Introduction
In 1983, the
callipyge phenotype was first identified in a Dorset ram transmitting an extremely heavily muscled phenotype to some of his offspring with
callipyge (from Greek calli-, beautiful; -pyge, buttocks) and the symbol
CLPG was proposed for this gene (
Cockett et al., 1994). The
CLPG locus was mapped to the terminal region of the ovine chromosome 18 and exhibits a unique form of parental imprinting and polar over-dominance, in which only individuals received the allele from their sire and expressed
callipyge phenotype (
Cockett et al., 1994,
1996;
Freking et al., 1998;
Lien et al., 1999;
Shay et al., 2001). The magnitude of expression of
callipyge gene depended upon the location of muscles in the body, and the increased muscle mass was concentrated in the legs and loins (
Jackson et al., 1997a). Lambs expressing the
callipyge gene had higher dressing percentage, leg and conformation scores, and larger longissimus muscle areas. Their carcasses had higher protein, moisture, and ash percentages and lower fat percentage, which suggested that ram lambs expressing the
callipyge had an advantage in retail yield and carcass conformation (
Jackson et al., 1997b). Use of
CLPG mutant allele in structured mating systems could dramatically increase the production of lean lamb (
Freking et al., 1998).
It was shown that the
CLPG phenotype of sheep resulted from the transition of 267A→G, by analyzing sheep STS (AF401294) (
Freking et al., 2002;
Smit et al., 2003). Similar region containing ovine
callipyge mutation was obtained for pig (AY167895 and AY682208), horse (AY167898), cattle (AY167897), rabbit (AY167900), dog (AY167901), goat (AY884304 and AY850925), Aoudad (AY167896), Gray squirrel (AY167902), Norway rat (AY167894), and Meadow vole (AY167893) (
Smit et al., 2003). The
CLPG gene of pig and cattle was also mapped on chromosome 7 (AY682208) and 21 (
Fahrenkrug et al., 2000). We analyzed the bioinformatics of partial
CLPG gene sequences among different species and predicted its position on goat chromosome (
Shen et al., 2007). Meanwhile, we found 184C→T variation site of goat
CLPG gene and suggested that there might be a relationship between 184C→T and the trait of
Callipyge in goat (
Wang et al., 2007). Boer goat is one of the most famous meat-oriented goat breeds in the world, with more obvious muscular hypertrophy condition at the neck, breast, shoulder, back, loin, and buttocks than other goat breeds. In this article, the 184C→T was confirmed again by sequencing. Then the distribution of this SNP in Boer goat and 13 other Chinese indigenous goat breeds, and its association with body weight at different ages were investigated for discovering its possible role in controlling the double muscle trait in goats.
Materials and methods
Samples and DNA extraction
A total of 584 blood samples from goats of 14 populations (13 Chinese indigenous goat breeds and 1 imported goat breed, Boer goat) were collected from 11 provinces and autonomous regions in China (Table 1). The samples were divided into three groups, South (243), North (270), and Foreign (71), according to their distributions (
Compiling Group of Sheep and Goat Breeds in China, 1989) (Table 1). The South group included Chengdu brown goat, Nanjiang brown goat from Sichuan Province, Guizhou white goat, and Guizhou black goat from Guizhou Province, Duan goat from Guangxi Province, Leizhou goat from Guangdong Province, and Yichang white goat from Hubei Province. The North group included Inner Mongolia Cashmere goat from Inner Mongolia Autonomous Region, Liaoning cashmere goat from Liaoning Province, Taihang mountain goat and Chengde polled goat from Hebei Province, Shaannan white goat from Shanxi Province, and Jining grey goat from Shandong Province. The Foreign group had Boer goat imported from South Africa and sampled from Hebei Province. The samples were collected and stored at -70ºC prior to DNA extraction, which was conducted by the Phenol extraction method.
Primer, PCR amplification, and sequencing
The primer used for amplifying goat CLPG gene was from that of sheep CLPG gene (AF401294). The forward and reverse primers were 5′-tgaaaacgtgaacccagaagc-3′ and 5′-ggcaggagagacggttaat-3′, respectively, and were synthesized by TaKaRa Biotechnology Co. Ltd. (Dalian, China). The theoretical length of an amplified goat CLPG gene should be 493 bp according to the sheep CLPG gene (AF401294).
PCR was carried out in Programmable Thermal Controller (German Biometra) with a total volume of 30 μL reaction system containing 4 μL (75 ng∙μL-1) of goat genomic DNA, 3 μL of 10 × PCR standard reaction buffer, 2.4 μL dNTPs (2.5 pmol∙L-1 of each deoxynucleotide), 1.2 μL (10 pmol∙L-1) of each forward and reverse primer, 0.3 μL (5U∙μL-1) of Taq DNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China) and 17.9 μL of distilled water. After pre-denaturation for 3 min at 94°C, the PCR profile consisted of a denaturation step at 94ºC for 45 s, an annealing step at 55ºC for 45 s, and an elongation step at 72ºC for 1 min for a total of 34 cycles, followed by a final extension of 10 min at 72ºC. The PCR products were run on 1.5% agarose gel including 0.5 mg∙mL-1 of ethidium bromide, and visualized and photographed with the help of a gel automatic photographer under UV light.
A total number of 23 goats from 10 breeds (five Boer goats, three Chengdu brown goats, three Nanjiang brown goats, three Leizhou goats, three Jining grey goats, three Liaoning cashmere goats, and three Chengde polled goats) were amplified first for sequencing in order to screen SNPs. The PCR products were sequenced by Shanghai Sangon Biological Engineering & Technology services Co. Ltd. (Shanghai, China).
PCR-RFLP of goat CLPG and genotype determination
In order to identify the SNP of goat CLPG gene, 23 sequenced individuals were aligned by BioEdit software (Version 7.0) with two Tibetan goat sequences (AY850925 and AY884304). The transition of 184C→T was defined after aligning and checking for sequencing results (A, C, D, and E in Fig. 1). The PCR-RFLP was carried out according to the variation site (184C→T). Fork I (TaKaRa Biotechnology Co. Ltd., Dalian, China) recognizing GGATG and cutting at positions 194 and 444 was selected according to the variation, using Webcutter 2.0 (http://users.unimi.it/~camelot/tools/cut2.html). The digestion solution with a total volume of 10 μL containing 6.5 μL of PCR products, 0.3 μL (5 U∙uL-1) of Fork I, 1.2 μL of distilled water, 1.0 μL 0.1% BSA, and 1.0 μL of 10 × buffer, was incubated at 37ºC for 2 h in the Programmable Thermal Controller. The genotype was detected by running digested products on 1.5% agarose gel including 0.5 mg∙mL-1 of ethidium bromide. Homozygote CC was defined when base C was present at position 184, forming GGACG but not recognized by Fork I, and forming 444 bp and 49 bp (B in Fig. 1, invisible on agarose gel for 49 bp fragment, same as below), homozygote TT was defined when base T was present at position 184 forming GGATG, recognized by Fork I, forming 250 bp, 194 bp, and 49 bp, and heterozygote CT was defined when C and T were both present at the same position on the homologous chromosome, forming 444 bp, 250 bp, 194 bp, and 49 bp.
Statistical analysis of the relationship between 184C→T and body weight
A total of 247 individuals (131 males and 116 females) including 71 Boer goats and another 176 upgrading offspring of the Boer goat to Tangshan dairy goat with body weight records and identified genotypes of CLPG gene using PCR-RFLP mentioned earlier were collected from the Boer goat farm in Qinhuangdao City, Hebei Province. According to birth types, all the individuals were sorted into single birth (66), twin births (139), and triplet births (42). The body weight records were marked at birth, and in one week, two weeks, three weeks, one month, two months, three months, and four months, respectively.
The statistical objective was to investigate the effect of paternal genotype, maternal genotype, interaction of paternal and maternal genotype, and individual genotype on body weight at different ages. According to the data type, some other factors also affected the dependent variable and body weight. Therefore, the general linear model, Y = μ + B + S + T + P + M + P*M + G + e, was selected according to the data type, where the cardinals B, S, T, P, M, P*M and G, and the fixed factors, represent the effect of breed type (Boer goat and its upgrading offspring), sex (male and female), birth type (single, twin, or triplet birth), paternal genotype, maternal genotype, interaction of paternal and maternal, and genotype of individuals. μ and e represent the population mean and error. SPSS software (version 13.0) was used to analyze the genotype effect.
The chi-square test for crosstables or Fisher’s exact test was performed to analyze differences in allele frequencies between two populations using SPSS software (version 13.0).
Results
SNP of goat CLPG gene and its distribution in different populations
The substitution of 184C→T was detected according to the alignment of 23 sequenced individuals with two Tibetan sequences from GenBank (AY850925 and AY884304). Genotyping results using PCR-RFLP with Fork I for individuals from different populations are listed in Table 1. The results indicated that the transition of 184C→T was monomorphic in Liaoning Cashmere goat, Chengde polled goat, Taihang Mountain goat, Shannan white goat, Chengdu brown goat, and Guizhou white goat. The populations which were polymorphic at 184C→T were all in the Hardy-Weinberg equilibrium. The gene frequency did not display a significant difference among Chinese indigenous goat breeds, but it was significant (P < 0.05 or P < 0.01) between Chinese indigenous goat breeds and the Boer goat imported from South Africa.
Genotype effects of goat CLPG gene on body weight at different ages
The general linear model was significant (P < 0.01) for body weight at different ages, and the adjusted R squared of the model ranged from 0.964 to 0.981 (Table 2). In this model, the effect of the individual on body weight at different ages was not significant (P > 0.05, Table 2). The effect of breed and sex on body weight was significant (P < 0.01, P < 0.05, Table 2) from birth to two weeks, but were not significant from three weeks to four months (P > 0.05, Table 2). It showed that the effect of breed and sex on body weight decreased with an increase in age. It could also be seen from Table 2 that there was a significant effect of birth type, genotype of paternal and maternal genotype, and the interaction of paternal and maternal genotype (P < 0.01, P < 0.05, Table 2). The pair wise comparisons between estimated marginal means of body weight at different ages from paternal, maternal, and individual genotypes are listed in Table 3, and the interaction between paternal and maternal genotype is shown in Table 4.
Discussion
It has been known that the
CLPG phenotype of sheep results from the transition of 267A→G, by analyzing the sheep STS (AF401294) (
Freking et al., 2002;
Smit et al., 2003). It can be seen from Table 1 that the populations with polymorphic at 184C→T were all in Hardy-Weinberg equilibrium, and most of the Chinese indigenous goat breeds had higher significant frequencies of C, from 0.9446 to 1.0000, but the Boer goat had a lower significant frequency of C (0.7535) and a higher frequency of T (0.2465). It is also known that the Boer goat from South Africa is a famous meat-oriented goat breed in the world, with more obvious muscular hypertrophy conditions at the neck, breast, shoulder, back, loin, and buttocks compared with other goat breeds. Therefore, it can be inferred that the transition of 184C→T may be related to the muscular hypertrophy of the Boer goat.
Our results of general linear model analysis showed that paternal genotype, maternal genotype, and their interactions affected their body weights significantly at different ages (P < 0.01, P < 0.05, Table 2). Table 3 shows that the effect of paternal genotype (CC) was significantly higher than that of CT (P < 0.05), whereas, the effect of maternal genotype (CT) was significantly higher than that of CC (P < 0.01, P < 0.05). Meanwhile, it is also seen from Table 4 that the interaction between paternal genotype (CC) and maternal genotype (CT), CC(P) × CT(M) was significantly higher (P<0.05) than that of CT(P) × CC(M) from birth to one-month-old, and significantly higher (P < 0.05) than that of other combinations, CC(P) × CC(M), CT(P) × CC(M), and CT(P) × CT(M). Therefore, the CC(P) × CT(M) is the best combination among the four kinds of mating.
This phenomenon was similar to sheep parental imprinting form, a polar over-dominance, in which only individuals having received the allele from their sire expressed the
callipyge phenotype (
Cockett et al., 1994,
1996;
Freking et al., 1998;
Lien et al., 1999;
Shay et al., 2001) . It could be inferred that the transition of 184C→T might be a paternal imprinting form, a polar over-dominance, in which only individuals having received the allele from their mothers expressed the
callipyge phenotype, the double muscle characteristics of the Boer goat might be related to the maternal genotype. However, the difference between the sire or dam CC genotype and CT genotype was so complicated that further analysis could hardly be done to date because only few individuals could be recognized for its allele in genotype from the parent.
Unfortunately, only several TT genotypes were observed in the Boer goat. Hence, another task needs to be done, that is, selecting special genotype individuals to mate with each other, to determine the offspring genotype easily, and to determine from which allele it comes. Fortunately, we are carrying out phenotype recording and genotyping of the CLPG gene of individual Boer goats to confirm the findings mentioned above. On the other hand, although there is a very low frequency of T allele in the Chinese indigenous goat breeds, it can be increased through genotyping and selection as T allele proved to be related to muscular hypertrophy.
Higher Education Press and Springer-Verlag Berlin Heidelberg
PDF
(143KB)
