Evaluation of the RYR1 gene genetic diversity in the Latvian White pig breed

K. Dokāne, D. Jonkus, T. Sjakste © 2016 K. Dokāne et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577 Evaluation of the RYR1 gene genetic diversity in the Latvian White pig breed


Introduction
RYR1 encodes the ryanodine receptor 1, which is a calcium ion channel in the sarcoplasmic reticulum of skeletal muscle.In humans multiple single nucleotide polymorphisms (SNPs) in the coding gene region (cSNPs) are associated with the malignant hyperthermia susceptibility [1], central core disease [2], and other myopathies [3].Remarkably, that a homologous missense mutations in human (Arg614Cys) and pig (Arg615Cys) originated from homologous C>T cSNPs (rs118192172 and rs344435545 for human and pig respectively) lead to the same malignant hyperthermia syndrome.Besides malignant hyperthermia the rs344435545T allele is associated in pig with the porcine stress syndrome (PSS, halothane susceptibility) and pale, soft and exudative meat (PSE) syndrome [4].It is suggested, ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 3. P 184-189 doi: http://dx.doi.org/10.7124/bc.00091Dhowever, that heterozygote carriers in some breeds may have some adaptive advantages and better parameters for the economically important meat traits [5].

Genomics, Transcriptomics and Proteomics
Besides the rs344435545 up today about 2000 SNPs and 150 cSNPs have been reported for the pig RYR1 gene (http://www.ncbi.nlm.nih.gov/snp).However, there are still no data on their genetic diversity and possible association with phenotype.
The aim of the current study was to genotype the rs344435545 (C1912T, Arg615Cys), rs196953058 (T8434C, Phe2769Leu) and rs323041392 (G12484A, Asp4119Asn) in Latvian local pig breed Latvian White and to evaluate in silico the potential effect of amino acid substitution on the protein function.

Materials and methods
Sample collections include the Primary (PR) collection of 8 samples of original Latvian White randomly collected from Latvia University of Agriculture (LLU) herd in 2006 within Latvian Ministry of Agriculture supported project and Representative (RP) collection of 103 samples collected in 2015 in three geographically distant private farms in frame of the "AgroBioRes" Latvian National Research Program (VPP) 2014-2017.DNA was isolated from 500 μl of blood of each animal by using a K0512 Genomic DNA Purification Kit (Thermo Scientific).Sequence information for swine (Sus scrofa) RYR1 gene available in GenBank (http://www.ncbi.nlm.nih.gov/nuccore/347616829?report=fasta&from=17 14228&to=1862032) was used for the genomic region reconstruction and PCR primer design.Figure 1 provides a detailed information on the conserved domain RYR1 protein architecture and location of the genotyped polymorphic loci.
All three polymorphisms were genotyped by a restriction fragment length polymorphism (RFLP) technology.Primer sequences and other PCR and RFLP related information are given in Table 1.
PCR was performed using DreamTaq polymerase (Thermo Scientific) with following DNA amplification parameters: 94 °C for 5 min; then 40 cycles of 94 °C for 30 s, appropriate annealing temperature (60.2-65.6 ˚C) for 30 s, 72 °C for 45 s and 72 °C for 5 min.DNA digestion with restriction enzymes was performed according to the producer protocol (Thermo Scientific).The PCR and restriction products were analysed by electrophoresis in 1-3 % agarose gel.Genotyping was performed in duplicate for each locus of each animal.The genotyping results were verified by sequencing in both directions (ABI Prism3100 Genetic Analyzer) of randomly chosen amplicons.
The prediction of potential effect of amino acid substitutions on the protein function was performed using online available tool SIFT (Separate Intolerant from Tolerant, http://sift.bii.a-star.edu.sg/)following the recommendations of developers [6,7].The pig RYR1 sequence with the accession P16960 used as a query was compared with the related sequences found by SIFT in SwissProt database.The sequence median conservation was set to 3.00 prior the analysis [6].For every amino acid in each position in the alignment, SIFT calculates normalized probabilities for all possible amino acid substitutions.The substitution is predicted as deleterious if normalized values are less than cut-off (0.05) [7].
The research was approved by the LLU review board.

Results and Discussion
All polymorphic loci studied are cSNPs located in the RYR1 gene evolutionary conserved domains (Figure 1, 2). Figure 2 illustrates a homology between the related sequences of human and pig proteins and a high level of homology in the positions and motifs of amino acid substitutions.
The conserved domains are thought to be responsible for binding different modulators of the protein function [9].Therefore, amino acid substitution in the RYR1 domains may significantly change the protein target affinity and response to the regulators.We performed an in silico analysis using SIFT online tool to detect, whether an amino acid substitutions in the loci studied may affect the RYR1 function.We found out that the R615C substitution potentially possessed a damaging influence on the RYR1 function (SIFT score 0.01) while the F2769L and D4119N substitutions appear to be neutral (SIFT scores 1.00 and 0.24, respectively).
Our findings on the R615C functionality is in good correlation with well-known data on the mutation phenotypic effect in both human [1,10] and pig [8].In    contrast to human where the mutation is dominant [11], the malignant hyperthermia, PSS and PSE syndromes in pig are associated with a homozygous genotype [12] and the adaptive advantages and better parameters for economically important meat traits were reported for the heterozygous animals [5,8].Figure 3 illustrates genotyping results.
The locus rs344435545 was found to be monomorphic in both PR and RP collections.Despite the fact, that a related unfavourable phenotype (malignant hyperthermia, PSS) had not been reported for Latvian White until now, the full absence of site polymorphism in this breed was not expected before our study.The rs344435545 minor (unfavourable) T allele had been detected in the pigs of Yorkshire breed [13] used in Latvian White initial selection (http://www.ccc.lv/lv/selekcija/latvijas_balta.htm) and often used in current breeding programs (http:// www.ccc.lv/lv/selekcija/latvijas_balta.htm).Heterozygote carriers of the rs344435545 T allele of Yorkshire breed could be supported by breeding programs as having adaptive advantages and better meat Fig. 3. Genotyping results given for two samples from PR collection.Panel A illustrates RFLP data; restriction enzymes and genotypes defined are indicated below and lower electrophoregrams respectively.Letter M indicates 100 bp DNA ladder (Thermo Scientific).Panel B provides examples of sequencing chromatograms: the rs344435545 and rs196953058 were found to be homozygous on the C and T alleles respectively, the rs323041392 was observed in the PR collection either as homozygote GG or as heterozygote GA.
quality parameters [5].However, this allele appears not to have been introduced in Latvian White.A permanent control using molecular markers is required to prevent introducing of unfavourable 1912T allele from Yorkshire pigs to Latvian White in the future breeding programs.
The locus rs196953058 was also observed as monomorphic in both PR and RP collections.
Instead, the rs323041392 showed in the PR a high level of polymorphism with the GG/GA/AA genotype correlation equal to 3/4/1.As the LLU herd (PR collection) had been under a strong selection control focusing on the Latvian White breed maintenance and conservation, polymorphism at the rs323041392 observed in the PR collection could be considered as a breed specific character.However, in the RP collection (animals from private farms) this locus was found to be monomorphic.So, full loss of the rs323041392 variability in Latvian White happened in nine years (from 2006 till 2015) of private farming.The loss of heterozygosity at the rs323041392 may signal to Latvian livestock industry that the A B breeding intensification and over-introgression of favourable alleles may lead to the loss of local breeds' specific features and decrease in the selective facilities and adaptability to the environmental challenges in the local breeds of small populations.

Fig. 1 .
Fig. 1.Pig (Sus scrofa) RYR1 protein sequence scheme given along with information on the mutations studied.Upper line illustrates the polypeptide chain architecture where the conserved domains are indicated in black.The conserved domains and exons encompassing the studied cSNPs are indicated below by abbreviation and number for the domains and exons respectively.SNPs' description include the SNP ID number and nucleotide and amino acid residue change in mRNA (NM_001001534.1) and polypeptide (NP_001001534.1)respectively given according to SNP database at NCBI (http://www.ncbi.nlm.nih.gov/snp).Information on the homologous mutation in human is given in the bottom of the picture.Abbreviations: RYDR_ITPRthe RIH extracellular domain found in both the ryanodine and the inositol-1,4,5-trisphosphate receptors types of calcium channels; RYR -the ryanodine receptor specific conserved domain of unknown function; C -EF-hand_8 -the conserved domain of the cl08302 superfamily, contains calcium sensors and calcium signal modulators; n/d -no data.
genotyped according toSilveira et al. (2011) [8] simultaneously by two restriction enzymes: HhaI cuts the sequence having major favourable allele C and Alw21I cuts the sequence encompassing unfavourable allele T; # ApaI cuts two and three restriction sites in the sequence encompassing the rs196953058 T ( #1 ) and C ( #2 ) alleles respectively.A polymorphic nucleotide is boxed in the restriction site description.

Fig. 2 .
Fig. 2. Partial alignment of the human (P21817.3/GI:108935904)and pig (P16960.2/GI:1173335)RYR1 protein sequences.Panels A, B and C illustrate alignment of the regions encompassing the rs344435545, rs196953058 and rs323041392 of the pig protein respectively.Arrows indicate a position of mentioned and neighbouring amino acid substitutions in the human (upper line) and pig (lower line) protein, motif description is given above the corresponding arrows.*there are two variants of amino acid substitution at the 628: besides indicated R628C substitution the R628H also has been reported.Abbreviations: aa -amino acid.