New genetic polymorphisms of KiSS-1 gene and their association with litter size in goats

Small Ruminant Research 96 (2011) 106–110

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New genetic polymorphisms of KiSS-1 gene and their association with litter size in goats J.X. Hou 1 , X.P. An 1 , J.G. Wang, Y.X. Song, Y.H. Cui, Y.F. Wang, Q.J. Chen, B.Y. Cao ∗ College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, PR China

a r t i c l e

i n f o

Article history: Received 2 July 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Available online 18 December 2010 Keywords: PCR-SSCP Base deletions SNP Candidate gene

a b s t r a c t In the present study, the polymorphisms of kisspeptin (KiSS-1) gene were analyzed in 652 individuals from three goat breeds: Xinong Saanen goats (SG), Guanzhong goats (GZ) and Boer goats (BG). Three alleles (T, C and G) and four genotypes (TT, TC, CC and TG) were detected in the intron 2 of KiSS-1 gene by PCR-SSCP analysis. In addition, two single nucleotide polymorphisms (SNPs) – T2643C and 8bp base deletions (2677AGTTCCCC) were identified by DNA sequencing. The SNPs loci were in Hardy–Weinberg disequilibrium in three goat breeds (P < 0.05). After comparing genotypic distribution within three goat breeds, BG had conspicuous differences from SG and GZ (P < 0.001). Association of polymorphism with litter size was done in SG and GZ breeds, which showed to be associated with litter size in both goat breeds. The SNP in goat KiSS-1 gene had significant effects on litter size (P < 0.05). Therefore, these results suggest that KiSS-1 gene is a strong candidate gene that affects litter size in goats. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Kisspeptins are a family of structurally related peptides, encoded by the kisspeptin (KiSS-1) gene, with ability to bind and activate the G protein coupled receptor (GPR54) (Roa et al., 2008; Li et al., 2009; Roseweir and Millar, 2009). The first biological actions attributed to kisspeptins were related to their capacity to inhibit tumor metastasis in several malignancies (Roa et al., 2008; Tena-Sempere, 2010). However, in late 2003, an unsuspected, fundamental role for KiSS-1 peptides in the control of the gonadotropic axis was suggested on the basis of the reproductive phenotypes of human and mouse models carrying null mutations of GPR54 (de Roux et al., 2003; Seminara and Kaiser, 2005). From that moment onwards, an ever growing number of research groups worldwide have pursuit the characteriza-

∗ Corresponding author. Tel.: +86 29 87092102; fax: +86 29 87092164. E-mail address: [email protected] (B.Y. Cao). 1 These authors equally contributed to this paper. 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.11.013

tion of the physiological roles and mechanisms of action of kisspeptins in the control of reproduction. In less than 5 years, in more than 150 original research articles, which have substantiated the crucial functions of this family of molecules in key aspects of reproductive maturation and function (Tena-Sempere, 2010). Gonadal sex steroids are major regulators of GnRH secretion through negative and positive feedback loops (Novaira et al., 2009). Irwig et al. (2004) and Navarro et al. (2004) have provided evidence in rats that kisspeptinexpressing neurons are targets for regulation by sex steroids, furthermore, these neurons are directly regulated by the negative and positive feedback actions of sex steroids in distinct regions of the forebrain (Gottsch et al., 2006; Smith et al., 2006). These observations suggest that kisspeptin/GPR54 signaling provides tonic stimulatory input to GnRH neurons, which could be governed by the feedback effects of sex steroids acting on kisspeptin secreting neurons (Gottsch et al., 2006). Investigations by many laboratories over the last 5 years have led to the general concept that kisspeptin neurons activate GnRH neurons

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(Kauffman et al., 2007; Caraty and Franceschini, 2008; Seminara and Crowley, 2008; Roa et al., 2009). Kisspeptinproducing neurons (or KiSS-1 neurons) have been localized to various regions of the forebrain in rodents, sheep and primates (Gottsch et al., 2004; Franceschini et al., 2006). Central and/or peripheral administration of kisspeptins could activate GnRH-dependent release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and induce the precocious activation of the gonadotropic axis in human (Dhillo et al., 2005; An et al., 2010a), mouse (Gottsch et al., 2004), rat (Matsui et al., 2004; Thompson et al., 2004), sheep (Messager et al., 2005; Estrada et al., 2006), goat (Hashizume et al., 2010), rhesus monkey (Plant et al., 2006), hamster (Revel et al., 2006; Greives et al., 2007) and fish (Filby et al., 2008). Kisspeptins stimulate GnRH release directly and uniquely through GPR54 to result in the initiation of puberty (Tena-Sempere, 2006; Navarro and Tena-Sempere, 2008). Based on the above considerations, the objective of the present study was to estimate the alleles and genotype frequencies of goat KiSS-1 gene polymorphisms and to determine associations between these polymorphisms and the litter size of these animals when submitted to an intensive production model. 2. Materials and methods 2.1. Animals and genomic DNA isolation All procedures involving animals were approved by Boer goat breeding center, Qianyang Xinong Saanen goat breeding center and Green Century Biology Development Company in Shaanxi Province, China. A total of 652 female goats were examined in this study, including 299 Xinong Saanen goats (SG), 120 Guangzhong goats (GZ) and 233 Boer goats (BG). Xinong Saanen and Guangzhong are the dairy breeds, while Boer is a very important breed for mutton production. 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 (Joseph and David, 2002; Wu et al., 2009). 2.2. Primers and PCR conditions According to goat KiSS-1 gene (GenBank accession no. GU142847.1), three pairs of primers were designed to amplify an 1189 bp product of KiSS-1 gene intron 2, and a 449 bp product of one of the primers has polymorphism (F: 5 -CAGACCGCATTTCCACCTAT-3 and R: 5 -TTCATCAGGCTCCTACTAGACC-3 ). Other primers with no polymorphism detected in their amplification regions are not listed in this article. The PCR was performed in a 25 ␮l reaction mixture containing 50 ng genomic DNA, 0.5 ␮M of each primer, 1× buffer (including 1.5 mM MgCl2 ), 200 ␮M dNTPs and 0.625 U Taq DNA polymerase (MBI Fermentas). The cycling protocol was 5 min at 95 ◦ C, 35 cycles of denaturing at 94 ◦ C for 30 s, annealing at 62.8 ◦ C for 40 s,

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extending at 72 ◦ C for 40 s, with a final extension at 72 ◦ C for 10 min.

2.3. Single strand conformation polymorphism (SSCP), cloning and DNA sequencing PCR products (3 ␮l) were mixed with 9 ␮l denaturing solution (95% formamide, 25 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue), heated for 10 min at 98 ◦ C and chilled on ice. Denatured DNA samples were subjected to PAGE (80 mm × 73 mm × 0.75 mm) in 1× TBE buffer and constant voltage (200 V) for 4 h. The gel (acrylamide:bis – 29:1) was stained with 0.1% silver nitrate (Ji et al., 2007; Chessa et al., 2010). After the polymorphisms were detected, DNA bands was extracted from the SSCP gel, and then the PCR products of different electrophoresis patterns were sent to sequence in both directions (repeated three times) in ABI 377 DNA analyzer (Applied Biosystems). In addition, the PCR products of heterozygotes were separated on 1% agarose gel, and then purified with Agarose Gel DNA Fragment Recovery Kit Ver. 2.0 (TaKaRa, Dalian, China) and inserted into pMD19TM -T vector (TaKaRa, Dalian, China) according to the provided protocols. The recombinant plasmids were transformed into competent Escherichia coli JM109 cells. At least five positive clones were sequenced in both directions for each individual using an ABI 377 DNA analyzer (Applied Biosystems). The sequences were analyzed with DNAstar software (version 7.1) and blast in NCBI (National Center for Biotechnology Information).

2.4. Statistical analysis The genotypic frequencies, heterozygosity (He) and polymorphism information content (PIC) were calculated using Cluster-analysis software (version 1.2), and Hardy–Weinberg equilibrium for each breed was analyzed using 2 -test, which was performed by SPSS software (version 16.0). The software SPSS (version 16.0) was also used to analyze the relationship between genotypes and litter size in goats. The adjusted linear model with fixed effects was established and included fixed effects of sire, parity, dam within sire, parity and genotype, as well as the interaction between sire and genotype, and the analysis was run within breed. Adjusted linear model: Yijklm =  + Si + Dij + Pk + Gl + (SG)il + (PG)kl + Eijklm , where Yijklm is the trait measured on each of the ijklmth animal,  is the overall population mean, Si is the fixed effect associated with the ith sire, Dij is the fixed effect associated with jth dam with sire i, Pk is the fixed effect due to the kth parity, Gl is the fixed effect associated with lth genotype, (SG)il is the interaction between the ith sire and lth genotype, (PG)kl is the interaction between kth parity and lth genotype, and Eijklm is the random error. Effects associated with farm and season of birth (spring vs. fall) 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 breeds.

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J.X. Hou et al. / Small Ruminant Research 96 (2011) 106–110 Table 2 2 and P value differences for genotypic frequencies between different goat breeds in the intron 2 of KiSS-1 gene. Locus

Breed

Intron 2

SG GZ BG

SG P = 0.53 P < 0.001

GZ

BG

2 = 2.20

2 = 145.20 2 = 132.00

P < 0.001

Note. SG: Xinong Saanen goats; GZ: Guanzhong goats; BG: Boer goats.

3.3. Association of polymorphism with litter size in three goat breeds Fig. 1. SSCP analysis of PCR products in three goat breeds: (1) CC genotype; (2–5) TT genotype; (7) TG genotype; (6 and 8) TC genotype.

3. Results 3.1. Polymorphisms of KiSS-1 gene in three goat breeds According to international practice and reference to (Gupta et al., 2009; An et al., 2010b; Li et al., 2010) about the naming of SSCP patterns, different SSCP patterns were named TT, TC, CC and TG genotypes (Fig. 1), GG genotype was not detected because of a lower frequency. TT, TC, CC and TG genotypes were observed in SG and GZ breeds, and only TT genotype was observed in BG breed. The frequencies of T allele ranged from 0.688 to 1, and frequencies of C allele ranged from 0 to 0.299, and frequencies of G allele ranged from 0 to 0.008, and the PIC ranged from 0 to 0.356 (Table 1). Genotypic frequencies, He, and the equilibrium 2 -test are shown in Table 1. The SNPs loci were in Hardy–Weinberg disequilibrium in SG and GZ breeds (P < 0.001). Statistical differences in genotypic frequencies of KiSS-1 gene intron 2 implied that the polymorphisms were remarkably associated with goat breeds by 2 -test (Table 2). After comparing genotypic distribution within three goat breeds, BG had conspicuous differences from SG and GZ (P < 0.001). 3.2. Analysis of KiSS-1 gene sequence The different SSCP patterns TT, TC, CC and TG of KiSS-1 gene intron 2 shown in Fig. 1 were sequenced in both directions. Comparisons between the two nucleotide sequences of TT and CC genotypes indicate that one T2643C mutation was detected in CC genotype (GenBank accession no. HM572231) (Fig. 2). Compared with the nucleotide sequence of TT genotype, 8bp deletions (2677AGTTCCCC) were detected in TG genotype (GenBank accession no. HM572230) (Fig. 2).

In SG and GZ breeds, the genotypes of 416 individuals were analyzed for correlation with litter size. In SG breed, the does with TC genotype had more litter size than those with TT genotype (P < 0.05) for the first, second, third and average parity, and no significant difference was found for litter size between TT and CC genotypes (P > 0.05). In GZ breed, the does with TC and CC genotypes had more litter size than those with TT genotypes (P < 0.05) for the third parity; the does with TC genotype had more litter size than those with TT genotypes (P < 0.05) for the fourth and average parity, and the litter size of the first and second parity have no significant difference among TT, TC and CC genotypes (P > 0.05) (Table 3). Because the does with TG genotype have only three, those individuals were not analyzed for correlation with litter size. 4. Discussion More and more studies about the litter size of livestock were performed, such as gonadotropin releasing hormone (GnRH) gene (Karaca et al., 2009), gonadotropin releasing receptor hormone (GnRHR) gene (An et al., 2009), inhibin alpha-subunit (INHA) gene (Wu et al., 2009), estrogen receptor (ESR) gene (Szreder and Zwierzchowski, 2007), bone morphogenetic protein 15 gene (BMP15), growth differentiation factor 9 gene (GDF9) (Vacca et al., 2010) and prolactin gene (Chu et al., 2009). Polymorphism scan of the KiSS-1 gene was performed for the first time by bidirectional resequencing of the whole gene in a subset of 272 Chinese Han girls and 43 unrelated African women diagnosed to be central precocious puberty (CPP) patients, and 288 unrelated normal Chinese Han girls, and the SNP (G54650055T) of exon 3 of KiSS-1 substituting one amino acid in kisspeptin (P110T) was found to be statistically related to the CPP in Chinese girls (P = 0.025) (Luan et al., 2007). Feng et al. (2009) detected the polymorphism in the exon 1 of goat KiSS-1 gene and did not find polymorphism. Cao et al. (2010) preliminarily indicated the associations between allele C of the 296 locus (intron 1) and allele

Table 1 Genetic structure of KiSS-1 gene intron 2 in three goat breeds. Breeds

Observed genotypes TT

SG GZ BG

161 63 233

TC 95 39 0

CC 42 16 0

Genotype frequencies

Allele frequencies

TG

TT

TC

CC

TG

T

C

G

1 2 0

0.538 0.525 1

0.318 0.325 0

0.140 0.133 0

0.003 0.017 0

0.699 0.688 1

0.299 0.296 0

0.002 0.008 0

Note. SG: Xinong Saanen goats; GZ: Guanzhong goats; BG: Boer goats.

He

PIC

Equilibrium 2 -test

0.422 0.439 0

0.335 0.356 0

P < 0.001 P < 0.001 –

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Fig. 2. Partial sequencing maps of different genotypes. Note: The purified PCR products of TG genotype were inserted into pMDTM 19-T vector, and transferred into competent E. coli JM109 cells. At least five positive clones were sequenced in both directions for each individual.

Table 3 Association of KiSS-1 genotypes with litter size (mean ± S.E.) in SG and GZ breeds. Breeds

Genotypes

SG

TT TC CC TT TC CC

GZ

Sample size 161 95 42 63 39 16

1st parity litter size 1.36 1.50 1.45 1.37 1.46 1.38

± ± ± ± ± ±

0.04a 0.05b 0.08ab 0.06a 0.08a 0.12a

2nd parity litter size 1.62 1.84 1.76 1.51 1.67 1.56

± ± ± ± ± ±

0.04a 0.05b 0.08ab 0.07a 0.08a 0.13a

3rd parity litter size 1.73 1.94 1.81 1.57 1.80 1.75

± ± ± ± ± ±

0.04a 0.06b 0.09ab 0.07a 0.08b 0.13ab

4th parity litter size 1.94 2.08 1.98 1.73 2.08 1.81

± ± ± ± ± ±

0.05a 0.06a 0.09a 0.07a 0.09b 0.15ab

Average litter size 1.66 1.84 1.75 1.54 1.75 1.63

± ± ± ± ± ±

0.03a 0.03b 0.05ab 0.03a 0.04b 0.07ab

Note. SG: Xinong Saanen goats; GZ: Guanzhong goats. Values with different superscripts within the same column differ significantly at P < 0.05.

deletion (–) of the 1960–1977 locus (intron 2) in KiSS-1 gene and high litter size in Jining Grey goats. Till now, the researches about the polymorphisms of KiSS-1 gene in human and other mammals were rare. In the present study, we found novel variant of KiSS-1 gene intron 2 in 419 goats by PCR-SSCP and DNA sequencing analysis. We assessed the associations between different KiSS-1 genotypes and litter size in SG and GZ breeds. The quantified results showed that TC genotype was associated with superior litter size in SG and GZ breeds. We consider that these associations can be explained by the following two possible reasons. (1) Although these mutations of intron 2 do not concern the coding region, it possibly influences the stability of the mRNA, can affect the mechanism of mRNA deadenylation and degradation (Gallie and Young, 1994; Xu et al., 1997, 1998; Clement et al., 2001). Therefore, the mutations (T2643C and 2677AGTTCCCC deletion) in the KiSS-1 gene intron 2 may directly or indirectly influence the stability of mRNA, and consequently, the amount of proteins produced, which needs further study. (2) Linkage disequilibrium with the causal mutation possibly affects the variation of the litter size in goats. If this polymorphism is in linkage disequilibrium with a gene affecting the variation of litter size, segregation based on marker alleles would result in phenotypic differences (van der Werf et al., 2007; Lan et al., 2009). The mutation found might not be the causal mutation by itself, but might be in linkage disequilibrium with the causal mutation which could affect either the KiSS-1 gene or other genes near to the KiSS-1 locus. These novel mutations provide further evidence that KiSS-1 gene is a key regulator of reproductive function. The female goats with TC genotype had remarkable litter size, and some of those with better performance could be used

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