complete mitogenome sequencing of andaman buffalo: an

Journal of Genetics (2019) 98:97 © Indian Academy of Scienceshttps://doi.org/10.1007/s12041-019-1140-z

RESEARCH ARTICLE

Complete mitogenome sequencing of Andaman buffalo: an endangeredgermplasm of Andaman and Nicobar Islands, India

ARUN KUMAR DE1,2, PERUMAL PONRAJ1, DHRUBA MALAKAR3, RAMACHANDRAN MUTHIYAN4,A. KUNDU1 and DEBASIS BHATTACHARYA1∗

1Animal Science Division, ICAR-Central Island Agricultural Research Institute, Port Blair 744 101, India2Bioinformatics Centre, ICAR-Central Island Agricultural Research Institute, Port Blair 744 101, India3Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132 001, India4GCC Biotech India Pvt. Ltd., Kirtankhola, Joychandipur 743 377, India*For correspondence. E-mail: [email protected].

Received 23 May 2019; revised 15 July 2019; accepted 22 July 2019; published online 24 October 2019

Abstract. Andaman buffalo is an indigenous buffalo of Andaman and Nicobar Islands, India. Over the last decade, it has witnesseda rapid decline in population, necessitating its immediate characterization and conservation. The present study reports the completemitogenome profile of Andaman buffalo which is 16,359 bp in length and comprised of 37 genes, including 13 protein-coding genes(PCGs), 22 transfer RNAs and two ribosomal RNAs. In addition, one A + T rich region (D-loop) was also present. A biasnesstowards A and T base was observed in all the genes. All the PCGs except ND6 were present on heavy strand. Start codons for allthe 13 PCGs were ATN codon and abbreviated/truncated stop codons were observed in ND1, ND2, COX3, ND3 and ND4. Thephylogenetic analysis revealed that the Andaman buffalo is closely related to buffalo from India and China. The results from thisstudy will help in sketching the conservation plan of the threatened breed.

Keywords. Andaman buffalo; mitogenome; illumina sequencing; Andaman and Nicobar Islands; Bubalus bubalus.

Introduction

Buffalo has immense economic importance as dairy, meatand draught animals in many countries worldwide (Kier-stein et al. 2004; Yindee et al. 2010). The total globalpopulation of water buffalo is around 168 million ofwhich majority (161 million) is located in Asia (FAO2000). It contributes more than 5% share of global milkproduction and of superior quality than the cow’s milkin terms of fat, protein, lactose and mineral content(Barłowska et al. 2011). Moreover, the buffalo meat con-tains less fat and cholesterol as compared to beef (Yindeeet al. 2010). The population of buffalo in India is 108.7million (19th Livestock Census 2012, http://dahd.nic.in/documents/statistics/livestock-census) and they con-tributes more than half (55%) of the total milk produced.Currently, there are 16 registered buffalo breeds in India(www.nbagr.res.in) and India is considered the home tractof some of the best buffalo breeds of the world. Besidesthe registered breeds, there are several native buffalo breeds

with huge genetic potential scattered throughout the coun-try, but due to negligence some of them are vulnerable toglobal extinction. It is very important to characterize thenative breeds and make proper conservation strategy toconserve the genetic pool.

Andaman and Nicobar Islands, an archipelago of 572islands, is situated in Bay of Bengal, and is about 1200 kmaway from mainland India. The islands are characterizedby hot and humid climate with temperature ranging from28◦C to 35◦C and with average annual rainfall of 3000 mm.Buffaloes are very well suited to sustain in this local con-dition making them a sustainable source of livelihood forthe farmers of Andaman and Nicobar Islands. Andamanbuffalo is an indigenous livestock of Andaman and thriveswell in minimal management and low input (Kundu et al.2010). These buffaloes are water buffalo and its populationis scattered in different islands. In recent years, introduc-tion of exotic breeds has made this breed very fragile andthe population has declined from 16 thousand in 2003 to7.86 thousand in 1012 (19th Livestock Census 2012, http://

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dahd.nic.in/documents/statistics/livestock-census) There-fore, characterization and conservation of this breed is theneed of the hour.

The genetic root and phylogenetic history of Andamanbuffalo are still unknown. Historical indenture suggeststhat Andaman and Nicobar Islands were the colonies ofDenmark, Austria, Britain and Japan before they becamea part of Indian Union in 1950 and declared UnionTerritory in 1956. During colonization period, the colo-nial people introduced domestic animals for milk andmeat purpose. Moreover, domestic animals have beenbrought to Andaman and Nicobar Islands from differ-ent parts of India in different phases of inhabitation andrehabilitation of migrated/settled people (Sunder et al.2018).

The mitochondrial DNA has emerged as a vital tool tostudy breed characterization, population genetics, phylo-geography and tracing genetic root of animals (Ludwiget al. 2013; Rissler 2016). For the first time, in thepresent work, the whole mitochondrial DNA of Andamanbuffalo was sequenced and characterized using next-generation sequencing-based methodology. The geneticroot and phylogenetic relationship of Andaman buffalowith buffalo breeds of different countries was also investi-gated.

Materials and methods

Sample collection and DNA extraction

Blood sample, 10 mL was collected from an adultAndaman buffalo maintained in the Institute farm ofICAR-Central Island Agricultural Research Institute,Port Blair, into a heparinized vacutainer following asepticmeasures. Total genomic DNA was extracted from bloodof Andaman buffalo using a commercial kit (DNeasyBlood and Tissue kit, Quagen, Caldwel) according to man-ufacturer’s instructions.

Illumina sequencing

The quality of the isolated DNA was checked by using aNanodrop Spectrophotometer and quantity of DNA wasassessed on a Qubit fluorometer with a commercial kit(dsDNA high-sensitivity kit, Invitrogen). Enrichment ofmitochondrial DNA was done by following the protocolof NEBNext Microbiome Enrichment kit (New EnglandBiolab). Further, library preparation was done by usingNextera XT DNA Library Preparation kit, Illumina. Thelibrary was sequenced on Illimina Nextseq platform withpaired-end sequencing (150 × 2 chemistry). A total of1,194,473 raw paired reads with average length of 150 baseswas generated.

Reads quality check and genome assemble

All the illumina pair end (PE) short reads were assignedfor base quality check using FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). ThePhred Quality scores were fixed as cut-off Q > 30 toremove bad quality base call sequences in the total readlength. Low quality bases, barcode and adapter sequenceswere trimmed using Timgalore v0.4.4 program (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Further, overlapping reads were identified and weresubsequently removed from the total reads by Picard2.18 algorithm (https://github.com/broadinstitute/picard).A total of 9664 high-quality reads were used to assemblethe mitochondrial genome of Andaman buffalo with aver-age coverage of 90.7572 with 20x read depth using Bwav0.7.17 (Li and Durbin 2009) with that of Bubalus bubalus(GenBank: KX758302) (Wang et al. 2017) as an initial ref-erence sequence.

Mitogenome annotation

Protein coding genes (PCGs) were identified by BLASTsearches of open reading frame (ORF). Transfer RNA(tRNA) genes were discriminated by tRNAscan-SE (Loweand Chan 2016). Further, noncoding regions and ribo-somal RNA (rRNA) genes were identified by BLASTsearches. The structure of the tRNAs was determined byMitos Web Server (Bernt et al. 2013).

Amino acid composition and relative synonymous codon usage(RSCU)

Amino acid composition of concatenated sequence infor-mation and RSCU were calculated by MEGAX (Kumaret al. 2018).

AT-/GC- skew

Skewness was determined manually as described ear-lier (Perna and Kocher 1995). AT-skew = (A-T)/(A +T) and GC-skew = (G-T)/(G + T).

Sequence alignment and phylogenetic analysis

Representative mitogenome sequences of Asiatic buffaloesof different countries (India, Burma, Vietnam, China,Thailand, Laos and Italy) and African Buffalo wereretrieved from GenBank. Phylogenetic analysis was con-ducted based on (i) concatenated amino acid sequences,and (ii) concatenated sequences of PCGs. Alignmentof sequence information was done using CLUSTAL W(Thompson et al. 1994) as implemented in MEGA X(Kumar et al. 2018). Further, molecular phylogenetic tree

Mitogenome analysis of Andaman buffalo of 8 97

Figure 1. Graphical representation of the complete mitochondrial genome organization of Andaman buffalo. tRNAs are labelledwith their corresponding amino acids. The physical map was generated by using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/)(Lohse et al. 2013).

was drawn by maximum likelihood (ML) method provid-ing 1000 bootstrap replications using MEGA X (Kumaret al. 2018). Best-fit nucleotide substitution model wasselected based on Bayesian information criterion (BIC)score. mtREV24 + G and HKY + G substitution modelswere selected as best fit models for phylogenetic analysisbased on concatenated amino acid sequences and concate-nated PCGs, respectively.

Results

Genome structure, organization and composition

The complete mitochondrial DNA was submitted to Gen-Bank with accession number MK234704. The mitogenomeis a circular DNA of 16359 bp in length (figure 1;table 1) and is comparable to mitogenomes of otherspecies under vertebrate. The mitogenome encoded 37genes including 13 PCGs, 22 tRNAs and two rRNAs.In addition, one A + T rich region (D-loop) was present(table 1). All the PCGs except ND6 were present on heavystrand. Among the tRNAs, 14 (tRNA-Phe, tRNA-Val,tRNA-Leu1, tRNA-Ile, tRNA-Met, tRNA-Trp, tRNA-Asp, tRNA-Lys, tRNA-Gly, tRNA-Arg, tRNA-His,tRNA-Ser2, tRNA-Leu2 and tRNA-Thr) were present onH strand whereas the rest of them (tRNA-Gln, tRNA-Ala,

tRNA-Asp, tRNA-Cys, tRNA-Tyr, tRNA-Ser1, tRNA-Glu and tRNA-Pro) were present on L strand. H strandencoded both the rRNAs. The nucleotide composition ofthe genes indicated a biasedness towards A+T nucleotides,ranging from 52.174% (tRNA-Met) to 78.261% (tRNA-Arg).

PCGs

The concatenated PCGs of Andaman buffalo was esti-mated to be 11,339 bp in length and formed 69.31% ofthe total mitogenome. The PCGs were AT rich with overallA+ T content as 58.96% ranging from 54.974% (COX3) to63.185% (ATP8) (table 1). ATN (ATG or ATA) start codonwas observed in all the 13 PCGs. Further, analysis of themitogenome revealed that eight of the 13 PCGs (COX1,COX2, ATP8, ATP6, ND4L, ND5, ND6 and CYTB)used a typical TAA termination codon whereas abbrevi-ated/truncated stop codon was evidenced for ND1, ND2,COX3, ND3 and ND4 (table 1). The AT-skew and GC-skew of all the 13 PCGs of Andaman buffalo mitogenomeis depicted in figure 2. Positive AT skewness was observedin most of the proteins except COX1, COX3, ND4L andND6 suggestive of adenine biased nucleotide composi-tion as compared to thymine. On the other hand, negativeGC skewness was observed in all the proteins indicating


Table 1. The organization and characterization of complete mitogenome of Andaman buffalo.

Gene Stand Location Size T/U% C% A% G% AT%Startcodon

Stopcodon Anticodon

Intergenicnucleotides

tRNA-Phe H 377–445 69 23.188 23.188 37.681 15.942 60.869 – – GAA 012S rRNA H 446–1402 957 22.153 23.406 36.468 17.973 58.621 – – – 0tRNA-Val H 1403–1469 67 26.866 20.896 40.299 11.940 67.165 – – TAC 016S rRNA H 1470–3038 1569 23.709 21.033 38.113 17.145 61.822 – – – 0tRNA-Leu1 H 3039–3113 75 28.000 22.667 33.333 16.000 61.333 – – TAA 2ND1 H 3116–4071 956 26.569 29.079 31.904 12.448 58.473 ATG TA-(*) – 0tRNA-Ile H 4072–4140 69 33.333 11.594 39.130 15.942 72.463 – – GAT −3tRNA-Gln1 L 4138–4209 72 36.111 11.111 23.611 29.167 59.722 – – TTG 2tRNA-Met H 4212–4280 69 26.087 27.536 26.087 20.290 52.174 – – CAT 0ND2 H 4281–5322 1042 25.624 28.695 36.180 9.501 61.804 ATA T-(*) – 0tRNA-Trp H 5323–5389 67 26.866 19.403 35.821 17.910 62.687 – – TCA 1tRNA-Ala L 5391–5459 69 39.130 10.145 28.986 21.739 68.116 – – TGC 1tRNA-Asn L 5461–5533 73 28.767 16.438 24.658 30.137 53.425 – – GTT 0Rep_origin L 5534–5564 32.258 29.032 9.677 29.032 41.935 – – – 1tRNA-Cys L 5566–5632 67 31.343 17.910 23.881 26.866 55.224 – – GCA 0tRNA-Tyr L 5633–5699 67 29.851 14.925 32.836 22.388 62.687 – – GTA 1COX1 H 5701–7245 1545 29.256 25.502 28.026 17.217 57.282 ATG TAA – −3tRNA-Ser1 L 7243–7313 71 33.803 15.493 25.352 25.352 59.155 – – TGA 4tRNA-Asp H 7318–7387 70 31.429 14.286 38.571 15.714 70 – – GTC 1COX2 H 7389–8072 684 26.170 25.146 33.333 15.351 59.503 ATG TAA – 3tRNA-Lys H 8076–8143 68 25.000 23.529 27.941 23.529 52.941 – – TTT 1ATP8 H 8145–8345 201 22.886 29.353 40.299 7.463 63.185 ATG TAA – −40ATP6 H 8306–8986 681 27.460 28.781 31.718 12.041 59.178 ATG TAA – −1COX3 H 8986–9769 784 28.571 29.337 26.403 15.689 54.974 ATG T- – 0tRNA-Gly H 9770–9838 69 28.986 21.739 33.333 15.942 62.319 – – TCC 0ND3 H 9839–10185 347 27.666 29.107 30.548 12.680 58.214 ATA TA-(*) – 0tRNA-Arg H 10186–10254 69 36.232 11.594 42.029 10.145 78.261 – – TCG 0ND4L H 10255–10551 297 31.650 25.253 29.630 13.468 61.28 ATG TAA – −7ND4 H 10545–11922 1378 28.592 28.229 31.713 11.466 60.305 ATG T-(*) – 0tRNA-His H 11923–11993 71 30.986 19.718 36.620 12.676 67.606 – – GTG 0tRNA-Ser2 H 11994–12053 60 25.000 26.667 31.667 16.667 56.667 – – GCT 1tRNA-Leu2 H 12055–12124 70 28.571 14.286 37.143 20.000 65.714 – – TAG 0ND5 H 12125–13945 1821 26.908 28.940 32.729 11.422 59.637 ATA TAA – 3ND6 L 13929–14456 528 41.288 7.576 21.591 29.545 62.879 ATG TAA – 0tRNA-Glu L 14457–14525 69 37.681 13.043 24.638 24.638 62.319 – – TTC 4CYTB H 14530–15669 1140 24.912 30.175 31.404 13.509 56.316 ATG AGA – 4tRNA-Thr H 15674–15742 69 24.638 23.188 37.681 14.493 62.319 – – TGT −1tRNA-Pro L 15742–15807 66 31.818 16.667 21.212 30.303 53.03 – – TGG

*Abbreviated stop codon; A + T rich region (D-loop) from 1–376 and 15792–16359.

that guanine occurred more frequently than cytosine. TheRSCU of the mitochondrial PCGs of Andaman buffalo ispresented in figure 3. A bias towards A/T ended codons wasobserved. The amino acid composition of Andaman buf-falo mitogenome indicated that the most frequent aminoacids were Leu (15.64%), Ile (8.65%), Thr (8.10%), Ser(7.9%) and Met (7.09%), whereas Cys was least frequent(0.63%).

rRNA and tRNA genes

The lengths of 12S and 16S rRNAs were 957 bp and1569 bp, respectively. The 12S rRNA was positionedbetween tRNA-Phe and tRNA-Val whereas 16S rRNA

Figure 2. Graphical representation of AT-skew and GC-skew inall the 13 PCGs of Andaman buffalo (MK234704).


Figure 3. The RSCU of the mitochondrial PCGs of Andaman buffalo (MK234704).

was between tRNA-Val and tRNA-Leu1. The base com-position of the two rRNA genes is presented in table 1 andboth were A+ T rich. Total content of A+ T of the smallerand larger subunit was 58.62% and 61.82%, respectively.

The length of the 22 tRNAs varied from 60 (tRNA-Ser) to 75bp (tRNA-Leu). The anticodons of the tRNAs(table 1) were similar to those of species under vertebrate.The secondary structure of the tRNAs (figure 4) was pre-dicted by Mitos Web Server and was found that all of themexcept tRNA-S2 formed a typical clover-leaf like structure.

Overlapping and intergenic spacer regions

In the complete mitogenome of Andaman buffalo, a totalof six regions of gene overlaps ranging from 1 bp to 40bp was observed. The largest overlap was located betweenATP8 and ATP6 (40 bp). The other five overlaps werebetween tRNA-Ile and tRNA-Gln1, COX1 and tRNA-Ser1, ATP6 and COX3, ND4L and ND4, tRNA-Thr andtRNA-Pro. A total of 13 intergenic spacer regions rangingfrom 1 bp to 4 bp were observed (table 1).

The A + T rich region

The noncoding A + T rich region or D-loop (1–376 and15808–16359 nt) was 928 bp in length and was positionedbetween tRNA-Pro and tRNA-Phe. The total A + Tcontent, AT skew and GC skew in this region was 60%,0.0467% and –0.478%, respectively.

Phylogenetic relationship

Phylogenetic analysis was performed to know the phylo-genetic relationship of Andaman buffalo with buffaloes ofdifferent countries. From the phylogenetic tree (figure 5),it was found that Asiatic buffaloes were grouped togetherand Andaman buffalo was phylogenetically closely relatedto buffaloes of India (AF547270) and China (KX758295).

Discussion

Water buffalo (B. bubalis) is adaptable to the hot humidclimate and are present in several Asian countries, servesas a good source of milk, meat and drought power used foragricultural purposes. Buffalo belong to the Bovidae fam-ily and two main species of buffalo, i.e. the Asiatic buffalo(B. bubalus) and the African buffalo (Syncerus caffer) havebeen reported (Lannuzzi and Di Meo 2009). Two majorsubspecies of domestic water buffalo, namely river buf-falo and draft swamp buffalo are available in Asia (Kumaret al. 2007) and are believed to be descended from the wildAsian buffalo (B. arnee) (Lei et al. 2007). The river buf-falo is mainly available in the Indian subcontinent, SouthAsia and the Mediterranean area whereas northeast India,China and Southeast Asia are the home tracts of swampbuffalo (FAO 2014). India is well known for its rich diver-sity of water buffalo and several elite germplasm of waterbuffalo are available here. Negligence has brought someof the native buffalo breeds at the edge of extinction andimmediate conservation efforts are required to maintain


Figure 4. Secondary structures of the 22 tRNA genes of the Andaman buffalo mitogenome. tRNAs are designated with theircorresponding amino acids. Standard one letter abbreviation for the respective amino acids has been used.

the genetic pool. Andaman buffalo is a native water buf-falo germplasm of Andaman and Nicobar Islands and areadaptable to the hot humid climate of the islands. The pop-ulation of this buffalo germplasm is declining rapidly dueto the unavailability of proper breeding strategy, makingthe species very vulnerable to extinction. It is important toevaluate their genetic structure to develop suitable breed-ing strategy for their conservation. Moreover, the geneticroot of Andaman buffalo needs to be understood. In thepresent study, the mitogenome of Andaman buffalo, anendangered water buffalo germplasm of Andaman andNicobar Islands has been characterized and the geneticroot of the breed has been unveiled.

Mitogenome analysis has emerged as an important toolin many research areas such as studying population genet-ics, evolutionary relationships, phylogenetic relationshipsand phylogeography (Behura et al. 2011). Additionally,whole mtDNA genome sequencing has improved the res-olution of phylogenetic analysis compared to single geneor single region studies (Simon et al. 2006; Santamariaet al. 2007). The complete mitogenome of Andaman

buffalo is 16,359 bp in length (figure 1) which is compa-rable to the mitogenome lengths of other buffalo breedsreported (Parma et al. 2004; Wang et al. 2017). The com-plete mitogenome of Andaman buffalo encodes a total of37 genes of which 13 were PCGs, 22 tRNA, two rRNAgenes and an A + T rich region (table 1), which is typicallyobserved in vertebrates (Sarvani et al. 2018). The order andorientation of the genes were similar to the mitogenomes ofother vertebrates (Sarvani et al. 2018). AT-skew, GC-skew,and A + T content are parameters that are frequentlyused to investigate the pattern of nucleotide compositionof mitochondrial genomes (Hassanin et al. 2005; Wei et al.2010). The genes in the buffalo mitogenome were A + Trich. The bias towards A and T is a common feature in themitogenome of other vertebrates.

The data generated in this study is also valuable in under-standing the genetic root of the Andaman buffalo breed.From the phylogenetic study, it is clear that this breed isphylogenetically close to north Indian buffalo (AF547270)indicating that it might be introduced in Andaman fromnorth India during rehabilitation programme. Historical


Figure 5. Molecular phylogenetic analysis of Andaman buffalo by maximum likelihood method based on (a) concatenated aminoacid sequence, (b) concatenated sequences of PCGs. Representative mitogenome sequences of buffaloes from different countries wereretrieved from GenBank. The name of the country buffalo breed belongs to is mentioned in parenthesis.

documents suggest that in the middle of the 19th century,people from eastern and northern parts of India came toAndaman and Nicobar Islands as per the colonization pol-icy of Government of India. They brought some livestockwith them and they most probably introduced this nativebuffalo to Andaman and Nicobar Islands.

The current study presents the mitogenome analysis ofAndaman buffalo; the results of this study will be helpfulto understand the genetic structure of the breed and forsketching the conservation plan of the local breed.

Acknowledgements

This work was supported by a grant from Department of Biotech-nology, Ministry of Science and Technology, Government ofIndia (grant no. BT/BI/04/066/2004). The Indian Council ofAgricultural Research (ICAR), India, also supported the study.

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Corresponding editor: H. A. Ranganath

complete mitogenome sequencing of andaman buffalo: an

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