Philippine Journal of Science 150 (3): 837-846, June 2021 ISSN 0031 - 7683 Date Received: 05 Oct 2020 Genetic Diversity of Philippine Carabao (Bubalus bubalis) Using Mitochondrial DNA D-loop Variation: Implications to Conservation and Management Lilian P. Villamor1,2*, Yukimizu Takahashi2, Koh Nomura2, and Takashi Amano3 1Department of Agriculture, Philippine Carabao Center National Headquarters and Gene Pool Science City of Muñoz, Nueva Ecija 3120 Philippines 2Tokyo University of Agriculture, Atsugi, Kanagawa 243-0034 Japan 3Yamazaki University of Animal Health Technology, Hachioji, Tokyo 192-0364 Japan Developing rational conservation programs for Philippine carabao (PC) (Bubalus bubalis) requires knowledge on the pattern of genetic variability within and between populations of swamp buffalo. Studying the genetic diversity and phylogeographic structure is essential to understand the distribution of the Philippine swamp buffalo maternal haplotypes towards the PC conservation. This study aimed to determine the genetic diversity and the phylogeographic structure of PC using molecular approaches. D-loop of the mitochondrial DNA (mtDNA) on the PC B. bubalis (n = 107) were randomly selected from 23 populations across the islands of Luzon, Visayas, and Mindanao as major islands sub-groups. DNA was isolated from the whole blood, and the D-loop region of the swamp buffalo was amplified using the polymerase chain reaction (PCR). Purified PCR products were sequenced with the Applied Biosystems Automated 3730. Results showed that the phylogenetic analyses detected 16 mtDNA haplotypes observed with 12 variable sites and haplotype and nucleotide diversities of 0.695 ± 0.042 and 0.004 ± 0.001, respectively. This study also identified the delineation of swamp buffalo populations into major and minor groups. Among the major groups, 14 haplotypes were included, and sequences were incorporated in the maternal lineage A. Minor groups which fell in the maternal lineage B identified two new haplotypes. Thus, the current findings revealed the moderate mtDNA haplotype diversity and weak phylogeographic structure of the PC. In addition, the results of this study will serve as a vital starting point in planning effective strategies and prioritizing the genetic resources for PC conservation and management programs. Keywords: genetic diversity, mtDNA D-loop, phylogenetic, Philippine carabao, conservation INTRODUCTION have a worldwide distribution in the Indian subcontinent, China, Brazil, Italy, and mostly across Southeast Asia Water buffaloes (Bubalus bubalis) are widely distributed (Lau et al. 1998; Kierstein et al. 2004; Cruz 2015). In the and economically important livestock species in many Philippines, swamp-type buffalo is locally known as native developing countries regarding their significant contribution carabao or PC. The animal is considered the farmer's great to agriculture and rural economy (Zhang et al. 2020). They partner in providing draft power in his farm works and has the potential for meat, milk, and hide sources. *Corresponding Author: [email protected] 837 Philippine Journal of Science Villamor et al.: Genetic Diversity of Philippine Carabao Vol. 150 No. 3, June 2021 The Philippine buffalo population has decreased by maternal origins: A and B (Lei et al. 2011). Previous 130,345 heads and at a rate of –0.216% annually between studies on mtDNA D-loop sequence variation have 1998–2018, and these animals are commonly reared in shown the genetic differentiation between the swamp the backyard or at commercial farms. However, they are and the riverine buffalo types in Southeast Asia (Lau et not distinguished between two types: riverine or swamp al. 1998; Lei et al. 2007b), Brazil, and Italy (Kierstein et (http://www.fao.org/faostat/en/#data/QA). Thus, this leads al. 2004). Similarly, the mtDNA D-loop was useful in the to a primary concern to conserve and manage buffalo genotypic analysis to characterize the Malaysian water breeds – particularly the swamp buffaloes – across the buffaloes and their crossbreds, which strongly indicated islands in the Philippines. The Philippine archipelago swamp maternal lineage (Shaari et al. 2019). In addition, is comprised of Luzon, Visayas, and Mindanao major Chinese swamp buffaloes shed light on the two distinct island groups. The Luzon island is in the northern part maternal lineages A and B. After the domestication in the of the country and considered the largest island group in China region, the domesticated Chinese swamp buffaloes terms of land area with dimensions of 740 by 225 km2 revealed two dispersal pathways: first, through Taiwan and covers approximately one-third of the land area of the and the Philippines to the eastern islands of Borneo and Philippines. The Visayas is situated in the central portion Sulawesi; and second, through the mainland Southeast of the country and the third major island group in terms of Asia and then to the western islands of Indonesia (Lau et land area. The Mindanao island group is in the southern al.1998; Lei et al. 2007a). part and the second-largest landmass group of the country (https://www.britannica.com/place/Philippines). The genetic diversity of Philippine swamp buffaloes has few published data and remained unresolved. The mtDNA The buffalo species are divided into swamp and riverine cytochrome c oxidase I (COI) gene was used to identify based on the differences of cytogenic and morphological the species of buffaloes in Calayan Island as swamp-type characterizations. There are no formally recognized breeds buffaloes and suitable for establishing the PC sanctuary and variation in breed characteristics among the swamp (Paraguas et al. 2018). Previous studies on mtDNA D-loop buffaloes (Qui 1986). For example, swamp buffaloes sequences of Philippine swamp buffaloes from the three in other countries such as China and the Philippines Philippine Carabao Center (PCC) institutional herds and are considered a single and homogeneous breed (Qui farms were all incorporated in the maternal lineage A (del 1986; Paraguas et al. 2018). However, swamp buffalo Barrio et al. 2009; Yue et al. 2013). It was reported that no is identified with 48 chromosomes, and riverine buffalo significant geographical structure was identified among with 50 chromosomes. The common physical features swamp-types in Luzon and Visayas islands using the of swamp buffaloes are the white or light gray markings mtDNA COI gene (Bondoc 2013). Lack of studies relating in the lower jaw and brisket (chevron) and the lighter to wide genetic diversity and phylogeographic analyses skin and coat color than the riverine buffaloes. The horn of Philippine swamp buffaloes with more individuals and is sickle-shaped, which extends backward compared to populations were reported. Assessment of the Philippine riverine buffaloes having heavily curled horns (Castillo swamp-type genetic variation with the rationale for 1998). However, the chromosome karyotyping and conservation and management of PC has been conducted morphological-based identification systems could be with the mtDNA control region (D-loop) of swamp buffalo limited to identify swamp buffaloes. The genetic diversity populations. Therefore, this study aimed to determine the assessment of PC could complement a more accurate genetic diversity and the phylogeographic structure using technique such as the DNA-based approach. This entails molecular approaches. using informative genetic markers that could be used to understand the genetic diversity and phylogenetic relationship of swamp buffalo in the country. MATERIALS AND METHODS In Asia, previous studies have been reported that genetic diversity and phylogenetic analyses of swamp buffaloes using the variable regions in the mtDNA markers (Lau Site Selection and Specimen Collection et al. 1998; Kierstein et al. 2004; Lei et al. 2011; Shaari The coordination of collection sites was identified and et al. 2019; Winaya et al. 2019). For instance, the assisted by various regional managers of the PCC and phylogenetic relationships of Indonesia's swamp buffalo local government units. Moreover, the selection of sites groups corresponded to their biogeographic origin and highly considered isolated areas such as mountainous their closer genetic affinity to the Philippines swamp and coastal parts of the country to ensure no introduction buffalo based on the cytochrome b (cyt b) gene partial of artificial insemination program of the river-type was sequences (Winaya et al. 2019). Genetic diversity of cyt done. In this study, 107 fresh blood samples of Philippine b gene in Chinese swamp buffaloes confirmed the two swamp buffalo were randomly selected from 23 different populations of Luzon, Visayas, and Mindanao islands 838 Philippine Journal of Science Villamor et al.: Genetic Diversity of Philippine Carabao Vol. 150 No. 3, June 2021 (Table 1). Approximately 5 ml of blood was collected DNA Extraction, Amplification, and Sequencing into heparinized tubes from the swamp buffalo's jugular Genomic DNA was extracted from the whole blood vein and was stored on ice before transportation to the samples using the commercially available DNA extraction laboratory for molecular analysis. All animal handling kit, and the manufacturer's recommended procedure and techniques involved were approved by the PCC Ethics with little modifications was followed (Promega Committee, Nueva Ecija, Philippines. ReliaPrep™). After the third washing using column wash solution (CWS), additional centrifugation (14,000 Nine reference D-loop sequences