Use of cattle microsatellite markers to assess genetic diversity of Thai Swamp Buffalo (Bubalus bubalis).
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 DNA 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 buffalo (Navani et al., 2001), the Asian water buffalo (Barker et al., 1997a, b) and the African 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), 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
A total of 105 Thai swamp buffalo (Bubalus bubalis) 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 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 extraction using QIAamp DNA blood kit (QIAGEN GmbH, Hilden, Germany) according to he manufacturers' instructions.
A total of 34 microsatellite loci 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) 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).
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 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 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.
This research was supported by the Department of Livestock Development, Bangkok, Thailand, the Institute of Molecular Biology 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
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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, 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.