Haplotype Diversity of Swamp Buffalo and River Buffalo Based on Cytochrome B Gene: A Study of Meta-Analysis

Buffalo ( Bubalus bubalis ) is well known as a domesticated buffalo in Asia. The genetic diversity of buffaloes in Asia needs to be studied to ensure a proper breeding program. A meta-analysis study on the cytochrome b gene of the mitochondrial genome from various published data was conducted to evaluate genetic variation and haplo-geography of Asian buffaloes. A meta-analysis is used to provide a comprehensive view of the data. A total of 1369 swamp buffaloes Cytochrome B sequences (from Indonesia (79), Bangladesh (98), China (909), India (4), Laos (96), Taiwan (29), Thailand (54), and Vietnam (100)) and 91 river buffaloes (from China (42), Nepal (42), and Pakistan (7)) were used in this study. Cytochrome B sequences (678 bp) of Syncerus caffer, Bubalus arnee , Bubalus depressicornis, Bubalus quarlesi, Bubalus mindorensis, swamp buffalo, and river buffalo were determined for their haplotypes using DnaSP v6 program. Haplotypes were analyzed by Principal Coordinate Analysis (PCoA) using Adegenet Package and Hierarchical Clustering on Principal Components (HCPC) methods using Factoextra and FactoMineR Package in R-4.0.0 program. Bayesian analysis of genetic differentiation was implemented in BAPS 6.0. Furthermore, we found 56 haplotypes for swamp buffaloes in eight Asian countries and 5 haplotypes for river buffaloes in Pakistan. We also found 5 haplotypes for outgroup ( B. arnee , S. caffer , B. depressicornis , B. quarlesi , B. mindorensis ). Therefore, we found 66 haplotypes in total with outgroup sequences. Based on the PCoA results, three clusters were found. However, the HCPC found eight clusters. Based on HCPC, countries in East and South Asia have four maternal lineages. This is evidence that buffalo domestication has first occurred in East-South Asia. In conclusion, we found four maternal lineages of swamp buffalo and two maternal lineages of river buffalo from ten countries. We also found one maternal lineage for Syncerus caffer and one maternal lineage for B. depressicornis , B. quarlesi , and B. mindorensis .


INTRODUCTION
Buffaloes are commonly used as livestock for producing meat and milk. However, buffaloes are still used as draught animals and have not been kept intensively like cattle and chickens. In Indonesia, swamp and river buffaloes get less concern than cows and chickens, which are strengthened by better research funding. Genetic diversity studies are needed to determine a good breeding program for buffaloes in the future.
Evaluation of genetic variability and genetic association in populations is essential to control the loss of genetic diversity for selective breeding (Mastrochirico-Filho et al., 2019). Breeding programs can be implemented if genetic diversity is observed and maintained. In the same way, genomic tools will allow breeding strategies to ensure the improvement of performance and preserve genetic diversity (Taberlet et al., 2011).
Genetic diversity in livestock is also useful for distinguishing between populations. To find out genetic differences between populations, phenotypic traits are generally used or based on gene diversity. Sequence data have been widely known to observe relationships between organisms (Konishi et al., 2019). To identify variations in the maternal lineage, the mitochondrial genomes are usually explored. In the mitochondrial genome, there are D-loop, Cytochrome b gene (Cyt B), and Cytochrome Oxidase I (COI) gene markers that are often used to evaluate kinship (Kumar et al., 2007;Saputra et al. 2013;Paraguas et al., 2018). Research conducted by Tobe et al. (2010) has shown that the diversity of Cyt B is higher than that of COI. Meta-analysis might contribute to a better understanding of the domestication history of animals (Guangxin et al., 2016). A recent study of cytochrome b in swamp buffalo by Sun et al. (2020) showed 5 maternal lineages of swamp buffalo using neighborjoining in six countries (China, Thailand, Vietnam, Laos, India, and Bangladesh). Kumar et al. (2007) suggested that swamp and river buffaloes were domesticated independently and the classification of swamp and river buffaloes as two related subspecies is more suitable. The evaluation of the genetic diversity of cytochrome B in swamp and river buffaloes has been widely carried out. However, a meta-analysis study has never been carried out to analyze the genetic diversity of swamp buffalo in Asia. Therefore, we identified cytochrome B variation in swamp and river buffaloes from various published data in this meta-analysis study.

MATERIALS AND METHODS
A total of 1369 swamp buffalo sequences (originating from Bangladesh, China, India, Indonesia, Laos, Taiwan, Thailand, Vietnam), 91 river buffalo sequences (originating from China, Nepal, and Pakistan), and five outgroup sequences (Bubalus arnee, Bubalus depressicornis, Bubalus quarlesi, Bubalus mindorensis, and Syncerus caffer) were used in this study. The data used in this study are published data (Table 1). The data used were base positions 70 to 747 (678 bp) of Cytochrome B gene based on GenBank reference (D82894). We used those base positions to accommodate the length of cytochrome B gene sequence of swamp buffalo from Indonesia based on the result reported by Rusdin et al. (2020). Sequences data were analyzed using the DnaSP v6 program to determine the haplotypes and haplotype diversity (Rozas et al., 2017). The haplotype data were further analyzed using the Principal Coordinate Analysis (PCoA) using the Adegenet Package (Jombart, 2008) in the R-4.0.0 program (R Core, 2020). Furthermore, Hierarchical Clustering on Principal Components (HCPC) was drawn using Factoextra (Kassambara & Mundt, 2020) and FactoMineR (Husson et al., 2015) packages from p-distance data in MEGA 7.0.26 (Kumar et al., 2016). Bayesian analysis of genetic differentiation was implemented in BAPS 6.0 (Cheng et al., 2013).

Bubalus bubalis
Indonesia 78 (Rusdin et al., 2020) 61 haplotypes, and the haplotype diversity was 0.7020 with 62 polymorphic sites. The smallest haplotype diversity was found in Taiwan (0.1970), and the highest was found in India (1.0000). The highest number of haplotypes was located in China (36 haplotypes), and the lowest number of haplotypes was found in Nepal (3 haplotypes) and Taiwan (3 haplotypes). By using data of S. caffer, B. arnee, B. quarlesi, B. mindorensis, and B. depressicornis, we found 66 haplotypes (Table 3). Haplotype 16 was the most common with large samples, i.e., 73.50% in China, 7.31% in Vietnam, 5.85% in Laos, 5.05% in Indonesia, 3.47% in Taiwan, 3.47% in Thailand, and 1.45% in Bangladesh (Table 4). We also found unique haplotypes in Bangladesh (6), China (24), India (1), Indonesia (8), Laos (1), Thailand (3), Pakistan (5), and Vietnam (1). Based on PCoA results, we found three clusters (Figure 1).  Figure   3) that were very similar to the results shown by the principal coordinate analysis. Interestingly, we found sequences of river buffaloes from Nepal and China in haplotypes 22 and 23 along with sequences from swamp buffaloes. This result is possible because we only took 678 bp to observe the genetic diversity of the buffalo in this study.

DISCUSSION
Haplotype diversity based on the mtDNA of Egyptian and Indian buffaloes ranged from 0.8236 ± 0.0488 to 0.9428 ± 0.0088 (Nagarajan et al., 2015). Lei et al. (2011) found that the haplotype diversity of Chinese buffaloes ranged from 0.469 ± 0.039 to 0.815 ± 0.033. Based on this haplotype diversity, Taiwan buffalo has lower haplotype diversity than buffaloes in the other countries. However, the gene diversity based on Cytochrome B in Indonesian buffalo is more diverse than the other studies on GH, GHR, GHRH, Pit1, COI, and microsatellite Misrianti et al., 2010;Saputra et al., 2013Saputra et al., , 2020Sumantri et al., 2010). The results we found with the HCPC showed that the haplotypes of B. depressicornis (Lowland Anoa), B. quarlesi (Mountain Anoa), and B. mindorensis (Tamaraw) were self-clustered, and our findings are different from result reported by Tanaka et al. (1996) that B. mindorensis is genetically closer to swamp buffalo than to anoa.
Data of B. arnee (Wild water buffalo) were obtained from Chikuni et al. (1995), showing a genetic closeness with swamp buffalo. Zhang et al. (2020) suggest the swamp and river buffaloes are descended from distinct populations of wild water buffalo (B. arnee). The river buffaloes originating from Pakistan have different haplotypes from the river buffaloes originating from China. Of the seven haplotypes of the Pakistani river buffalo (Saif et al., 2012), only five haplotypes are unique in this meta-analysis. Most likely, the domestication of river buffalo occurs in South Asia. Youssef et al. (2021) found three maternal lineages for river buffaloes in five countries (Bangladesh, China, Egypt, India, and Italy) based on the mitochondrial genome. The study also found that Indian river buffaloes had more maternal lineage. Nagarajan et al. (2015) suggested that the river buffalo was first domesticated in the Northwestern region of India and spread to other parts of the world. In other    words, the domestication of wild water buffalo occurred on the border of South and East Asia. Rusdin et al. (2020) found 10 haplotypes and 9 polymorphic sites for swamp buffalo in Indonesia. Based on this meta-analysis study, we found 8 unique haplotypes of Indonesian swamp buffalo. Lei et al. (2007) found two maternal lineages in Chinese Native Swamp Buffaloes. We got different results because we added sequences carried out by Zhang et al. (2016) so that we found four maternal lineages of swamp buffalo in China and Bangladesh. Based on the complete mitochondrial genome of swamp buffalo revealed eight maternal lineages and this evidence support initial major domestication of swamp buffalo, probably between southern China and Vietnam (Wang et al., 2017). River and swamp buffaloes were domesticated independently based on these findings. Using the whole genome sequencing, Luo et al. (2020) found swamp and river buffaloes shared a common ancestor 1.1-3.5 million years ago. Based on the results of this meta-analysis, it is possible that the domestication of swamp buffalo occurred in East-South Asia.

CONCLUSION
Based on this study, we found four maternal lineages and unique haplotypes for swamp buffaloes, also two maternal lineages for river buffaloes as an outgroup in this study. Based on this research, we suggested that domestication of swamp buffalo occurred in East-South Asia, and domestication of river buffalo occurred in South Asia. Further research with a large sample size in Asian countries is needed to determine the exact number of maternal lineages. Meta-analysis can contribute to a good understanding of the overall data that has been studied to provide comprehensive conclusions.

CONFLICT OF INTEREST
Cece Sumantri serves as an editor of the Tropical Animal Science Journal, but has no role in the decision to publish this article. The authors declare that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.