Genetic Variation and Genetic Relationship of Seventeen Chinese Indigenous Pig Breeds Using Ten Serum Protein Loci *

Seventeen Chinese indigenous pig breeds and three introduced pig breeds had been carried out by means of vertical polyacrylamide gel electrophoresis (PAGE). According to the results, eight serum protein loci were highly polymorphic except Pi-2 and Cp. The polymorphism information content (PIC) of Hpx was the highest (0.5268), while that of Cp was the lowest (0.0257). The population genetic variation index showed that about 84% genetic variation existed in the population, and the rest of 16% distributed between the populations. The genetic variation of Yimeng black pig and Duroc were the highest and the lowest, respectively. The genetic variation of Chinese indigenous pig breeds was much more than that of exotic groups. Genetic distance results showed that Chinese indigenous pig breeds were classified into four groups with the three introduced pig breeds clustered into another group. The results also supported the geographic distribution of Chinese indigenous pig breeds in certain extent. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 7 : 939-945)


INTRODUCTION
Serum protein polymorphism has the characteristics of genetic stability and lifetime constancy, which are usually controlled by autosomal codominant alleles.Therefore serum protein polymorphism is significant in studying the origin, differentiation and genetic distances between animal populations (Li et al., 1993).With massive land and complex natural environment, China has abundant resources of pig breeds.Additionally, in the domestic animal diversity information system (DAD-IS) of the FAO, 128 Chinese pig breeds have been reported, which take up almost one-third of the total pig breeds in the world.The Chinese indigenous pig breeds are known for their strong adaptability, high reproductive capacity and excellent taste.The reproductive capability of Taihu pig is the highest in the world (Zhang et al., 1986); Wuzhishan pig is one of the special and rare small pig breeds in the world and can be used as experimental animals.Much work has been devoted to the study on domestic animal serum proteins and enzymes after the introduction of molecular silver electrophoresis.Especially, zone electrophoresis in starch gels described by Smithes (1955a, b) provided a great impetus to the study of polymorphic proteins (Hesselhol, 1969;Zuozuomu, 1982); But in China, the study on indigenous pig breeds using serum protein loci has not been performed systematically, and their work was restricted in a few provinces (Huang et al., 1989;Lin et al., 1998).Analysis of 17 indigenous pig breeds and three introduced pig breeds has been carried out using 10 serum protein polymorphisms in the present study in order to study revealing present genetic variation and relationships among breeds.

Serum protein electrophoresis
Serum protein was measured by means of vertical discontinuous PAGE in this study.Ten proteins loci for examination, the number of observed alleles and their sign were listed in the Table 2. Gel concentration, buffer system component and other electrophoresis conditions concerned were listed in the Table 3.

Data analysis
Genotype was judged according to the opposite migration rate of protein.Gene frequency, Number of the effective alleles (Ne), PIC and genetic similarity coefficient were obtained by programming in Microsoft Excel.GENEPOP ver. 3.3 (Raymond et al., 1995) computer package was employed in the calculations of the expected heterozygosity, observed heterozygosity of each population, Wright's Fst static in each locus and the test of Hardy-Weinberg equilibrium.The D A distance (Nei's et al., 1983), gene diversity (Ht) and their associated parameters such as Hs and Gst were performed by the DISPAN program (Ota, 1993).Phylogenetic trees were constructed by using the unweighted pair group-method with arithmetic mean (UPGMA) (Sneath and Sokal, 1973) from matrix of D A distances.

Serum protein polymorphism in swine
The allele frequencies showed that Pi-2 in all breeds was monomorphic and the other 9 loci were polymorphic, in which Cp allele represented a homozygote almost, too.The very low frequencies of Cp A and Cp C were later ascertained in the breeds of Wuzhishan pig, Huai pig, Laiwu pig, Mashen pig, large white and Landrace.It indicated that Cp was homozygotic and was losing its practical significance gradually in studying consanguineous relationship among pig breeds, which was similar as Pi-2.On basis of the phenotypic heterozygosity of locus Tf in these populations, it was reasonable to suggest that the variations observed were controlled by three codominant alleles: Tf A , Tf B and Tf C , which were consistent with the results of other studies in China (Huang and Zhou, 1989;Lin et al., 1998).These three alleles were observed in all the breeds studied except Duroc, in which the allele of Tf A was not observed.Resulted from the comparison 3 alleles' frequencies of Po-2, it was concluded that Po-2 C and Po-2 A were the dominant alleles in Chinese indigenous pig breeds whereas the allele of Po-2 B was dominant in the introduced three pig breeds.In the three protein loci (Po-1, Alb, Pi-1), the frequency of allele A was higher in the examined pig breeds except Laiwu pig, Yimeng pig, large white and Landrace.In addition, we observed the existence of Po-1 monomorphism in large black-white pig, Lantang pig and the introduced three pig breeds.
It's found that the allele Am B was primary in most breeds, and the result of study on polymorphism of Am was consistent with Hesselhol (1969) and Zuozuomu (1982).The allele Am BF , which was very rare, occurred in Dongshan pig, Chenghua pig, Baixiang pig and Hetao large-ear pig.In this investigation, four alleles of Akp A , Akp B , Akp C and Akp D were observed in Akp loci.It was easy to discover that the primary allele of Akp C was in Chinese indigenous pig breeds and Akp D was in most of the introduced pig breeds.The Hpx components occured in these populations suggested that the polymorphism was under genetic control of six alleles of Hpx 0 , Hpx 1F , Hpx 1 , Hpx 2 , Hpx 3 and Hpx 4 with a codominant mode of inheritance.Among these alleles, Hpx 2 was possessed in all pig breeds studied, but allele Hpx 1 was not observed in Chenghua pig and Hpx 3 lacked in Huai pig and Mashen pig.
It can be seen from the Table 5 that the increasing/ decreasing magnitude order of heterozygosity among populations(Dst) in the 10 loci was listed as following: Hpx, Po-2, Am, Tf, Akp, Pi-1, Po-1, Alb, Cp, Pi-2 whereas the magnitude order of heterozygosity within population (Hs) is: Hpx, Tf, Pi-2, Po-2, Akp, Alb, Am, Po-1, Pi-1, Cp.From the above, it could be concluded that there was discrepancy of alleles of locus Hpx between breeds as much as within breeds in the constitution and content.As a result, Hpx can be used as an important locus for studying the

Population genetic variations and genetic differentiation
As described in Table 4, it showed that most of PIC are middle polymorphism except Duroc whose is low (0.2496).As for mean expected heterozygosity, it ranged from 0.3084 to 0.4423 and Duroc's was the lowest.The value of mean observed heterozygosity distributed between 0.3518 and 0.4891, and it was higher than that of former.With the three indexes (Ne, PIC, H), it can be directly compared not only genetic polymorphism and selective potential of the same gene loci in different populations, but also variation extent of the same population at different gene loci.The more the mean genetic heterozygosity is, the more alleles are, and the higher the genetic diversity is.It also results in the higher selection potential and the higher selection pressure.
Taking a comprehensive view of the whole gene frequency, in the protein loci with more alleles such as Am, Akp, Hpx, etc., the number of alleles in Chinese indigenous pig breeds is often more than that of introduced pig breeds.Moreover, much more variation exists within breeds.As it is concerned, this kind of phenomena indicated that many genes in exotic pig breeds had been lost resulted from the long-term natural selection.On the other hand, many indigenous pig breeds still kept relatively higher genetic variations because of the conditional restrictions of the complex survival environments and the relatively lower crossing.In addition, the genetic differentiation coefficient (Gst) and the fixed index (Fst) between the populations indicated that 84% of the total genetic variation was between populations in all the 20 pig breeds.As for genetic differentiation, Hpx is maximal (0.2598) while Pi-2 is minimal (0.0000).The numbers of pig breeds deviating from equilibrium are different in variant loci.Am and Hpx are the least (5, p<0.05); on the other hand, that of Pi-2 is the most (20, p<0.05), which according to the value of X 2 resulted from fisher's method.
With the advancement of reforming and opening in China, and the continuous improving of artificial insemination and crossing, the genetic variation of many breeds became smaller and some excellent breeds had almost lost their breeding potentiality.So protecting the present breeds should be the important strategic selection in the 21 st century for all the countries in the world.

Population genetic relationships and clustering analysis
Table 6 showed that the value of genetic distance ranged from 0.0193 to 0.2773, and the genetic distance between Donglan pig and Baixiang pig was the closest whereas that of Lantang pig and Landrace was the farthest.Figure 1 showed that the Chinese indigenous pig breeds studied in this work were divided into three branches.In the first branch, there are Donglan pig, Baixiang pig, Dongshan pig, Wuzhishan pig, Huai pig and Longlin pig; large black-white pig and Lantang pig grouped the second branch.In the third branch, it contained the North China type pig and Southwest China type pig, which distributed according to the book of pig Breeds in China.In addition, Mashen pig was far away from other breeds.Three introduced pig breeds were used as the out-group.
From above, it was not fully consistent with the previous division.The Donglan pig in Guangxi province and the Baixiang pig in Guizhou province had the near geographic location and there were frequent exchanges, therefore they had the nearest relationship and they formed into a small cluster with the Dongshan pig in Guangxi province.Then the cluster was merged with the Wuzhishan pig, Huai pig and Longlin pig, which were long distance away, into a larger cluster.This formed the South China type pig.This result was easily comprehended and it simultaneously indicated the geographic isolation had certain influence on genetic differentiation of breeds.
To be surprised, the Guanlin pig in Guizhou province of Southwest type and the Chenhua pig in Sichuan province, Putian pig in Fujian province of Central China type clustered into a branch with North China type pig such as Yimeng pig, Min pig.In fact, they had relatively farther geographic location.We thought the causes might be that the serum samples from the backward of farmers and they might be mixed by the blood of Min pig and exotic pig breeds, which had a good crossing effect with them in recent years.The possibility of gene mixture was larger and might be affected by the genetic erosion.According to the book of pig Breeds in China, the immigrant people from Shanxi province carried the Bamei pig into the Hetao area, so it had large influences on the forming of Hetao large-ear pig in Hetao area of Inner Mongolian autonomous region.
The results of our study indicated that the classification according to body stature, appearance features, producing functions and geographic distribution could not reflect the basic genetic difference of breeds and genetic relationship between them.Mashen pig in Shanxi province existed in the remote mountain areas and had the less exchange with the outer spaces, so it formed a branch itself.Of course the Chinese indigenous pig breeds originated from Asian wild pig had a far relationship with the three exotic pig breeds from Europe and America.

ACKNOWLEDGMENTS
This project was supported by the Center of Preservation & Utilization of Germplasm Resource of Animal Husbandry & Forage Grass, Nation Animal Husbandry & Veterinary Service (NAVS), Ministry of Agriculture, P.R. China, the National Outstanding Youth Science Foundation of China (39925027) and the Key Project of National Basic Research and Developmental Plan (G2000016103) of China.This Project also belongs to the EU-China collaboration project (QLRT-2001-01059).

Figure 1 .
Figure 1.UPGMA phylogenetic tree showing the genetic structure of 17 Chinese indigenous pig breeds and three introduced pig breeds using Nei's D A distance.

Table 1 .
17 Chinese indigenous pig breeds and 3 introduced pig breed included in the study and their type, sampling location, sample size, number of males and females

Table 3 .
Gel concentration, buffer system and other electrophoresis conditions concerned Predicates the condition is same as that ahead of it in the same row.

Table 5 .
Loci variation at pig breeds studied including coefficient of gene differentiation (Gst), total genetic heterozygosity (Ht), heterozygosity within population (Hs), heterozygosity between populations (Dst), Polymorphism information content (PIC), fixation indices (Fis, Fit and Fst) in each loci and number of breeds deviated from Hardy-Weinberg equilibrium (H-W) which resulted from X 2

Table 6 .
The D A genetic distance (below diagonal) and resemble coefficient in 20 pig breeds (above diagonal) a The abbreviation of pig breeds.