Subscribe Now Subscribe Today
Research Article

Study on Population Genetic Structure of Liangshan Semi-Wool Sheep Using Microsatellite Markers

X.Y. Zhang, M.L. Zhou, X.H. Zhang and D.J. Wu
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

To evaluate the genetic polymorphisms and to search for available molecular markers for Liangshan semi-wool sheep, 15 microsatellite markers of 187 samples were amplified by multiplex PCR. A total of 133 alleles were detected, with the number of alleles ranging from 6 (BM2830) to 15 (McM130), giving a mean No. of 8.87 alleles per locus. The total and mean effective allele No. were 64.29 and 4.29, respectively. The observed heterozygosity and expected heterozygosity were from 0.4486 (McM130) to 0.8877 (BMS1678) and 0.5704 (BMS0887) to 0.8373 (McM130), respectively. Mean observed heterozygosity and mean expected heterozygosity were 0.672 and 0.7536. Polymorphism information content values were from 0.5202 (BMS0887) to 0.8183 (McM130) and mean polymorphism information content of the 15 microsatellite loci was 0.7184. All 15 microsatellite loci were highly polymorphic, which showed that there were rich genetic polymorphisms at these detected microsatellite loci in Liangshan semi-wool sheep. Comparison of allele distributions among loci did not reveal consistent shapes. Distributions were centralized in some cases, whereas in others some kind of skewness was evident. Complex evolution at these loci is an important factor in the irregularity of microsatellite distributions.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

X.Y. Zhang, M.L. Zhou, X.H. Zhang and D.J. Wu, 2008. Study on Population Genetic Structure of Liangshan Semi-Wool Sheep Using Microsatellite Markers. Pakistan Journal of Biological Sciences, 11: 2423-2427.

DOI: 10.3923/pjbs.2008.2423.2427



In the past years, sheep breed registrations have led to genetic isolation of many sheep breeds. The selection of a few highly productive breeds has caused the decline of numerous other diverse breeds. This is the reason why the evaluation and the preservation of cattle genetic resources have already become a major and common problem that has attracted global concern. The genetic polymorphism and diversity found in the domestic breeds allow farmers to develop new characteristics in response to changes in environment, or market conditions. So, the importance of increasing, maintaining and conserving the genetic diversity in these animals for the future has been recognized. Gradually attention has been turned in this direction, with every tool including phenotypic parameters and biochemical and molecular genetic techniques having been used to assess the genetic diversity of the animal. DNA microsatellite sequences are valuable genetic markers due to their dense distribution in the genome, great variation, co-dominant inheritance and easy genotyping. In recent years, they have been extensively used in parentage testing, linkage analysis, population genetics and other genetic studies (Goldstein and Pollock, 1997; Calvo et al., 2006). Although, they show some limitations in the analysis of phylogenetically distant organisms, due to their irregular mutation processes involving range constraints and asymmetries, they have proved very useful in sheep population studies (Sun et al., 2007; Álvarez et al., 2008). This breed originated from Tibetan sheep in China, where intensive cross-breeding programme started in 1958. To improve wool production the crossing with wide range of breeds (Xinjiang fine-wool sheep Lincoln and Boioder Leicestler sheep) was attempted. The new breed, with name Liangshan Semi-wool Sheep, was announced in 1987. The number of animals is relatively high with 350,000 animals in China. Up to the last decade, microsatellite have been used in parentage testing, linkage analysis, association analysis and QTL detection of Liangshan Semi-wool Sheep (Gao and Wu, 2005; Zhang and Wu, 2005; Wang and Wu, 2006; Zhou et al., 2007). The purpose of this study was to reveal the genetic polymorphisms of Liangshan Semi-wool Sheep by examining the microsatellite DNA and to provide some theoretical basic for conservation and improvement of this sheep.


A total of 187 blood samples were randomly collected from genetically unrelated sheep across their breeding tract of Liangshan Semi-wool Sheep (Liangshan, of Sichuan, China) to make them representative of population. Blood samples (8-10 mL) were obtained from the jugular vein using vacuitaners treated with 15% Ethylene Diamine Tetra Acetic (EDTA) acid as an anticoagulant. This study has been done in China in Autumn 2004.

Fifteen microsatellite markers were selected in respect of polymorphisms and a non-linkage criterion for syntenic markers, based on the recommendations of either of the following organizations: United Staes Department of Agriculture (USDA); Australian Gene Mapping Web Site; Food and Agricultural Organization (FAO); International Society for Animal Genetics (ISAG).

Genomic DNA was isolated as per the method described by Sambrook et al. (l999) with minor modifications. Polymerase Chain Reactions (PCRs) were carried out to amplify microsatellite loci using fluorescent-labelled primers. The amplified products were separated using POP-4 polymer (Applied Biosystem). Genetic variants were visualized using ABI PRISM 310 DNA sequencer (Applied Biosystem). Results of the capillary electrophoresis were read directly with the Genescan software and interpreted with Genotyper.

Breed genetic variation of the microsatellites and breed structure was quantified using the allele frequencies, observed No. of alleles, effective No. of alleles (Kimura and Crow, 1964) observed heterozygosity and expected (Levene, 1949) heterozygosity. Within breed heterozygosity deficit (Wrights, 1978) was estimated using Wrights fixation index. All the estimates were derived using POPGENE, Version1.31 program (Yeh et al., 1999). Polymorphism information content was calculated as per Botstein et al. (1980).


The study of genetic diversity is the basis for any animal breeding program. The first step in a effective breeding or conservation program is accurate evaluation of available genetic resources and microsatellite analysis is a well tool for measuring the genetic polymorphisms in a population. Genotypic data from 15 microsatellites was used to assess the genetic structure of Liangshan Semi-wool Sheep population. All 15 microsatellite loci were polymorphic in the Liangshan Semi-wool Sheep population (Table 1). Total 133 alleles were detected from the 15 microsatellite loci examined. The No. of alleles per locus ranged from 6 (BM2830) to 15 (McM130) with an average of 8.8667. The total effective allele number and mean effective allele No. per locus were 64.2905 and 4.286. High mean number of observed alleles per locus showed high genetic variation in the sheep. At every microsatellite locus, allele size range was distinctive. And at every locus, there was a most frequent allele. At BMS0887 and BM1577, the most frequent allele was 149 and 184, which had an allele frequency of 0.6043.

Observed heterozygosity, expected heterozygosity, observed homozygosity, expected homozygosity, mean heterozygosity, Polymorphism Information Content (PIC) and fixation index (F) in Liangshan Semi-wool Sheep population were shown in Table 2.

Table 1: Allele No. allele size ranges, effective allele No. and most frequent alleles and their frequencies of each markers

Table 2: Observed heterozygosity, expected heterozygosity and observed homozygosity, expected homozygosity, Polymorphic Information Content (PIC) and fixation indices (F) in Liangshan Semi-wool Sheep population

Observed heterozygosity and expected heterozygosity at the 15 microsatellites in Liangshan Semi-wool Sheep population were from 0.4486 (McM130) to 0.8877 (BMS1678) and from 0.5704 (BMS0887) to 0.8373 (McM130), respectively. Mean observed heterozygosity and mean expected heterozygosity were 0.672 and 0.7536, respectively. The average genetic variation (0.672) observed in this study was higher than that of Muzzafarnagri 0.652 (Arora and Bhatia, 2004), Nali 0.651 and Chokla 0.657 (Sodhi et al., 2006) as well as this is higher than Swiss sheep breed Mouflon 0.450 (Stahlberger-Saitbekova et al., 2001), Northern Spanish sheep-Latxa 0.661, Rubin del Molar 0.600, Churra 0.661, Xalda 0.572 (Alvarez et al., 2004). The high mean heterozygosity values could be attributed to low selection pressure and large number of alleles present in a population. The present estimates of gene diversity (Ho) were, however, lower than that of Spanish sheep breeds (Mean Ho = 0.713-0.771) and Awassi sheep from Turkey (Ho = 0.750) as reported by Arranz et al. (1998). Thus, differences in gene diversity (Ho) values observed might be ascribed to choice of microsatellite loci typed as well as existing population structure.

Another parameter also indicative of the genetic variation is Polymorphism Information Content (PIC). PIC was from 0.5202 (BMS0887) to 0.8183 (McM130) and mean PIC was 0.7184. PIC is a parameter indicative of the degree of informativeness of a marker. Following the criteria of Botstein et al. (1980), in this study, all 15 microsatellite loci appeared to be highly informative (PIC>0.5), According to the selective standard of the microsatellite loci, microsatellite loci ought to have at least four alleles to be considered useful for the evaluation of genetic diversity. Based on this criterion, the 15 microsatellite loci used in this study were useful for the evaluation of genetic diversity in Liangshan Semi-wool Sheep. These results imply that abundant genetic polymorphisms exist in the Liangshan Semi-wool Sheep. These high estimates of PIC substantiated the suitability of used set of markers to applications such as parentage control, linkage-mapping programs in addition, to genetic polymorphism studies in the sheep too. Of the 15 microsatellite loci, the fixation indices of BMS835, BMS0887, OarCP34, BMC1009, BMS1678 microsatellite loci were negative and others were positive. The mean fixation indices was 0.0991, reflecting that the degree of heterozygote surplus at these loci was not high and deviations from Hardy-Weinberg equilibrium were not significant.

Figure 1a-o reveal that for a few markers (e.g., BM2830, ILSTS0049) a narrow range of variation was found in the number of observed alleles. When this situation is found the most frequent alleles are likely to be the oldest, the others being the result of mutation process through insertion-deletion mechanisms. We can point out loci such as McM130 with a large number of alleles with low frequencies leading to flat distributions. The allele frequency distributions of 15 microsatellite loci show asymmetry. In several cases, the most frequent alleles showed an intermediate size (e.g., BMS0887, BMS1126), producing a centralised distribution. In other markers (e.g., OarFCB11, BMC1009) the variants with the greatest frequencies showed an extreme size. However, in most cases a common shape was not found. Even when some kind of skewness was evident it did not follow a consistent pattern, either to the right or to the left, in the populations. The distributions of allele frequency are in accordance with data previously reported for microsatellites. As an example, Forbes et al. (1995) analysed different domestic and bighorn sheep and found that distributions at some loci were highly centralised, while others were skewed, flat. Asymmetries of microsatellite mutation distributions have been found in different studies. Goldstein and Pollock (1997) reviewed data in the literature and indicated that a positive asymmetry (a tendency to mutate to alleles of larger size) was first observed for large alleles at trinucleotide-expansion loci, but further investigations revealed asymmetry towards mutations that decrease size. They concluded that apart from differences among loci, allele size is an important factor in the determination of asymmetry effects. Another aspect that contributes to the irregularity of microsatellite distributions is the complex evolution at these loci. The high level of genetic variation was evident in the breed. The abundant genetic polymorphisms affords an opportunity to improve the defects that exist in Liangshan Semi-wool Sheep.

Fig. 1: Distributions of allele frequencies at 15 microsatellite markers in Liangshan Semi-wool Sheep, (a) BMS2263, (b) BMS1636, (c) ILSTS0049, (d) ILSTS004, (e) BMS835, (F) BM2830, (g) Oar FOB11, (h) Oar OP0079, (I) BMS1126, (j) BMS0887, (k) OarOP0034, (l) BMC1009, (m) BM4513, (n) BMS1678 and (o) McM130
1:  Alvarez, I., L.J. Royo, I. Fernandez, J.P. Gutierrez, E. Gomez and F. Goyache, 2004. Genetic relationships and admixture among sheep breeds from Northern Spain assessed using microsatellites. J. Anim. Sci., 82: 2246-2252.
PubMed  |  Direct Link  |  

2:  Alvarez, I., L.J. Royo, J.P. Gutierrez, I. Fernandez, J.J. Arranz and F. Goyache, 2008. Relationship between genealogical and microsatellite information characterizing losses of genetic variability: Empirical evidence from the rare Xalda sheep breed. Livest. Sci., 115: 80-88.
CrossRef  |  

3:  Arora, R. and S. Bhatia, 2004. Genetic structure of Muzzafarnagri sheep based on microsatellite analysis. Small Rumin. Res., 54: 227-230.
Direct Link  |  

4:  Arranz, J.J., Y. Bayon and F. San Primitivo, 1998. Genetic relationships among Spanish sheep using microsatellites. Anim. Genet., 29: 435-440.
CrossRef  |  PubMed  |  Direct Link  |  

5:  Botstein, D., R.L. White, M. Skolnick and R.W. Davis, 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet., 32: 314-331.
PubMed  |  Direct Link  |  

6:  Calvo, J.H., J.A. Bouzada, J.J. Jurado and M. Serrano, 2006. Genetic substructure of the Spanish Manchega sheep breed. Small Rumin. Res., 64: 116-125.
CrossRef  |  Direct Link  |  

7:  Forbes, S.H., J.T. Hogg, F.C. Buchanan, A.M. Crawford and F.W. Allendorf, 1995. Microsatellite evolution in congeneric mammals: Domestic and bighorn sheep. Mol. Biol. Evol., 12: 1106-1113.
PubMed  |  Direct Link  |  

8:  Gao, A.B. and D.J. Wu, 2005. Microsatellite markers for paternity testing of Liangshan semi-fine wool sheep (Chinese). Yi Chuan, 27: 85-90.
PubMed  |  Direct Link  |  

9:  Goldstein, D.B. and D.D. Pollock, 1997. Launching microsatellites: A review of mutation processes and methods of phylogenetic interference. J. Hered., 88: 335-342.
PubMed  |  Direct Link  |  

10:  Kimura, M. and J.F. Crow, 1964. The number of alleles that can be maintained in a finite population. Genetics, 49: 725-738.
PubMed  |  Direct Link  |  

11:  Levene, H., 1949. On a matching problem in genetics. Ann. Math. Stat., 20: 91-94.
CrossRef  |  Direct Link  |  

12:  Sambrook, J., E.F. Fritsch and T.A. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA., ISBN-13: 9780879695774, Pages: 397.

13:  Sodhi, M., M. Mukesh and S. Bhatia, 2006. Characterizing Nali and Chokla sheep differentiation with microsatellite markers. Small Rumin. Res., 65: 185-192.
CrossRef  |  Direct Link  |  

14:  Stahlberger-Saitbekova, J., G. Schlapfer Dolf and C. Gaillard, 2001. Genetic relationships in Swiss sheep breeds based on microsatellite analysis. J. Anim. Breed. Genet., 118: 379-387.
CrossRef  |  Direct Link  |  

15:  Sun, W., H. Chang, D. Ji, X. Liao, L. Du, S. Lu and T. Kenji, 2007. Analysis on genetic diversity and isolation mechanism by distance of different ecological type sheep breeds in Mongolia sheep group. J. Genet. Genom., 34: 1001-1009.
CrossRef  |  Direct Link  |  

16:  Wang, G.F. and D.J. Wu, 2006. Correlation analysis of microsatellite DNA markers with wool traits in Liangshan semi-fine wool sheep. Yi Chuan, 28: 1505-1512.
CrossRef  |  PubMed  |  Direct Link  |  

17:  Wrights, S., 1978. Evolution and the Genetics of Populations: Variability Within and Among Nature Populations. Vol. 4., Chicago University Press, Chicago, ISBN: 9780226910529.

18:  Yeh, F.C., R. Yang and T. Boyle, 1999. A Microsoft Window Based Freeware for Population Genetic Analysis. Version 1.31, University of Alberta, Canada.

19:  Zhang, M.Y. and D.J. Wu, 2005. A linkage map of mirosatellite on chromosome 1 in Liangshan semi-wool sheep. Yi Chuan, 27: 575-578.
PubMed  |  Direct Link  |  

20:  Zhou, M.L., D.J. Wu and X.Y. Zhang, 2007. Construction of genetic linkage map and QTL detection of chromosome 3 in sheep. Yi Chuan, 29: 1475-1482.
PubMed  |  Direct Link  |  

©  2021 Science Alert. All Rights Reserved