Introduction

Rapid development in economy and living standards has remarkably changed the human dietary structure. The current demand for mutton has been gradually increasing due to its low cholesterol and high contents of protein, amino acid, and trace elements.

Dazu black goat, originated in Dazu County, Chongqing, China, is formed by breeding and selection with excellent meat quality. In addition, Dazu black goats are also well known for reproduction traits with high fecundity, big litter size, and precocious sexual maturity. Thus, it can be used as a valuable material to search for genetic markers related to economic traits. Litter size is widely recognized as one of the most important economic parameters for mutton sheep farming. Fortunately, some major genes and genetic variations associated with litter size, including growth differentiation factor 9 (Wang et al. 2019), bone morphogenetic protein receptor, type IB (Shokrollahi and Morammazi 2018), and gonadotropin-releasing hormone receptor (Bemji et al. 2018), have been identified. The development of the whole-genome resequencing technique provided a useful tool to elaborate on genetic factors underlying the formation of economic traits at the whole-genome level. Several studies have been carried out on goat litter size with the whole-genome resequencing (E et al. 2019; Zhang et al. 2019). However, studies on the polymorphisms in untranslated regions (UTRs) are limited (Zhang et al. 2018; Kang et al. 2019; Quan et al. 2019; Yang et al. 2019), and the associations with litter size remain unclear.

UTRs, including 5′ and 3′ UTRs, play crucial roles in regulating gene expression at multiple levels including transcriptional regulation, pre-mRNA processing, mature mRNA transportation, mRNA stability, and translational control. Variations in UTR sequences can affect gene expression and thus phenotypic expression. The association between the polymorphisms in the UTR and goat litter size should be evaluated to further understand the relevant molecular mechanisms. Follistatin-like 4 (FSTL4) is a member of the follistatin gene family and it belongs to transforming growth factor (TGF)-β superfamily inhibitors. FSTL4, ubiquitously expressed in tissues including heart, lung, kidney, testis, neurons, muscle, and intestine, plays multiple roles in biological processes (Tsuchida et al. 2000). Recent studies showed that FSTL4 was a candidate gene for the reproduction ability in dairy cows (Lu et al. 2021). Scavenger receptor class B member 1 (SCARB1), also known as SR-BI, plays an important role in reproduction activity. SCRAB1 is expressed in cells of developing follicles such as theca, granulosa, and cumulus cells. Mice carrying null mutation of the SCARB1 gene (SCARB1^−/−) had ovaries with small corpora lutea (Jiménez et al. 2010).

In this study, based on the data of the published article (E et al. 2019) and expanded the sample size, we explore the association of 26 single nucleotide polymorphisms (SNPs) and one deletion in the UTRs with litter size in Dazu black goats and determine the potential for these polymorphisms as genetic markers in the marker-assisted selection of goat.

Materials and methods

Statistical analyses

Although the limitations of using a small sample size for case-control studies were reported (Clarke et al. 2011), the goat litter size is a strong associated trait with high heritability. Many studies have been proved that relative small sample size could be used to detect key candidate markers (Lai et al. 2016; Islam et al. 2020; Tao et al. 2020; Smołucha et al. 2021). In accordance with the Genome Variant Map data (GVM000039) and litter size record of Dazu goats (E et al. 2019), the genotype frequencies of 27 variants located at the 5′ and 3′ UTRs were measured by Fisher’s test with R script. The genetic effects (GEs) of minimum allele frequency (MAF) were measured via a general linear model with efficient mixed-model association eXpedited (EMMAX, Kang et al. 2010; Legarra et al. 2018). On the basis of the limited number of markers in this study, P < 0.05 was used to filter significant markers. All markers were placed into fixed effect estimated matrix to explain the phenotype variances:

where Y is a vector of phenotype values; μ is the average vector; the length is the same as the number of individuals (n); α is a vector of the markers’ estimated effect; X is a designed matrix in which the dimension is n × m, where m is the number of markers; and e is the residual matrix.

Deviations of SNPs from the Hardy–Weinberg equilibrium (HWE) within populations were identified using Arlequin software version 3.5.1.3 (Excoffier and Lischer 2010).

Results and discussion

In this study, the genotype frequencies of 27 variants located at the 5′ and 3′ UTRs were significantly different between high and low yields of litter size by Fisher's test according to previously published wide-genome data set (Table 1, Genome Variant Map data: GVM000039). This finding suggests that the series of SNPs have a strong correlation with goat litter size. In addition, the genotype data of 27 variants of all individuals were uploaded to European Variation Archive (Project_PRJEB35784, Analyses_ERP118896). Based on the data analysis, we further explore the correlation between 27 variant loci and litter size by expanding the sample size of Dazu goat. Analysis of all 27 variant loci showed that the majority of variants carry high levels of polymorphism, as revealed by H_E, H_O, and PIC. Among these variants, SNP_chr11-70706856 and SNP_chr12-74985766 were homozygous and excluded from the further study (Table 2). According to HWE, seven SNPs within the population deviated from equilibrium, thus suggesting that these SNPs may be subject to artificial selection. The dominant genotype frequencies of the relevant trait-determining sites were fixed due to artificial selection within the population during long-term breeding.

Table 1.

Twenty-seven variants with significant divergence between high and low yields of litter size in Dazu black goats based on a genome-wide selection data set.

Table 2.

Polymorphisms of 27 variants within the Dazu black goat population.

Correlation analysis showed that two SNP loci, namely, SNP_chr17-20182525 (P = 0.0472, GE [MAF] = −0.1908) and SNP_chr7-65652612 (P = 0.0493, GE [MAF] = −0.2860), which were located at the 3′ UTR of SCARB1 (XM_018061401.1: c.*403G > A) and FSTL4 (XM_018050393.1: c.*741C > G), respectively (Table 3), were significantly correlated with the litter size of first parity goats (LF). No SNP was significantly associated with the litter size of second-born animals (LS). The sum litter size of first and second parity animals (SL) was evaluated, and correlation analysis of all SNPs with the first and second parity litter size was performed. The results showed that SNP_chr7-65652612 was significantly associated with SL (P = 0.0118, GE (MAF) = −0.6357, Table 3).

Table 3.

Correlation analysis of 24 SNP variations with litter size in Dazu black goats.

SCARB mediates the liver uptake and bile secretion of high-density lipoprotein cholesterol, thereby promoting reverse cholesterol transport and slow lipoprotein aggregation; thus, SCARB has antiatherosclerotic effects (Takiguchi et al. 2018; Lee et al. 2019). Previous studies have shown that the SNP rs5888 in the SCARB1 gene is associated with serum lipid levels in a sex-specific manner (Morabia et al. 2004). SCARB1 has been involved in osteoblast differentiation (Tourkova et al. 2019), the molecular immune mechanism of allicin (Toma et al. 2019), and reproduction performance (Jiménez et al. 2010). However, no direct evidence supports the involvement of SCARB1 in goat litter size.

SNP_chr7-65652612 in the 3′ UTR of FSTL4 was not only significantly associated with the litter size of first parity goats and the total litter size of first and second parity goats but it was also a factor promoting significant differences in litter size between different genotypes (Table 4). FST is a mono-affinity glycoprotein that binds to many members of the TGF-β superfamily via paracrine and autocrine signaling, hence critical to female animal reproduction activity (Passos et al. 2013; Cannarella et al. 2019). A series member of FSTL has been found to play a role in reproduction. FSTL3 levels could be attributed to pre-eclampsia, and they are associated with an increased likelihood of developing pre-eclampsia (Founds et al. 2015). Moreover, FSTL3 could limit the age-related degeneration and size of testicles (Oldknow et al. 2013). FSTL4 was recently shown to be a candidate for the reproduction performance in dairy cows (Lu et al. 2021).

Table 4.

Genotype frequency and litter size differences between genotypes of candidate SNPs.

Natural selection is the main cause of genetic polymorphism. The diversity of genetic variation in population is the basis for phenotype difference. In this study, 27 gene polymorphisms may lead to changes in protein function, resulting in differences in litter size of Dazu black goat. At present, many studies have concluded that genetic variation is related to economic traits of animals. IGF1R c.654g > a variation had significant effects on many economic traits of Polish colored Merino sheep (Grochowska et al. 2020). The variation of FABP4 is related to the milk production traits of Greek Sfakia sheep (Ibrahim et al. 2019). The sample size in the study is a key parameter for detection of candidate marker. Although the sample size in this study is much larger than other reported studies (Lai et al. 2016; Islam et al. 2020; Tao et al. 2020; Smołucha et al. 2021), expansion of sample size will surely enhance detection power and reduce false positive rate.

In conclusion, this study explored 26 SNPs and one deletion variation in the UTR and found that two SNPs were associated with high fecundity of Dazu black goats. The results could contribute to revealing the genetic mechanism underlying the high reproduction performance of Dazu goats.