Evaluation of the Genetic Relationship among Ten Chinese Indigenous Pig Breeds with Twenty-six Microsatellite Markers

The genetic diversities and relationships of 10 Chinese indigenous pig breeds and three exotic pig breeds have been evaluated using 26 microsatellites recommended by the Food and Agriculture Organization & the International Society of Animal Genetics (FAO-ISAG). The allele frequencies, genetic heterozygosity (H) and polymorphism information content (PIC) have been calculated. The results showed that genetic diversity of Chinese indigenous pig breeds is higher than that of the introduced pig breeds. The clustering of 10 breeds is generally consistent with their geographical distribution. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 4 : 441-444)


INTRODUCTION
Chinese indigenous pig breeds possess the most abundant genetic resource of pig breeds in the world.118 Chinese indigenous pig breeds were documented in the domestic animal diversity information system (DAD-IS), which take up one-third of the world total.Chinese indigenous pig breeds, given their own special characters, such as prematurity, prolific and delicious pork, should be a source of great interest, appeal and curiosity to pig breeders of the world.However, in the past twenty years, most indigenous pig breeds have been facing the challenge of modern commercial pig breeds.The population size of many breeds has become very small or even being effectively lost.Therefore, evaluation and conservation have been recognized as an essential task for breeders and geneticists.
In recent years, the genetic variations of some Chinese indigenous pig breeds have been evaluated by cytogenetic and biochemical methods, randomly amplified polymorphic DNA (RAPD) and microsatellite markers (Li et al., 2000).
Microsatellite markers, with the characters of high polymorphism, evenly distributed in the whole genome, easy for genotyping, had been recommended by the Food and Agriculture Organization and the International Society for Animal Genetics (FAO-ISAG) to study the diversity of global pig breeds, and the method was used extensively to construct structure of the closely related populations and breed allocating of cattle, pig and sheep (MacHugh et al., 1998;Diez-Tascón et al., 2000;Li et al., 2000;Martínez et al., 2000;Fan et al., 2002).

Methods
Primers of 26 microsatellites recommended by ISAG-FAO were synthesized by Beijing Sangon Biotechnology Co.. DNA amplifications were performed with PE480 and Amp9600 Thermal Cycler (PE Company, USA).PCR conditions were optimized according to the referenced protocols; the denatured PCR products were analyzed on 8% polyacrylamide denaturing sequencing gel and then dye the gel in AgNO 3 solution and scanned the gel by Gel Analyzer of Fotodyne (Incorporated Hartland Company, USA).To ensure the accuracy of allele calling, in our studies, microsatellites products of two samples were cloned and sequenced, which were used as the control animals (DR and LD pigs).The control DNAs, F9110010 and F9110012, kindly provided by Dr Denis Milan of INRA, France, were also used as the control animals (DR and LD pigs) to adjust our microsatellite data.

Statistical analyses
Allele frequencies and observed heterozygosity (Ho), expected heterozygosity (He) at each locus for each population, polymorphic information content (PIC) and effective numbers of alleles, fixation indices (F IT , F IS , F ST ), total population heterozygosity (Ht) and Nei's standard genetic distance were obtained using GENEPOP ver.3.3 package (Raymond et al., 1995).The Neighbor-Joining tree was constructed using DISPAN (Ota. 1993) andTreeView (Page 1996).

RESULTS
The electrophoresis results, allele frequencies (the part of gene frequency is shown in Table 2) and the number of alleles are available from the corresponding author upon request.All loci were polymorphic and the number of alleles per locus ranged from 11 (SW951) to 42 (CGA) in whole population, and the mean number of the alleles observed in 26 microsatellite loci was 20.2.The Fixation indices (Fit, Fst, Fis) in all populations, total population heterozygosity (H T ) and sub-population heterozygosity (H S ) were shown in Table 3.The total heterozygosity per locus ranged form 0.4892 (SW951) to 0.9308 (CGA) and fixation indices of F ST per locus varied from 0.0327 (SW951) to 0.3725 (CGA).The average F ST of all loci is 0.1626, which means most of the genetic variation generated within breeds and only a few existed among populations (Table 3).Table 3 also gives other Fixation indices (Fit, Fis) and subpopulation heterozygosity (H S ).The mean of unbiased expected heterozygosity (H) and polymorphism information content (PIC) of all breeds were showed in Table 4.The mean heterozygosity (H) of all breeds varied from 0.8114 (Dongshan, DS) to 0.8803 (Donglan, DL).The mean PIC of Chinese indigenous pig breeds was prominent higher than exotic pig breeds.The change trends of the mean H of all breeds were identical with that of the mean PIC of all pig breeds.
The Neighbor-Joining (NJ) tree was constructed based on Nei's standard genetic distances (Table 5).The clustering result is shown in Figure 1.Three exotic breeds used as outgroups, are grouped into a branch and the Chinese indigenous pig breeds into another branch.Among all the Chinese indigenous pig breeds, the two pig breeds (LT and DH) in Guangdong province are firstly clustered with the two pig breeds (HA and PT) in Fujian province, they have formed the first branch.The second branch is the cluster of the four pig breeds (LL, DS, LC and DL) distributed in Guangxi province.And then, the first branch and the second one form a large branch.However, the only exception is that the two pig breeds (LA and YM) in Shandong province are not firstly clustered together.

Genetic variation within breed
The genetic variation within breed was estimated by the number of alleles, their frequencies, H, PIC and fixation indices (Fit, Fst, Fis).The results illustrated that all loci were deviated from Hardy-Weinberg equilibrium in at least 1 population (p<0.05).Seven loci (IGFI, S0090, S0227, S0386, SW122, SW911, SW951) were disequilibria in all populations.The number of the loci deviated from equilibrium ranged from 4 to 11 in each population.This might be caused by population subdivision owing to genetic drift, the effect of a bottleneck through the reproductive isolation of rare populations or sample collection (Li et al., 2002).
The average F ST of all loci was 0.1626, it means 83.7% of the genetic variation is within breeds, only 16.3% of the genetic variation exists between populations and this might be resulted from the natural and artificial selection, inbreeding cross, and mutation.

Genetic variation among breeds
NJ tree derived from Nei's (1978) standard genetic distance was used for describing Genetic relationship among the populations.The cluster result was consistent with their geographical distribution, but was not completely consistent with their classification in the book of Pig Breeds in China.DH of the Central China Type in Guangdong province and LT of the South China Type in Guangdong province did not belong to the same type as classified by The Pig Breeds in China were firstly clustered together and then they clustered with HA and PT of the Central China Type in Fujian province.The DL, DS, LC and LL of South China Type in Guangxi province were clustered together.This research also showed that the genetic distances among Chinese indigenous breeds were quite close, which may indicate that Chinese indigenous breeds had a common ancestor.But according to the classification in The Pig Breeds in China, Chinese indigenous pig breeds had several originators.The two different conclusions implied that there would be further related research to do for understanding the real genetic relationship of Chinese indigenous pig breeds.

Figure 1 .
Figure 1.The Neighbor-Joining dendrogram of 13 breeds based on Nei's genetic distances.

Table 1 .
The detail information of 10 Chinese indigenous pig breeds and 3 introduced pigs included in the study

Table 2 .
The gene frequency of 10 Chinese indigenous pig breeds and 4 exotic pig breeds

Table 3 .
The Fixation indices (Fit, Fst, Fis), heterozygosity of total population (H T ) and subpopulation (H S )

Table 4 .
The mean heterozygosity (H) and polymorphism information content (PIC) of 13 breeds in 26 microsatellite loci