AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-63-417-2020The effect of single-nucleotide polymorphisms within heat shock protein beta 1 on beef quantity in Korean native steersThe effect of SNPs within HSPB1 on beef quantitySuhJung-KeunLeeJae-SungKongHongsikLeeYoonseokyoonseok95@hknu.ac.krLeeHong-GuDepartment of Animal Science and Technology, Sanghuh College of Life
Sciences, Konkuk University, Seoul, 05029, Republic of KoreaTeam of An Educational Program for Specialists in Global Animal
Science, Brain Korea 21 Plus Project, Sanghuh College of Life Sciences,
Konkuk University, Seoul, 05029, Republic of KoreaDepartment of Biotechnology,
Hankyung National University, Anseong-si, Gyeonggi-do, 17579, Republic of
KoreaCenter for Genetic Information, Hankyung National University, Anseong-si, Gyeonggi-do, 17579, Republic of Korea
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, 2014).
Position of SNPs within the HSPB1
gene. (a) Black and grey boxes represent UTR and exon, respectively. (b) Position of g.2352 SNP (*) in RNA splicing site. Grey boxes represent exon 2
and exon 3 of HSPB1 gene. The italic letters represent the intron 2 region. Underscores refer to the donor and
acceptor sites of the RNA splicing site.
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 (Kim et al., 2018). 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.
Materials and methodsAnimals, 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 (Lee et al., 2010), 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.
The single effect of g.111 T > C SNP and g.2352
T > C SNP within HSPB1 gene on carcass
traits in a commercial Korean native steer population.
Carcass traitsGenotype (number of animals) g.111 T > C SNP g.2352 T > C SNP CC (6)CT (66)TT (118)p valueCC (42)CT (91)TT (57)p valueBack-fat thickness, mm12.5 ± 1.6513.15 ± 0.5114.45 ± 0.380.08412.09 ± 0.63a14.48 ± 0.42b14.31 ± 0.53b0.004Rib-eye area, cm298 ± 3.6494.92 ± 1.1194.23 ± 0.840.56296.31 ± 1.493.96 ± 0.9594.41 ± 1.180.362Carcass weight, kg465.18 ± 17.2456.29 ± 5.27464.69 ± 3.990.428459.64 ± 6.65464.92 ± 4.5461.64 ± 5.630.776Meat ratio*65.17 ± 1.2364.58 ± 0.3863.50 ± 0.290.0465.37 ± 0.47a63.44 ± 0.32b63.68 ± 0.4b0.002
a,b Means with the same superscript in the same row for each
quality are not significantly different (P < 0.05).
* Meat ratio = 68.184–[0.625 × back-fat thickness (mm)] + [0.13 × rib-eye area (cm2)]–[0.024 carcass
weight (kg)]. The Korean native cattle meat ratio has an additional 3.23 added
after calculation.
The multiple effect of genotype combination of pairwise
SNPs on carcass traits in commercial Korean native steer population.
a,b Means with the same superscript in the same row for each
quality are not significantly different (P < 0.05).
* Meat ratio = 68.184–[0.625 × back-fat thickness (mm)] + [0.13 × rib-eye area (cm2)]–[0.024 carcass
weight (kg)]. The Korean native cattle meat ratio has an additional 3.23 added
after calculation.
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:
Yijkl=μ+Pi+GSj+SNPk+βagel+eijkl,
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 combination 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 combination 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 back-fat
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.
Primer information used in this study.
SNPdbSNP no.Primer sequence TmProductPrimer sequence for Fluidigm SNP genotyping*(∘)size (bp)g.111 T > Crs208395876FAAGGTTCCAGATGTGGGCAG62781ASP1CGCCCGCCACTTCTCCRACCAGGGGTTGGGCGAAGAGASP2CGCCCGCCACTTCTCTLSPGGCCATGCTGGCTGGTCSTACCATAAAAGCGCTGCGGGg.2352 T > Crs110832311FCAGTCTCGGGCACCCAGATC60783ASP1CTACCCTCTTTGCCCGTCTRAGTGACGGATGGCACTGCACASP2ACCCTCTTTGCCCGTCCLSPGGGTGGGGTCCACACCGSTAGACGCCCTTGTGTGTAACTg.2548 C > Grs483014585FCAGTCTCGGGCACCCAGATC60783ASP1AACAGCCGGAAAACAAGTAAAGACRAGTGACGGATGGCACTGCACASP2GAACAGCCGGAAAACAAGTAAAGAGLSPTGGGCCGCTGGGCTAASTACCCCGAAGCCGGGAAG
* ASP1: allele specific primer 1, ASP2: allele specific primer 2, LSP: locus specific primer, STA: specific target amplification.
Data availability
The original data of the paper are available from the corresponding author
upon request.
Author contributions
JKS performed the research. JKS and YL performed the data analyses and wrote
the manuscript. JSL and HK revised the manuscript. YL and HGL designed the
experiment. All authors reviewed and approved the final paper.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by a grant from the Next-Generation BioGreen 21
Program (project no. PJ013322012019), Rural Development Administration,
Republic of Korea.
Financial support
This research has been supported by the Next-Generation BioGreen 21 Program (grant no. PJ013322012019).
Review statement
This paper was edited by Steffen Maak and reviewed by Jaedon Oh and one anonymous referee.
ReferencesArrigo, A.-P.: Mammalian HspB1 (Hsp27) is a molecular sensor linked to the physiology and environment of the cell, Cell Stress Chaperon., 22,
517–529, 2017.
Charge, S. B. and Rudnicki, M. A.: Cellular and molecular regulation of
muscle regeneration, Physiol. Rev., 84, 209–238, 2004.
Dubińska-Magiera, M., Jabłońska, J., Saczko, J., Kulbacka, J.,
Jagla, T., and Daczewska, M.: Contribution of small heat shock proteins to
muscle development and function, FEBS Lett., 588, 517–530, 2014.
Hamelin, M., Sayd, T., Chambon, C., Bouix, J., Bibé, B., Milenkovic, D.,
Levéziel, H., Georges, M., Clop, A., and Marinova, P.: Proteomic
analysis of ovine muscle hypertrophy, J. Animal Sci., 84,
3266–3276, 2006.
He, H., Chen, S., Liang, W., and Liu, X.: Genome-wide proteomics analysis on
longissimus muscles in Qinchuan beef cattle, Anim. Genet., 48, 131–140,
2017.Jung, U., Kim, M., Wang, T., Lee, J., Jeon, S., Jo, N., Kim, W., Baik, M.,
and Lee, H.: Upregulated heat shock protein beta-1 associated with caloric
restriction and high feed efficiency in longissimus dorsi muscle of steer,
Livest. Sci., 202, 109–114, 2017.
Kim, N.-K., Lim, D., Lee, S.-H., Cho, Y.-M., Park, E.-W., Lee, C.-S., Shin,
B.-S., Kim, T.-H., and Yoon, D.: Heat shock protein B1 and its regulator
genes are negatively correlated with intramuscular fat content in the
Longissimus Thoracis muscle of Hanwoo (Korean cattle) steers, J.
Agr. Food Chem., 59, 5657–5664, 2011.Kim, Y.-S., Lee, J.-S., Lee, Y., Kim, W.-S., Peng, D.-Q., Bae, M.-H., Jo,
Y.-H., Baik, M., and Lee, H.-G.: Effect of glutamine on heat-shock protein
beta 1 (HSPB1) expression during myogenic differentiation in bovine embryonic
fibroblast cells, Food Sci. Biotechnol., 27, 829–835, 2018.
Lee, Y.-S., Oh, D., Kim, J.-J., Lee, J.-H., Park, H.-S., and Yeo, J.-S.: A
single nucleotide polymorphism in LOC534614 as an unknown gene associated
with body weight and cold carcass weight in Hanwoo (Korean cattle),
Asian-Austr. J. Animal Sci., 23, 1543–1551, 2010.
Oh, D.-y., Nam, I., Hwang, S., Kong, H., Lee, H., Ha, J., Baik, M., Oh, M.
H., Kim, S., and Han, K.: In vivo evidence on the functional variation
within fatty acid synthase gene associated with lipid metabolism in bovine
longissimus dorsi muscle tissue, Genes Genom., 40, 289–294, 2018.
Perng, M. D., Cairns, L., van den IJssel, P., Prescott, A., Hutcheson, A.
M., and Quinlan, R. A.: Intermediate filament interactions can be altered by
HSP27 and alphaB-crystallin, J. Cell Sci., 112, 2099–2112, 1999.
Rajesh, R. V., Kim, S.-K., Park, M.-R., Nam, J.-S., Kim, N.-K., Kwon, S.,
Yoon, D., Kim, T.-H., and Lee, H.-J.: Proteomic Functional Characterization
of Bovine Stromal Vascular Cells from Omental, Subcutaneous and
Intramuscular Adipose Depots, Asian Austral. J. Anim.,
24, 110–124, 2010.
Sugiyama, Y., Suzuki, A., Kishikawa, M., Akutsu, R., Hirose, T., Waye, M.
M., Tsui, S. K., Yoshida, S., and Ohno, S.: Muscle develops a specific form
of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during
myogenic differentiation, J. Biol. Chem., 275, 1095–1104,
2000.
Sweetman, D.: The myogenic regulatory factors: critical determinants of
muscle identity in development, growth and regeneration, Intech, 31–39, 2012.
Tucker, N. R. and Shelden, E. A.: Hsp27 associates with the titin filament
system in heat-shocked zebrafish cardiomyocytes, Exp. Cell Res.,
315, 3176–3186, 2009.
Zhang, Q., Lee, H.-G., Han, J.-A., Kang, S. K., Lee, N. K., Baik, M., and
Choi, Y.-J.: Differentially expressed proteins associated with myogenesis
and adipogenesis in skeletal muscle and adipose tissue between bulls and
steers, Mol. Biol. Rep., 39, 953–960, 2012.
Zhang, Q., Lee, H.-G., Kang, S. K., Baik, M., and Choi, Y.-J.: Heat-shock
protein beta 1 regulates androgen-mediated bovine myogenesis, Biotechnol.
Lett., 36, 1225–1231, 2014.