Journal Pre-proof

Association of two single nucleotide polymorphisms in the calpastatin

gene with tenderness under varying lengths of meat ageing in two
native Spanish cattle breeds

Laura Pilar Iguácel , Jorge Hugo Calvo , Isabel Casasús ,

Malena Serrano , Guillermo Ripoll , Pilar Sarto , Daniel Villalba ,
Mireia Blanco

PII: S1871-1413(19)30304-X
DOI: https://doi.org/10.1016/j.livsci.2019.103820
Reference: LIVSCI 103820

To appear in: Livestock Science

Received date: 28 February 2019

Revised date: 3 October 2019
Accepted date: 4 October 2019

Please cite this article as: Laura Pilar Iguácel , Jorge Hugo Calvo , Isabel Casasús ,
Malena Serrano , Guillermo Ripoll , Pilar Sarto , Daniel Villalba , Mireia Blanco , Association
of two single nucleotide polymorphisms in the calpastatin gene with tenderness under vary-
ing lengths of meat ageing in two native Spanish cattle breeds, Livestock Science (2019), doi:
https://doi.org/10.1016/j.livsci.2019.103820

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HIGHLIGHTS

 Beef shear force was affected by the interaction between the management and aging

 In Parda de Montaña, shear force was affected by SNP rs210072660 of calpastatin

 In Pirenaica, shear force was only affected by SNP rs109221039 of calpastatin

 The effects of both SNPs were aging-dependent

1
Association of two single nucleotide polymorphisms in the calpastatin gene with

tenderness under varying lengths of meat ageing in two native Spanish cattle breeds

Laura Pilar Iguácela, Jorge Hugo Calvoa b*, Isabel Casasúsa, Malena Serrano c, Guillermo

Ripolla, Pilar Sarto a, Daniel Villalbad, Mireia Blancoa

a
Unidad de Producción y Sanidad Animal, CITA, Instituto Agroalimentario de Aragón - IA2

(CITA-Universidad de Zaragoza), 59059 Zaragoza, Spain;

b
ARAID, 50018 Zaragoza, Spain.

c
Departamento de Mejora Genética Animal, INIA, 28040 Madrid, Spain
d
Departamento de Producciò Animal, ETSEA, UdL, 251798 Lleida, Spain

* Corresponding author: jhcalvo@aragon.es

2
ABSTRACT

The objective of this study was to validate the effect of the SNPs rs210072660 and

rs109221039 of the calpastatin (CAST) gene on meat toughness in Longissimus thoracis

muscle aged for 1 d (± 0; short-term ageing), 8 d (± 1; medium-term ageing) and 15 d (± 2;

long-term ageing). The samples were obtained in experiments with different management

strategies in Parda de Montaña (grazing and concentrate-fed bulls, grazing steers, and steers

finished on a total mixed ration) and Pirenaica (concentrate-fed bulls, steers and heifers, and

steers on a total mixed ration) cattle. In the Parda de Montaña breed, the Warner-Bratzler

Shear Force (WBSF) was similar among management strategies under short-term aging, but

it was lower for concentrate-fed bulls than for grazing bulls under medium-term and long-

term aging (P<0.05), presenting both groups of forage-fed steers intermediate values.

Similarly, there were no differences in the short-term ageing, but there were differences

observed thereafter in the Pirenaica breed. Regarding the effect of SNPs of CAST in the Parda

de Montaña breed, WBSF was only affected by the interaction between the SNP rs210072660

and ageing (P<0.05). Shear force did not differ among genotypes (P>0.05) under short-term

ageing, but the GG genotype had 14-16% greater WBSF than the GA and AA genotypes

under medium-term ageing (P < 0.001), and a trend was found under long-term ageing

(P=0.07). In Pirenaica, WBSF was only affected by SNP rs109221039 (P<0.001). The GG

genotype showed higher WBSF than the GA and AA genotypes under medium-term and

long-term ageing (P<0.01). Haplotype analyses confirmed the association results. In the

Parda de Montaña breed, animals carrying no copies of haplotype AA had a greater WBSF

under medium-term ageing than animals with 1 or 2 copies of this haplotype (P < 0.05). In

the AG haplotype, animals with 0 copies of this haplotype had greater toughness than animals

with 1 copy (P < 0.05). In Pirenaica breed, animals with 2 copies of the GG haplotype had

greater toughness than animals with 1 or 2 copies in both ageing periods (P < 0.001). These

3
findings suggest that SNPs rs210072660 and rs109221039 could be useful markers for

effective marker-assisted selection to increase meat tenderness in the Parda de Montaña and

Pirenaica breeds, respectively.

Keywords: beef, management, CAST, shear force, Parda de Montaña, Pirenaica

4
1. Introduction

There is abundant evidence about the close relationship between the calpastatin (CAST)

gene and meat tenderness in meat of livestock (Casas et al., 2006); however, the associations

are breed-dependent. A study was carried out to determine whether there were associations

between described and newly detected SNPs of CAST and beef toughness in Parda de

Montaña and Pirenaica breeds (Calvo et al., 2014). These two beef cattle breeds, selected in

recent decades for beef production, have different origins. The former breed has been

selected from the dual-purpose old Brown Swiss breed, and the latter breed is a local hardy

breed from the Pyrenees. The SNP rs210072660 located in exon 7 (BTA7: g.98535683A>G

in UMD3.1 genome assembly) of CAST affected toughness measured in Longissimus thoracis

muscle with medium-term ageing (7 d) in Parda de Montaña cattle (Calvo et al., 2014),

having the GG genotype greater values than GA and AA genotypes. This effect was

confirmed later in Bos indicus and Bos indicus x Bos Taurus cattle (Enriquez-Valencia et al.,

2017). On the other hand, the SNP rs109221039 located in the 3’UTR region (exon 30;

BTA7: g.98579663A>G in UMD3.1 genome assembly) of CAST and described by Barendse

(2002) has been related to tenderness in different cattle breeds, but it was not evident in Parda

de Montaña breed (Calvo et al., 2014). These authors have also found more evident effects

with medium-term ageing. Therefore, some authors have described that the effects of the

SNPs of the CAST gene depend on the breed and ageing of the meat (Morris et al., 2006;

Schenkel et al., 2006; Curi et al., 2009; Allais et al., 2011). Because the effect of genetic

markers on phenotypes are intrinsic factors of each population and environment, it could be

hypothesised that rs210072660 and rs1092212039 could differentially affect meat toughness

depending on ageing and breed. Thus, the aim of the study was to evaluate whether the SNPs

5
affected the toughness of meat with short-term (1 d) medium-term (8 d) and long-term (15 d)

ageing in Parda de Montaña and Pirenaica cattle breeds.

2. Materials and Methods

Data used in the current study were obtained from previous experiments (2003-2010) in

CITA Research Station, and those obtained in 2012-2013 were directly collected from the

slaughterhouse. The management and slaughtering of the animals followed the guidelines of

EU legislation on the protection of animals used for scientific purposes (Council Directive

86/609/EEC, European Communities, 1986; Directive 2010/63/EU of the European

Parliament and of the 28 Council of 22 September 2010) and supervised by the Animal

Welfare Committee of the institution.

2.1. Animals, samples and ageing

A database updated from that used in Calvo et al. (2014) was used in the analyses. The

database included samples of Longissimus thoracis et lumborum (LTL) muscle from Parda de

Montaña and Pirenaica cattle from different experiments conducted in the research centre

between 2003 and 2010 [Table 1: Exp. 1-8 & 10-11, only samples with medium-term ageing

were used in Calvo et al. (2014)] and samples from concentrate-fed Parda de Montaña young

bulls (Exp. 9) and Pirenaica young bulls and heifers (Exp. 12) of different origins collected in

2012-2013. There were four managements in the Parda de Montaña breed (grazing bulls,

grazing steers, TMR-fed steers and concentrate-fed bulls) and five in the Pirenaica breed

(grazing steers, TMR-fed steers, concentrate-fed bulls, concentrate-fed steers and

concentrate-fed heifers).

Cattle were slaughtered in EU-licensed commercial abattoirs. After the 24-h chilling

period (at 4 ºC in total darkness), LTL muscle was removed, excised and sliced into 3.5-cm

steaks. Samples were aged for different ageing periods, depending on the experiment, and

were grouped into short-term (average: 1 ± 0 d; n=110), medium-term (7-10 d; average: 8 ± 1

6
d; n=321) and long-term (14-20 d; average: 15 ± 2 d; n=258) ageing groups. The steaks were

vacuum-packaged and stored in total darkness in a cooler at 4 °C for ageing and were then

frozen at -20 °C until instrumental texture analyses. Table 1 shows the management of the

animals, the age at slaughter, hot carcass weight, ageing, and Warner-Bratzler shear force

(WBSF) for the whole database.

2.2. Warner-Bratzler shear force measurement

The thawed steaks were boiled in a water bath to an internal temperature of 70 °C. When

the steaks cooled off, ten strips (10×10 mm cross-sections) were cut with the fibre direction

parallel to a long dimension. The samples were sheared using a Warner–Bratzler meat Instron

shear machine (Model 5543, Instron Limited, Barcelona, Spain) with a crosshead speed of

150 mm/min. Values for WBSF (maximum load per unit of cross-section) were determined.

The WBSF value for each animal was calculated by averaging the results of those 10 probes

per steak.

2.3. SNP genotyping

DNA samples were used from previous studies, except those from Exp. 9 and 12. Genomic

DNA from these experiments was extracted using the SpeedTools DNA Extraction kit

(Biotools, Madrid, Spain). The SNPs rs210072660 and rs109221039 [called CAST_2 and

CAST_5, respectively] were genotyped by PCR-RFLP, as detailed in Calvo et al. (2014).

Briefly, PCR was carried out individually for each SNP in a final PCR volume of 25 l

containing 5 pmol of each primer (5’-TGCATCATGTCCAGCAGA-3’ and 5’-

CACGTGTGCTCACCTTGTCAGCTGGTA-3’ amplify a fragment of 238 pb of exon7 for

the SNP rs210072660; and 5’-ACATTCTCCCCACAGTGCC -3’ and 5’-

GACAGAGTCTGCGTTTTGCTC-3’ amplify a fragment of 375 pb of exon30 for the SNP

rs109221039), 200 nM dNTPs, 2.0 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, 0.1%

TritonX-100 and 1 U Taq polymerase (Biotools, Madrid, Spain). Cycling conditions were as

7
follows: an initial denaturation step of 94ºC for 3 min, 35 cycles of [94ºC for 45 s, annealing

temperature for 45 s, and 72ºC for 45 s] and a final extension step of 72ºC for 10 min. The

annealing temperatures were 55 and 62ºC for the SNPs rs210072660 and rs109221039,

respectively. The SNPs rs210072660 and rs109221039 were detected with HhaI and DdeI

restriction enzymes (New England Biolabs, Beverly, MA, USA), respectively. The HhaI

RFLPs have two alleles: G (178 and 60 bp) and A (238 bp), whereas the DdeI RFLPs also

have two alleles: A (247 and 128 bp) and G (375 bp). For each SNP, 10 l of the PCR

product was digested with the corresponding enzyme for 4 h at 37ºC in a total volume of 15

l. The PCR-RFLP bands were visualised on 3% agarose gels stained with SYBR Safe

(Invitrogen, Carlsbad, CA, USA).

2.4. Statistical analyses

The Hardy–Weinberg equilibrium exact test values for the SNP rs210072660 and

rs1092212039 were calculated using PLINK 1.9 software (Purcell et al., 2007). The statistical

analyses were run separately for each breed. Phenotypic and animal variances on meat WBSF

were estimated by fitting a Linear Mixed Model using the High-Performance Mixed

procedure (HPMIXED). The model included the management, the ageing period, and its

interaction as fixed effects; the hot carcass weight and slaughter age as covariates; and the

animal (A) from which records were collected and the residual (e) as random effects. When

the effect of the covariate was not significant, it was deleted from the model. Heterogeneous

animal variance was considered for the ageing period (A ~ N (0, σ2 AB)) and homogeneous

variance for the residual (e ~N (0, σ2)).

The effects of the management and the SNPs on WBSF in the different ageing periods

were evaluated with a mixed model with repeated measurements for each breed separately.

The degrees of freedom used for testing were adjusted with the Satterthwaite correction to

account for possible unequal number of observations (Kaps and Lamberson, 2009). Within

8
each breed, the management, the SNP, the ageing period (short-term, medium-term and long-

term), and the interaction of the ageing period with the SNP and management were

considered fixed effects, and the animal and the experiment were considered to be random

effects. Hot carcass weight, fat score and slaughter age were included in the models as

covariates. When the effect of the covariate was not significant, it was deleted from the

model. Different variance-covariance matrices were tested to model the error. The one with

the lowest Aikake (AIC) and Bayesian Information Citeria (BIC) values was chosen. To test

differences between managements and genotypes, the least square means (LSMs) for each

pair-wise comparison were estimated. Bonferroni correction was applied to consider multiple

tests.

For haplotype association studies, SNPs were phased with PLINK1.9 using the

expectation–maximisation (E–M) algorithm to assign individual haplotypes (Purcell et al.,

2007). The haplotype for each animal was encoded with 0, 1 or 2 to indicate the numbers of

copies of each haplotype. The effect of the haplotype on the WBSF was estimated by the

MIXED procedure separately for each breed. The model was similar to genotype association,

but in this case, the interaction between ageing and haplotype was included in the model

instead of the interaction between ageing and SNP.

3. Results

In the Parda de Montaña breed, the phenotypic mean values for WBSF were 88.4 (± 14.1),

66.3 (± 19.4) and 57.3 (± 17.4) N/cm2 for short-term, medium-term and long-term ageing,

respectively. In the Pirenaica breed, the phenotypic mean values for WBSF were 49.6 (±

15.8) and 50.8 (± 16.5) N/cm2 for the medium-term and long-term ageing, respectively. The

phenotypic and animal variances and the residual for each ageing and breed are reported in

Table 2.

3.1. Effect of the management strategy and ageing on Warner-Bratzler shear force

9
 Warner-Bratzler shear force was affected by the interaction between the management and

the ageing period (P < 0.001) in both breeds (Fig. 1). In the Parda de Montaña breed, there

were no differences in WBSF at short ageing (P > 0.05). Shear force decreased between

short-term and medium-term ageing periods by 27% and 22% for concentrate-fed young bulls

and pasture-fed steers (P < 0.001) but only 6% and 7% for pasture-fed bulls and TMR-fed

steers (P > 0.05), respectively. Pasture-fed bulls had greater WBSF than concentrate-fed

bulls (P < 0.001) in the medium-term ageing period, whereas both groups of steers presented

intermediate values. Between the medium-term and long-term ageing period, WBSF

decreased (P < 0.001). In the long-term ageing period, pasture-fed bulls and TMR-fed and

pasture-fed steers had higher WBSF than concentrate-fed young bulls (P < 0.001).

In Pirenaica breed, WBSF was also affected by the interaction between the management

strategy and ageing (P < 0.05). As in the Parda de Montaña breed, WBSF was not different

between pasture and TMR-fed steer at short ageing (P > 0.05). Pasture-fed steers had greater

values than their counterparts under medium-term ageing (P < 0.05) but only greater values

than concentrate-fed heifers and steers under long-term ageing (P < 0.05). Shear force

decreased in grazing- and TMR-fed steer between the short-term and medium-term ageing

but did not decrease between medium-term and long-term ageing periods (P > 0.05).

3.2. Effect of SNP rs210072660 and rs109221039 genotypes on shear force with different

lengths of ageing

The number of genotypes of SNPs rs210072660 and rs109221039 in each ageing period

and breed is reported in Supplementary Table 1. The SNPs were in Hardy-Weinberg

equilibrium in each ageing and considering the whole population for each breed. The

interaction between the SNP rs210072660 and ageing (P < 0.01) affected WBSF in Parda de

Montaña cattle but not in the Pirenaica breed (Table 3). In the Parda de Montaña breed,

10
WBSF did not differ among genotypes under short-term ageing (P > 0.05). However, under

medium-term ageing, the GG genotype had 14-16% higher WBSF than the GA and AA

genotypes in the Parda de Montaña breed (P < 0.01) but only tended to be different in the

Pirenaica breed (P < 0.10). Under long-term ageing, only a trend was found for WBSF in the

Parda de Montaña breed (P=0.07).

Shear force was not affected by the SNP rs109221039 in the Parda de Montaña breed, but

it was affected in the Pirenaica breed ageing (P = 0.001) (Table 3). In this sense, the GG

genotype had 22% and 29-35% higher WBSF than the GA and AA genotypes under medium-

term and long-term ageing (P < 0.01).

The results of the analyses of linkage disequilibrium of the haplotypic frequencies of the

SNPs rs210072660 and rs109221039 in the Parda de Montaña and Pirenaica populations are

shown in Supplementary Tables 2 and 3. The most frequent haplotype was AA followed by

GG and GA in both breeds.

3.2. Effect of the haplotypes on WBSF with different lengths of ageing

In the Parda de Montaña breed, WBSF was not affected by the GG and GA haplotypes,

but it was affected by the interaction between the AA and the AG haplotypes and ageing

(Table 4). There were differences among the number of copies of each haplotype (AA and

AG) under medium-term ageing (P < 0.01) but not under short-term and long-term aging (P >

0.05). Animals carrying no copies of haplotype AA had a greater WBSF than animals with 1

or 2 copies of this haplotype (P < 0.05). In the AG haplotype, there were no animals with 2

copies. Animals carrying 1 copy of this haplotype had more tender meat than animals with 0

copies (P < 0.05).

Concerning the Pirenaica breed, WBSF was affected by the GG haplotype (P < 0.001).

Animals with 2 copies of the GG haplotype had greater toughness than animals with 1 or 2

copies in both ageing periods (P < 0.001). Shear force tended to be affected by the AA

11
haplotype (P < 0.10). Animals with 0 copies of the AA haplotype had a greater WBSF than

animals with 1 or 2 copies of the haplotype.

4. Discussion

4.1. Effect of the management strategy

The decrease of WBSF between the medium-term and long-term ageing period in the

Parda de Montaña breed agrees with the findings of previous experiments (Morgan et al.,

1993; Claus et al., 2010). The similar WBSF between 7 and 14 d agrees with results reported

in Pirenaica and Rubia Gallega cattle (Campo et al., 2000). In the current experiment, the

greater WBSF of pasture-fed animals compared to concentrate-fed cattle could be attributed

to several aspects: i) differences in the deposition of soluble and insoluble collagen and their

cross-links (Muir et al., 1998). However, the results in the literature are not clear, and it is

difficult to elucidate whether the results obtained in these studies are due to differences in the

energy level, differences in exercise, which affects collagen content (Jurie et al., 1998), or

differences due to only grazing per se. ii) to differences in weight gains as increased protein

turnover in rapidly growing cattle resulted in higher concentrations of proteolytic enzymes in

the carcass tissues at slaughter (Muir et al., 1998); iii) to differences in carcass fat cover.

There is a shortening of the muscle fibres during the cooling of the carcasses after slaughter

when subcutaneous fat cover is narrow, which could lead to a lower proteolysis during rigor

mortis (Bowling et al., 1977) and a lower reduction in the calpastatin activity (Morgan et al.,

1993). When cattle had similar carcass weight and fatness, concentrate- and forage-fed cattle

had similar Warner-Braztler shear forces in 7-d- and 14-d-aged beef (French et al., 2000).

4.2. Effect of the SNP and haplotype genotypes

The frequencies of the genotypes of the SNP rs210072660 and rs109221039 in each

ageing period and breed are similar frequencies to those described in Calvo et al. (2014), but

they were different from those described in Nellore beef cattle (Bos indicus) and their crosses

12
with Bos taurus, Baladi, Limousine, Holstein, Simmental and Brahman cattle (Enriquez-

Valencia et al., 2017; Shor-Shimoni et al., 2017).

The absence of an effect of SNP rs210072660 at short ageing could be possible due to the

rigor mortis status. The greater WBSF of the GG genotype compared to the GA and AA

genotypes in the Parda de Montaña breed confirmed the effect reported with fewer animals

(Calvo et al., 2014). The tendency found in Pirenaica breed was probably due to the lower

number of samples. In Bos indicus and Bos indicus x Bos taurus cattle, the effect of the SNP

was slightly different, as the GA had greater WBSF than the AA genotype, and the GG

genotype was not included in the analyses due to its low presence in the studied population

(Enriquez-Valencia et al., 2017). The suggestive trend for WBSF in the Parda de Montaña

breed during the long ageing period could be explained by the lower WBSF and the lower

difference in the WBSF between the homozygous genotypes AA and GG in the long-term

ageing period (10%). Similarly, an interaction between SNP rs110955059 of CAST and

ageing was reported by Schenkel et al. (2006), who found an association between the SNP,

which is located in intron 6, and the measures of Longissimus muscle tenderness at 2, 7 and

21 d but not for 14 d in crossbred cattle.

The effect of SNP rs109221039 in the Pirenaica breed is confirmed by previous results

that associated the G allele with tougher meat aged for 14 d in different populations of

crossbred cattle (Van Eenennaam et al., 2007), Bos indicus x Bos taurus cattle (Casas et al.,

2006), and Charolais, Limousin and Blonde d'Aquitaine bulls (Allais et al., 2011), as well as

in meat of crossbred cattle derived from Hereford, Angus, Red Angus, Limousin, Charolais,

Gelbvieh, and Simmental (Barendse, 2002). In the current study, animals that were

homozygous for the unfavourable allele were under-represented (GG; n=8 and n=2 for

medium-term and long-term ageing, respectively), as found in other previously published

studies. However, it is important to note that we observed an unbalanced distribution of

13
genotypes and a small number of animals in this population (n = 72 and n=42 for medium-

term and long-term ageing, respectively). However, the effect was not evident in Parda de

Montaña cattle, confirming the relevance of the studies that evaluate the effect of SNPs in the

different breeds.

The effects of the haplotype association studies on WBSF at medium-term ageing agree

with the effect found at genotype association of the SNPs rs210072660 and rs109221039. In

the Parda de Montaña breed, we found the AA and AG haplotypes associated with lower

WBSF under medium-term ageing (alleles significantly associated with greater WBSF for

each SNP in each haplotype according to the SNP association results are shown in bold).

These haplotypes have the A allele for the SNP rs210072660 (associated with lower WBSF)

and the allele A and G for rs109221039 (associated with lower and greater WBSF), which

could indicate that the found effect could be attributed to SNP rs210072660 and not to

rs109221039. In the Pirenaica breed, only the GG haplotype was associated with higher

WBSF, with the two alleles of both SNPs being associated with greater WBSF. However, we

believe that the effect found could be due to the G allele of SNP rs109221039 because the

SNP rs109221039 was the only significant SNP in the Pirenaica breed. Furthermore, the

estimated effects on the WBSF trait were similar between the homozygous animals for the G

allele (in SNP association studies; Table 3) and animals with two copies of the GG haplotype

(in haplotype association studies (Table 4). However, these results should be interpreted with

caution because there were only 8 samples with medium-term ageing and 2 samples with

long-term ageing with 2 copies of this haplotype. To confirm the effects of this

polymorphism in the Pirenaica population, it will be necessary to increase the number of

animals analysed.

The putative functional effects on meat toughness of these SNPs have been described in

Calvo et al. (2014) and Leal-Gutiérrez et al. (2018) for the SNPs rs210072660 and

14
rs109221039, respectively. The SNP rs210072660 produces an amino acid change at position

p.Thr182Ala (NM_174003). This amino acid substitution could affect the stability of the

complex calpain-calpastatin that depends on the electrostatic charges localised in the

interacting regions where the SNP is located (in the calpastatin L-domain). The SNP

rs109221039 is located in the 3’UTR region. In this sense, Leal-Gutierrez et al. (2018)

predicted using bioinformatics tools that this SNP could affect the stability and molecular

folding of the mRNA and could decrease the mRNA translational rate. Another possible

mechanism that could affect the final amount of calpastatin mRNA and protein may be

observed because this SNP modifies a putative target site of a bovine miRNA (bta-miR-542-

5p) (Calvo et al., 2014).

In conclusion, the present results show that the effect of the SNP rs210072660 of the

CAST gene on meat WBSF depends on ageing, showing the greatest effect at the medium

ageing period in the Parda de Montaña breed. A trend was observed in the longer ageing

period; therefore, more research should be performed to study the most adequate length of

ageing to minimise the effect on toughness to be able to obtain a meat that can be

commercialised under a quality label of tender meat. The GG genotype of the SNP

rs109221039 had higher WBSF than the GA and AA genotypes at medium-term and long-

term ageing in the Pirenaica breed, although this association is based on a small population.

Haplotype analyses confirmed the association results, indicating that the found effect could

be attributed to SNPs rs210072660 and rs109221039 in Parda de Montaña and Pirenaica

breeds, respectively. These polymorphisms can be useful for industry as genetic markers to

identify tough meat and design adequate handling procedures for these carcasses. In this

sense, a long ageing period might be advisable in these breeds for GG genotypes of the SNPs

rs210072660 and rs109221039 for Parda de Montaña and Pirenaica breeds, respectively, to

obtain a more tender meat and to reduce the variability of this parameter as is demanded by

15
the consumers. Currently, breeding programmes of Parda de Montaña and Pirenaica breeds

are based on BLUP genetic evaluations, while the use of DNA markers is restricted to major

genes (MTSN gene in Parda de Montaña breed) or parentage assignments. The SNPs

rs210072660 and rs109221039 provide an additional resource as a potential genetic marker in

breeding programmes, considering the size of the effects found and the relatively high

frequency of the favourable alleles (A alleles for both SNPs) across Parda de Montaña and

Pirenaica animals. Furthermore, the breeding programmes of both breeds are only beginning

to implement genomic selection in their breeding programmes. In this sense, including

functional mutations can improve the performance of genomic predictions (MacLeod et al.,

2016).

Conflict of interest

The authors have no conflicts of interest.

Acknowledgements

The authors wish to thank the staff of CITA Research Station, ARAPARDA and ASAPI

for their help in sampling, as well as FRIBIN. This study was supported by funds from the

Research Group of the Aragón Government (A14_17R), ARAPARDA and INIA (RZP2017-

00001-00-00 & RZP2015-001-00-00). M. Blanco is supported by INIA-EFS.

References

Allais, S., Journaux, L., Leveziel, H., Payet-Duprat, N., Raynaud, P., Hocquette, J.F., Lepetit,
J., Rousset, S., Denoyelle, C., Bernard-Capel, C., Renand, G., 2011. Effects of
polymorphisms in the calpastatin and mu-calpain genes on meat tenderness in 3 French beef
breeds. Journal of Animal Science 89, 1-11.
Barendse, W.J., 2002. DNA markers for meat tenderness. International Patent Application p.
35.
Bowling, R.A., Smith, G.C., Carpenter, Z.L., Dutson, T.R., Oliver, W.M., 1977. Comparison
of Forage-Finished and Grain-Finished Beef Carcasses. Journal of Animal Science 45, 209-
215.

16
Calvo, J.H., Iguácel, L.P., Kirinus, J.K., Serrano, M., Ripoll, G., Casasús, I., Joy, M., Pérez-
Velasco, L., Sarto, P., Albertí, P., Blanco, M., 2014. A new single nucleotide polymorphism
in the calpastatin (CAST) gene associated with beef tenderness. Meat Science 96, 775-782.
Campo, M.M., Santolaria, P., Sañudo, C., Lepetit, J., Olleta, J.L., Panea, B., Alberti, P., 2000.
Assessment of breed type and ageing time effects on beef meat quality using two different
texture devices. Meat Science 55, 371-378.
Casas, E., White, S.N., Wheeler, T.L., Shackelford, S.D., Koohmaraie, M., Riley, D.G.,
Chase, C.C., Johnson, D.D., Smith, T.P.L., 2006. Effects of calpastatin and -calpain markers
in beef cattle on tenderness traits. Journal of Animal Science 84, 520-525.
Claus, H.L., Dikeman, M.E., Murray, L., Brooks, J.C., Shook, J., Hilton, G.G., Lawrence,
T.E., Mehaffey, J.M., Johnson, B.J., Allen, D.M., Streeter, M.N., Nichols, W.T., Hutcheson,
J.P., Yates, D.A., Miller, M.F., Hunt, M.C., Killefer, J., 2010. Effects of supplementing
feedlot steers and heifers with zilpaterol hydrochloride on Warner-Bratzler shear force
interrelationships of steer and heifer longissimus lumborum and heifer triceps brachii and
gluteus medius muscles aged for 7, 14 and 21 d. Meat Science 85, 347-355.
Curi, R.A., Chardulo, L.A.L., Mason, M.C., Arrigoni, M.D.B., Silveira, A.C., de Oliveira,
H.N., 2009. Effect of single nucleotide polymorphisms of CAPN1 and CAST genes on meat
traits in Nellore beef cattle (Bos indicus) and in their crosses with Bos taurus. Animal
Genetics 40, 456-462.
Enriquez-Valencia, C.E., Pereira, G.L., Malheiros, J.M., de Vasconcelos Silva, J.A.I.I.,
Albuquerque, L.G., de Oliveira, H.N., Chardulo, L.A.L., Curi, R.A., 2017. Effect of the
g.98535683A &gt; G SNP in the CAST gene on meat traits of Nellore beef cattle (Bos
indicus) and their crosses with Bos taurus. Meat Science 123, 64-66.
French, P., O'Riordan, E.G., Monahan, F.J., Caffrey, P.J., Vidal, M., Mooney, M.T., Troy,
D.J., Moloney, A.P., 2000. Meat quality of steers finished on autumn grass, grass silage or
concentrate-based diets. Meat Science 56, 173-180.
Jurie, C., Picard, B., Geay, Y., 1998. Influences of the method of housing bulls on their body
composition and muscle fibre types. Meat Science 50, 457-469.
Kaps, M., Lamberson, W., 2009. Biostatistics for animal science. Cabi International,
Oxfordshire, UK.
Leal-Gutiérrez, J. D., Elzo, M. A., Johnson, D. D., Scheffler, T. L., Scheffler, J. M.,
Mateescu, R. G., 2018. Association of μ-Calpain and Calpastatin Polymorphisms with Meat
Tenderness in a Brahman-Angus Population. Frontiers in genetics, 9, 56.

17
MacLeod, I. M., Bowman, P. J., Vander Jagt, C. J., Haile-Mariam, M., Kemper, K. E.,
Chamberlain, A. J., Schrooten, C., Hayes, B. J., Goddard, M. E., 2016. Exploiting biological
priors and sequence variants enhances QTL discovery and genomic prediction of complex
traits. BMC Genetics17, 44.Morgan, J.B., Wheeler, T.L., Koohmaraie, M., Savell, J.W.,
Crouse, J.D., 1993. Meat tenderness and the calpain proteolytic system in longissimus muscle
of young bulls and steers. Journal of Animal Science 71, 1471-1476.
Morris, C.A., Cullen, N.G., Hickey, S.M., Dobbie, P.M., Veenvliet, B.A., Manley, T.R.,
Pitchford, W.S., Kruk, Z.A., Bottema, C.D.K., Wilson, T., 2006. Genotypic effects of calpain
1 and calpastatin on the tenderness of cooked M. longissimus dorsi steaks from Jersey x
Limousin, Angus and Hereford-cross cattle. Animal Genetics 37, 411-414.
Muir, P.D., Deaker, J.M., Bown, M.D., 1998. Effects of forage- and grain-based feeding
systems on beef quality: a review. New Zealand Journal of Agricultural Research 41, 623-
635.
Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A.R., Bender, D., Maller, J.,
Sklar, P., De Bakker, P.I.W., Daly, M.J., Sham, P.C., 2007. PLINK: A tool set for whole-
genome association and population-based linkage analyses. American Journal of Human
Genetics 81, 559-575.
Schenkel, F.S., Miller, S.P., Jiang, Z., Mandell, I.B., Ye, X., Li, H., Wilton, J.W., 2006.
Association of a single nucleotide polymorphism in the calpastatin gene with carcass and
meat quality traits of beef cattle. Journal of Animal Science 84, 291-299.
Shor-Shimoni, E., Shabtay, A., Agmon, R., Cohen-Zinder, M., 2017. Detection of allelic and
genotypic frequencies of polymorphisms associated with meat quality in the mediterranean
baladi cattle. Open Agriculture Journal 11, 1-10.
Van Eenennaam, A.L., Li, J., Thallman, R.M., Quaas, R.L., Dikeman, M.E., Gill, C.A.,
Franke, D.E., Thomas, M.G., 2007. Validation of commercial DNA tests for quantitative beef
quality traits. Journal of Animal Science 85, 891-900.

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Figure Legends

Figure 1. Effect of the management on Warner-Bratzler Shear Force (WBSF) shear force

through aging in Parda de Montaña and Pirenaica breeds.

The authors (L.P. Iguácel, J.H. Calvo, I. Casasús, M. Serrano, G. Ripoll, P. Sarto, D. Villalba

and M. Blanco ) of the manuscript “The effect of SNPs rs210072660 and rs109221039 of

calpastatin gene on toughness through aging differs depending on the beef cattle breed”,

submitted for its publication to Livestock Science state that they have no conflicts of interest

Fig.1

● grazing bull, ◊ grazing steer; TMR-fed steer, concentrate-fed steer; ■ concentrate-

fed bull, ∆ concentrate-fed heifer

Within a breed and ageing time, means with different letter differ at P<0.05

TMR: total mixed ration

19
Table 1. Hot carcass weight (HCW), age at slaughter, number of samples (n) and Warner-

Bratzler Shear Force (WBSF) in the short (1 d), medium (7 d) and long (15 d) term ageing in

each experiment according the management and the breed

WBSF, N/cm2
HCW, Short- Medium- Long-
age, d
kg ageing ageing ageing
Ex mean mean mean mean mean
Breed Management n n n
p. ± SD ± SD ± SD ± SD ± SD
Parda de Concentrate-fed 285 ± 362 ± 1 62 ±
1
Montaña bulls 23 5 5 16
Concentrate-fed 259 ± 297 ± 1 61 ±
2
bulls 12 20 9 15
Concentrate-fed 262 ± 336 ± 1 92 ± 1 73 ± 1 58 ±
3
bulls 15 23 6 13 6 21 6 13
256 ± 350 ± 90 ± 71 ± 61 ±
Grazing bulls 7 7 7
11 18 9 12 20
309 ± 594 ± 1 93 ± 1 71 ± 1 66 ±
4 Grazing steers
26 23 8 12 8 21 8 19
Concentrate-fed 289 ± 439 ± 84 ± 62 ± 51 ±
5 7 7 7
bulls 40 46 8 19 15
293 ± 569 ± 97 ± 75 ± 67 ±
Grazing steers 8 8 8
31 45 17 19 15
Total mixed 280 ± 539 ± 85 ± 77 ± 69 ±
8 8 8
ration-fed steers 22 37 8 11 9
293 ± 609 ± 91 ± 76 ± 60 ±
6 Grazing steers 8 8 8
28 16 11 20 10
278 ± 372 ± 1 96 ± 1 100 1 81 ±
7 Grazing bulls
32 13 6 15 6 ± 16 6 15
Concentrate-fed 136 ± 185 ± 1 70 ± 1 51 ± 1 43 ±
8
bulls 18 23 4 7 4 9 4 6
122 ± 225 ± 79 ± 78 ± 81 ±
Grazing bulls 8 8 8
9 13 9 13 12
1 1
Concentrate-fed 319 ± 378 ± 60 ± 50 ±
9 0 0
bulls 35 38 16 14
5 4
Concentrate-fed 267 ± 328 ± 1 52 ±
Pirenaica 2
bulls 13 23 2 12
292 ± 614 ± 96 ± 79 ± 74 ±
10 Grazing steers 8 8 8
32 19 6 20 20
Total mixed ration 292 ± 587 ± 86 ± 53 ± 53 ±
8 8
-fed steers 26 18 8 10 8 9
Concentrate-fed 289 ± 384 ± 46 ± 50 ±
11 8 8
bulls 19 20 12 14
Concentrate-fed 271 ± 444 ± 41 ± 40 ±
8 8
heifers 32 13 6 6
Concentrate-fed 289 ± 433 ± 47 ± 41 ±
8 8
steers 21 8 10 6

20
 Concentrate-fed 314 ± 338 ± 1 44 ± 45 ±
12 4
bulls 59 43 6 8 11
Concentrate-fed 226 ± 344 ± 30 ±
4
heifers 5 14 5

Table 2. Phenotypic and animal variances and residuals for each ageing

Short Medium Long

Parda de Montaña
variance (σ2) 198 376 316
Animal variance (σ2a) 139 254 211
(σ2a)/(σ2), % 71 68 67
residual 1.4 1.0 1.0
Pirenaica
variance (σ2) - 248 272
Animal variance (σ2a) - 90 144
(σ2a)/(σ2), % - 36.1 53.0
residual - 1.0 0.01

Table 3. Effect of the SNPs on the Warner-Bratzler Shear Force (WBSF) measured in

different ageing (A) periods for Parda de Montaña and Pirenaica breeds

P-value
SNP Ageing n Genotype
SNP SNPxA

rs210072660 AA GA GG
Parda de Montaña 0.04 0.01 Short 110 88 ± 2.8 87 ± 3.0 89 ± 4.5
Medium 249 72 ± 2.7a 75 ± 2.8a 87 ± 3.7b
Long 214 65 ± 2.7 65 ± 2.9 72 ± 3.8
Pirenaica 0.10 0.78 Medium 72 48 ± 3.8 46 ± 3.7 55 ± 4.2
Long 44 48 ± 4.1 45 ± 3.8 52 ± 5.0
rs109221039 AA GA GG
Parda de Montaña 0.71 0.77 Short 110 88 ± 2.2 91 ± 2.5 85 ± 7.0
Medium 249 73 ± 2.0 75 ± 2.2 76 ± 5.7
Long 214 65 ± 2.0 65 ± 2.3 66 ± 5.6
Pirenaica 0.001 0.09 Medium 72 47 ± 3.4b 47 ± 3.5b 61 ± 4.8a
Long 44 48 ± 3.5b 43 ± 3.8b 67 ± 6.5a

21
Means with different letter differ at P < 0.01 with Bonferroni correction

Table 4. Effect of the haplotypes on the Warner-Bratzler Shear Force (WBSF) measured in

different ageing (A) periods and number of animals for each copy of the haplotype (indicated

in parenthesis) for Parda de Montaña and Pirenaica breeds.

P-value nº copies of the haplotype

Haplotype A
Hap Hap x A 0 1 2
AA
Parda 0.03 0.02 short 87 ± 3.7 (12) 87 ± 2.5 (54) 87 ± 2.5 (44)
medium 84 ± 3.7 (29)a 73 ± 2.7 (119)b 73 ± 2.7 (101)b
long 70 ± 3.4 (27) 64 ± 2.5 (95) 64 ± 2.5 (92)
Pirenaica 0.07 0.84 medium 59 ± 4.2 (17) 50 ± 3.6 (34) 53 ± 3.8 (21)
long 55 ± 5.0 (6) 48 ± 3.6 (25) 52 ± 4.0 (13)
GG
Parda 0.12 0.23 short 86 ± 2.4 (75) 87 ± 2.9 (32) 90 ± 6.9 (3)
medium 72 ± 2.7 (169) 77 ± 3.1 (74) 81 ± 7.2 (6)
long 64 ± 2.5 (151) 65 ± 2.9 (57) 70 ± 6.3 (6)
Pirenaica 0.002 0.08 medium 51 ± 3.4 (39)b 52 ± 3.6 (25)b 65 ± 5.0 (8)a
long 50 ± 3.5 (27)b 47 ± 3.7 (15)b 70 ± 6.6 (2)a
GA
Parda 0.15 0.13 short 88 ± 2.5 (85) 86 ± 3.6 (22) 84 ± 7.9 (3)
medium 73 ± 2.4 (185) 76 ± 3.1 (56) 86 ± 5.9 (8)
long 65 ± 2.5 (158) 65 ± 3.2 (48) 76 ± 5.8 (8)
Pirenaica 0.47 0.90 medium 54 ± 2.5 (50) 50 ± 3.2 (19) 51 ± 7.2 (3)
long 52 ± 2.6 (28) 49 ± 3.4 (14) 48 ± 8.0 (2)
AG
Parda 0.04 0.02 short 88 ± 2.5 (98) 87 ± 4.8 (12) -
medium 76 ± 2.5 (230)a 64 ± 4.3 (19)b -
long 66 ± 2.5 (198) 62 ± 4.5 (16) -
Means with different letter differ at P < 0.05 with Bonferroni correction

Table 5. Effect of the haplotypes on the maximum stress measured in different ageing (A)

periods and number of animals for each copy of the haplotype (indicated in brackets) for

Parda de Montaña and Pirenaica breeds.

P-value nº copies of the haplotype

Haplotype
Hap Hap x A A 0 1 2
AA
Parda 0.03 0.02 short 87 ± 3.7 (12) 87 ± 2.5 (54) 87 ± 2.5 (44)
medium 84 ± 3.7 (29)a 73 ± 2.7 (119)b 73 ± 2.7 (101)b

22
 long 70 ± 3.4 (27) 64 ± 2.5 (95) 64 ± 2.5 (92)
Pirenaica 0.07 0.84 medium 59 ± 4.2 (17) 50 ± 3.6 (34) 53 ± 3.8 (21)
long 55 ± 5.0 (6) 48 ± 3.6 (25) 52 ± 4.0 (13)
GG
Parda 0.12 0.23 short 86 ± 2.4 (75) 87 ± 2.9 (32) 90 ± 6.9 (3)
medium 72 ± 2.7 (169) 77 ± 3.1 (74) 81 ± 7.2 (6)
long 64 ± 2.5 (151) 65 ± 2.9 (57) 70 ± 6.3 (6)
Pirenaica 0.002 0.08 medium 51 ± 3.4 (39)b 52 ± 3.6 (25)b 65 ± 5.0 (8)a
long 50 ± 3.5 (27)b 47 ± 3.7 (15)b 70 ± 6.6 (2)a
GA
Parda 0.15 0.13 short 88 ± 2.5 (85) 86 ± 3.6 (22) 84 ± 7.9 (3)
medium 73 ± 2.4 (185) 76 ± 3.1 (56) 86 ± 5.9 (8)
long 65 ± 2.5 (158) 65 ± 3.2 (48) 76 ± 5.8 (8)
Pirenaica 0.47 0.90 medium 54 ± 2.5 (50) 50 ± 3.2 (19) 51 ± 7.2 (3)
long 52 ± 2.6 (28) 49 ± 3.4 (14) 48 ± 8.0 (2)
AG
Parda 0.04 0.02 short 88 ± 2.5 (98) 87 ± 4.8 (12) -
medium 76 ± 2.5 (230)a 64 ± 4.3 (19)b -
long 66 ± 2.5 (198) 62 ± 4.5 (16) -
Means with different letter differ at P < 0.05 with Bonferroni correction

23