D-loop Analysis in 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

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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 , 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

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Table 1. Information of 140 cattle samples taken from six locations in Myanmar. Sagaing Mandalay Magway Naypyitaw 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).

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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. 2017). The possible relationship in five Myanmar cattle populations identified 26 variants, between haplotypes was estimated using the neighbor- including 9 singletons and 17 parsimony-informative sites. joining (NJ) tree (Saitou & Nei 1987), drawn by the MEGA Based on these variants, 27 haplotypes (MYAH01 to 27) 7.0 software (Kumar et al. 2016), in which the evolutionary were observed in Myanmar cattle (Table 2). distances were computed using the Kimura 2-parameter For distribution of the 27 haplotypes in Myanmar method (Kimura 1980). We used the haplotypes obtained in cattle, a single haplotype (MYAH10), which was identical to this study, and the sequences belonged to the representative the reference sequence (L27733), accounted for 68.57% of haplogroups (I1, I2, R, P, Q, T, E and C) and outgroups the whole and ranged from 40% of Cro to 90% of Sha in (Banteng, Gaur, Yak and Bison) in the NJ tree. The GenBank frequency. High frequency of the reference sequence was accession numbers of the representative sequences were consistent with data from indicine populations of other Asian L27722 for the haplogroup I1, EU177869 for the haplogroup countries (Mannen et al. 2000; Lai et al. 2006; Chen et al. I2, HQ184045 for the haplogroup R, DQ124389 for the 2010). The frequency of the remaining haplotypes ranged haplogroup P, EU177867 for the haplogroup Q, V00654 for from 3.5% to 0.71% in 140 animals. Especially, 16 the haplogroup T, DQ915576 for the haplogroup E, haplotypes were singletons in Myanmar cattle, whose KF525852 for the haplogroup C, JN632605 for the Banteng distribution ranged from one in Shw and Ngw to eight in (Bos javanicus), DQ319905 for the Gaur (Bos gaurus), Pya. These results suggest that Myanmar cattle may GQ464312 for the Yak (Bos grunniens), EU177871 for the originate from various maternal lineages but is strongly Bison (Bison bison), L27733 for the haplotype I1, influenced by one maternal lineage. In addition, the AB268565 for the haplotype I1a, EU177869 for the BLASTN search (Altschul et al. 1997) revealed that 15 of haplotype I2 and FJ492612 for the haplotype I2a. The the 27 haplotypes were novel in this study (data not shown). robustness of the tree was evaluated by resampling the data This finding suggests that Myanmar cattle may possess by the bootstrap test (Felsenstein 1985) with 500 replicates novel polymorphisms, including gene variants. (Hedges 1992). The median-joining (MJ) network using a maximum parsimony calculation was constructed with the Assignment of the 27 haplotypes to haplogroups Network 5.0.0.3 software (www.fluxus-engineering.com/ Fig. 3 shows the NJ tree of 27 haplotypes, with the sharenet.htm) to investigate the possible relationships among representative sequences of the D-loop haplogroups and the haplotypes and the distribution of the haplotypes among outgroups (I1, I2, R, P, Q, T, E, C, Banteng, Gaur, Bison and populations. The population genetic structure indexes were Yak). In Fig.3, the 27 haplotypes belonged to two indicine estimated by analyses of molecular variance (AMOVA, haplogroups (I1 and I2). Other haplogroups and the Excoffier et al. 1992) implemented in the Arlequin, version sequences of Banteng (JN632605, Bos javanicus), Gaur 3.5.1.2 (Excoffier & Lischer 2010). Pairwise FST values of (DQ319905, Bos gaurus), Yak (GQ464312, Bos grunniens) genetic distance (Slatkin 1995) were also generated using and Bison (EU177871, Bison bison) were becoming the the Arlequin, version 3.5.1.2 (Excoffier & Lischer 2010) outgroup of Myanmar cattle. These results were consistent with 10,100 replicates. To understand the population with the earlier D-loop study on Myanmar cattle (Chen et al. expansion, mismatch pairwise distribution and neutrality 2010). In addition, no taurine haplotype was obtained from tests (Tajima’s D test, Fu and Li’s D test and Fu and Li’s F the 140 animals, including the crossbred animals in this

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Table 2. 27 haplotypes of mtDNA D-loop and their distribution in local breeds and crossbred population. Nucleotide positiona Population 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 9 0 0 0 0 0 0 0 1 1 1 1 1 2 2 2 2 3 3 3 1 1 2 2 3 5 1 2 4 5 7 8 8 0 1 2 2 4 2 3 3 6 0 0 2 3 7 7 4 4 5

Haplotypes 3 9 2 9 0 4 4 5 1 3 4 7 1 9 1 2 0 0 1 6 9 3 8 1 6 3 Shwe Ni Pyar Sein Shan Ngwar Pu Ngwar pyar Ni Crossbred Total Haplotype% Sahiwalb G T A T C C T T T C T C T G C C C G T G C G A T T C MYAH1 - - - C ------0 3 1 0 1 5c 3.57 MYAH2 - C - C - T - - - - C - - - T ------0 1 0 1 0 2 1.43 MYAH3 - C - - - T - - - - C ------1 0 0 0 0 1 0.71 MYAH4 - - - - T T ------0 0 0 0 3 3 2.14 MYAH5 - - G - - T ------0 0 0 0 1 1 0.71 MYAH6 - - - - - T ------A - - - - 0 1 0 1 1 3 2.14 MYAH7 ------A ------0 1 0 1 1 3 2.14 MYAH8 ------A ------A - - - - 0 0 0 0 1 1 0.71 MYAH9 ------G 0 0 1 0 0 1 0.71 MYAH10 ------21 20 27 20 8 96 68.57 MYAH11 ------G - - - 0 0 0 0 1 1 0.71 MYAH12 ------T ------G - - - 0 0 0 0 1 1 0.71 MYAH13 ------A ------1 0 0 0 0 1 0.71 MYAH14 A ------1 0 0 0 0 1 0.71 MYAH15 ------C ------1 0 0 0 0 1 0.71 MYAH16 ------T ------0 1 0 0 0 1 0.71 MYAH17 ------C ------1 0 0 0 0 1 0.71 MYAH18 ------T ------0 0 0 2 0 2 1.43 MYAH19 ------T ------0 2 0 0 0 2 1.43 MYAH20 ------A ------1 0 0 0 0 1 0.71 MYAH21 - - - - - T ------1 0 0 0 0 1 0.71 MYAH22 ------C ------1 1 0 0 0 2 1.43 MYAH23 ------C ------1 0 0 0 0 1 0.71 MYAH24 ------C C - - - - C - - T - A - - T - - - - - 0 0 0 1 1 2 1.43 MYAH25 ------C C - - - - C - - T - - - - T - - - - - 0 0 1 0 0 1 0.71 MYAH26 ------C C - - - - C - - T - - - - T A - - - - 0 0 0 2 2 4 2.86 MYAH27 ------C C - - - - C - - T - - - - T A - A A - 0 0 0 1 0 1 0.71 Hyphen indicates the identical nucleotide with reference sequences. aNumbers written vertically show nucleotidebase positions of mtDNA genome bReference sequence (L27733) determined by Loftus et al. (1994). cNumbers in parentheses indicate number of animals observerd in Myanmar cattle population study. Earlier reports using Asian native cattle described that (I1 and I2). In this analysis, we added four representative some taurine introgressions were observed from the mtDNA indicine haplotypes – I1, I2, I1a and I2a (Chen et al. 2010). D-loop sequence data (Loftus et al.1994; Bhuiyan et al. The MJ network represented the relationship of the indicine 2007). In Myanmar, there were two official importations of haplotypes observed in the NJ tree. This network shows a pregnant Holstein cows from New Zealand and Australia to star-like skeleton; the center of the star corresponds to the Myanmar for improvement and distribution of quality breeds haplotype with the highest frequency, while a number of in 1978 (National Consultative Committee, Myanmar 2002). peripheral haplotypes were dispersed. A network map Furthermore, 15,000 doses of imported semen were showed that the two main haplogroups (I1 and I2) were introduced from North America and Europe (U Than Hla et separated by one median joining vector. The 23 haplotypes al. 1999; National Consultative Committee, Myanmar assigned to the I1 haplogroup accounted for 94.3% of the 2002). Our finding suggests that genes of Holstein may have whole. In addition, the sequences of the I1a haplotype were been inherited from crossbred bulls although the sample size observed in the populations except for Sha, although the I1a was small. We plan to investigate this matter in the next haplotype was not observed in the previous study (Chen et study using variations of the nucleus genome. al. 2010). The remaining four haplotypes (MYAH24 to 27) Fig. 4 is the MJ network of two indicine haplogroups assigned to the I2 haplogroup accounted for 5.7% of the

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MYAH27 MYAH26㻌 MYAH25㻌 MYAH24㻌 I2㻌 EU177869-I2 MYAH22 MYAH1㻌 L27722-I1㻌 MYAH23 MYAH20 MYAH18 MYAH12㻌 MYAH11 MYAH19 MYAH16 MYAH17 MYAH15 MYAH10 I1㻌 MYAH14 MYAH13 MYAH9㻌 MYAH7㻌 MYAH8㻌 MYAH6㻌 MYAH4㻌 MYAH21 MYAH2㻌 MYAH3㻌 MYAH5㻌 HQ184045-R DQ124389-P EU177867-Q Other V00654-T DQ915576-E Haplogroup KF525852-C㻌 JN632605-Bos javanicus DQ319905-Bos gaurus EU177871-Bison bison Outgroup㻌 GQ464312-Bos grunniens

0.01 Fig. 3 NJ tree of the 27 haplotypes with the representative sequences of the haplogroups. As the representative sequences of the haplogroups, L27722 (Loftus et al. 1994) and EU177869 (Achilli et al. 2008) were used for the zebu haplogroups I1 and I2, respectively. The GenBank accession numbers, V00654 for haplogroup T (Anderson et al. 1982), HQ184045 for haplogroup R (Bonfiglio et al. 2010), DQ124389 for haplogroup P (Achilli et al. 2008), EU177867 for haplogroup Q (Achilli et al. 2008), DQ915576 for haplotype E (Edwards et al. 2007) and KF525852 for haplotype C (Zhang et al. 2013), were used in this study. Moreover, the sequences of JN632605 for Bos javanicus, (Hassanin et al. 2012), DQ319905 for Bos gaurus, GQ464312 for Bos grunniens (Wang et al. 2010) and EU177871 for Bison bison (Achilli et al. 2008) were also used as outgroup of Myanmar cattle. All 27 haplotypes were separated into two zebu haplogroups.

Fig. 4 MJ network of the haplotypes from 140 Myanmar cattle. The size of the node is proportional to the haplotype frequency in the 140 animals. For haplotype differentiation, L27733 (Loftus et al. 1994), AB268565 (Lin et al. 2007), EU177869 (Achilli et al. 2008) and FJ492612 (Chen et al. 2010) were used for I1 haplotype, I1a haplotype, I2 haplotype and I2a haplotype, respectively.

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Table 3. Genetic diversity estimates of the local breeds and crossbred population in Myanmar cattle. Neutrality Test Population Code n 1K 2H 3 兀 Tajima’s D Fu &Li’s D Fu & Li’s F 0.554 0.00105 Shwe Ni Shw 30 8 ± ± -1.802 -2.123 -2.367 0.011 0.00034 0.517 0.00080 Pyar Sein Pya 30 10 ± ± -2.252** -3.439* -3.596* 0.112 0.00024 0.556 0.00252 Ngwar Pyar Ni Ngw 30 8 ± ± -1.472 -1.055 1.393 0.107 0.00073 0.193 0.00051 Shan Ngwar Pu Sha 30 4 ± ± -2.174** -3.601* -3.699* 0.095 0.00034 0.832 0.00334 Crossbred Cro 20 10 ± ± -0.848 -0.305 -0.538 0.075 0.00070 0.528 0.00157 Total 140 27 ± ± -2.012** -1.700 -2.189 0.053 0.00026 1K: number of haplotypes; 2H: mean ± standard deviation of haplotype diversity; 3 兀 : mean ± standard deviation of nucleotide diversity; **p < 0.01 and *p < 0.05 whole and was observed in the populations, except Shw and a lower genetic diversity, when compared with those of other Pya. No sequence of the I2a haplotype was observed in this Bos indicus populations of China (Lai et al. 2006), India study, which agreed with the previous study (Chen et al. (Chen et al. 2010) and the Red Chittagong cattle of 2010). Bangladesh (Bhuiyan et al. 2007).

Genetic diversity of Myanmar local breeds and crossbred Genetic differentiation and relationship among the local population breeds and crossbred population Table 3 shows the genetic diversity estimates of five To reveal a genetic differentiation and relationship cattle populations obtained in this study. The haplotype and among the breeds and population, we calculated pairwise nucleotide diversities of the five populations ranged from FST distances using the D-loop sequences. The pairwise FST 0.193 in Sha to 0.832 in Cro and from 0.00051 in Sha to values ranged from -0.0083 to 0.1320 (Table 4). The breeds 0.00334 in Cro. The Cro population had the highest genetic and population except Sha were closely related to each other diversity among all populations. This resulted from lower in terms of the pairwise FST values, but Sha was MYAH10 haplotype frequency (40%) and higher haplotype significantly different from others (Table 4). AMOVA also frequency of the I2 haplogroup (15%) and suggests the showed that a variation among the populations was 1.4% of composite population of Holstein and various Myanmar the total variation (Table 5) and was not significant (P = native breeds. The Shw and Pya breeds were much the same 0.197). These results suggest no artificial selection in as Ngw in haplotype diversity but lower nucleotide diversity Myanmar cattle. Because Myanmar cattle populations are than Ngw, reflecting the difference of haplogroup vital for agricultural cultivation, commercial trade and distribution between them. Sha showed the lowest genetic extensive transport along the human migratory paths, it diversity because of the highest MYAH10 frequency (90%) might cause low or high genetic differentiation in the and a few haplotype numbers. The possible reason is that Myanmar cattle breeds. Sha has been reared only in the Shan state and farmers of Sha were fewer than the other states. In total, Myanmar Population expansion of Myanmar cattle breeds cattle showed 0.528 ± 0.053 in haplotype diversity, 0.00157 In Fig. 4, the star-like pattern of the MJ network map ± 0.00026 in nucleotide diversity and 1.423 in the mean suggests that a recent population expansion event may have number of nucleotide differences. These estimates indicated occurred for the Myanmar cattle (Ferreri et al. 2011). Fig.5

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Table 4. Pairwise FST values (below diagonal) and FST p values (above diagonal; *p < 0.05, **p < 0.01) between the local breeds and crossbred population. Population Shw Pya Sha Ngw Cro Shw 0.612 0.000** 0.721 0.297 Pya -0.0285 0.027* 0.550 0.396 Sha 0.0982 0.1320 0.009** 0.027* Ngw -0.0268 -0.0228 0.0932 0.7748 Cro -0.0211 -0.0083 0.0859 -0.0327

Table 5. Results of AMOVA in Myanmar cattle.

Source of variation d.f. Sum of squares Variance components Percentages of variation FST P value Among populations 4 47.1 0.12 1.4 0.014 0.197 Within populations 135 1130.3 8.37 98.6 Total 139 1177.4 8.49

FST = fixation index

(a)I1 haplogroup presents the mismatch distribution (pairwise number of differences) for the two maternal haplogroups using the complete dataset, to infer recent demographic events, such as population growth (Slatkin & Hudson 1991; Rogers & Harpending 1992). The mismatch distributions for the haplogroups I1 and I2 were bimodal distributions: One major peak with maximum frequency at mismatch number of 2 in haplogroup I1 and two major peaks in the maximum frequency at mismatch numbers of 1 and 2 in haplogroup I2 (Fig.5). These results suggest that recent population expansion events may occur in both I1 and I2 haplogroups. Neutrality tests (Tajima’s D test, Fu and Li’s D test, Fu and Li’s F test) were performed to detect population growth (Tajima 1989; Fu & Li 1993). The complete dataset of the (b)I2 haplogroup 140 Myanmar cattle had a significantly negative Tajima’s D value in Table 3, suggesting a population expansion event in Myanmar cattle. Among the local breeds and crossbred population, Pya and Sha had significantly negative values in the neutrality test and may have had a population expansion event. The remaining three were not significant (Table 3). Pya may spread more rapidly than the other populations because it’s the most popular local breed in Myanmar (National Consultative Committee, Myanmar 2002). Concerning Sha, which is reared only in the Shan state, the reason for the population expansion was unclear.

CONCLUSION

Fig.5 Mismatch distribution (pairwise number of Myanmar consists of abundant cattle in the world. differences) for the two maternal haplogroups (a: I1 Here, the mtDNA D-loop sequence analysis provided and b: I2). Mismatch distribution shows the expected and observed pairwise differences between the evidence for genetic diversity and differentiation of the sequences with the respective frequency. popular local breeds and the crossbred cattle population in

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