT) and SC10 (GGTTAATT) in the second parity of SN breed. In addition, individuals with SC1 (AATTAATT) had higher litter size than those with SC10 (GGTTAATT) in average parity of SN breed. The litter size at second kidding is often a valuable index to determine whether a goat is prolific [26]. Therefore, SC1  Note: Values with different superscripts within the same column and mutation locus in particular breed differ significantly at P < 0.05.
(AATTAATT) can be used in marker-assisted selection to select the individuals with higher litter size. Accumulating evidence further showed that central or peripheral administration of kisspeptin stimulates GnRH-dependent luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in various mammalian species from rodents to humans [27][28][29], suggesting that kisspeptin plays an essential role in governing reproductive functions throughout species. The biochemical and physiological functions, together with the results obtained in our study, indicate that KISS1 gene could be as a molecular breeding marker in goats.

Conclusions
This study explored the genetic polymorphism of KISS1 gene, and indicated that four SNPs may play an important role in litter size. Their genetic mechanism of reproduction in goat breeds should be further investigated. The female goats with SC1(AATTAATT) and BC7 (TTAATT) had higher litter size than those with other combination genotypes in average parity and could be used for the development of new breeds of prolific goats. Further research on a large number of animals is required to confirm the link with increased prolificacy in goats.

Animals and genomic DNA isolation
Blood samples were obtained from 680 goats belonging to three breeds: Xinong Saanen goat (SN; n=257), Guanzhong goat (GZ, n=231) and Boer goat (BG; n=192). They were reared in Qianyang, Zhouzhi and Liuyou county of Shaanxi province, respectively. All diets were based on alfalfa, corn silage, and a combination of concentrates including corn, soya meal, and bone meal. Health, fertility and production records were maintained by the dairymen and veterinarians. The litter size from the first to fourth parity was obtained from production records. Five milliliters blood per goat were collected aseptically from the jugular vein and kept in a tube containing anticoagulant ACD (citric acid:sodium citrate: dextrose -10: 27: 38). All samples were delivered back to the laboratory in an ice box. The genomic DNA was extracted from white blood cells using standard phenol-chloroform extraction protocol. All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

SNPs investigation and genotyping
According to caprine KISS1 gene (GenBank accession no. GU142847), Seven pairs of primers were designed to amplify caprine KISS1 gene. Their optimal annealing temperatures are showed in Additional file 1: Table S7.
Herein we screened them for identifying SNPs of this gene by DNA pooling sequencing assay [30]. Five microliters DNA (100ng/μl) per sample were collected to create a DNA pool for each goat breed. PCR products were sent to Beijing Genomics Institute (Beijing, China) to sequence in both directions. Discovery of SNPs was conducted using Chromas 2.31 and DNAstar 7.0 software. The SNP in 5'UTR (89-409bp) of KISS1 gene was genotyped using primer-introduced restriction analysispolymerase chain reaction (PIRA-PCR) [31]. Other SNPs of KISS1 gene were genotyped with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The 25 μL volume contained 50 ng genomic DNA, 12.5 μL 2 × reaction mix (including 500 μM dNTP each; 20 mM Tris-HCl; pH 9; 100 mM KCl; 3 mM MgCl2 ), 0.5 μM of each primer, and 0.5 units of Taq DNA polymerase. The cycling protocol was 5 min at 95°C, 35 cycles of denaturing at 94°C for 30 s, annealing at X°C (Additional file 1: Table S7) for 30 s, extending at 72°C for 35 s, with a final extension at 72°C for 10 min. PCR products (5μl) of different primer pairs were mixed with 0.7 μl 10 × Buffer, 2.5 U restriction enzyme (NEB, Ipswich, Britain) and 3.8 μl sterilized ddH 2 O, and then incubated for 1.5 h at 37°C. The restriction enzymes were showed in Additional file 1: Table S1. Digestion products were subjected to 3.5% horizontal agarose gel electrophoresis or 12% polyacrylamide gel electrophoresis (PAGE). The agarose and polyacrylamide gels were stained with ethidium bromide and 0.1% silver nitrate, respectively, and then the genotypes were observed.
The allelic frequencies, heterozygosity (He) and polymorphism information content (PIC) were calculated using Popgene (version 1.31). The linkage disequilibrium was performed by SHEsis software [19]. Association analysis of combinative genotypes of different loci in KISS1 gene with litter size was done in three goat breeds. SC n , GC n and BC n represented different combinative genotypes of SN, GZ and BG breeds, respectively. Statistical analysis was performed using univariate analysis in the general linear model procedure of SPSS 16 statistical software. Multiple comparisons of the means were performed using the least significant difference method or Dunnett's T3. The model applied was: Y ikm = μ + G i + S k + E ikm , where Y ikm is the trait measured on each of the ikm th animal, μ is the overall population mean, G i is the fixed effect associated with i th genotype or combinative genotype, S k is the fixed effect associated with the k th sire, and E ikm is the random error. Effects associated with farm, birth year and season of birth are not matched in the linear model, as the preliminary statistical analyses indicated that these effects did not have a significant influence on variability of traits in the analyzed populations.