The effect of single-nucleotide polymorphisms within heat shock protein beta 1 on beef quantity in Korean native steers

Abstract Heat shock protein beta 1 (HSPB1), a member of the heat-shock family of protein, is a relatively small (27 kDa) molecular chaperone protein associated with cellular development. The relationship between HSPB1 expression and muscle growth in beef cattle has previously been reported, but there have been no reports of DNA markers related to meat quantity in Korean native steers. Therefore, the aim of this study was to evaluate the relationship of single-nucleotide polymorphisms (SNPs) within HSPB1 in terms of the carcass traits related to muscle growth in Korean native steers. Through direct sequencing, we discovered three SNPs: g.111 T > C SNP (rs208395876) and g.2548 C > G SNP (rs483014585) were respectively located in 5′ UTR (untranslated region) and 3′ UTR. Further, g.2352 T > C SNP (rs110832311) was located in the adjacent region of the RNA splicing site. The least square means of steers with a CC genotype of g.111 T > C SNP had a significantly higher meat ratio (P = 0.04), while the least square means of steers with a CC genotype of g.2352 T > C SNP had a significantly higher meat ratio (P = 0.002) and lower back-fat thickness (P = 0.004) than those of the other genotype. Moreover, although the least square means of steers with CC-CC, CT-CC, and TT-CC genotypes were significantly decreased for back-fat thickness, they were significantly increased for the meat ratio. Therefore, our results suggested that g.111 T > C SNP and g.2352 T > C SNP could be a causal mutation related to an adipose metabolism in Korean cattle steer.


Introduction
Recently, several studies have evaluated the relationship between heat-shock protein beta 1 (HSPB1) and muscle growth (Dubińska-Magiera et al., 2014;Hamelin et al., 2006). HSPB1 is a 27 kDa small heat-shock protein that is expressed in many vertebrate tissues, particularly muscle. The general functions of HSPB1 in muscle are to protect cells from physiological stress, inhibit cell death, and chaperone activity (Arrigo, 2017). In previous studies, HSPB1 has also been shown to be relevant in terms of feed efficiency (Jung et al., 2017). HSPB1 is also involved in muscle development in many species, including those reported in Dubińska-Magiera et al. (2014). HSPB1 has been shown to be related to muscle hypertrophy in vivo (Hamelin et al., 2006). In a recent study, the expression of HSPB1 was found to be significantly increased during bovine myogenic differentiation compared to in the un-differentiation stage in myogenic cells, and it was also shown to be regulated by androgen-mediated myogenesis (Zhang et al., 2012(Zhang et al., , 2014. In the beef industry, efficient muscle growth and development in cattle is critical for meat production (He et al., 2017).
Myogenesis is regulated by several myogenic genes, such as MyoD, Myogenin, MRF4, and Desmin (Charge and Rudnicki, 2004). These myogenic marker genes are known to be upregulated during myogenic differentiation (Sweetman, 2012). HSPB1, a major factor of actin polymerization in muscle, is known to play a role in maintaining muscle structure (Sugiyama et al., 2000). HSPB1 also shows chaperone function, including preventing protein degradation, inhibiting muscle atrophy, and stabilizing muscle protein (Tucker and Shelden, 2009). It has been speculated that HSPB1 enhances muscle development by protecting muscle proteins (Perng et al., 1999). In a recent study, the inhibition of HSPB1 expression during myogenesis was shown to repress the expression of MyoD, Myogenin, and Desmin; formation of myotubes; and protein synthesis . A relationship has also been reported between HSPB1-10 expression and muscle growth in beef cattle, but there have been no reports of DNA markers related to meat quantity in Korean native steers. Therefore, the aim of this study was to evaluate the relationship of SNPs within HSPB1 with carcass traits related to muscle growth in Korean native steers.

Animals, DNA extraction, SNP discovery
A total of 192 Korean cattle (body weight BW = 464.68 ± 45.09 kg; 32.18 months old [SD1.0]) raised in Pyeongchang (Gangwon, Republic of Korea) were used in this study. All of the steers were maintained under constant environmental conditions, such as having access to two types of commercial feeds at six feedlots. Muscle tissues were sampled from slaughtered individuals. Therefore, there is no certificate of animal ethics. Genomic DNA was extracted from longissimus dorsi muscle tissue using a LaboPass™ tissue mini kit (Cosmo Genetech, Seoul, Korea). In order to discover SNPs, the bovine HSPB1 sequence was obtained from the NCBI database (AC_000182.1). The primer sequence was designed using NCBI Premer-BLAST based on the selected polymorphism sites, and the primer information is shown in Table 1. The sequencing was performed as outlined in a previous study , and SNPs were discovered using the Sequencer v5.2.4 program (Gene Codes Corp., Ann Arbor, MI). In order to map the functional SNPs on DNA, mRNA, and protein sequences, they were aligned using the Graphical View Legend on the NCBI database.

SNP genotyping and statistical analysis
Large-scale SNP genotyping was performed commercially using the Fluidigm ® SNP™ Type assay as described in a previous study (Oh et al., 2018). In order to evaluate the association SNPs and carcass traits, the data were analyzed using the general linear model (GLM) of SPSS v. 22 (IBM, USA). The model is as follows: where Yijkl is a phenotype of the carcass trait, µ is the overall mean for each trait, Pi is the feed type in farms, GSj is the random effect of the sire, SNPk is the fixed effect of SNP genotype, βagelis the covariance of days bred, and eijkl is the random error.

Results and discussion
Bovine HSPB1 has been shown to be closely associated with cell growth and bovine myogenesis in certain types of muscles. Thus, based on the results reported by Kim et al. (2018), we found that SNPs, which are directly regulated by gene expression, were discovered, and then we identified the relationship of their SNPs with beef quantity. The position of SNP in DNA, RNA, and the protein sequence of the HSPB1 gene as well as their alignments are shown in Fig. 1. In the present study, we discovered three SNPs using direct sequencing. These SNPs were located in 5 UTR, intron 2, and 3 UTR; g.111 T > C SNP (rs208395876) and g.2548 C > G SNP (rs483014585) are in 5 UTR and 3 UTR, while g.2352 T > C SNP (rs110832311) is in the region adjacent to the RNA splicing site. However, SNPs were not discovered in the exon region.
The multiple and single effects of SNPs within HSPB1 on carcass traits, such as beef quantity, are shown in Tables 1 and 2. A previous study (unpublished data) demonstrated that the g.2548 C > G SNP (rs483014585) had no effects on carcass traits, and we thus removed the related data from the present study, as we could not use it as evidence to support our result. As shown in Table 1, g.111 T > C SNP was significantly associated with meat ratio (P > 0.05). The least square mean in the steer group with a CC genotype of g.111 T > C SNP was significantly higher than that in the steer group with other genotypes. Moreover, g.2352 T > C SNP was significantly associated with back-fat thickness and meat ratio (P < 0.01). Regarding back-fat thickness, the least square mean in the steer group with the CC genotype of g.2352 T > C SNP was significantly lower than that in the steer group with other genotypes. On the other hand, in terms of meat ratio, this group was significantly higher than that in the steer group with other genotypes.
As shown in Table 2, consistent with the results of a single effect, the combination genotype of g.111 T > C and g.2352 T > C SNPs was significantly associated with back-fat thickness and meat ratio (P < 0.01). The steer group with combi-  nation genotypes of CC-CC, CT-CC, and TT-CC had a low least square mean for back-fat thickness compared to the steer group with other combinations. On the other hand, the steer group with the combination genotype had the highest least square mean for the meat ratio. HSPB1 is expressed in many vertebrate tissues, particularly muscle. In a previous study, Zhang et al. (2012) demonstrated that the HSPB1 expression level was higher in the skeletal muscle of bulls than that of steers. Thus, in this study, the steer group in Korean cattle was used. As shown in Tables 1 and 2, although there were no significant differences between these SNPs and the rib-eye area, the mean in the group with the CC genotype of g.2352 T > C SNP and combi-nation genotypes of CC-CC, CT-CC, and TT-CC were found to be numerically higher than that in other groups.
The cellular development associated with adipose tissue growth involves both cellular hypertrophy (increase in size) and hyperplasia (increase in number). Rajesh et al. (2010) reported that HSPB1 interact with insulin-like growth factor receptor 1 and its signal transducer, the serine/threonine kinase Akt, which together modulate an adipocyte metabolism. Specifically, the previous results suggested that HSPB1 was negatively correlated with an adipose metabolism (Kim et al., 2011). Therefore, our results regarding the least square mean of back-fat thickness and the rib-eye area were found to be similar. In a previous study, it was reported that g.111. T > C SNP (rs208395876) was functional. In particular, our results identified that g.2352 T > C SNP was located in the acceptor site of the RNA splicing region.
Therefore, our results suggested that g.111 T > C SNP and g.2352 T > C SNP could be causal mutations related to an adipose metabolism in Korean cattle steer.

Conclusions
We discovered three SNPs, including g.111 T > C, g.2352, and g.2548, which are respectively located in 5 UTR, intron 2, and 3 UTR of the HSPB1 protein. Animals with a CC genotype of g.111 T > C SNP had a significantly higher meat ratio, and animals with a CC genotype of g.2352 T > C SNP had a significantly higher meat ratio and lower backfat thickness than those of other genotypes. Moreover, for the combination of g.111 T > C and g.2352 T > C SNPs, the CC-CC, CT-CC, and TT-CC genotypes' back-fat thicknesses were found to decrease while the meat ratios increased. In particular, a g.2352 T > C SNP was found to be located in the acceptor site of the RNA splicing site. Therefore, our results indicate that it could be a causal mutation related to an adipose metabolism in Korean cattle steer.