Genetic Differentiation between Sheep and Goats Based on Microsatellite DNA

The 7 sheep microsatellite markersOarFCB48, OarAE101, MAF33, OarFCB11, MAF70, OarFCB304 and OarFCB128, which were located on chromosomes 2, 4, 6, 9, 17 and 19, were selected to PCR in Hu sheep, Tong sheep and their closely related species,the goat. They were studied with the amplifying result of 7 microsatellite sites of Hu Sheep, Tong Sheep and goats, the data of allele number and range of allele's size of amplifying were analyzed with ANOVA. The results showed that there were no significant differences (p<0.05) in microsatellite DNA sites among 3 populations. Concerning the conservation of microsatellites in closely related species, selecting microsatellite sites located on the chromosome where the Robertsonian fusion was caused between sheep and goat, may be used in research into genetic differentiation and evolutionary relationships between sheep and goats. (Asian- Aust. J. Anim. Sci. 2004. Vol 17, No. 5 : 583-587)


INTRODUCTION
The karyotype of sheep and goats is 2n=54 and 60, respectively.They belong to the genera Ovis and Capra of the familyCaprinae.Archaeological and morphological research indicates that the sheep and the goat originated from the same ancestor: Rupicaprids, goat-antelopes in the Pleistocene era.Cellular genetic research showed that the sheep and the goat were evolved from a common ancestor: Rupicaprids and the karyotype of the goat is similar to the ancestral form (Li et al., 2000).It can be seen that the two species have a close relationship, but it is still necessary to study their genetic differentiation using on modern molecular technology.There are some reports about genetic differentiation between the sheep and the goat (Upholt et al., 1977;Li et al., 2000), but there are no reports on genetic differentiation of the two species based on microsatellite DNA.In the last 10 years, research on polymorphic markermicrosatellite DNA markers has been greatly advanced because of new techniques, especially PCR.The usefulness of microsatellites for the analysis of genetic relationships among closely related populations has been documented by numerous studies (Buchanan et al., 1994;Bancroft et al., 1995;Arranz et al., 1998;Joseph et al., 1999).The first genetic linkage map of the sheep genome was published in 1995 (Crawford et al., 1995), the second genetic linkage map of the sheep genome was published by de Gortari in 1998 and the first genetic linkage map of the goat genome was published by Vaiman in 1996 (Wu, 1999).This paper is concerned with Hu sheep, Tong sheep and their closely related speciesthe goat.We discuss the probability of studying the genetic differentiation between sheep and goats based on 7 sheep microsatellites, so as to provide a basis for the data bank of sheep (goat) microsatellites, and also put forward theoretical grounds for genetic differentiation among closely related species using microsatellite DNA.

Materials and sampling
The Hu and Tong sheep studied were from Lianshi Town of Huzhou city in Zhejiang province and Baishui country in Shannxi province of China, respectively.The sample size was 63 and 65 respectively.Blood sampling was performed by the "Random sampling in typical colonies of central area" method and we tried to avoid sampling two (or more) individuals that had traceable genetic relationships.Some external morphological characteristics were also investigated (Sun et al., 2002).At the same time, 49 Yangtse River Delta White Goats were sampled by the same method as the contrast population from the suburb of Yangzhou city in Jiangsu province of China.

Microsatellites, PCR conditions and fragment analysis
The genomic DNA was separated according to procedures described by Sun (2002).The 7 sheep microsatellites studied and their characteristics are shown in Table 1.Each 20 µl reaction contained: 0.4 µl dNTP (10 m mol/l), 2 µl 10×buffer, 25 m mol/l MgCl 2 (shown in Table 1)，1 µl GT and CA primer (8 µmol/µl) (2 µl for OarFCB11), 0.2 µl of Taq polymerase (5 U/µl) and 2 µl template DNA (50 ng/µl), then super purified water was added.After an initial denaturation at 94°C for 5 min, the PCR was performed with 30 cycles: denaturation at 94°C for 1 min, anneal at temperatures in Table 1 for 1 min, extension at 72°C for 1 min; the final cycle was followed by an extension at 72°C for 5 min.Amplified fragments were analyzed on 8% denaturation-polyacrylamide gel and detected with EB.Fragment sizes were calculated by Kdak Digital Science ID Image Analysis Software according to pBR322/Msp Marker.Ⅰ

Statistical analysis
Allele frequencies were computed by the gene counting method.Heterozygosity (H), polymorphism information content (PIC) (Bostein et al., 1980) and effective allele number (Ne) (Kimura et al., 1974) were calculated using the SAS package (Sun, 2002).The Nei's genetic distances were calculated using PAPP (Guo et al., 1996).The results of 7 sheep microsatellite primers amplifying in goat and sheep were analyzed with ANOVA procedure using the SPSS package.

RESULTS
From Table 2-7, we saw that the PIC and H of each microsatellite site is more than 0.7, the genetic information was abundant; and we also found that more than 7 alleles could be detected at each sheep microsatellite site in 3 populations.According to the protocol for the estimation of the global animal breeds distance, Barker put forward the selection standard for microsatellite DNA: only if there are more than 4 can the microsatellite site be used (Barker et al., 1994).At the same time, we compared the genetic distances obtained from 7 sheep microsatellites and found that the distance between goat and sheep was longer than the distance between Hu sheep and Tong sheep, thus, OarFCB48, OarAE101, MAF33, OarFCB11, MAF70, OarFCB304 and OarFCB128 could be used in the research of the genetic differentiation between sheep and goats.And we also could see from the tables the difference in gene amount and gene frequencies in the same microsatellite site in the different breeds (species), which could show the difference in heredity among them.,This also indicated that the 7 microsatellite sites could be used to distinguish the breeds and species.The common alleles were the most antique and conservative, the other alleles were the result of insertions, deletionsand mutations.Table 8-9 also shows the conservation of each microsatellite among 3 breeds (species): there were no significant differences in allele number of amplifying (p>0.05) and allele size of amplifying (p>0.05) in each microsatellite site.Sun (2002) also used 20 biochemical markers to detect the Tong sheep, Hu sheep and goats, and concluded that the microsatellite sites provided more accurate values and more abundant information than protein markers, for example, the PIC, H and Ne of each protein marker was smaller than those of microsatellite sites.The 20 biochemical markers indicated that the genetic distance between Hu sheep and Tong sheep, Hu sheep and goats, Tong sheep and goats was 0.0268, 0.2411, 0.2476, respectively, which is also smaller than those of the 3 populations based on microsatellite sites, but the dendrogram of the 3 populations based on the microsatellites is similar to that of the 3 populations based on biochemical markers.Thus, the microsatellites might be a better indicator than protein polymorphisms of evolutionaryrelationships among populations or between closely related species.

DISCUSSION
Nei thought that the time polymorphism alleles existed in a population were longer than the time the breed existed, so that gene diversity (heterzygosity) in one locus in one breed is relative with the gene diversity of its closely related species (Nei et al., 1983).For the conservation of microsatellite profile sequences, microsatellites could be used in many species, the microsatellite primer of one breed might be used in its relative species, and for example, the sheep microsatellite could amplify in goats, which provided the possibility of expediting the obtaining of microsatellite primers and speeding up the comparative genome map.Because of the slow research progress of goat microsatellites, the first genetic linkage map of the goat published by Vaiman (Wu et al., 1999) only reported 10 goat microsatellite sites.So in this study we adopted sheep microsatellite to study the genetic diversity of goats.
In general, during the evolution of mammalian chromosomes, the difference in chromosomes of closely related species was caused by the Robertsonian translocation (fusion or fission) near the centromere.Comparing the G band type between Capra and Ovis could yield significant homology.Some M chromosomes or SM chromosomes of Ovis: M1, M2, M3 and M4 were caused from the Robertsonian fusion of Capra 1/5, 3/10, 4/9 and 11/17 chromosomes.Thus, it was believed that the sheep and the goat originated from the same ancestor, the goat karyotype was similar to the ancestral, the telocentric chromosome of goat caused the Robertsonian fusion, the number of goat chromosomes was reduced from 2n=60 to 2n=54 and differentiated into the sheep karyotype (Chang, 1995).
Concerning the conservation of microsatellites and the similar origin of closely related species, selecting microsatellite sites located on the chromosome where the Robertsonian fusion occurred between sheep and goat, may be used in the study of genetic differentiation and evolutionary relationships between sheep and goats.

Table 1 .
Microsatellite primer sequences, chromosome assignment and part of PCR conditions of the microsatellites used in this study

Table 2 (
ii).Estimates of gene frequencies of the microsatellite DNA sites

Table 4 .
Effective number of alleles of each microsatellite DNA

Table 6 .
The standard genetic distance among 3 populations

Table 7 .
The comparison of 7 microsatellite primers amplifying in Goat, Hu sheep and Tong sheep

Table 8 .
The comparison of microsatellite allele number and range of allele's size in Goat, Hu sheep and Tong sheep Allele number of amplifying

Table 9 .
The comparison of microsatellite allele number and range of allele's size in goat, Hu sheep and Tong sheep Allele size of amplifying Electrophoresis photograph of some rnicrosatellite sites.