Effect of the calpain small subunit 1 gene (CAPNS1) polymorphism on meat quality traits in sheep

Accepted Manuscript Title: Effect of the calpain small subunit 1 (CAPNS1) gene polymorphism on meat quality traits in sheep Authors: E. Grochowska, B. Borys, E. Grze´skowiak, S. Mroczkowski PII: DOI: Reference:

S0921-4488(17)30058-5 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.02.022 RUMIN 5438

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

27-2-2016 16-11-2016 27-2-2017

Please cite this article as: Grochowska, E., Borys, B., Grze´skowiak, E., Mroczkowski, S., Effect of the calpain small subunit 1 (CAPNS1) gene polymorphism on meat quality traits in sheep.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2017.02.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of the calpain small subunit 1 (CAPNS1) gene polymorphism on meat quality traits in sheep

E. Grochowskaa,*, B. Borysb, E. Grześkowiak c, S. Mroczkowskia

a

Department of Genetics and General Animal Breeding, UTP University of Science and

Technology, Poland National Research Institute of Animal Production, Experimental Station Kołuda Wielka,

b

Poland c

Institute of Agricultural And Food Biotechnology, Poland

*Corresponding author. Ewa Grochowska, Department of Genetics and General Animal Breeding, UTP University of Science and Technology, Mazowiecka 28 St., 85-084, Bydgoszcz, Poland. E-mail address: [email protected] (E. Grochowska).

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HIGHLIGHTS 

Polymorphism of the ovine CAPNS1 and its effect on meat quality were investigated.

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Two C/T substitutions, one in intron 5 and one in exon 6, were found.

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The CAPNS1 genotype had highly significant effect on natural drip loss.

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Effect of CAPNS1 genotype on lightness was significant.

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Effect of CAPNS1 genotype on intramuscular fat content was significant.

Abstract The aims of this study were to investigate the polymorphism in the calpain small subunit 1 gene (CAPNS1) and evaluate the associations of this gene’s variants with meat quality traits in Coloured Polish Merino sheep. A 190-bp fragment of exons 5 and 6 including intron region of the CAPNS1 gene was amplified and subjected to MSSCP method to screen for polymorphisms. Traits of interest for this study were: muscle pH 24 hours post mortem (pH24h), reflectance coordinates (L*, lightness; a*, redness; b*, yellowness), water content (WC), intramuscular fat content (FC), total protein content (PC), water-holding capacity (WHC), Warner Bratzler shear force (WBSF), drip loss (DL), and cooking loss (CL). Two C/T substitutions, one in intron 5 and one in exon 6, were found. Three alleles named A1, B1 and C1 and six CAPNS1 genotypes: A1A1, A1B1, B1B1, A1C1, B1C1 and C1C1 were detected. The A1 allele and A1B1 genotype were predominant with the frequencies of 0.467 and 0.419, respectively. The CAPNS1 genotype was found to have highly significant (P=0.006) and significant (P=0.037 and P=0.039) effects on natural drip loss, lightness and intramuscular fat content, respectively. The CAPNS1 gene variants also tended to associate (P=0.056) with cooking loss. Results of this study confirmed that CAPNS1 gene could be considered as a candidate gene of meat quality traits in sheep and may affect lamb quality parameters.

Keywords: calpain small subunit 1, CAPNS1, sheep, meat quality traits, MSSCP, polymorphism

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1. Introduction Meat quality parameters, such as colour, intramuscular fat content or marbling, and water-holding capacity, are not only factors influencing customer’s decision about purchasing a meat cut but also important traits for the meat processing industry. Therefore the improvement of meat quality traits should be one of the most important tasks in lamb, beef and pork production. Among many factors affecting meat quality, effects of candidate genes variations still need to be investigated especially in sheep. Identification of associations between candidate genes polymorphisms and meat quality attributes can provide useful markers for the selection of economically favourable traits. Calpains are calcium-dependent intracellular cysteine proteases. Two main isoforms: μ-calpain (calpain I, CAPN1) and m-calpain (calpain II, CAPN2) are heterodimers consisting of distinct large 80-kDa catalytic subunits and identical small 28-kDa regulatory subunits (calpain small subunit 1, CAPNS1), which are required to maintain stability and activity of both calpains (Goll et al., 2003). Generally calpains play a role in cell spreading, migration, proliferation and apoptosis. The calpain system is well known for its effect on the post mortem muscle proteolysis and the meat tenderization processes (Melody et al., 2004). Moreover, calpains participate in the muscle development and in muscle fiber determination (Goll et al., 1992; Sultan et al., 2000). Therefore genes coding for different calpains (CAPN1, CAPN3 and CAPNS1) and calpastatin (CAST), a specific inhibitor of calpains, are considered as functional candidates for meat quality traits. The CAPNS1 gene encodes the protein of 263–269 amino acids, with the share of more than 90% amino acids identity in humans, mice, pigs, and cattle (Juszczuk-Kubiak et al., 2010). Ovine calpain small subunit 1 gene (CAPNS1), also known as CAPN4, is located on chromosome 14 and contains 12 exons (Chung and Davis, 2014). While there is little known about this gene in sheep, Drögemüller and Leeb (2002) reported that CAPNS1 gene is located

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on SSC6 q1.1–q1.2 in pig. Interestingly, a QTL affecting drip loss trait was found within this region (de Koning et al., 2001). The bovine CAPNS1 gene is located on chromosome 18 and contains 11 exons (McCelland et al., 1989). To date, only few polymorphism analyses regarding ovine CAPNS1 gene have been undertaken. Chung and Davis (2014) investigated genetic variants in whole CAPNS1 gene consisting of 12 exons. They detected SNPs, which positions were assigned to: g.45923178G>C, g.45923421G>A, g.45924950A>G, and g.45924969A>T for CAPN445 (nt 115), CAPN456 (nt 71),CAPN478 (nt 25), and CAPN478 (nt 44), respectively (Chung and Davis, 2014). Azari et al. (2012), Dehnavi et al. (2012), Nassiry et al. (2007) and Shahroudi et al. (2006) analysed polymorphisms in exons 5 and 6 (including intron region) of the CAPNS1 gene in sheep; however, they presented only the SSCP patterns without any sequence information, therefore positions and types of polymorphisms in this DNA fragment remained unknown. Arora et al. (2014) investigated genetic variability in the same fragment of the CAPNS1 gene in Indian sheep. They sequenced PCR amplicons and observed SNP in exon 5 of this gene (CAPNS1 g.44C>T). Kumar et al. (2015) showed in the same fragment of the ovine CAPNS1 gene the addition of an adenine in the B allele at 89bp position of PCR product. Furthermore, in cattle there is also limited knowledge about genetic variants in the CAPNS1 gene. Juszczuk-Kubiak et al. (2010) and Zhang et al. (1996) detected C/T substitutions in exon 11 and intron 6, respectively. Moreover, to our knowledge effects of CAPNS1 variations on meat quality traits have never been evaluated in sheep. Only Chung and Davis (2014) analysed associations of SNPs in the ovine CAPNS1 gene with growth traits. Also little is known about effect of this gene on meat quality parameters in cattle. For example, Chung and Davis (2011) reported an association of the CAPNS1 polymorphism with meat quality traits in cattle.

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As there is little information on the calpain small subunit 1 (CAPNS1) gene polymorphism in sheep, and no previous association studies between meat traits and CAPNS1 genotypes in sheep have been conducted, the aims of the present study were to investigate the polymorphism in the CAPNS1 gene and evaluate the associations between these polymorphisms and meat quality traits in Coloured Polish Merino sheep.

2. Materials and methods The research was carried out on 105 purebred Coloured Polish Merino male lambs. Coloured Polish Merino is wool and meat type sheep breed included in the Programme of Farm Animal Genetic Resources Conservation supervised by the National Research Institute of Animal Production (NRIAP) in Poland. Animals were kept indoors; however, they grazed on pasture six times a week in the NRIAP Experimental Station Kołuda Wielka. Lambs were produced during three years: 2011, 2012 and 2013 and were sired by nine different rams. Suckling lambs were fed dry granulated mash and meadow hay ad libitum. Procedures involving animals were approved by the Local Animal Research Ethics Committee and the Local Veterinary Service. In order to produce lambs approximately at the same age slaughters took place three (2011 and 2013) or four (2012) times a year from March to April in the abattoir of the NRIAP Experimental Station Kołuda Wielka. Mean age at slaughter was 105 days (s.d. 4.2, range 92119 days). Lambs were electrically stunned, exsanguinated and skinned. Subsequently carcasses were maintained in a chilling room, where the temperature was held at approximately 4ºC for 18h. After chilling carcasses were dissected according to the methodology of Krupiński et al. (2009). Loin muscles from the right carcass side were vacuum-packed and transported to the Institute of Agricultural and Food Biotechnology (Poland) for meat quality analyses. Traits of interest for this study were: muscle pH 24 hours

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post mortem (pH24h), reflectance coordinates (L*, lightness; a*, redness; b*, yellowness), water content (WC), intramuscular fat content (FC), total protein content (PC), water-holding capacity (WHC), Warner Bratzler shear force (WBSF), drip loss (DL), and cooking loss (CL). The pH measurements (three measurements per one sample) were taken 24 hours post mortem (pH24h) on samples of the longissimus lumborum (LL) muscle using the German pHmeter type Mettler Toledo 1140 with an integrated Mettler Toledo electrode (ISO 2917, 2001). An instrumental evaluation of meat colour was performed on samples of the longissimus lumborum. Steaks (thickness approximately 10mm) were cut crosswise in the direction of muscle fibres and exposed to daylight or electric light for 15 min. The values of L* (lightness), a* (redness) and b* (yellowness) were determined using the Minolta Chronometer CR-400. In order to measure the drip loss value of the LL muscle, samples of this muscle (weighing approximately 100g) were weighted, individually packed in plastic bags and stored at 4ºC for 48h. Subsequently samples were weighted again. The value of drip loss was defined as the percentage weight loss calculated based on the difference between the initial and final (after 48h of storing) weight of the sample. The water, intramuscular fat and total protein percentage content were determined according to the methods described in ISO 1442:2000 (water) 1442:2000 (fat) and PN-75/A–04018 (protein). Compositional analyses with regard to water, intramuscular fat and total protein percentage content were carried out on minced samples of the LL muscles using the Kjeltec System 1002 Distilling Unit and the Soxtherm device manufactured by the Gerhardt Laboratory System. Water-holding capacity (WHC) was determined in minced LL muscle samples according to the method devised by Grau and Hamm (1952) as modified by Pohja and Ninivaara (1957). To determine cooking loss, samples of the longissimus dorsi (LD) muscle were weighted, packed in plastic bags and then heated in water until they reached 75ºC in the centre of the sample. After that, samples were weighted again. The value of cooking loss was calculated based on the difference in

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sample weights recorded before and after the process of heating and were expressed as a percentage. Moreover, in order to determine shear force, samples of cooked lamb loin were cooled down. Subsequently cylinder shaped samples of the LD muscle (2.5cm diameter) were extracted parallel to the direction of muscle fibres. Shear force (N/cm2) was measured with the use of the Zwick Roell ZO with a 0.5kN head and Warner Bratzler device with blade speed of 100 mm/min. Total genomic DNA was extracted from whole blood with the use of the MasterPure™ DNA Purification Kit for Blood Version II (Epicentre). A 190-bp fragment of exons 5 and 6 including intron region of the CAPNS1 gene, was amplified using primers reported by Chung et al. (2001). DNA amplifications were performed in 25μl reaction volume containing: 150ng of genomic DNA, 5pmol of each primer, forward and reverse, 200μM of each dNTP, and 1.5U DreamTaq DNA polymerase (Fermentas, Lithuania) in one fold DreamTaq reaction buffer. The temperature profile of the reaction was: denaturation at 94°C for 2min, 35 amplification cycles including denaturation at 94°C for 30 s, annealing at 59°C for 30s, and extension at 72°C for 30 s, followed by a final 7-min extension step at 72°C. Amplification was carried out in the Mastercycler Pro (Eppendorf AG, Germany). 4μl of PCR product was mixed with 10μl of loading dye containing 98% formamide, 10mM EDTA, 0.025% bromophenol blue and 0.025% xylenecyanol (Zhou et al.,2007). Samples were denatured at 95ºC for 7min and rapidly cooled on ice. Then amplicons were subjected to MSSCP (multitemperature single strand conformation polymorphism) method to screen for polymorphisms using 9% acrylamide:bisacrylamide gels (Kucharczyk T.E., Poland) with the addition of 5% glycerol. Electrophoresis was performed using the DNAPointer® System (Kucharczyk T.E., Poland), at thermal profile of 15ºC-10ºC-5ºC for 800vh, for each thermal phase in a 0.5 TBE buffer. Gels were silver stained. The DNA bands were visualized by using UV transilluminator with a visible light converter screen and archived by the G:Box Chemi

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XR5 using GeneSys software (Syngene, United Kingdome). Furthermore, bands were recognized visually. PCR products representing unique MSSCP banding patterns were cleaned up with the use of the ExoSAP-IT® Affymetrix (United States) and sequenced in both directions in Genomed, Poland. The obtained B1 allele sequence was deposited into GenBank with accession no KU759704. The BLAST algorithm was applied to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences. Allele and genotype frequencies in the CAPNS1 locus were calculated. Furthermore, observed and expected heterozygosity as well as the Hardy-Weinberg equilibrium test calculations were made in Arlequin 3.5.1.2 (Excoffier et al., 2010). An association analysis was performed between the observed CAPNS1 genotypes and traits of interest using MIXED procedure of the SAS software package (SAS Institute Inc., 2008). The following mixed effect model was applied: Yijkl = μ + ai + bj + ck + dl + eijkl where: Yijkl – is the performance of the nth individual lamb (n=1, 2, 3, … 104) for each trait of interest, μ – is the general mean for each trait of interest, ai – is the fixed effect of the ith CAPNS1 genotype (i=A1A1, A1B1, B1B1, A1C1, B1C1), bj – is the fixed effect of the jth litter size (j=1 (single), 2 (twin and only one lamb born with triplet litter size was also included), ck - is the fixed effect of the kth year of observation (k=2011, 2012, 2013) , dl - is the random effect of the lth sire (l= sire 1,2, … 9), eijkl – random error. When a genotype was shown to be statistically significant, the significance of deviations was verified with the Tukey-Kramer test. One individual with C1C1 genotype was not included in statistical analysis. Moreover, Pearson's correlation coefficients between water-holding capacity, drip loss, cooking loss and meat colour (L*, lightness; a*, redness; b*, yellowness) were calculated using the CORR procedure of the SAS software package (SAS Institute Inc., 2008).

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3. Results SSCP analysis of the CAPNS1 gene fragment (exons 5 and 6 including intron region) revealed a total of three unique MSSCP patterns representing three CAPNS1 alleles, named A1, B1 and C1. One in three detected patterns was observed for homozygous genotypes: A1A1, B1B1 or C1C1, while a combination of two of these patterns was characteristic for heterozygous genotypes: A1B1, A1C1 and B1C1 (Fig. 1). A sequence analysis revealed that these MSSCP patterns represented three CAPNS1 haplotypes, resulting from two C/T dimorphisms at positions 44 and 154 (nucleotide positions are relative to the first nucleotide in sequence, GenBank no KU759704). Positions 44 and 154 were placed in intron 5 and exon 6, respectively. Variation at position 154 covered the third nucleotide in the codon GGC for glycine, and was synonymous. Variations in these two positions (44 and 154) resulted in the occurrence of three alleles, named A1, B1 and C1 (Table 1). Consequently, the analysis revealed six CAPNS1 genotypes: A1A1, A1B1, B1B1, A1C1, B1C1 and C1C1. Moreover, a search of the NCBI GenBank database, with the sequences obtained from sequencing with the use of the BLAST algorithm, showed that the A1 allele identified in this research was identical to sequences with GenBank nos. AF309634.1, KT377436.1, KT377437.1 and KT377438.1 for Ovis aries calpain 4 gene (former name of CAPNS1), while the C1 allele was identical to sequence with GenBank no KT37742.1. The allelic variant B1 was not found in NCBI, therefore its sequence was deposited into the NCBI GenBank with accession no KU759704. Moreover, a query sequence (the A1 allele) revealed 97% identity with the bovine calpain small subunit 1 (CAPN1S) gene (GenBank no EF139087.1) in region 2758-2894nt. CAPNS1 allele and genotype frequencies in the investigated breed are shown in Table 1. The A1 allele was predominant (0.467), while C1 occurred with the lowest frequency of

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0.152. The most frequent group were A1B1 heterozygotes (0.419). Only one lamb (0.01) carried the C1C1 genotype. The value of observed heterozygosity (Ho) was equal 0.70, and was higher than the value of expected heterozygosity (He), which amounted to 0.62. The population was in the Hardy–Weinberg equilibrium (P=0.30). Table 1 Table 2 The association of CAPNS1 genotype with meat quality traits (LSM±Std Error) in Coloured Polish Merino sheep is presented in Table 3. The CAPNS1 genotype was found to have highly significant effect (P=0.006) on natural drip loss. A1C1, A1A1 and A1B1 genotypes were associated with decreased natural drip loss, when compared with B1B1 and B1C1 genotypes. The results of the Tukey-Kramer test showed that A1A1 and B1B1, A1B1 and B1B1, A1C1 and B1B1 animals differed significantly in terms of natural drip loss (Table 3). A significant associations (P=0.037 and P=0.039) between CAPNS1 genotype and lightness and intramuscular fat content were detected. Meat from A1C1 lambs had the highest values of lightness; whereas meat from B1B1 animals was characterized by the lowest value of this trait. The results of the Tukey-Kramer test showed that A1C1 and B1B1 lambs differed significantly (Table 3). As regards intramuscular fat content, meat form B1C1 lambs had the highest value of this trait, while meat from A1A1 homozygotes was characterized by the lowest fat content. The results of the Tukey-Kramer test showed that A1A1 and B1C1 lambs differed significantly in terms of intramuscular fat content (Table 3). In contrast, no association was observed between CAPNS1 genotype and pH24h, reflectance coordinates: redness, yellowness, WC, PC, WHC, WBSF. Moreover, CAPNS1 genotypes tended to associate (P=0.056) with cooking loss. Table 3

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Positive and significant correlations was found between water-holding capacity and L*lightness (r=0.25, P=0.01), whereas correlations between water-holding capacity and redness a* were negative and also significant (r=-0.45, P<0.0001). Water-holding capacity was not correlated to yellowness b*. Moreover, drip loss as well as cooking loss were not correlated to meat colour.

4. Discussion In the present study, polymorphism in the CAPNS1 gene in exons 5 and 6 (including intron region), and its association with meat quality traits in the Coloured Polish Merino sheep were identified. Sequencing results revealed two SNPs, one in exon 5 and one in intron 6, and consequently new genotypes of the CAPNS1 gene were found. In total three allelic variants (A1, B1, C1) occurring in six different combinations were detected. The A1 allele was identical to the sequences nos. AF309634.1, KT377436.1, KT377437.1 and KT377438.1 deposited in GenBank. The B1 allele (GenBank no KU759704) differs from A1 with the nucleotide C/T substitution in intron 5. Arora et al. (2014) also observed this SNP in Indian sheep. This genetic variation is in non-coding DNA, therefore it is difficult to conclude how this polymorphism may affect the CAPNS1 gene activity. It should be mentioned that it may affect mRNA splicing, or may be linked to polymorphism elsewhere in the coding sequence, which may have an impact on expression of this gene. The C1 allele is characterized also by C/T substitution in intron 5 and additional synonymous C/T substitution in exon 6, which does not change amino acid sequence in this region of peptide; however, this variation may be linked to a polymorphism in another fragment of the gene, which may influence gene expression and/or function. In contrast, Dehnavi et al. (2012), , Nassiry et al. (2007), and Shahroudi et al. (2006) found only two, while Azari et al. (2012) three, CAPNS1 genotypes. However, neither of them presented sequencing results, therefore position and type of

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polymorphism in their studies were unknown. Arora et al. (2014) observed also two alleles and three genotypes, while Kumar et al. (2015) found two alleles and two genotypes. In the present study, new genotypes were detected only for one breed the Polish Merino sheep, therefore it is possible that genotyping of other sheep breeds will show new nucleotide substitutions and alleles. Among six CAPNS1 genotypes, A1B1 heterozygotes was found to be the most common (0.419), whereas only one lamb (0.010) carried the C1C1 genotype. Although authors of the abovementioned studies i.e., Azari et al. (2012), Dehnavi et al. (2012), Shahroudi et al. (2006), Nassiry et al. (2007), used the same primers to amplify the fragment of the CAPNS1 gene, it is difficult to compare results because of different methods used for band separation as well as the lack of sequencing information in the abovementioned research. Shahroudi et al. (2006), who investigated Karakul sheep, detected only two SSCP band patterns, representing two genotypes named AA and AB. The most common was AA genotype with the frequency of 0.79. Nassiry et al. (2007) also found only two genotypes in Kurdi sheep: AA and AB, with AA being the predominant one (0.92). Similarly, Dehnavi et al. (2012) reported two alleles (A and B) and two genotypes (AA and BB) in Zel sheep. The A allele and AA genotype had the highest frequencies: 0.845 and 0.69, respectively. Azari et al. (2012) identified three genotypes, named G1, G2 and G3, in a population of Dalagh sheep with the following frequencies: 0.082, 0.891 and 0.027. Arora et al. (2014) observed two alleles, named allele 1 and allele 2, with the frequencies: 0.603 and 0.397, respectively, and three genotypes, named 11, 12 and 12, with the frequencies: 0.388, 0.429 and 0.183, respectively. Kumar et al. (2015) detected two alleles: A and B and two genotypes: AA and AB. The A allele and AA genotype had the highest frequencies: 0.820 and 0.672, respectively. The level of polymorphism detected in the present study was higher than previously reported by Azari et al. (2012), Dehnavi et al. (2012), Nassiry et al. (2007) and Shahroudi et al. (2006).

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Genetic diversity in the CAPNS1 locus in Coloured Polish Merino was high. Three alleles contributed to this heterozygosity, since the least frequent allele had the frequency equal to 0.152. There were no large discrepancies between the values of HO and HE; however, the value of the observed heterozygosity (0.70) was higher than the expected heterozygosity (0.62). Arora et al. (2014) observed two alleles in the CAPNS1 locus and lower values of HO and HE: 0.435 and 0.569, respectively. Kumar et al. (2015) also detected two alleles in this locus; however, values of HO and HE were lower comparing with our and Arora et al. (2014) results (0.360 and 0.295, respectively.) In this study population was in the Hardy–Weinberg equilibrium (P=0.30), indicating no selection for investigated CAPNS1 locus. To our knowledge, this was the first analysis of the association between meat quality traits and C/T polymorphisms at positions 44 and 154 of the calpain small subunit 1 gene in sheep. The effect of CAPNS1 genotype on natural drip loss was highly significant (P=0.006). Sheep carrying A1C1, A1A1 and A1B1 genotypes had lower natural drip loss compared with B1B1 and B1C1 genotypes. Moreover, CAPNS1 genotype tended to associate with cooking loss values. Sheep carrying B1C1 and A1A1 genotype had the lowest values of cooking loss compared with the other genotypes. Gandolfi et al. (2011), who investigated the effects of CAPNS1 polymorphisms on meat quality traits in pigs, did not confirm the association between polymorphism c.429A>C and cooking or natural drip loss. However, Gandolfi et al. (2011) observed the tendency towards association between mRNA expression in the longissimus dorsi muscle and drip loss (P=0.06), which was not confirmed in the correlation analysis (r=0.06; n.s.). Furthermore, Ribeca et al. (2013) observed an association between natural drip loss and the calpain I large subunit (CAPN1) gene polymorphism in Piemontese cattle. On the other hand, Pinto et al. (2011) did not observe any association between the CAPN1 gene polymorphism and cooking and natural drip loss in Nellore cattle. Moreover, Li et al. (2013a) found that E4-2 locus of the CAST gene was significantly correlated to cooking

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loss in Yanbian cattle. Little is known about association of the CAPNS1 gene with meat quality traits, i.e. water-holding traits in sheep. However, Melody et al. (2004), who investigated three porcine muscles, found correlations between water-holding capacity and calpain activity, calpain autolysis and protein degradation in pigs. This fact could be in accordance with hypothesis of Kristensen and Purslow (2001), who claimed that waterholding capacity in the early post slaughter period may be influenced positively by retained water in the muscle cell if there is rapid degradation of intermediate filaments such as desmin at this time. Desmin is a calpain 1 large subunit substrate. Autolysis and activation of this subunit may potentially explain a portion of the variation of desmin degradation and activation/deactivation of drip channels, thus affecting the extent of drip loss (Ribeca et al., 2013). Moreover, Huff-Lonergan and Lonergan (2007) support that greater μ-calpain activity lead to the cleavage of specific myofibrillar proteins, such as desmin and vinculin, reducing drip loss. Calpain small subunit 1 is a small 28-kDa regulatory subunit required to maintain stability and activity of μ- and m-calpains (Goll et al., 2003). Therefore SNPs in the CAPNS1 gene may have direct or indirect effect on activity of calpains and also on water-holding traits. Drip and cooking losses are important meat quality traits as they may reduce muscle weight. Moreover, water loss during cooking has a great effect on the final product quality, affecting mostly the juiciness and tenderness (Pinto et al., 2011). In the present study an effect of the CAPNS1 gene polymorphism on meat colour was investigated. A significant association (P=0.037) between CAPNS1 genotype and lightness was detected. Lambs carrying A1C1 genotype had the highest values of lightness compared with the other genotypes. Gandolfi et al. (2011) showed that the CAPNS1 gene was associated with meat lightness (L*, P=0.02) and meat yellowness (b*, P<0.001) in the Italian Large White pig. Moreover, Ribeca et al. (2013) observed the association between yellowness and the CAPN1 gene polymorphism in Piemontese cattle. Pinto et al. (2011) detected an effect of

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the CAPN1 gene variants on meat colour (in particular redness and yellowness) in Nellore cattle. Xin et al. (2011) found associations between the CAPN1 gene and colour scores in Yanbian Yellow cattle of China. Additionally, Mazzucco et al. (2010) detected effects of CAPN1 316 on L* and CAPN1 4751 on a* and b* in Brangus steers. For CAPN1 316 genotype GG presented less brightness than GC and CC; however, for CAPN1 4751, significantly higher values in a* and b* for CT were found only when comparing it with CC (Mazzucco et al., 2010). Furthermore, Rearon et al. (2010) reported the highly significant

association between the CAST gene polymorphism and colour parameters in cattle. Moreover, Li et al. (2013a) found that E4-2 locus of the CAST gene was significantly correlated with brightness, degree of redness, degree of yellowness, colour, and colour angle in Yanbian cattle. Interestingly, in pork, colour (in particular lightness) was associated with water-holding capacity estimated by filter paper test and drip loss test (Joo et al., 1995). In the present study water-holding capacity was also correlated to lightness and redness; however, correlations between drip loss, cooking loss and meat colour were not found. The relationship between calpastatin and colour and water-holding capacity traits could be explained by the biological mechanisms that involve a connection through Ca2+ ion concentration and muscle contraction rate (Ciobanu et al., 2004; Rearon et al., 2010). Furthermore, colour and water-holding capacity may be influenced by calpastatin if this enzyme has an impact on rate and extent of glycolysis and pH decline (Rearon et al., 2010). Considering abovementioned information, worth mentioning is a relationship between pHu and both water-holding capacity (estimated by filter paper test and drip loss test) and colour (L*, C* and h) reported by Joo et al. (1995). Calpastatin is an inhibitor of calpain, therefore there is a possibility that SNPs detected in the CAPNS1 gene are not causative mutations for the investigated effects on meat colour, but are markers in linkage with the causative mutations in the other gene i.e. CAST. Moreover, Melody et al. (2004) indicated that some meat quality traits, i.e.: tenderness, drip loss may be

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influenced by the post mortem muscle myofibril degradation, controlled mainly by the calpain activity. Noteworthy is the fact that visual assessment of meat colour is believed to be influenced by the intramuscular fat content (Fiems et al., 2000). In the present study, the significant association (P=0.039) between CAPNS1 genotype and intramuscular fat content was detected. Meat form B1C1 lambs had the highest value of this trait, while meat from A1A1 homozygotes was characterized by the lowest fat content. To our knowledge, it was the first analysis of the CAPNS1 gene polymorphism association with intramuscular fat content in sheep. Moreover, to date only few analyses have described the effects of the CAPNS and CAPN1 genes mainly on marbling in cattle. For example, Chung and Davis (2011) observed the relationship between CAPNS genotype and marbling score in Angus bulls. Animals carrying the B allele showed higher marbling values comparing with animals carrying the A allele. Li et al. (2013) reported on a CAPN1 gene polymorphism that was associated with intramuscular fat (IMF) content and subjective marbling score in cattle. Animals carrying the CC genotype had higher IMF content and subjective marbling score than those with CG and GG genotypes. It was observed that high fat content accelerates muscle lipid oxidation (Ahn et al., 1998). Meat discoloration is an effect of myoglobin oxidation, which is in turn linked to lipid oxidation. Myoglobin and lipid oxidation are linked through the chemical compounds formed in the oxidation process (Faustman et al., 2010). The protease μ-calpain (CAPN1) and its inhibitor calpastatin (CAST) play an important role in the process of post mortem meat tenderization (Koohmaraie, 1996). In the present study an effect of the CAPNS1 gene polymorphism on Warner Bratzler shear force measured on samples of the LD muscle was investigated; however, the relationship was not significant (P=0.17). Contrary, Knight et al. (2012) found three SNPs within CAST and CAPN2, which showed significant association with shear force in Australian lamb and each of these SNPs 16

had an unfavourable effect on the investigated trait. Moreover, Tait et al. (2014) reported significant associations between CAPN1 and CAST and slice shear force in Angus cattle. Interestingly, Tait et al. (2014) also observed that the tender allele of the CAST (CASTaT) gene was associated with higher yield of red meat in Angus cattle. Page et al. (2004) and White et al. (2005) reported non-synonymous and intronic SNPs in the CAPN1 gene, which have been significantly associated with meat tenderness in Bos indicus and Bos taurus. Furthermore, several studies have shown that SNP located in the 3’ UTR region of the CAST gene is significantly associated with meat tenderness in Bos taurus and Bos taurus x Bos indicus beef cattle (Casas et al. 2006; Curi et al. 2009; Morris et al. 2006). Abovementioned studies showed that meat tenderness is influenced by polymorphisms in calpain and calpastatin genes in sheep and cattle. Lack of association between the CAPNS1 gene polymorphism and WBSF in present study may be explained by several reasons: 1) breed effect; 2) the examined polymorphism was not causative but in linkage disequilibrium with the associated trait; 3) the trait is polygenic and the detected polymorphism explain only a part of the total effect. Taking into consideration abovementioned reasons further investigation should be carried out to evaluate relationship between CAPNS1 polymorphism and meat tenderness in other sheep breeds.

5. Conclusion In the present study, two SNPs in the ovine CPANS1 gene have been detected. Taking into consideration that not much information about polymorphism was found in this gene, genotyping of other sheep breeds is recommended, which may result in detection of new nucleotide substitutions. Moreover, the associations found for CAPNS1 genotype with natural drip loss, intramuscular fat content and meat colour, provided the first information on this marker in relation with lamb quality. Results of the association analysis confirm that the

17

CAPNS1 gene could be considered as a candidate gene of meat quality traits in sheep. Furthermore, the CAPNS1 gene could be used as marker for the improvement of the abovementioned meat quality traits in Coloured Polish Merino sheep. However, as the obtained results are the first information gathered on the CAPNS1 gene effects on meat quality parameters in sheep, further investigation should be carried out to evaluate potential breed-specific effects of this gene’s polymorphism on lamb quality. It is also worth mentioning that the CAPNS1 gene may be pleiotropic, therefore before using CAPNS1 polymorphism in production system, association analyses regarding reproductive and other important production traits should be conducted, to ensure that selection will not have negative consequences for the breed.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments Project was founded by National Science Centre, Poland, project no. NN311521440

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Fig. 1. Polymorphism in exons 5 and 6 (including intron region) of the ovine CAPNS1 gene identified using mulitemperature single-strand conformational analysis. Representative MSSCP patterns for the three unique haplotypes (A1, B1 and C1) in both homozygous and heterozygous genotypes are shown.

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Table 1 Ovine CAPNS1 alleles detected in exons 5 and 6 (including intron region). Nucleotide SNP

Allele

a

position

A1

B1

C1

44

C/T

C

T

T

154

C/T

C

C

T

a

Nucleotide positions are relative to the first nucleotide in sequence no KU759704 (GenBank)

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Table 2 Allelic and genotypic frequencies in the CAPNS1 gene (n=105) in Coloured Polish Merino. Allele/genotype

Allele

Frequency A1

0.467

B1

0.381

C1

0.152

A1A1

0.181

A1B1

0.419

B1B1

0.105

A1C1

0.152

B1C1

0.133

C1C1

0.010

Genotype

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Table 3 The association of CAPNS1 genotype with meat quality traits (LSM±Standard error) in Coloured Polish Merino sheep. LSM1±Standar error

Trait

P(abbreviation, A1A1 (n=19)

A1B1 (n=44)

A1C1 (n=16) B1B1 (n=11)

B1C1 (n=14)

value

5.67±0.02

5.68±0.01

5.67±0.02

5.67±0.03

5.67±0.02

0.993

L*, lightness

39.63±0.58ab

40.10±0.38ab

41.13±0.65a

38.05±0.75b

39.98±0.68ab

0.037

a*, redness

12.75±0.28

12.97±0.20

12.46±0.30

13.26±0.34

13.02±0.31

0.306

b*, yellowness

2.52±0.34

2.26±0.22

2.93±0.36

1.70±0.43

2.24±0.38

0.220

73.80±0.41

73.04±0.26

73.24±0.45

73.29±0.53

73.10±0.48

0.607

content 1.80±0.12a

1.99±0.08ab

1.99±0.13ab

2.12±0.15ab

2.36±0.14b

0.039

23.27±0.37

23.84±0.24

23.59±0.41

23.41±0.47

23.38±0.43

0.675

34.78±0.69

34.97±0.44

33.91±0.77

32.93±0.89

33.36±0.81

0.150

unit) muscle pH 24 hour

post

mortem (pH24h) reflectance coordinates:

water content (WC,%) intramuscular fat (FC,%) total

protein

content (PC,%) water holding capacity

28

(WHC,%) Warner Bratzler shear 46.65±3.66

42.42±2.40

46.92±4.07

52.63±4.73

38.81±4.29

0.171

2.24±0.39a

2.39±0.26a

2.23±0.42a

4.11±0.50b

3.30±0.45ab

0.006

14.41±0.92

15.62±0.60

16.20±1.00

17.67±1.18

13.55±1.06

0.056

force (WBSF, N/cm*2)

drip

loss

(DL,%) cooking

loss

(CL,%) 1

LSM=Least Squares Mean; values with superscripts in common within a row are not

significantly different (P>0.05)

29