analysis of polymorphisms in candidate ’ s genes for meat quality in Lidia cattle

The aim of this paper was to analyze the segregation of some polymorphisms in three genes (CAPN1, CAST and DGAT1) related to meat quality in the Lidia cattle breed and some of its main lineages. To that effect, 119 individuals from the Lidia breed were analyzed. Although the association between the polymorphisms and the phenotype has never been demonstrated in this breed, the absence of fixed genotypes for these polymorphisms in the studied population makes the Lidia cattle a good candidate to develop selection objectives. The clear differentiation among lineages for most of the genes studied reinforces the high reproductive isolation presented in the Lidia cattle as revealed by previous studies on the structure of the population within the Lidia breed using microsatellite markers. Considering both issues in the design of breeding schemes will be necessary to save the lineages and not to lose this valuable genetic resource. Finally, it would be necessary to carry out an in depth search for new polymorphisms in genes associated with meat quality and to perform needed association analyses between the SNPs segregating in Lidia cattle and traits of economic interest. additional key words: bullfighting; calpastatin; single nucleotide polymorphisms. abbreviations used: MNA (mean number of alleles per locus); SNP (single nucleotide polymorphisms). authors’ contributions: Conceived and designed the experiments, and critical revision of the manuscript for important intellectual content: MV and AM. Performed the experiments: RP. Analyzed the data and wrote the paper: RP and CA. citation: Pelayo, R.; Valera, M.; Molina, A.; Avilés, C. B. (2016). Short communication: Analysis of polymorphisms in candidate’s genes for meat quality in Lidia cattle. Spanish Journal of Agricultural Research, Volume 14, Issue 4, e04SC02. http://dx.doi. org/10.5424/sjar/2016144-9279. Supplementary material (Figs. S1, S2, S3, S4 and S5) accompanies the paper on SJAR’s website. received: 11 Jan 2016. accepted: 11 Nov 2016. copyright © 2016 inia. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: Lidia cattle breed association (“Agrupación Española de Ganaderos de Reses Bravas”). competing interests: The authors have declared that no competing interests exist. correspondence should be addressed to Rocío Pelayo: rociopega55@hotmail.com carded. In these cases, the economic value and income from the meat is essential for the producer and breeder of Lidia cattle. The meat from excitable animals has lower quality than more docile animals as assessed by behavioral tests (Haskell et al., 2014). An interesting strategy to enhance the profitability of the farm would be to assess the possibility of improving the characteristics of the meat, to widen the selection criteria of the Lidia cattle breed and look for alternative uses of a breed mainly reared in extensive systems which could provide an organic and healthy dietary source. Given the fact that sensory and nutritional quality of meat are becoming more and more important, different studies related to this topic have recently been carried out on Lidia cattle (Beriain et al., 2011; Horcada-Ibáñez et al., 2012). These authors characterized this breed from its Spanish Journal of Agricultural Research 14(4), e04SC02, 5 pages (2016)

carded.In these cases, the economic value and income from the meat is essential for the producer and breeder of Lidia cattle.The meat from excitable animals has lower quality than more docile animals as assessed by behavioral tests (Haskell et al., 2014).An interesting strategy to enhance the profitability of the farm would be to assess the possibility of improving the characteristics of the meat, to widen the selection criteria of the Lidia cattle breed and look for alternative uses of a breed mainly reared in extensive systems which could provide an organic and healthy dietary source.Given the fact that sensory and nutritional quality of meat are becoming more and more important, different studies related to this topic have recently been carried out on Lidia cattle (Beriain et al., 2011;Horcada-Ibáñez et al., 2012).These authors characterized this breed from its 2 minion ® MBL kit following the manufacturer's instructions.Four different DNA fragments were analyzed.A set of PCR primers were designed in two regions (CAPN1 segment 1, from intron 8 to intron 9 and CAPN1 segment 2, from exon 11 to exon 22) of the CAPN1 gene (GenBank Acc.No. AH009246), the CAST gene (AY008267), and DGAT1 gene (JQ897351-53).The primers used in the analyses are shown in Table 1.PCR reactions were carried out in a thermocycler (Eppendorf® AG, Germany) in a total volume of 25 μL containing 2 μL (80 ng) of bovine genomic DNA, 2.5 μL of PCR buffer, 0.75 μl of MgCl 2 (50 mM), 1.2 μL of dNTPs (4μM), 2 μL of each primer (5 mM), 0.1 μL of Taq DNA polymerase (5U/μL) and 14.45 μL of MQ H 2 O.The thermal profiling consisted of a hot start step at 96 °C for 3 min, followed by 40 cycles of 30 s at 96 °C, 30 s at the annealing temperature of 72 °C (all genes), 4 min at 72 °C and a final extension step of 10 min at 72 °C.The obtained amplicons were purified and visualized on 2% agarose electrophoresis gels stained with ethidium bromide.Segments 1 and 2 of the CAPN1 gene, CAST and DGAT1 genes were sequenced.All sequences were edited, assembled and aligned using the program Sequencher v.4.6 software (Gene Codes Corporation).After alignment, polymorphic sites were determined and different genotypes assigned by visual examination of the electropherograms (Fig. S1 [suppl.]).The variability parameters across loci and populations for the whole population and for each different lineage were computed using the Genetix 4.2 program (Belkhir et al., 2004).Fisher's exact test was performed to assess the possible association between the frequency distribution of the different polymorphisms studied and the five lineages of our population (Statistica v.6.0 software).

results and discussion
Allele frequencies for each polymorphic locus are presented in Table 2. Six SNPs that were previously reported (Avilés et al., 2009) were found in the CAPN1 segment 1.According to published research, the C allele from the CAPN1: Ex9-g.316 C>G was associated phenotypic make up.Conversely, no studies have been performed to determine the heritability of these traits, but limited to behavior trait studies (Pelayo et al., 2016).Polymorphisms in genes associated with meat quality are one example of technology used to predict traits like tenderness or marbling.However, these associations must be validated in each particular breed.
The calpain-calpastatin complex (CAPN1 and CAST) is an endogenous, calcium-dependent proteinase system (Goll et al., 2003) involved in the post-mortem tenderization process since they regulate post-mortem proteolysis.The CAPN1 gene has been mapped to chromosome 29 and several single nucleotide polymorphisms (SNPs) in this gene have been reported to be associated with meat tenderness in beef cattle (Page et al., 2004), two of which produce glycine to alanine and valine to isoleucine substitutions in exons 9 and 14, respectively.According to Avilés et al. (2013) and Calvo et al. (2014) and the effect of the SNPs of the CAST on instrumental meat tenderness is evident in different beef populations reared in Spain.The diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis (Winter et al., 2002).This gene has been mapped to chromosome 14.A lysine to alanine substitution (K232A) in the DGAT1 gene has been associated with increased milk yield and milk fat content in dairy cattle (Casas et al., 2005).The aim of this paper was to analyze the frequencies of several polymorphisms in three genes related to meat quality (CAPN1 and CAST) for tenderness and intramuscular fat, back fat thickness or sensory flavor and juiciness (DGAT1), in the whole population of Lidia cattle as well as in the main lineages currently being reared.

material and methods
Blood samples of 119 individuals from the Lidia breed (79 males and 40 females), from 5 different lineages were analyzed (28 Domecq, 22 Marqués de Albaserrada, 24 Murube Urquijo, 20 Núñez and 25 Santa Coloma).Genomic DNA was obtained from 200 μL of whole blood using the extraction protocol of the Dotable 1. Amplified fragments and primers used in the analysis.presented a frequency of 0.57 of the allele associated with tender meat (Schenkel et al., 2006).Van Eenennaam et al. (2007) found frequencies for the C allele ranging from 0.63 (Angus breed) to 0.73 (Limousin breed).Finally, remarkable differences were found in Lidia cattle between lineages for DGAT1: Ex8-g.232 AAAA>GCGC.The lysine variant of the marker has been associated with a higher lipid content in different tissues (Thaller et al., 2003;Avilés et al., 2013).In our population, the lysine allele frequency ranged from 1.00 in Marqués de Albaserrada lineage to 0.04 in Domecq lineage.Overall, the population in Lidia cattle breed presented low frequency of the KK genotype (0.28).This result was consistent with those published in Lidia cattle breed (0.21) by Kaupe et al. (2004).

Length
Our results showed that there are statistically significant differences in the distribution of frequencies of the studied markers and the different lineages.The genetic variability was low in all lineages (Table 3) which is partitioned into very few farms that impose a high reproductive isolation, with a higher heterozygosity in the Murube Urquijo lineage (0.409) and a lower heterozygosity (0.197) in the Marqués de Albaserrada lineage (in accordance with the frequencies mentioned previously).This higher reproductive isolation of all the Lidia lineages (with regards to other cattle breeds) imposed by breeders with the objective to maintain their branded features (Sanz et al., 2014) may be the cause of the heterozygosity deficit observed in the five lineages.The effective size within the Lidia cattle lineages is very low.Therefore, it with lower shear force values (Page et al., 2004;Schenkel et al., 2006).Highlighted differences between lineages for this SNP were found in the Lidia cattle breed.The C allele frequency ranged from 0.71 to 0.14 for Domecq and Marqués de Albaserrada lineages, respectively.Overall, the population in the Lidia cattle breed presented a frequency of the C allele of 0.45.Avilés et al. (2009) found in the Spanish maternal beef breeds frequencies ranging from 0.11 (Avileña breed) to 0.29 (Retinta breed) while Van Eenennaam et al. (2007), showed in European continental breeds, frequencies between 0.05 for Charolais breed and 0.13 for Simmental breed.Three SNPs were detected in the CAPN1 segment 2, two of which were not published before: In13-g.4506 C>G and In14-g.4685 C>T, see Figs.S1-S5 [suppl.].Page et al. (2004) associated the G allele from CAPN1: Ex14-g.4558 G>A with lower shear force values.The G allele frequency ranged between 1.00 in Marqués de Albaserrada lineage to 0.90 in Domecq and Murube Urquijo lineages.Overall, the frequency of the G allele for the population in the Lidia cattle breed is 0.94, while Allais et al. (2011) published frequencies in European continental breeds ranging from 0.76 (Charolais breed) to 0.64 (Limousin breed).However, it would be interesting to perform an in depth study of the two new markers; CAPN1: In13-g.4506 C>G and In14-g.4685 C>T.Frequencies for the C allele from the CAST: In8-g.282 C>G SNP ranged between 1.00 in Marqués de Albaserrada lineage to 0.46 in Murube Urquijo lineage.Overall, the population table 2. Allele frequencies for the identified SNPs in CAPN1, CAST and DGAT1 genes and Fisher's exact association test with the five lineages of Lidia cattle breed.In parenthesis, number of samples of each lineage. [1] allele [2]   [1] Nucleotide positions are deduced from reference sequences EU386166-83 for CAPN1 segment 1, AF248054 CAPN1 segment 2, AY008267 for CAST and JQ897351-53 for DGAT1. [2]"Allele" shows the allele that is reported to have positive effect on tenderness (decreases shearforce).The results of this study in the Lidia cattle breed show the frequency of the alleles previously associated with more tender meat and higher fat content.Finally, it would be interesting to carry out an in depth search for new polymorphisms in genes associated with meat quality and the need of performing association analyses between the SNPs segregating in Lidia cattle and traits of economic interest.

acknowledgements
We acknowledge the collaboration of the Lidia Cattle Breed Association (AEGRB).

table 3 .
Expected (He)and observed (Ho) heterozygosity and mean number of alleles per locus (MNA) values in the five lineages of Lidia breed for the CAPN1, CAST and DGAT1 genes analysed.Standard deviation in parenthesis.