Association of Calpain 1 ( CAPN1 ) and HRSP12 allelic variants in beef cattle with carcass traits

Calpain 1 ( CAPN1 ) and its activator HRSP12 are evaluated as candidate gene for quantitative trait loci (QTLs) affecting meat tenderness. In this study, SNPs were detected by sequencing in 323 cattle from 9 breeds. The association results showed that the A3553G and T824C loci individually related with marbling and tenderness, and CAPN1 / HRSP12 double homozygote and heterozygote/homozygote pairs (AA/TT, AA/CC, AG/TT and GG/TT) had higher marbling score than the other groups. Our findings suggest that polymorphisms in CAPN1 and HRSP12 might be the important genetic factor influencing meat quality in carcass trait.


INTRODUCTION
Meat tenderness is an important component of palatability, whereas the difficulty in obtaining phenotypic data makes it hard for it to be selected for this trait.Establishing the genetic basis for variation in meat tenderness would likely aid in the development of selection criteria for improving meat tenderness in cattle.
Protein hydrolysis is closely related to muscle growth during the development and meat tenderness after slaughter.Calpain I (CAPN1) is an important protease that hydrolyzes proteins in myofibrils (Koohmaraie, 1996).The micromolar calcium-activated neutral protease (CAPN1) gene encodes a cysteine protease, µ-calpain, which degrades myofibrillar proteins under postmortem conditions and appears to be the primary enzyme in the postmortem tenderization process (Koohmaraie, 1992(Koohmaraie, , 1994)).Hundreds of allelic variants (SNPs) of CAPN1 investigated as potential candidate gene on BTA29 affecting meat tenderness are identified in bovine (Smith et al., 2000;Casas et al., 2003;Page et al., 2002), and some of them are found to have significant effects on tenderness (Casas et al., 2003;Juszczuk-Kubiak et al., 2004;Page et al., 2004;White et al., 2005;Morris et al., *Corresponding author.E-mail: simmenta@yahoo.com.cn. 2006; Rincon et al., 2006;Van et al., 2007).Current studies have shown that CAPN1 is a potential gene or linked with a major gene which controls meat tenderness (Casas et al., 2005(Casas et al., , 2006)).HRSP12 (UK114) is a Calpain1 activator of low molecular weight that is distinct from CaM.This heat responsive protein stimulates the hydrolysis activity of Calpain1 (Melloni et al., 1998).Under low calcium concentration, HSRP12 can activate Calpain1 that participates in muscle growth (Fox et al., 1985).HRSP12 gene is located at BTA 19 and the relationship between its polymorphisms and carcass traits in bovine has not been reported yet.
In order to identify the candidate genetic markers for commercial traits in cattle, we investigated polymerphisms in two carcass trait candidate genes CAPN1 and HRSP12, and explored the relationship between genotypes and carcass traits.

MATERIALS AND METHODS
The samples were collected from Inner Mongolian Sanyuan Cattle Farm (46 head of Angus sires, 30 head of Hereford sires and 59 head of Simmental sires), Hebei Dachang Cattle Farm (24 head of Jinnan sires, 25 head of Limousin sires, 29 head of Qinchuan sires, 28 head of Charolais sires and 30 head of Luxi sires) and Inner Mongolian Baolongshan cattle farm (52 head of Simmental × Menggu crossbreds).Carcasses were stored in refrigerated rooms at a temperature of 0 to 4°C for 24 h.Then, all the carcass traits were measured based on the criterion of GB/T17238-1998 cutting standard of fresh and chilled beef of U.S. (U.S.Standard Publishing House).Blood samples (10 ml each) were collected from the vein with anticoagulant ACD and stored in -70°C.DNA was extracted from 1 ml whole blood with the Tiangen DNA Blood Kit (Tiangen Biotech Co., Ltd., Beijing) by the manufacturer's directions.

Data analysis
Genotype and allele frequencies were calculated for all the SNPs of the CAPN1 and HRSP12 gene, and the overall sample was set with Genepop (Raymond et al., 1995).
Evaluation of association between the two SNP genotypes at each polymorphic locus and carcass traits were carried out with the general linear model (GLM) in SAS (Version 9.1, SAS Inst., Inc., Cary, NC, 2002to 2003).The linear model is as follows: YijK = µ + BFi + Monthj + Gk + eijk , where, Yijk is the observed value; µ is the overall mean for each trait; BFi is the fixed effect of i th breed and farm; Monthj is the fixed effect of j th month of slaughtering; Gk is the fixed effect of k th single SNP marker genotype and eijk is the random error.

Genotyping and frequencies
Sequencing result showed that there was A/G substitution at position 3553 of CAPN1 and missense T/C substitution at position 824 of HRSP12 which replaced lysine acid with argnine acid.Among the 9 breeds of cattle, G allele was predominant at A3553G locus, while genotype AA was not found in Hereford, Limousin, Luxi and Charolais cattle.For T824C locus, allele T was predominant in all breeds except Jinnan and Luxi cattle, whereas no genotype TT was observed in these two breeds (Table 1).

Association of SNPs with carcass traits
Correlations of the CAPN1 and HRSP12 SNPs genotypes Hou et al. 13715 with 14 carcass traits were analyzed with GLM procedure in SAS, and F test was used to generate P value.Table 2 indicated trait values for each genotype in the 9 breeds.For locus A3553G, genotype AA had higher marbling score than genotypes AG and GG (P<0.05);genotype AA had higher tenderness value than genotype GG (P<0.01).There were no significant differences among genotypes at locus A3553G in all other traits.For locus T824C, genotype CC was heavier in live weight and meat weight than genotype TC (P<0.05), and the same circumstances emergence in carcass weight (P<0.01), while genotype TT has higher marbling score than genotypes TC and CC (P<0.01).No distinct difference was found between HRSP12 genotypes and other meat parameters.
When both CAPN1 and the HRSP12 genotypes of each sample were considered together as a compound genotype (AA/TT, AA/TC, AA/CC, AG/TT, AG/TC, AG/CC, GG/TT, GG/TC and GG/CC), double homozygote alleles samples (AA/TT and AA/CC) were found to have significantly higher marbling scores (P<0.01,Table 3).Those who were CAPN1 heterozygous and HRSP12 homozygous (AG/TT) or other double homozygous (GG/TT) also had significantly higher marbling score (P<0.05).No significant association was found between the compound CAPN1/HRSP12 genotypes and tenderness (P>0.10).

DISCUSSION
Meat quality is of commercial importance for the animal husbandry industry.It is affected by genetic background, management, nutrition and meat processing.Calpain proteases participate in protein metabolism during animal development and muscle maturation after slaughter; therefore the genes encoding these enzymes have been studied widely as candidates for meat tenderness.In cattle, the relationship between CAPN1 and carcass traits is investigated intensively.More and more studies demonstrates that CAPN1 is remarkably correlated to beef tenderness (Koohmaraie, 1992(Koohmaraie, , 1994;;Smith et al., 2000;Casas et al., 2003;Page et al., 2002;Juszczuk-Kubiak et al., 2004).
Population genetic analysis indicated that allele G was predominant for A3553G locus among 9 breeds.Genotype GG was predominant in all breeds except Angus and Simmental.Genotype AA was not found in Hereford, Limousin, Luxi and Charolais populations, which may result from genetic background and sample size analyzed.For the T824C locus, allele T sample frequently existed in Angus, Hereford, Simmental and Charolais populations; sample with genotype TT was the highest in these breeds.Allele C emergence was more frequent in local breeds (Jinnan and Luxi populations).The results illustrated that the foreign breeds were opposite to the local breeds in the predominant genotype at T824C locus, which might be related to the origin and breeding.
The marbling property was directly linked to meat tenderness and texture, which was a very important indicator of meat quality.Our results showed that A3553G locus was correlated to marbling and tenderness.Genotype AA was different from AG and GG genotype in marbling trait (P<0.05), and markedly different from GG in tenderness (P<0.01).These results are consistent with other reports ( (Koohmaraie, 1992(Koohmaraie, , 1994;;Smith et al., 2000;Casas et al., 2003;Page et al., 2002;Juszczuk-Kubiak et al., 2004;Morris et al., 2006).Significant differences were also found among different genotypes at T824C locus in marbling index (P<0.01).
Therefore, both CAPN1 and HRSP12 are correlated to marbling trait.On the other hand, the homozygous/ homozygous (AA/TT and AA/CC) and heterozygous/ homozygous (AG/TT and GG/TT) compound CAPN1/HRSP12 SNP genotypes that were associated with marbling parameters presented higher score than samples with other genotypes.The two genes may be potentially functional genes influencing carcass traits, and the identification of such susceptibility genes may therefore be important marks in beef cattle breeding projects.

Conclusion
An experimental analysis in our study indicated that polymorphism in CAPN1 and HRSP12 was moderate and the genotype distributed in nine breeds was found to have marked differences in meat tenderness and marbling properties.This study would also be a foundation for further research of the beef cattle molecular assistant selection.Research Program (grant no.nyhyzx07-035-07).

Table 1 .
Genotype frequencies of A3553G and T824C loci in 9 cattle breeds.

Table 2 .
Analysis of the relationship between genotypes and productive performances using the least square method.
Data are least square means ± standard error.Values with different lower case scripts in the same row are significantly different at P<0.05, with extremely different at P<0.01.*: P<0.05, **: P<0.01.

Table 3 .
Significance of association of marbling score and tenderness with combined genotypes.