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Use of cattle microsatellite markers to assess genetic diversity of Thai Swamp Buffalo (Bubalus bubalis).



ABSTRACT : In this study, cattle microsatellite See miniaturized satellite.  markers recommended for diversity studies of cattle by the EU AIRE 2066 Concerted Action Group were used to study the genetic diversity of 105 Thai swamp buffalo which were randomly selected from eight different research stations of the Department of Livestock Development, Thailand. Of 34 primer pairs, 16 were successfully amplified while the rest showed non-specific amplification. The lowest number of alleles was two while the highest was nine, with an average of 4.7 alleles per locus. The average unbiased heterozygosity heterozygosity /het·ero·zy·gos·i·ty/ (het?er-o-zi-gos´i-te) the state of possessing different alleles at a given locus in regard to a given character.heterozy´gous

het·er·o·zy·gos·i·ty
n.
 for all eight populations was 0.5233, with a low of 0.4772 (Samui) and a high of 0.5616 (Burirum). The genetic distance ranged from 0.0574 to 0.2575. Populations from Lopburi and Burirum showed the closest relationship, whereas Srisagat and Samui were the most divergent. The results generated with the primers recommended by the EU AIRE 2066 Concerted Action Group are at a slight variance from our previous study, possibly as a result of the number of specific amplification products obtained, suggesting that cattle markers may not be optimal for studies of the genetic diversity of the Thai swamp buffalo. (Key Words : Genetic Diversity, Microsatellite, Thai Swamp Buffalo)

INTRODUCTION

Microsatellites have been identified and used for genetic studies of many organisms including several livestock species (Selvi et al., 2004; Chen et al., 2005; Osman et al., 2005; Girish et al., 2007) but only a few genetic studies have been devoted to the Thai swamp buffalo (Triwitayakorn et al., 2006). Moreover, no systematic studies have been undertaken to develop polymorphic polymorphic - polymorphism  DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 markers specific to this species. However, comparative genome studies have shown that microsatellite primer sequences are often conserved across related species and can be used for the development of markers in related species (Navani et al., 2001). Recently, cattle microsatellite markers have been randomly selected to study the genetic diversity of the Thai swamp buffalo (Triwitayakorn et al., 2006) riverine riv·er·ine  
adj.
1. Relating to or resembling a river.

2. Located on or inhabiting the banks of a river; riparian: "Members of a riverine tribe ...
 buffalo (Navani et al., 2001), the Asian water buffalo water buffalo: see buffalo.
water buffalo
 or Indian buffalo

Any of three subspecies of oxlike bovid (species Bubalus bubalis). Two have been domesticated in Asia since the earliest recorded history.
 (Barker et al., 1997a, b) and the African buffalo African buffalo: see cape buffalo.  (Van Hooft et al., 2000). In this study we applied cattle microsatellite markers that have been approved for diversity studies of cattle by the EU AIRE 2066 Concerted Action Group and recommended by the MoDAD program (FAO FAO,
n See Food and Agriculture Organization.
), to analyze the genetic variation and diversity of the Thai swamp buffalo from eight locations in Thailand. The results of this study were compared with a previous study in order to evaluate the results obtained.

MATERIALS AND METHODS

Experimental animals

A total of 105 Thai swamp buffalo (Bubalus bubalis Bubalus bubalis

the domestic buffalo of Europe; black cattle-like ruminants with very short hair, back-swept horns and a liking for marshes in which they wallow. Called also water buffalo, carabao, arna, Indian buffalo.
) were randomly selected from eight research stations of the Department of Livestock Development, Thailand, located in Payao, Lopburi, Burirum, Srisagat, Surin and Suratthani provinces, Samui island and Akha Tribe which is a hill tribe A hill tribe is any one of around twenty ethnic groups living in Northern Thailand, Laos, and Myanmar. Most of these groups also exist outside Thailand, but the term is most often used to refer to the Thai groups. The hill tribes together number approximately 550,000.  folk who live in Maechan District, Chiangrai Province. Most samples used in this study were the same samples used previously (Triwitayakorn et al., 2006) except the samples from Payao and the new additional group from the Akha Tribe. Blood samples were collected from each animal for genomic DNA genomic DNA
n.
The full complement of DNA contained in the genome of a cell or organism.
 extraction using QIAamp DNA blood kit (QIAGEN GmbH, Hilden, Germany) according to he manufacturers' instructions.

Microsatellite analysis

A total of 34 microsatellite loci loci

[L.] plural of locus.

loci Plural of locus, see there
 which were approved for diversity studies of cattle by the EU AIRE 2066 Concerted Action Group (Table 1) were used to analyze individual samples. Polymerase chain reaction (PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
) was performed according to Triwitayakorn et al. (2006) in a total volume of 20 il containing 50 ng of genomic DNA, 10 pmole each of forward and reverse primers, 200 iM dNTP (Promega), 1xPCR Buffer, 1.5 mM Mg[Cl.sub.2], and 1.5 U Taq polymerase (Promega). PCR was accomplished by 1 min at 94[degrees]C, 1 min at primer annealing temperature (Table 1), and 1 min at 72[degrees]C for 30 cycles. The PCR products were separated on 5% denaturing polyacrylamide gels and a 100 bp DNA standard ladder was loaded in parallel with the samples in order to estimate sizes of the PCR products. The gels were visualized by silver staining according to Sambrook and Russell (2001).

Data analysis

The genotypes were scored manually. The genotypic results of all individual groups were analyzed as described in Triwitayakorn et al. (2006) using TFPGA 1.3 (Miller, 1997) according to location.

RESULTS AND DISCUSSION

A total 16 of the 34 tested microsatellite primers were successfully amplified. The number of alleles per locus ranged from 2 (D9S30, D0S009 and D13S32) to 9 (D21S28) with an average of 4.7 (Table 1). Unbiased heterozygosity of each locus varied from 0.1654 (D0S009) to 0.8577 (D21S28) as shown in Table 1. The unbiased heterozygosity for all eight populations varied between 0.4772 (Samui) and 0.5616 (Burirum) with an average of 0.5233. The percentage of polymorphic loci using the 95% criterion varied from 87.50 (Payao) to 100.00% (Suratthani, Lopburi and Akha Tribe). The genetic distance according to NEI's (1972; 1978) ranged from 0.0574 to 0.2575. Considering all distances measured, the closest populations were found to be the populations from Lopburi and Burirum, with the populations from Samui and Srisagat being the most divergent. In support of this analysis, the data with UPGMA UPGMA Unweighted Pair Group Method, Arithmetic Mean  also is shown in Figure 1A.

[FIGURE 1 OMITTED]

In this study, we found that 16 of 34 (47%) cattle microsatellite markers gave polymorphisms when screened with B. bubalis. This similar results was also found by Navani et al. (2002), who reported that 56% cattle microsatellite markers provided polymorphic band patterns when tested with 25 buffalo. Comparing the results of this study to that of our previous study (Triwitayakorn et al., 2006), which used randomly selected microsatellites that were tested for polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile.  in riverine buffalo by Navani et al. (2002), both studies report that the populations from Samui and Suratthani exhibit a close relationship, while the rest of the results are different. The genotype of individuals were re-screened with 26 microsatellite markers, ten from Triwitayakorn et al. (2006), and 16 from this study. The results showed that the populations from Surin and Burirum, Srisagat and Lopburi, and Samui and Suratthani are in the same clusters as reported previously (Triwitayakorn et al., 2006) and as shown in Figure 1B. This indicates that the ten microsatellite markers previously used in the study of genetic diversity of the Thai swamp buffalo have more utility than the microsatellite loci for diversity studies of cattle approved by the EU AIRE 2066 Concerted Action Group. However, only 16 of the 34 (47%) markers that are recommended by the EU AIRE 2066 Concerted Action Group for genetic diversity analysis could be used in this study which may result in an inaccurate analysis. This suggests that the development of buffalo specific marker will greatly aid genetic diversity studies of the Thai swamp buffalo and other buffalo species.

ACKNOWLEDGMENTS

This research was supported by the Department of Livestock Development, Bangkok, Thailand, the Institute of Molecular Biology molecular biology, scientific study of the molecular basis of life processes, including cellular respiration, excretion, and reproduction. The term molecular biology was coined in 1938 by Warren Weaver, then director of the natural sciences program at the Rockefeller  and Genetics, Mahidol University and the Thailand Research Fund. We would like to thank the Department of Livestock Development for providing blood samples that were used in this project.

Received June 12, 2007; Accepted September 9, 2007

REFERENCES

Barker, J. S., S. G. Tan, O. S. Selvaraj and T. K. Mukherjee. 1997a. Genetic variation within and relationships among populations of Asian water buffalo (Bubalus bubalis). Anim. Genet genet: see civet. . 28:113.

Barker, J. S., S. S. Moore, D. J. Hetzel, D. Evans, S. G. Tan and K. Byren. 1997b. Genetic diversity of Asian water buffalo (Bubalus bubalis): microsatellite variation and a comparison with protein-coding loci. Anim. Genet. 28:103-115.

Chen, G. H., X. S. Wu, D. Q. Wang, J. Qin, S. L. Wu, Q. L. Zhou, F. Xie, R. Cheng, Q. Xu, B. X. Liu, Y. Zhang and O. Olowofeso. 2004. Cluster analysis Cluster analysis

A statistical technique that identifies clusters of stocks whose returns are highly correlated within each cluster and relatively uncorrelated across clusters. Cluster analysis has identified groupings such as growth, cyclical, stable, and energy stocks.
 of 12 Chinese Native chicken populations using microsatellite markers. Asian-Aust. J. Anim. Sci. 17:1047-1052.

Girish, H., S. N. Sivaselvam, S. M. K. Karthickeyan and R. Saravanan. 2007. Molecular characterisation of Nilagiri sheep (Ovis aries) of south India based on microsatellites. AsianAust. J. Anim. Sci. 20:633-637.

Miller, M. P. 1997. Tools for population genetic analysis. Version 1.3. Dept of Biological Sciences, Northern Arizona Univ., Flagstaff Flagstaff, city (1990 pop. 45,857), seat of Coconino co., N Ariz., near the San Francisco Peaks; inc. 1894. Lumbering, ranching, and a lively tourist trade thrive in the region, where many ruined pueblos, numerous state parks, several lakes, and large pine forests , AZ.

Navani, N., P. K. Jain, S. Gupta, B. Sisodia and S. Kumar. 2001. A set of cattle microsatellite DNA markers for genome analysis of riverine buffalo (Bubalus bubalis). Anim Genet. 33:149-154.

Nei, M. 1972. Genetic distance between populations. Am. Nat. 106:283-292.

Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics. 89:58392.

Osman, S. A.-M., M. Sekino, M. Nishibori, Y. Yamamoto and M. Tsudzuki. 2005. Genetic variability and relationships of native Japanese chickens assessed by microsatellite DNA profiling focusing on the breeds established in Kochi Prefecture, Japan. Asian-Aust. J. Anim. Sci. 18:755-761.

Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual. Cold Springs Harbour Laboratory Press, Cold Springs Harbour, NY.

Selvi, P. K., J. M. Panandam, K. Yusoff and S. G. Tan. 2004. Molecular characterisation of the Mafriwal Dairy Cattle of Malaysia using microsatellite markers. Asian-Aust. J. Anim. Sci. 17:1366-1368.

Triwitayakorn, K., B. Moolmuang, S. Sraphet, A. Na-Chiangmai, S. Panyim and D. R. Smith. 2006. Genetic diversity in Thai swamp buffalo (Bubalus bubalis) using cattle microsatellite DNA markers. Asian-Aust. J. Anim. Sci. 19:617-621.

Van Hooft, W. F., A. F. Groen and H. H. Prins. 2000. Microsatellite analysis of genetic diversity in African buffalo (Syncerus caffer Syncerus caffer

a wild black ruminant, up to 6 ft high, with horns that drop down beside the head then wave out and up. There are a number of subspecies; the most common is the Cape buffalo of southeastern Africa. Called also South African buffalo.
) population throughout Africa. Mol. Ecol. 9:2017-2025.

Supajit Sraphet (1), Benchamart Moolmuang (1), Ancharlie Na-Chiangmai (2), Sakol Panyim (1, 3) Duncan R Smith (1) and Kanokporn Triwitayakorn (1), *

* Corresponding Author: Kanokporn Triwitayakorn. Tel: +66-2800-3624 (1259), Fax: +66-2-441-9906, E-mail: mbktw@ mahidol.ac.th

(1) Institute of Molecular Biology and Genetics, Mahido (l) University, Salaya, Nakhonpathom, 73170, Thailand

(2) Department of Livestock Development, Phayathai Rd., Bangkok, 10400, Thailand.

(3) Department of Biochemistry, Faculty of Science, Mahidol University The Faculty of Science was founded as a Premedical School in 1958 by Prof. Dr. Stang Mongkolsuk, and took the name of Faculty of Science, Mahidol University in 1969. The Faculty is located on Rama VI Road, Phaya Thai District, Bangkok, Thailand. , Phayathai, Bangkok, 10400, Thailand.
Table 1. Characteristics of cattle microsatellite markers
approved for diversity studies by the EU AIRE 2066
Concerted Action Group tested in Thai swamp buffalo

Loci             Tm ([degrees]C)     No. of alleles

D14S15                 55            Multiple bands
D14S16                 55            Multiple bands
D11S26                 58            Multiple bands
D19S10-1               59            Multiple bands
D19S10-2               60            Multiple bands
DXS11                  55                 1-4
D29S7                  58                 1-3
D10S27                 60                 1-5
D0S001                 58            Multiple bands
D5S000                 60            Multiple bands
D17S40-1               61            Multiple bands
D17S40-2               60                 1-4
D2S42                  63                 1-3
D5S54                  60            Multiple bands
D11S59                 60                 1-8
D25S24                 58                 1-7
D14S001                60            Multiple bands
D9S30                  58                 1-2
D11S62                 58                 1-8
D10S43                 58                 1-4
D0S009                 61                 1-2
D25S20                 61                 1-3
D10S41                 59            Multiple bands
D11S61                 60                 1-4
D20S24                 59            Multiple bands
D13S17                 58                 1-7
D21S28                 60                 1-9
D10S31                 60            Multiple bands
D21S29                 58            Multiple bands
D24S12                 60            Multiple bands
D1S41-1                58            Multiple bands
D1S41-2                58            Multiple bands
D13S32                 60                 1-2
D1S44                  59            Multiple bands

Loci            Allelic range (1)    Heterozygosity

D14S15                 --                  --
D14S16                 --                  --
D11S26                 --                  --
D19S10-1               --                  --
D19S10-2               --                  --
DXS11                200-220             0.7280
D29S7                175-180             0.2275
D10S27               140-150             0.7161
D0S001                 --                  --
D5S000                 --                  --
D17S40-1               --                  --
D17S40-2             170-175             0.6422
D2S42                150-160             0.4721
D5S54                  --                  --
D11S59               160-170             0.7923
D25S24               160-170             0.6220
D14S001                --                  --
D9S30                130-135             0.4154
D11S62               160-170             0.5969
D10S43               160-170             0.4790
D0S009               200-210             0.1654
D25S20               160-170             0.5222
D10S41                 --                  --
D11S61               130-140             0.4361
D20S24                 --
D13S17               165-170             0.7169
D21S28               180-200             0.8577
D10S31                 --                  --
D21S29                 --                  --
D24S12                 --                  --
D1S41-1                --                  --
D1S41-2                --                  --
D13S32               175-180             0.4739
D1S44                  --                  --

(1) Allelic range was estimated using 100 bp standard DNA ladder.
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Author:Sraphet, Supajit; Moolmuang, Benchamart; Na-Chiangmai, Ancharlie; Panyim, Sakol; Smith, Duncan R.; T
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9THAI
Date:Feb 1, 2008
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