A Comparison of Two Kinds of Markers Applied in Analysis of Genetic Diversity in Sheep and Goat Populations

A genetic examination using 14 structural loci and 7 microsatellite markers was carried out among random samples of Hu sheep (Hu), Tong sheep (Tong) and Yantse River Delta White goat (YRD); The mean heterozygosity (H), mean polymorphism information contents (PIC) and mean effective numbers of alleles (Ne) calculated based on the data from the above two types of genetic markers were compared. The standard genetic distances among the three populations based on two types of gene frequencies were calculated and compared. The results show that the mean heterozygosity (H), mean polymorphism information contents (PIC) and mean effective numbers of alleles (Ne) based on 7 microsatellite markers are greater than those based on the structural loci. The standard genetic distances based on structural loci among the three populations are: 0.0268-0.2487, the standard genetic distances based on microsatellite markers are: 0.2321-1.2313. The study indicates that structural and microsatellite markers reflect the genetic variation of the three populations consistently: Tong>Hu>YRD. The differentiation between related species or interpopulations can be expressed more effectively by microsatellite markers than structural markers. Oar FCB11, MAF33, Oar AE101, Oar FCB128 and OarFCB304 can be used as representative loci for research on genetic differentiation between sheep and goat. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 7 : 892-896)


INTRODUCTION
Since the development of enzyme electrophoresis (Hunter and Markert, 1957), numerous natural animal populations have been investigated for genetic variation by using a range of protein loci (Nevo et al., 1984).More recently, many new DNA based methods, usually offering much greater resolution of differences between individuals and populations, have become available.These include mitochondria DNA variation (Cann et al., 1987), restriction fragment length polymorphism (RFLP) (Quinn et al., 1987), random amplified polymorphic DNA (RAPD) (Williams et al., 1990) and microsatellite DNA variation (Tautz, 1989).This paper attempts to compare the results from the application of structure loci and microsatellite markers in the analysis of genetic differentiation in sheep and goat populations.
With regards to sheep and goat, there are rich resources in China.Hu sheep distributed along the Taihu valley, and it is famous for its high fertility and beautiful lambskin.Tong sheep distributed in Baishui county Shanxi province.Tang Dynasty ago, the area had been empire pasture (Lei, 1999).Now the total number is less than 1 thousand.Yantse River Delta white goats (YRD) distributed along lower Yantse River valley of Jangsu province with total number of about 10 millions.This study also wants to describe the genetic constitution diversity of the three populations, so as to provide a basis for sheep and goat husbandry and the genetic resources protection.

Sampling methods and experiment materials
The 63 Hu and 65 Tong sheep were from Lianshi Town of Huzhou city in Zhejiang province and Baishui countryside of Shanxi province of China respectively.The 49 Yantse River Delta white goats (YRD) were from the suburb of Yang Zhou city of Jiangsu province of China.The method of "Random sampling in typical colonies of central area" was performed and we tried to avoid sampling two (or more) individuals that have traceable genetic relationship.

Collecting and treatment of blood samples
16 ml of blood was collected from the cervical vein of each animal.8ml of the total was put into a centrifuge tube using heparin as an anticoagulant for separating blood cells and serum (Sun et al., 2002).Another 8ml mixed with SDS-EDTA was used to extract DNA (Xiong et al., 1999;Li et al., 2000).

ABSTRACT :
A genetic examination using 14 structural loci and 7 microsatellite markers was carried out among random samples of Hu sheep (Hu), Tong sheep (Tong) and Yantse River Delta White goat (YRD); The mean heterozygosity (H), mean polymorphism information contents (PIC) and mean effective numbers of alleles (Ne) calculated based on the data from the above two types of genetic markers were compared.The standard genetic distances among the three populations based on two types of gene frequencies were calculated and compared.The results show that the mean heterozygosity (H), mean polymorphism information contents (PIC) and mean effective numbers of alleles (Ne) based on 7 microsatellite markers are greater than those based on the structural loci.The standard genetic distances based on structural loci among the three populations are: 0.0268-0.2487, the standard genetic distances based on microsatellite markers are: 0.2321-1.2313.The study indicates that structural and microsatellite markers reflect the genetic variation of the three populations consistently: Tong>Hu>YRD.The differentiation between related species or interpopulations can be expressed more effectively by microsatellite markers than structural markers.Oar FCB11, MAF33, Oar AE101, Oar FCB128 and OarFCB304 can be used as representative loci for research on genetic differentiation between sheep and goat.(Asian-Aust.J. Anim.Sci. 2004. Vol 17, No. 7 : 892-896) dehydrogenase (MDH).Lysine (Ly) was analyzed by cellulose acetate electrophoresis and potassium (Ke) in red cell was tested with a Na/K/Cl Analytical Instrument (MEDICA, USA).The methodologies used and the types of the variations present were determined according to the universally accepted methods (Tsunoda et al., 1998(Tsunoda et al., ,1999)).
The amplified fragments were electrophoresed on an 8% polyacrylamide gels in 1×TBE with 150-180 v of running voltage.Then the gels were detected by ethidium bromide staining.The fragment sizes were calculated by using the Kodak Digital Science ID Image Analysis Software.

Statistical analyses
The allele frequencies for the polymorphisms of blood proteins and non-protein systems were computed by the gene counting or square root methods.Microsatellite allele frequencies were determined by direct counting.

Structural and microsatellite loci characterization
The allele frequency data for 7 microsatellite and 14 structure loci are shown in Table 2.The number of alleles observed at a single microsatellite locus ranged from 19 (MAF70) to 36 (OarFCB304), with an average number of alleles per locus of 26.3.For each population, there were at least 7 alleles per locus.This showed that microsatellite markers could give a lot of genetic information.Of the 14 structural gene loci, polymorphisms were seen at 12 loci in Hu and Tong sheep populations (Al and EsD loci did not show polymorphism).In the Yantse River Delta white goat population, polymorphisms were seen at only 7 loci (Al, Po, Ary-Es, Hb-β, CA, Cat and Ly did not show polymorphism).

Diversity analysis
Diversity measures were calculated on the basis of allele frequencies.Table 3 shows mean heterozygosity, information content of polymorphism and number of effective alleles for each population.The diversity measures calculated based on microsatellite markers were obvious higher than those based on structural loci.When comparing the diversity indices of the three populations, Tong sheep was highest, while Yantse River Delta white goat was lowest.
One-Way ANOVA indicates that Hu sheep and Tong sheep only had significant greater PIC than Yantse River Delta white goat (p<0.microsatellite and structural loci showed significant difference (p<0.01).Ds genetic distances based on structural loci data ranged from 0.0268 to 0.2487, while those calculated on the basis of microsatellite markers ranged from 0.2312 to 1.2313.Thus, the latter values were much larger than the former (see Table 4).Barker et al. (1997) analyzed genetic diversity of Asian water buffalo using two kinds of genetic markers.As expected, the microsatellite loci showed very high levels of genetic diversity.This study show that heterozygosity for each population ranged from 0.8906-0.9184.These estimates are approximately three times of those derived from assay of 14 structural loci in the same three populations, which show an expected heterozygosity for each population ranging from 0.1519-0.3081.Our study has demonstrated far greater genetic variability at microsatellite compared with structural markers.This higher variability of microsatellite DNA is an obvious advantage over protein variability when applied to inter population or inter species genetic differentiation studies if the same loci can be screened.So, this observation indicates the superiority of microsatellite over protein markers for many population genetic studies.Tsunoda et al. (1999) divided the Asian sheep into three groups: Mongolian group, Indo-Pakistani group and Tibetan group.Hu sheep is generally recognized as belonging to the Mongolian group (Sun et al., 2002;Geng et al., 2003).Hu sheep and Tong sheep are famous breeds in agricultural area of China.According the origin, breeding history (Lei, 1999) and Yang et al. (2002), there were closed genetic relationships among Tong sheep, Hu sheep and Mongolia sheep populations.This study shows that the genetic distances between Hu sheep and Tong sheep were 0.2312 (based on microsatellite loci) and 0.0268 (based on structural loci) and confirms that just as Hu sheep, Tong sheep could belong to the Mongolian group.

DISCUSSION AND CONCLUSION
Our observations with sheep and goat also show that the conservation of microsatellite markers between these two related species existed in all microsatellite loci detected in this study.With regard to the distribution of microsatellite alleles in three populations, the allele co-shared percentages between sheep and goat were more than 50% for locus Oar FcB48 and MAF70, and less than 50% for locus Oar FcB11, MAF33, Oar AE101, OarFCB128 and Oar FCB304.Thus genetic differentiation between species leads to inter specific microsatellite polymorphism allowing OarFCB11, MAF33, OarFCB128, OarAE101 and OarFCB304 to be used as representative loci for research on genetic differentiation between sheep and goat.

A Comparison of Two Kinds of Markers Applied in Analysis of Genetic Diversity in Sheep and Goat Populations
Animal Science and Technology College, Yangzhou University, Yangzhou, 225009, Jiangsu Province, P. R.China Z. P. Yang*, H. Chang, W. Sun, R. Q. Gen, Y. J. Mao and K. Tsunoda 1

Table 1 .
The primer sequence, chromosome assignment, annealing temperature and MgCl 2 volume

Table 2 (
ii).Alleles frequencies of 7 microsatellite and 14 structural loci in 3 sheep and goat populations

Table 4 .
The standard genetic distances among 3 populations (upper right: based on structural loci data, lower left: based on microsatellite marker data)