Genetic variations of the porcine PRKAG3 gene in Chinese indigenous pig breeds

Four missense substitutions (T30N, G52S, V199I and R200Q) in the porcine PRKAG3 gene were considered as the likely candidate loci affecting meat quality. In this study, the R200Q substitution was investigated in a sample of 62 individuals from Hampshire, Chinese Min and Erhualian pigs, and the genetic variations of T30N, G52S and V199I substitutions were detected in 1505 individuals from 21 Chinese indigenous breeds, 5 Western commercial pig breeds, and the wild pig. Allele 200R was fixed in Chinese Min and Erhualian pigs. Haplotypes II-QQ and IV-QQ were not observed in the Hampshire population, supporting the hypothesis that allele 200Q is tightly linked with allele 199V. Significant differences in allele frequencies of the three substitutions (T30N, G52S and V199I) between Chinese indigenous pigs and Western commercial pigs were observed. Obvious high frequencies of the "favorable" alleles 30T and 52G in terms of meat quality were detected in Chinese indigenous pigs, which are well known for high meat quality. However, the frequency of the "favorable" allele 199I, which was reported to have a greater effect on meat quality in comparison with 30T and 52G, was very low in all of the Chinese indigenous pigs except for the Min pig. The reasons accounting for this discrepancy remain to be addressed. The presence of the three substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations. A novel A/G substitution at position 51 in exon 1 was identified. The results suggest that further studies are required to investigate the associations of these substitutions in the PRKAG3 gene with meat quality of Chinese indigenous pigs, and to uncover other polymorphisms in the PRKAG3 gene with potential effects on meat quality in Chinese indigenous pigs.


INTRODUCTION
The protein kinase adenosine monophosphate-activated γ3-subunit (PRKAG3) gene encodes the muscle specific isoform of the regulatory γ3 subunit of adenosine monophosphate activated protein kinase (AMPK), which plays a key role in regulating energy homeostasis in eukaryotes. A nonconserved substitution (R200Q) in the PRKAG3 gene was initially characterized in Hampshire pigs [6], resulting in an approximate 70% increase in muscle glycogen content with large effects on meat quality and processing yields [2,3]. Three additional economically important missense substitutions (T30N, G52S and V199I) in the porcine PRKAG3 gene were recently identified with additive effects on the glycogen content in muscle and meat quality traits [1]. The PRKAG3 gene was therefore supposed to be a functional gene significantly affecting meat quality traits in pigs. China has abundant indigenous pig breeds that are well known for high meat quality. Here we report on the genetic variations of four missense substitutions (T30N, G52S, V199I and R200Q) in the PRKAG3 gene in Chinese indigenous pigs in order to provide new evidence of the effects of the PRKAG3 gene on meat quality.

Animals
Three single nucleotide polymorphisms (SNP) of the porcine PRKAG3 gene (T30N, G52S and V199I) were genotyped using 1505 individuals from 21 Chinese indigenous pig breeds, 5 Western commercial pig breeds and wild pigs (Tab. I). Samples were chosen avoiding full sib animals. Allele frequencies at the R200Q mutation site were estimated in 37 purebred unrelated Hampshire individuals, 13 unrelated Min pigs and 12 unrelated Erhualian pigs. SNP screening was performed using 18 founder animals (2 White Duroc and 16 Chinese Erhualian) of a three-generation intercross between White Duroc × Chinese Erhualian. Genomic DNA were isolated from ear tissues by phenol/chloroform extraction [4].

DNA sequencing and SNP identification
PCR products representing different genotypes at the four PRKAG3 SNP loci were purified using a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), and were bi-directionally sequenced with the respective PCR primers using the ABI PRISM  BigDye TM Terminator Cycle Sequencing Kit (v3.0) and an ABI PRISM  3100 Genetic Analyzer (Applied Biosystems, Foster City, USA). The sequence identity was compared by the Gapped BLAST program (http://www.ncbi.nih.gov), and the sequences of 18 founder animals were aligned by the application of the program SeqMan (DNAStar software package, http://www.lynnon.com) to identify the putative SNP.

T30N, G52S and V199I substitutions
Allele frequencies at the T30N, G52S and V199I polymorphic loci in 21 Chinese indigenous pigs, 5 Western commercial pigs and the wild pig are summarized in Table I. Chinese indigenous pig breeds belonging to the same ecotype had more similar allele frequency distribution. Western commercial pigs had higher polymorphism information content (PIC) at the three SNP loci compared with most of the Chinese indigenous pigs. The differences of allele frequencies between most of the Chinese indigenous pigs and Western commercial pigs were significant (P < 0.05, data not shown). The genetic variations at the three SNP loci in Landrace, Large White and Duroc studied here were similar to those reported previously [1], although some tiny deviations were observed, presumably due to the smaller sample size in this study.
The 30T and 52G alleles were reported to be favorable in terms of meat quality [1]. As expected, the 30T and 52G alleles were the predominant alleles in all Chinese indigenous pigs, which was consistent with the fact that Chinese indigenous pigs generally have flavorful meat quality [5].
Amongst the T30N, G52S and V199I substitutions, the largest effects on meat quality were obtained with the V199I substitution with the 199I allele being the favorable allele for high meat quality in the association study [1]. A high frequency of the 199I allele was observed in Berkshire pigs which are known for good meat quality in Western commercial pigs [1]. Surprisingly, the frequencies of the favorable allele 199I were very low in all of the Chinese indigenous pigs except for Min pigs. The explanations for the discrepancy might be the following: (1) the effects of V199I substitution on the meat quality of Chinese indigenous pigs are different from those of Western commercial pigs; (2) other unrevealed substitutions in the PRKAG3 gene or other functional genes with significant favorable effects in meat quality exist in Chinese indigenous pigs, which ameliorate the negative effects of the 199V allele in terms of meat quality in Chinese indigenous pigs. Further SNP screening and identification of the porcine PRKAG3 gene are worthwhile.
All three substitutions were observed in Chinese Tibetan pigs. Few exotic pig breeds have been introduced into the Qinghai-Tibet plateau, the habitat of Chinese Tibetan pigs, because of geographical isolation. The existence of T30N, G52S and V199I substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations.
Berkshire pigs were initially developed in Britain in the middle of the nineteenth century by crossing local pigs in England (the basic Berkshire stock) with Siamese and Chinese Guangdong spotted pigs, which were introduced into Britain in the 1770s [5]. The 199I allele was not found in the 57 Guangdong spotted pigs, indicating that tracing the high frequency of the 199I allele in Berkshire pigs probably originated from local pigs in England or Siamese pigs.

R200Q substitution
The R200Q substitution in the PRKAG3 gene was shown to be a causative mutation underlying the large differences in meat quality and processing yield in Hampshire pigs [6]. The allele frequencies of 200R and 200Q in the Hampshire population in this study were 65% and 35%, respectively (Tab. I). The ratio of RN − carriers (RQ and QQ individuals) was 57%, which was less than that of 86% evaluated by a glycolytic potential in American Hampshire populations [7]. The II-QQ and IV-QQ haplotypes were not found in the Hampshire population in this study (data not shown), supporting the hypothesis that 200Q is tightly linked with 199V [1]. No 200Q allele was present in the Chinese Min and Erhualian pigs, consistent with the fact that the 200Q allele is observed mainly in Hampshire pigs [1,6].

SNP identification
A novel SNP, A/G transversion at position 51 in exon 1, was identified in the PCR1 amplicon; the synonymous mutation does not alter the encoding amino acid. The A/G substitution was verified by the alignment of two porcine PRKAG3 gene sequences deposited in the database (Genbank accession no. AF214521 and AX398341.1, data not shown).