Association between INHA gene polymorphisms and litter size in Hainan black goats

Introduction

As one of the earliest domesticated livestock species, goats are closely related to human activities (Tabbaa & Al-Atiyat, 2009). Hainan black goats are a unique local breed in Hainan and the only breed on the tropical island in China (Hu et al., 2016). Hainan black goats have a good tolerance to hot and humid environment, and their meat is delicious and popular among people in southern China (Shi et al., 2020). Hainan black goats usually attain sexual maturity at 4–6 months of age, and first breed at 7–8 months of age. They normally produce a single kid per year and sometimes three kids in 2 years (Hua et al., 2019). Due to their poor reproductive performance, their development is currently limited.

Kidding traits are affected by several factors (genetics, environment, nutritional status, etc.). Genetic factors are the most critical factors (González-Rodríguez et al., 2018; Ofori & Hagan, 2020; Zheng et al., 2020). The INHA gene has been shown to be associated with the kidding ability of many animals, such as the Suhuai pigs and Dazu black goats (Liu et al., 2017a; Wang et al., 2021). INHA gene is the main gene controlling the biological activity of inhibin. Inhibin is mainly secreted by male testicular cells and female ovarian granulosa cells (Cai et al., 2011; Cui et al., 2020). Inhibin regulates reproductive functions through endocrine, paracrine, and autocrine modes, specifically acting on the pituitary gland. It inhibits the secretion of follicle-stimulating hormone (FSH), and locally regulates the production of estrogen (E) (Burger, 1988). High levels of FSH and E will lead to greater ovulation in animals and thus more chances of multiple births, which may be an important reason why INHA affects litter size. However, INHA gene polymorphism in Hainan black goats has not been studied.

When considering candidate genes involved in reproductive performance, it is important to detect single nucleotide polymorphisms (SNPs) and to analyze the associations between these SNPs and reproductive traits. Therefore, this study aims to detect INHA gene polymorphisms in Hainan black goats by sequencing and analyzing the relationship between INHA gene variants and litter size.

Materials and Methods

Results

INHA gene specific fragment

Four pairs of primers were used to amplify the INHA gene of Hainan black goats, and the four DNA sequence fragments of 605, 600, 506 and 432 bp were obtained (Fig. 1). The amplified fragment was consistent with the target fragment and could be analyzed directly by sequencing.

SNPs identified by sequencing

According to the pooled sequencing results, we found seven SNPs. One SNP was located in exon 1, and the other six SNPs were located in exon 2. Since the six SNPs in exon 2 span fragments of primers INHA-2, 3, and 4, we redesigned primer INHA-5 (Material S1) to amplify these SNPs. The PCR products were sequenced, and the sequences of different genotypes are shown (Fig. 2). Among them, g.28315021G>A, g.28317295G>A and g.28317663A>C resulted in amino acid sequence changes (Table 2).

Table 2:

Information content of seven polymorphism loci in the INHA gene of Hainan black goats.

Mutation type	Mutation location	Mutation region	Amino acid change
G/A	g.28315021	Exon 1	(Arg→His)
G/A	g.28317284	Exon 2	(Ala→Ala)
G/A	g.28317295	Exon 2	(Arg→His)
G/A	g.28317368	Exon 2	(Pro→Pro)
C/T	g.28317623	Exon 2	(Pro→Pro)
A/C	g.28317663	Exon 2	(Thr→Pro)
G/A	g.28317758	Exon 2	(Glu→Glu)

DOI: 10.7717/peerj.15381/table-2

Bioinformatics analysis of the INHA g.28317663A>C

It is noteworthy that the mutation at g.28317663 loci leads to the change of amino acid sequence at the 316 site of inhibin A protein, which is Thr change Pro. PRABI was used to predict the protein secondary structure of alleles A and C at locus 316. The secondary structure of the alpha helix of allele A inhibin protein was 18.13%, that of the extended strand was 16.62%, that of the beta turn was 5.44%, and that of the random coil was 59.82%. The secondary structure of the alpha helix of allele C inhibin protein was 17.52%, that of the extended strand was 16.01%, that of the beta turn was 5.44%, and that of the Random coil was 61.03%. It was found that the missense mutation site changed the alpha helix, random coil, and extended strand in the secondary structure of the protein (Fig. 3). Using SWISS to predict the changes in the tertiary structure of the proteins of alleles A and allele C at locus 316, significant changes with g.28317663A>C mutation were observed (Fig. 4).

Linkage disequilibrium and haplotype frequency

In order to explore the linkage relationship among the seven SNPs in the INHA gene of Hainan black goats (Fig. 5), linkage disequilibrium parameters (D′ and r2) were used. The results of the linkage disequilibrium are showed in Table 5. Linkage disequilibrium was found between g.28317368G>A, g.28317623C>T, and g.28317663A>C of the INHA gene. In these three SNPs, the D′ values ranged from 0.817 to 1.000 and the r2 values from 0.079 to 0.264. When subject to a 1% haplotype frequency threshold (Haploview4.2), a total of four haplotypes were identified within three SNPs of the Hainan black goat INHA gene. With regard to haplotype frequency, the ACA haplotype was found to be the most common, observed at a frequency of 0.595, while the GTC haplotype displayed the lowest frequency, occurring at a frequency of 0.042 (Table 6).

Table 5:

Estimated linkage disequilibrium for the SNPs identified in the INHA gene.

	D′	r2
g.28317368G>A/g.28317623C>T	1.000	0.079
g.28317368G>A/g.28317663A>C	0.921	0.264
g.28317623C>T/g.28317663A>C	0.817	0.220

DOI: 10.7717/peerj.15381/table-5

Table 6:

Estimates of haplotypes frequencies observed in the INHA gene.

Haplotype	g.28317368G>A	g.28317623C>T	g.28317663A>C	Haplotype frequency
Hap1 (ACA)	A	C	A	0.595
Hap2 (GCA)	G	C	A	0.260
Hap3 (GCC)	G	C	C	0.089
Hap4 (GTC)	G	T	C	0.042

DOI: 10.7717/peerj.15381/table-6

Discussion

Inhibin is an important inhibitor of pituitary gonadotropin secretion of FSH (Cai et al., 2015). Inhibin can competitively bind activing (ACT) receptor with ACT to weaken the action of ACT, thereby reducing the effect of ACT on the positive feedback regulation of pituitary FSH synthesis and secretion, and achieving the effect of negative regulation of FSH secretion (Gaccioli et al., 2018). FSH can stimulate the growth and development of follicles (Casarini & Crepieux, 2019). Decreased inhibin induces multiple ovulation and increase pituitary secretion of FSH, which appears to be the primary mechanism for stimulating additional follicle growth (Wang et al., 2012). Hence, the polymorphism of INHA gene involving the functional center of inhibin has the potential to affect the reproductive performance of animals. Previous studies have found that a large number of INHA gene mutations are closely related to reproductive system problems such as male infertility (Li et al., 2015), premature ovarian failure (Yoon et al., 2012), superovulation (Luo et al., 2019), follicular cysts (Li et al., 2016), and sperm quality (Rafaqat et al., 2020). Considering the above research results, we believe that the INHA gene is an important candidate gene for improving kidding in does. However, it is still unknown whether INHA gene polymorphism has an effect on the reproductive traits of Hainan black goats. Therefore, the relationship between SNPs of the INHA gene and the kidding of Hainan black goats was analyzed, helping to find useful molecular markers for breeding selection Hainan black goats.

Polymorphisms of the INHA gene have been verified in different animals. In this study, we found seven SNPs in this gene, including three new SNPs, which were g.28315021G>A, g.28317368G>A, and g.28317758G>A. Wu et al. (2009) identified 12 SNPs of the INHA gene in Boer goats, four of which were consistent with our study. In addition, studies in other animals have also found that this gene has higher polymorphisms, such as 12 SNPs being detected in the INHA gene of chickens (Cui et al., 2019) and three SNPs in the INHA gene in Murrah bulls (Chandra et al., 2020). Combined with our results, the INHA gene has abundant polymorphisms.

Polymorphisms of the INHA gene are related to reproductive traits in different animals. Studies have found that polymorphisms in the INHA gene are closely related to the reproductive traits of Holstein cows (Sang et al., 2011; Tang et al., 2011). INHA gene polymorphisms have been shown to be significantly correlated with litter size in several goat breeds. For example, Jining Grey goat does with the genotype GA having 0.79 (p < 0.01) more kids than those with the genotype GG in the g.28315680 locus of the INHA gene (Liu et al., 2017b). For g.28318073C>T of the INHA gene, Nigerian goat does with the genotype CT had 0.33 (p < 0.05) more kids than those with the genotype CC (Isa et al., 2017). These results are similar to those in the present study. In our study, for g.28317663A>C of the INHA gene, the litter size of the AC genotype was significantly higher than that of the AA genotype.

In this study, g.28317663A>C, which is a missense mutation, was significantly associated with litter size in Hainan black goats. INHA missense mutations have also been found to be significantly associated with litter size in Malabari goats (Pillai & Venkatachalapathy, 2020). Missense mutations cause amino acid changes that can alter protein properties (e.g., binding, expression, and protein stability) (Norn, André & Theobald, 2021). For example, studies have found that mutations in the ASMT and ADAMTS1 genes can cause changes in protein structure and affect protein function, which may affect the litter size of goats (Hu et al., 2020). We speculate that the change of amino acids from Thr to Pro may affect the function of INHA protein by impacting the structure of secondary and tertiary proteins. This change may affect the secretion of pituitary FSH, and then affect the concentration of FSH, which may ultimately increase the litter size of Hainan black goats. However, the effect still needs further verification.

In this study, three SNPs of the INHA gene were found to be in linkage disequilibrium in Hainan black goats (Table 4) which include g.28317368G>A, g.28317623C>T, and g.28317663A>C of INHA genes. To the best of our knowledge, our study is the first to analyze the linkage disequilibrium of the SNPs of the INHA gene in Hainan black goats. In addition, linkage disequilibrium in the SNPs of this gene has also been found in Luhua chickens (Cui et al., 2019). If these polymorphisms are in linkage disequilibrium with genes that influence variation in reproductive traits, segregation based on marker alleles will result in phenotypic differences. Therefore, the linkage disequilibrium phenomenon needs to be considered when this gene polymorphism is applied to the breeding of goats with a high litter size.

Due to the ancestral structure captured in the distribution of haplotypes, the haplotypes show more influence than the single SNPs for important trait associations (Akey, Jin & Xiong, 2001). Although no significant correlation was found between haplotype and litter size in the haplotype analysis, we found that H3 and H4 enriched with the g.28317663 C allele had a higher litter size than H1 and H2. The g.28317663 C allele may be related to litter size, which supports g.28317663A>C being a useful genetic marker affecting the litter size of Hainan black goats.

Conclusions

In this study, a total of seven SNPs loci were found in the INHA gene of Hainan black goats, among which g.28317663A>C was significantly associated with litter size. Mutation at this site will cause changes in amino acids from Thr to Pro, affecting the secondary and tertiary structure of INHA protein. The INHA gene is one of the genes that affect the reproductive capacity of Hainan black goats, and the g.28317663A>C loci may be a potential genetic marker for future breeding. It will be nevertheless necessary to expand the sample size to further study the impact of INHA gene polymorphism on the litter size of Hainan black goats.

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