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Genetic Diversity of Myanmar Cattle Breeds Using Complete Mitochondrial D-Loop Sequence

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Genetic Diversity of Myanmar Cattle Breeds Using Complete Mitochondrial D-Loop Sequence D-loop Analysis in Myanmar cattle 〔Original Paper〕 Genetic diversity of Myanmar cattle breeds using complete mitochondrial D-loop sequence Moe LWIN1, Su Lai Yee MON2, Yukio NAGANO1, 3, Kotaro KAWABE4, Hideyuki MANNEN5, Shin OKAMOTO1, 2, Takeshi SHIMOGIRI1, 2 1The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Kagoshima, Japan 2Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima, Japan 3Analytical Research Center for Experimental Sciences, Saga University, Honjo, Saga, Japan 4Education Center, Kagoshima University, Korimoto, Kagoshima, Japan 5Graduate School of Agricultural Science, Kobe University, Kobe, Japan ABSTRACT In Myanmar, native cattle are mainly used for draught. Currently, the available genetic information about them is limited. In this study, complete mtDNA D-loop sequences were analyzed for genetic diversity and differentiation of four popular local breeds – Shwe Ni, Pyar Sein, Ngwar Pyar Ni and Shan Ngwar Pu – and the crossbred population (Holstein Friesian X Myanmar native cattle) among Myanmar’s cattle. From the complete D-loop sequences, 26 polymorphic sites and 27 haplotypes were obtained. All haplotypes (MYAH01 to 27) belonged to two zebu haplogroups of I1 and I2 by the NJ tree and MJ network. A MYAH10 haplotype was major (68%) and common in all breeds and population. Fifteen haplotypes were novel. The haplotype diversity and nucleotide diversity of the four local breeds and crossbred population ranged from 0.193 in Shan Ngwar Pu to 0.832 in the crossbred, and from 0.00051 in Shan Ngwar Pu to 0.00334 in crossbred, respectively. Genetic differentiation among the breeds and population was quite low in the D-loop of Myanmar cattle because the genetic variation among populations (1.4%) was not significant in AMOVA. However, Shan Ngwar Pu was significantly different from other breeds, according to the pairwise FST values. These results provided the genetic diversity and relationship in the popular local breeds and crossbred population of the Myanmar cattle. Key words: crossbred population, genetic diversity, mitochondrial D-loop, local breeds, Myanmar cattle INTRODUCTION local breeds, such as Pyar Sein, Shwe Ni, Ngwar Pyar Ni, Cattle are one of the most economically important Shan Ngwar Pu (Shan), Katonta, Kyauk Pyu, Yenbye and livestock in Myanmar. In 2016, Myanmar owned 16.6 Kadarta, have been developed (Than Daing 2004). In the million head of cattle (about 1.1% of world cattle local breeds, Pyar Sein, Shwe Ni, Ngwar Pyar Ni and Shan population), based on the FAOSTAT database (http://www. Ngwar Pu (Shan), shown in Fig. 1, are popular because of fao.org/faostat/en/#data/QA), and was among the top twenty their usefulness as excellent draught animals. Pyar Sein countries rearing cattle head. Historically, cattle in Myanmar (Pya; Fig.1a) is blue in coat color. Shwe Ni (Shw; Fig.1b) is were used as a source of power in agriculture, for ploughing red in coat color, hooves, eyes, nose, tail and horns. Ngwar the fertile fields of the country’s river valleys. Native cattle Pyar Ni (Ngw; Fig.1c) is red in coat color, but black in are the main source of animal power for cultivation (U Khin hooves, eyes, nose, tongue and horns. Shan Ngwar Pu (Sha; Win 1991). Fig.1d) is variable in coat color but has the smallest body Myanmar native cattle are the zebu type (Bos indicus) and adapted to the harsh native environment, resistant to Correspondence: Takeshi SHIMOGIRI, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan tropical diseases and external parasites, and can sustain with (e-mail: [email protected]) low-quality roughages and grasses. Based on them, several Accepted April 11, 2018 The Journal of Animal Genetics (2018) 46, 57–67 57 LWIN et al. a) Pyar Sein (Pya) b) Shwe Ni (Shw) c) Shan Ngwar Pu (Sha) d) Ngwar Pyar Ni (Ngw) Fig. 1 Myanmar native cattle breeds used in this study. weight among the local breeds (Maeda et al. 2004). These and modern Asian cattle populations (Achilli et al. 2008). breeds, except Sha, have been reared throughout the country Haplogroup Q is closely related to the haplogroup T but are more concentrated in the central part of Myanmar, (Bonfiglio et al. 2010) and was found in ancient cattle such as the Mandalay, Sagaing and Magway regions (Bollongino et al. 2006) and modern cattle in Eurasia and (National Consultative Committee, Myanmar 2002). Shan Africa (Achilli et al. 2008; Achilli et al. 2009). Haplogroup Ngwar Pu is reared only in the Shan state. Furthermore, R has only been detected in modern Italian cattle (Bonfiglio cattle crossbred between exotic breeds and native cattle are et al. 2010). Haplogroup C was observed in ancient reared in Myanmar. The crossbreds between Holstein northeast Chinese cattle dated to 10,660 BP (Zhang et al. Friesian and native cattle are the most popular because of 2013). Haplogroup E was detected in an aurochs (<6,000 high milk productivity. BP) from Germany (Edwards et al. 2007). Clear phylogenetic bifurcation in bovine mitochondrial To date, some genetic studies on Myanmar native cattle DNA (mtDNA) has been reported (Loftus et al. 1994; have been reported (Maeda et al. 2004; Nomura et al. Bradley et al. 1996): major haplogroups T and I in Bos 2004; Tanaka et al. 2004; Chen et al. 2010; Shimogiri et taurus and Bos indicus, respectively. In the mtDNA D-loop al. 2010). Maeda et al. (2004) stated that Myanmar native sequences, B. taurus was categorized into five sub- cattle have nine patterns of coat color. From the studies on haplogroups (T, T1, T2, T3 and T4) and B. indicus into two blood protein and SRY gene polymorphisms, Myanmar sub-haplogroups I1 and I2 (Achilli et al. 2008; Chen et al. native cattle generally belong to the zebu type (Nomura et 2010). In addition, five major haplogroups P, Q, R, C and E al. 2004; Tanaka et al. 2004). Also, Chen et al. (2010) have been identified in the D-loop sequences: Haplogroup P showed that 30 Myanmar native cattle had zebu haplotypes was found in ancient European aurochs (Bos primigenius) using mitochondrial D-loop sequence. However, there is no The Journal of Animal Genetics (2018) 46, 57–67 58 D-loop Analysis in Myanmar cattle Table 1. Information of 140 cattle samples taken from six locations in Myanmar. Sagaing Mandalay Magway Naypyitaw Yangon Shan Population Total M F M F M F M F M F M F Shwe Ni 3 2 3 2 4 5 3 3 4 1 – – 30 Pyar Sein 7 – 7 – 5 2 1 5 2 1 – – 30 Ngwar Pyar Ni 2 2 3 4 5 4 1 3 2 4 – – 30 Shan Ngwar Pu – – – – – – – – – – 21 9 30 Crossbred 1 5 – 6 – – – – – 8 – – 20 Total 13 9 13 12 14 11 5 11 8 14 21 9 140 M is for male and F is for female. molecular genetic study focusing on local breeds in MATERIALS AND METHODS Myanmar. The present study was undertaken to assess the Sample collection, DNA extraction mtDNA variation and the genetic diversity of popular local Blood samples of 30 Shwe Ni (Shw), 30 Pyar Sein breeds and the crossbred population in Myanmar. The (Pya), 30 Ngwar Pyar Ni (Ngw), 30 Shan Ngwar Pu (Sha) resulting information can be used in formulating national and 20 crossbred cattle (Cro) were collected from the plans and strategies for sustainable improvement and venipuncture of jugular vein during April to May 2016 conservation of cattle genetic resources in Myanmar. (Table 1). The sampling locations are shown in Table 1 and Fig. 2. Each sample was unrelated and taken from one head per farmer, after interviewing about the sex, age and relatedness of the cattle. Genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. Polymerase Chain Reaction (PCR) amplification and sequencing The complete mtDNA D-loop was amplified using a pair of primers: 5’-AAACTGCAGTCTCACCATCAAC-3’ and 5’-GATTATAGAACAGGCTCCTCTA-3’. These primers were slightly changed from those of the previous paper (Loftus et al. 1994). The PCR protocol was as follows: Each 10 µl reaction contained 20 ng of genomic DNA, 20 µmoles of each primer, 200 µM each of dNTPs, 1× Ex Taq buffer including 2 mM Mg2+, and 1.0 unit of Ex Taq HS DNA polymerase (TAKARA BIO Inc, Otsu Japan). Thermocycling (Veriti Thermal Cycler, Applied Biosystems) consisted of 2 min denaturation at 94°C, 40 cycles of 30 s at 94°C, 30 s at 60°C and 60 s at 72°C, and a final extension for 5 min at 72°C. The amplified products were visualized on a 2.0 % agarose gel, which was stained with GelGreenTM (Biotium, CA, USA). The amplified products were purified with VIOGENE Gel/PCR DNA Isolation System (Viogene- Bio Tek Corporation, Taiwan) and directly sequenced using a Big Dye Terminator v3.1 cycle sequencing ready reaction kit and ABI-PRISM 3730 genetic analyzer (Applied Fig. 2 The map of Myanmar and sampling locations. Biosystems, CA, USA). The Journal of Animal Genetics (2018) 46, 57–67 59 LWIN et al. Data analysis of bovine mtDNA D-loop sequences test) were calculated by the DnaSP software. The bovine Identification of polymorphic sites and haplotypes was mtDNAs D-loop sequences obtained in this study have been performed after aligning the D-loop sequences of 140 deposited in DNA Data Bank of Japan (DDBJ) under Myanmar cattle with a reference sequence (GenBank accession nos. LC377275 to LC377301. accession number L27733; Loftus et al. 1994). The multiple sequence alignment program ClustalW (http://www.genome. RESULTS AND DISCUSSION jp/tools-bin/clustalw) was used. Estimation of genetic Variation of complete mtDNA D-loop sequences in Myanmar diversity parameters, including haplotype diversity and cattle nucleotide diversity, was performed by the DnaSP software Comparison of the complete mtDNA D-loop sequences V6.10.1 (Rozas et al.
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