J. Anim Sci. 2008. 86:1495-1502. doi:10.2527/jas.2007-0321
© 2008 American Society of Animal Science
Genetic relationship and diversity analysis of Indian water buffalo (Bubalus bubalis)
R. K. Vijh1,
M. S. Tantia,
B. Mishra and
S. T. Bharani Kumar
National Bureau of Animal Genetic Resources (NBAGR), Post Box 129, Karnal 132 001, India
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Abstract
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The water buffalo (Bubalus bubalis) is an important dairy animal on the Indian subcontinent and in Southeast Asian countries. The diversity and differentiation among 12 populations or breeds of buffalo were studied. Data were generated and analyzed from 527 animals belonging to 10 recognized breeds and 2 additional populations of Indian buffalo by using 22 microsatellite loci. Relationships among buffalo breeds and populations were estimated based on genetic distances. The Bayesian analysis grouped 12 populations into 8 distinctive clusters. Geographically close breeds clustered together, except for the Jaffarabadi and Murrah, which were not in geographic contiguity. The Mantel test revealed nonsignificant correlations between genetic and geographic distances. This supports the hypothesis that buffaloes have been domesticated at different places for specific purposes. The phylogenetic relationship based on microsatellite loci supported the breed classification based on body size. The Toda breed, which is considered to be endangered, had genotypes similar to those of the surrounding buffalo populations.
Key Words: buffalo • diversity analysis • microsatellite
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INTRODUCTION
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The water buffalo (Bubalus bubalis) contributes significantly to the agricultural economy and food security of countries on the Indian subcontinent and in Southeast Asia. It contributes through milk, meat, hides, and draught power. The buffalo is known to have been domesticated at a very early date, but exactly when and where is unknown (Cockrill, 1974
). Representative and probably domesticated buffaloes appear on seals of both the Indus Valley and Mesopotamia from the second millennium BC. They have typical crescentric horns of the wild swamp buffaloes (Zeuner, 1963). In China domesticated buffaloes were known in the second millennium BC and appear to have been introduced from Southeast Asia (Epstein, 1969).
River buffaloes are the mainstay of the dairy industry in India and are fast replacing indigenous cattle (Bos indicus) in milk pockets or areas of concentrated milk production. India has 97.9 million buffaloes (Department of Animal Husbandry and Dairying, Government of India, 2003). Their milk is preferred over cow’s milk because of its whitening properties and higher fat and protein percentage. Buffalo milk is considered to be more economical for the production of casein, caseinates, whey protein concentrate, and fat-rich dairy products. The buffaloes have been grouped into morphologically distinctive breeds, namely, the Murrah, Nili-Ravi, Bhadawari, Toda, Mehsana, Jaffarabadi, Surti, Nagpuri, Pandharpuri, and Marathwada. There are also 2 large populations, 1 found in the foothills of the Himalayan ranges (Tarai buffaloes) and 1 in the southern part of India (Kalasthi buffaloes). Amplification of heterologous markers of cattle in buffalo was reported previously (Navani et al., 2002). Data on 3 buffalo populations (Bhadawari, Kerala, and Tarai) were analyzed for genetic relationships, diversity analysis, and structuring (Vijh et al., 2005; Tantia et al., 2006). Kumar et al. (2006) analyzed microsatellite data for phylogenetic relationships of 8 buffalo breeds. In the present study, variation at the microsatellite loci was studied to infer the relationship among 10 recognized breeds, which included the 8 breeds studied by Kumar et al. (2006) and 2 additional populations of Indian riverine buffalo.
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MATERIALS AND METHODS
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All procedures involving animals followed guidelines of the Animal Ethics Committee of the National Bureau of Animal Genetic Resources.
Blood samples of 527 buffaloes were collected from the home tract of 10 defined buffalo breeds and 2 local populations of Uttarakhand (Tarai) and Andhra Pradesh (Kalasthi; Figure 1). The animals were selected at random and represented the breeds or populations. The 10 recognized buffalo breeds of India include the 8 breeds studied by Kumar et al. (2006). The blood samples collected were from the home tract of the breeds but with a time gap of 7 to 8 yr between the 2 sample collections. Eight to 10 mL of whole blood was collected from the jugular vein in EDTA-coated Vacutainer tubes (BD Vacutainer Systems, Plymouth, UK) and transported to the NBAGR laboratory at 0 to 5°C.
Deoxyribonucleic acid was extracted from whole blood by using a standard protocol (Sambrook and Russell, 2001). The concentration of DNA was judged by comparison with the standard DNA marker concentration on agarose gels. The quality of DNA was checked on 0.8% agarose gels prepared in Tris-acetate EDTA buffer (Ogden and Deborah, 1987).
A total of 22 heterologous microsatellite loci were chosen for the study. These loci were BM1818, CSSM29, ILSTS11, ILSTS52, ILSTS59, CSSM43, CSSM45, ILSTS05, ILSTS49, ILSTS58, ILSTS72, ILSTS30, ETH152, CSSM47, CSSM33, CSSM08, CSRM60, CSSM06, CSSM19, CSSM57, ILSTS38, and ILSTS29 (NCBI GenBank). The criterion for selection of the heterologous microsatellite loci was based on their polymorphism in buffaloes, polymorphism information content value, and number of alleles (Navani et al., 2002). The 5' ends of the forward primers were labeled with either VIC, NED, or PET (Applied Biosystems, Foster City, CA) dyes. The 22 loci were located on 18 autosomal chromosomes of cattle (Bos taurus).
The PCR conditions were standardized for all of the 22 primer pairs selected for the study. Polymerase chain reaction amplification was carried out in a 20-µL reaction containing 50 ng of genomic DNA, 150-µM dNTP, 4 pmol each of forward (labeled) and reverse primers, 1 U of Taq DNA polymerase, and 1x reaction buffer (containing 1.5-mM MgCl2). Amplification was carried out by using an ABI 9700 instrument (Applied Biosystems) with initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 57 to 60°C (primer specific) for 45 s, and extension at 72°C for 45 s. The final cycle was followed by an extension step at 72°C for 10 min. The PCR products were visualized on 2% agarose gels with 1x Tris-acetate-EDTA buffer containing 200 ng/mL of ethidium bromide.
Genotyping was carried out on an ABI 3100 Avant automated DNA sequencer, with LIZ 500 (Applied Bio-systems) as the internal lane standard (size standard). Post-PCR multiplexing was used to simultaneously genotype 3 or 4 loci, depending on the PCR product size and dye label of the primers used. Sizing and allele calling were performed by using Genotyper version 2.0 software (Applied Biosystems). The allele data thus generated were used for further statistical analyses.
Statistics
The allele frequency, number of alleles, observed and expected heterozygosity, population differentiation (FST) (Weir and Cockerham, 1984), and genetic distances (Nei’s standard genetic distances and Chord distances) were calculated using Microsatellite Analyzer version 4.05 (Dieringer and Christian, 2003). Tests for deviation from the Hardy-Weinberg equilibrium (HWE) at each locus for each breed were performed using GenePop version 3.1 (Raymond and Rousset, 1995). The P-values were corrected for multiple comparisons by applying a sequential Bonferroni correction (Rice, 1989). Chord distances among breeds were used to construct a neighbor-joining tree using PHYLIP version 3.5 (Felsenstein, 1993), and the tree was visualized with TreeView version 1.6.6 software (Page, 1996). The FST values between all possible breed pairs were displayed by multidimensional scaling (MDS) using NTSYS software version 2.02e (stress value 0.12; Exeter Software Inc., Setauket, NY).
Breed differentiation was further investigated by using a Bayesian clustering approach implemented in Structure version 2.2 (Pritchard et al., 2000). This program generates clusters of individuals based on their multilocus genotypes. We used an admixture model with a burn-in of 50,000 iterations and 150,000 Markov chain Monte Carlo repetitions to estimate the probable number of genetic clusters (K). An analysis of molecular variance was performed using ARLEQUIN version 3.11 (Excoffier et al., 2005). A hierarchical ANOVA was carried out to partition total variance into variance components attributable to interindividual or interbreed differences, or both. Variance components were then used to compute fixation indices and their significance was tested at 1,000 permutations, as described by Excoffier et al. (1992). Under a stepping-stone model of migration (Kimura, 1953; Kimura and Weiss, 1964), isolation by distance is generally expected if the change in diversity is balanced by genetic drift and migration. Researchers have suggested several methods for identifying isolation by distance. In the present study, we tested isolation by distance by plotting Nei’s standard genetic distances against geographic distances. The significance of the correlations was determined by using Mantel tests (Mantel, 1967), as implemented in isolation by distance (IBD version 2.1; Jensen et al., 2005).
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RESULTS AND DISCUSSION
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A total of 380 alleles were observed in the 12 populations or breeds. The total number of alleles per breed ranged from 129 in Pandharpuri buffaloes to 207 in Nagpuri buffaloes (Table 1). The average number of alleles per locus per breed ranged from 5.86 in Pandharpuri buffaloes to 9.41 in Nagpuri buffaloes. The number of alleles per locus ranged from 11 (CSSM29) to 26 (BM1818). The observed and expected heterozygosity ranged from 0.53 to 0.70 and 0.63 to 0.73, respectively. The lowest values for observed heterozygosity were in Toda buffaloes and the highest values were in Nagpuri buffaloes. The HWE at 22 loci was tested in 12 populations for deviations. It was observed that a total of 50 locus-population combinations were not in the HWE. Loci BM1818 and ILSTS38 were not in the HWE in 8 and 11 populations, respectively (Table 2). The Toda and Jaffarabadi had 7 loci deviating from the HWE. Five loci deviated from the HWE in the Bhadawari, Tarai, Murrah, and Kalasthi.
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Table 2. Microsatellite loci deviating from the Hardy-Weinberg equilibrium in different breeds of Indian water buffalo
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Nei’s standard genetic distances and FST values between pairs of populations were estimated (Table 3). The significance of FST values was tested by using a permutation test. All pairwise FST values were significant. The smallest FST value, which was 0.0213, was obtained between Mehsana and Surti buffaloes. Low FST values were also obtained between Marathwada and Pandharpuri buffaloes (0.0468) and between Toda and Kalasthi buffaloes (0.0553). The largest FST was obtained between Jaffarabadi and Pandharpuri buffaloes (0.1797), followed by Jaffarabadi and Bhadawari buffaloes (0.1744). Nei’s standard genetic distance was lowest between the Mehsana and Surti breeds (0.0442) and highest between the Jaffarabadi and Pandharpuri (0.5982) breeds.
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Table 3. Population differentiation (FST) values (below diagonal) and Nei’s standard genetic distance (above diagonal) between various Indian water buffalo breeds
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The FST values were used for display of the MDS (Figure 2). The MDS display exhibited Kalasthi and Toda buffaloes as one cluster, and Pandharpuri buffaloes as another, whereas Nagpuri, Murrah, and Jaffarabadi buffaloes also formed distinctive clusters. The other 6 buffalo populations (Bhadawari, Nili-Ravi, Marathwada, Tarai, Surti, and Mehsana) formed a single cluster.
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Figure 2. Multidimensional scaling plot constructed on the basis of pairwise FST values for Indian water buffalo breeds.
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The Bayesian clustering analysis using the software Structure was prepared and 8 clusters were inferred based on log-likelihood ratios (Figure 3). The proportion of membership coefficient of Indian buffalo breeds revealed the first cluster as having a contribution mainly from the Nagpuri, whereas the second cluster had representation mainly from the Bhadawari, the third cluster had a contribution mainly from the Mehsana and Surti, and the fourth was from the Toda and Kalasthi. The fifth cluster included the Pandharpuri and Marathwada, the sixth cluster was the Jaffarabadi and Murrah, and the seventh and eighth clusters predominantly had contributions from the Nili-Ravi and Tarai, respectively.
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Figure 3. Eight inferred clusters based on Bayesian analysis of Indian water buffalo breeds. The inferred clusters are on the x-axis and the proportion of membership coefficient is on the y-axis.
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Cockrill (1981) classified the buffalo breeds of India on the basis of geographical distribution and phenotype. He described 15 breeds in 5 groups based on their geographical distribution in India. Groups included i) Murrah group: the Murrah and Nili-Ravi; ii) the Gujarat group: Surti, Mehsana, and Jaffarabadi; iii) the Uttar Pradesh group: Bhadawari and Tarai; iv) the India group: Nagpuri, Manda, Jerangi, Kalahandi, Sambalpuri, and Pandharpuri; and v) the South India group: Toda and South Kanara. Of these listed breeds the Murrah, Nili-Ravi, Surti Marathwada, Mehsana, Jaffarabadi, Bhadawari, Nagpuri, Pandharpuri, and Toda are the breeds listed in India primarily on the basis of morphology. Cockrill (1981) grouped these recognized breeds into 2 categories: 1) the Bhadawari, Nagpuri, Surti, Pandharpuri, and Toda and 2) the Nili-Ravi, Murrah, Mehsana, and Jaffarabadi. We performed an analysis of molecular variance, taking no grouping into account. Among-breed variation was 9.69% and the remaining variation (90.31%) was within the breeds. We did not observe significant differences between the 2 groups when data were analyzed by using Cockrill’s (1981) classification.
Nei’s standard genetic distance between breeds and log of geographical distance were used to determine the evidence for isolation by distance. The correlation between genetic distance and log of geographical distance was not significant (r = 0.181), indicating a lack of evidence for isolation by distance. This supports the hypothesis that the buffalo has been domesticated for specific purposes at different places and that breeds have evolved as a result of selection. The neighbor-joining tree using Chord (Figure 4) genetic distances for phylogenetic relationships revealed results similar to those inferred by the Bayesian analysis.
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Figure 4. Neighbor-joining tree based on pairwise Chord genetic distance among 12 Indian water buffalo breeds.
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This study presents comprehensive genetic relationships among Indian riverine buffaloes based on microsatellite markers. The genetic analyses of 22 microsatellite loci revealed that all the breeds or populations had reasonably high levels of diversity (average heterozygosity of 0.53 to 0.70). This was in contrast to a heterozygosity of 0.4 to 0.58 in swamp buffaloes (Barker et al., 1997). The mean observed heterozygosity values in the present study were lower than those obtained by Kumar et al. (2006) for the 8 breeds, except for Nagpuri buffaloes, for which it was similar (0.7). The observed heterozygosity was less than the expected heterozygosity in all 12 breeds or populations of buffaloes. A total of 50 locus-breed combinations deviated from the HWE, and the deviation was on account of a deficiency of heterozygotes. This deficiency may be on account of the presence of null alleles, the small sample size, or the Wahlund effect. The Wahlund effect refers to the reduction of heterozygosity in a population caused by the subpopulation structure. Several loci deviated from the HWE in Toda and Jaffarabadi buffaloes. The reanalysis of data obtained in the present study for 8 breeds revealed 37 locus-breed combinations deviating from the HWE, compared with 26 reported by Kumar et al. (2006). The decrease in observed heterozygosity and increased number of loci deviating from the HWE in the present study, compared with values obtained by Kumar et al., (2006), may be due to the changing genetic constitution of various breeds over a period of 7 to 8 yr. The spread of milk cooperatives, especially during the last 7 to 10 yr, has led to the provision of a basic infrastructure, including veterinary care, the acceptance of AI by farmers, and the availability of these facilities at the farmers’ doorstep, coupled with selection of sires of the breeds, and their large-scale use may be reasons for the reduced heterozygosity and increased inbreeding levels in the breed home tracts. The number of breeding males in the Toda is limited, and the total population consists of only 3,500 animals (Nivsarkar et al., 2000). In the Jaffarabadi, the sires have been selected for behavioral and economic traits and few breeding sires are being used for AI in the breeding tract. The genetic differentiation among breeds was 9.69%. This value was quite high compared with the 3.4% reported by Kumar et al. (2006), but was in agreement with studies on other livestock species (7 to 11%) using microsatellites (Ritz et al., 2000; Vijh et al., 2007). The increase occurred even though 8 of the 12 breeds were the same as those used by Kumar et al. (2006). Analysis of the present data for the 8 breeds (Kumar et al., 2006) further increased the genetic differentiation to 10.38%.
The genetic differentiation among different breeds or populations was due to selection, drift, and local inbreeding effects. The neighbor-joining tree constructed from the Chord genetic distance matrix and MDS of pairwise FST values revealed clusters consisting of Toda and Kalasthi; Murrah, Nagpuri, and Jaffarabadi; and the remaining 7 breeds in another cluster. The Bayesian clustering analysis generated 8 clusters. Four clusters had only 1 breed, whereas the remaining 4 had 2 each (Toda and Kalasthi; Pandharpuri and Marathwada; Surti and Mehsana; and Jaffarabadi and Murrah). The Murrah and Jaffarabadi are well-recognized dairy breeds in India and selection in both breeds is for dairy traits. This might have resulted in similar genotypes. Another possible explanation is the use of Murrah semen in the Jaffarabadi tract to overcome management difficulties because of their large horn size and downward horn orientation, hindering feeding under stall-fed conditions. Kumar et al. (2006) also reported the Jaffarabadi as a distinctive cluster from the Surti and Mehsana, despite the fact that the breeding tracts of the Jaffarabadi, Mehsana, and Surti are in geographic proximity. Our genetic classification was more or less in agreement with Cockrill’s (1981) classification of buffaloes based on size. The Toda buffaloes, reared by Toda tribes in the Nilgiri hills, are considered to be a threatened breed because of their small population size. In the present study, the local population of buffalo in southern India (Kalasthi) revealed close similarity with genotypes of the Toda, which was attributed to their geographic contiguity with Toda buffaloes.
This study provides a comprehensive view of the riverine buffaloes of India, covering the major milkshed areas. Some of the geographically contiguous breeds revealed very similar genotypes, and this knowledge can have a bearing on progeny testing and bull selection programs. The Toda is considered a threatened breed of buffalo, but similar genotypes are present in the areas surrounding its breeding tract. This genetic diversity analysis of buffalo breeds will help in conservation prioritization and in making plans that reconcile their genetic improvement with maintenance of genetic variation.
1 Corresponding author: rameshvijh{at}yahoo.com
Received for publication June 2, 2007.
Accepted for publication March 4, 2008.
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Abstract
INTRODUCTION
