The Agouti signaling protein (ASIP) has been described as a secreted protein expressed mainly in the skin and regulated pigmentation in wild mice (Bultman et al., 1992; Voisey and van Daal, 2002). Mutations in ASIP, such as lethal yellow (Ay) and viable yellow (Avy/a), can lead to an overexpression in various tissues, including adipose and muscle tissue (Bultman et al., 1992; Morgan et al., 1999). The ectopic ASIP expression in mice caused not only yellow fur but also obesity (Klebig et al., 1995). Therefore, another biological function of ASIP was suggested in the regulation of obesity in mice.

In human and farm animals, ASIP is ubiquitously expressed. ASIP mRNA could be detected in human adipose tissue, skin, heart, testis, ovary, liver, and kidney (Wilson et al., 1995). In pigs, the expression of ASIP could be found in liver and muscle (Zhao et al., 2015). Norris and Whan (2008) detected ASIP mRNA in the liver, kidney, skin, heart, and spleen of sheep. Several bovine studies had reported on ASIP mRNA expression in adipose tissue, M. longissimus, skin, heart, testis, ovary, and kidney (Girardot et al., 2005; Albrecht et al., 2012; Liu et al., 2018). Studies on ASIP in farm animals mainly focused on coat color (Kim et al., 2004; Norris and Whan, 2008; Mao et al., 2010; Li et al., 2014; Han et al., 2015; Zhang et al., 2017). Both Girardot et al. (2005) and Royo et al. (2005) found that there was no variant in coding exons of ASIP in different breeds of cattle with different coat color. A full-length element (L1-BT element; line 1) inserted at 5′ upstream of the ASIP gene was identified to give rise to an overexpression of ASIP in the skin of Normande cattle (Girardot et al., 2006). Girardot et al. (2006) concluded that the insertion of the L1-BT element may be the reason for the brindle coat color of the Normande cattle. The authors also presumed that the overexpression of ASIP caused by L1-BT element insertion may influence the meat production in cattle, as mice with ectopic expression of ASIP were obese. Liu et al. (2018) reported that in bulls of an F2 generation (Charolais × Holstein) an extremely high level of ASIP mRNA was observed due to the L1-BT element insertion at ASIP in 17 bulls, but no association with fat-related traits was found. However, there were significant correlations between the ASIP mRNA level of subcutaneous fat and traits related to fat deposition in bulls without an L1-BT element (Liu et al., 2018). Up to now, there is no research yet for L1-BT element insertion at the ASIP locus in Chinese cattle populations. Also, association analyses between single nucleotide polymorphism (SNP) of bovine ASIP and carcass traits and fatty acid composition in cattle have not been reported. Therefore, in this study we scanned for the L1-BT element and further SNPs at the ASIP locus in a population consisting of 363 Chinese Simmental steers. Here we focused on the traits related to fat deposition (e.g., marbling score, back fat thickness) and fatty acid composition, which can provide important information for meat production and quality in cattle. Carcass traits like liver weight and thigh meat thickness were also analyzed, as ASIP, a secreted protein, may circulate and act in the different tissues and parts of the body. The correlation between SNPs and traits was determined to illustrate the potential effect of ASIP in cattle, especially for meat production and meat quality.

In mice, there are five dominant mutations causing ectopic expression of ASIP (Voisey and van Daal, 2002). Insertions of different sizes upstream of first coding exon are the cause for intracisternal A-particle yellow (Aiapy), intermediate yellow (Aiy), sienna yellow (Asy), and viable yellow (Avy) mutation (Duhl et al., 1994; Michaud et al., 1994b; Siracusa et al., 1995). In the case of the Ay mutation, ASIP is controlled by the RALY promoter due to the deletion at both the RALY and the ASIP locus. It happened only in the Ay mutation that the homozygosity of Ay resulted in embryonic lethality (Michaud et al., 1994a). Long interspersed nuclear elements (lines; L1s) known as autonomously mobile DNA sequences belong to autonomous retrotransposon lacking long terminal repeats (LTRs) (Kazazian and Moran, 1998). This is one of the most abundant retrotransposon in eukaryotes (Kazazian and Moran, 1998; Walsh et al., 2013). Girardot et al. (2005) observed L1-BT element insertion upstream of ASIP in bulls. The insertion of the L1-BT element formed a new non-coding exon which resulted in an overexpression of ASIP (Girardot et al., 2005). Both the homozygote and heterozygote of the L1-BT element indel were detected in bulls of the Montbéliarde breed (Girardot et al., 2006). Albrecht et al. (2012) found heterozygote insertion of this element, which gave rise to an ectopic expression of ASIP in different tissues of Japanese Black and Charolais cattle. In our study, three genotypes of the L1-BT element indel were also detected. It is obvious that homozygosity of L1-BT element insertion is not a lethal factor in cattle. Liu et al. (2018) reported that steers with a heterozygote of the L1-BT element indel had an extremely high ASIP mRNA level in various tissues, but it was not associated with any traits related to fat deposition. In the current study, there was no significant difference between GL and GG genotype Chinese Simmental steers in fat-related traits. However, steers with the LL genotype had lower perirenal fat weight, thinner back fat, and less carcass fat coverage percentage compared with other genotypes (P<0.05). According to previous knowledge that ASIP is highly expressed in L1-BT-inserted bulls (Girardot et al., 2006), it can be presumed that in this Chinese Simmental population overexpression of ASIP in steers with the LL genotype showed a negative association with traits related to fat deposition. In a previous study, we found that a heterozygous insertion of L1-BT led to an ectopic expression of ASIP mRNA but not protein (Liu et al., 2018). Hence, further experiments should focus on the expression pattern and effect of ASIP in cattle with the LL genotype.

In this study, we detected the L1-BT element indel and three SNPs at the bovine ASIP locus. Among them, indel of the L1-BT element at g. -14643 nt, g. -568A > G, and g. 4805A > T showed significant associations with traits related to fat deposition (e.g., back fat thickness and carcass fat coverage rate) in 363 Chinese Simmental steers. The mutations of g. -568A > G and g. 4805A > T occurred in introns and did not alter the amino acid sequence of ASIP. However, mutations in introns may also influence the expression and function of a gene by aberrant splicing (Komar, 2007; Shastry, 2009). Coding exons were scanned for SNPs in this study, but no variant of ASIP could be found (data not shown). Likewise, Girardot et al. (2005) and Royo et al. (2005) did not find any SNP of bovine ASIP in coding exons. This suggested that the coding region of bovine ASIP is highly conserved. The analysis of haplotypes is an important component of genetic association analysis. A haplotype consists of a group of linked SNP markers, and it may increase the coverage value of genotype over single SNP analysis (Stram, 2017). The association analysis between haplotype combinations and traits of Chinese Simmental steers indicated that the H1H2 genotype was positively correlated to carcass and meat quality traits, e.g., with a higher carcass weight and thicker back fat. But the H3H3 genotype had a negative association with fat-related traits such as a lower marbling score and perirenal fat weight. Moreover, linoleic acid and α-linolenic acid are essential fatty acids which can only be obtained from the diet (Spector and Kim, 2015). Our results showed that H1H2 steer individuals had a lower content of linoleic acid and α-linolenic acid than the H1H3 and H2H2 genotypes, respectively. These results may illustrate that Chinese Simmental steers with an H1H2 genotype may have more fat deposition in the body but less essential fatty acids, e.g., linoleic acid and α-linolenic acid, in longissimus dorsi.

In summary, both polymorphisms and haplotype analysis of ASIP showed significant correlations with fat-related traits in Chinese Simmental steers. This suggests that ASIP is involved in fat deposition at different stages of the growth and fattening of beef cattle, such as intramuscular fat, back fat, and perirenal fat deposition. The haplotype combinations of H1H2 and H3H3 may be molecular markers for traits related to fat deposition and meat quality (e.g., fatty acid composition). However, further studies are necessary to unravel the mechanism of how the ASIP gene affects meat production and quality in cattle.