Abstract
Background
Mithun (Bos frontalis), also called gayal, is an endangered bovine species, under the tribe bovini with 2n = 58 XX chromosome complements and reared under the tropical rain forests region of India, China, Myanmar, Bhutan and Bangladesh. However, the origin of this species is still disputed and information on its genomic architecture is scanty so far. We trust that availability of its whole genome sequence data and assembly will greatly solve this problem and help to generate many information including phylogenetic status of mithun. Recently, the first genome assembly of gayal, mithun of Chinese origin, was published. However, an improved reference genome assembly would still benefit in understanding genetic variation in mithun populations reared under diverse geographical locations and for building a superior consensus assembly. We, therefore, performed deep sequencing of the genome of an adult female mithun from India, assembled and annotated its genome and performed extensive bioinformatic analyses to produce a superior de novo genome assembly of mithun.
Results
We generated ≈300 Gigabyte (Gb) raw reads from whole-genome deep sequencing platforms and assembled the sequence data using a hybrid assembly strategy to create a high quality de novo assembly of mithun with 96% recovered as per BUSCO analysis. The final genome assembly has a total length of 3.0 Gb, contains 5,015 scaffolds with an N50 value of 1 Mb. Repeat sequences constitute around 43.66% of the assembly. The genomic alignments between mithun to cattle showed that their genomes, as expected, are highly conserved. Gene annotation identified 28,044 protein-coding genes presented in mithun genome. The gene orthologous groups of mithun showed a high degree of similarity in comparison with other species, while fewer mithun specific coding sequences were found compared to those in cattle.
Conclusion
Here we presented the first de novo draft genome assembly of Indian mithun having better coverage, less fragmented, better annotated, and constitutes a reasonably complete assembly compared to the previously published gayal genome. This comprehensive assembly unravelled the genomic architecture of mithun to a great extent and will provide a reference genome assembly to research community to elucidate the evolutionary history of mithun across its distinct geographical locations.
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Background
Mithun (Bos frontalis) is a rare bovine species living under free-range conditions inside tropical rainforest ecosystems of India, Bangladesh, Bhutan, China, and Myanmar [1]. It is a unique animal having a massive body, with characteristic ‘white stockings’ on their stout legs. This animal efficiently converts grass, forage, tree leaves as well as various agricultural by-products into highly nutritious meat. Moreover, mithun holds a unique place in the evolution of bovines. Mithun, having a specific chromosomal pattern, 2n = 58 is distinguishable from that of cattle (2n = 60) and yak (2n = 60) [2]. However, the origin of mithun is an on-going debate with no well-supported conclusion [3,4,59] flagstat were used to investigate the correctness of assembly. BUSCO v3 [20, 60] with the mammalian database was used to assess the completeness of genes presented by assembly. Nineteen-mers was counted from PE data with Jellyfish [61]. Genome size was estimated by dividing the total number of k-mers by the peak value of the k-mer frequency distribution [77] pathway information of [77] pathway information of the mithun gene set.
Genome alignment
Both mithun and gayal genomes were soft-masked and aligned to the soft-masked cattle genome (ARS-UCD1.2) [21] by Large Scale Genome Alignment Tools (LASTZ) [78]. The pairwise genome alignment was chained according to their location in both genomes by axtChain program [79]. The netting process chooses for the reference species the best sub-chain in each region. The statistics of different size of synteny block was done by a custom script. We only used block size larger than 100 kb to investigate how many cattle chromosomes the mithun scaffold can span.
Availability of data and materials
All data generated and analyzed during this current study are available in the Data Cell of ICAR-NRC on Mithun, Nagaland, India with permission from the Competent Authority. Illumina whole genome sequence and RNA-seqdata of mithun were submitted in NCBI Database having BioProject ID PRJNA241403 and BioProject ID PRJNA307305, respectively.
Abbreviations
- BTA 7:
-
Bos taurus genome assembly v.7
- BUSCO:
-
Benchmarking Universal Single-Copy Orthologs
- CNVs:
-
Copy number variations
- DBG:
-
De Bruijn Graphs
- dNTPs:
-
Deoxyribonucleotide triphosphates
- eggNOG:
-
Evolutionary genealogy of genes: Non-supervised Orthologous Groups
- EVM:
-
EVidence Modeler
- Gb:
-
Gigabyte
- KAAS:
-
KEGG Automatic Annotation Server
- KEGG:
-
Kyoto Encyclopedia of Genes and Genomes
- LASTZ:
-
Large-Scale Genome Alignment Tool
- LINEs:
-
Long Interspersed Nuclear Elements
- LoRDEC:
-
Long Read DBG Error Correction
- LTR:
-
Long terminal repeat
- MaSuRCA:
-
Maryland Super-Read Celera Assembler
- MB:
-
Megabyte
- Mt.:
-
Mitochondria
- NGS:
-
Next-generation sequencing
- PacBio:
-
Pacific BioSciences
- PASApipeline:
-
Program to Assemble Spliced Alignments Pipeline
- PBJelly 2:
-
Software for Long-Read Sequencing Data from PacBio
- Pfam:
-
Protein families database
- PRINTS:
-
Protein fingerprints database
- QUAST:
-
Quality Assessment Tools
- RIN:
-
RNA Integrity Number
- SINEs:
-
Short Interspersed Nuclear Elements
- SMRT:
-
Single-molecule real-time (SMRT)
- SNPs:
-
Single-Nucleotide Polymorphisms
- SSPACE:
-
Scaffolding Pre-Assemblies AfterContig Extension
- SVs:
-
Structural variations
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Acknowledgements
The authors would like to thank the Director of the Institute and Indian Council of Agricultural Research for funding the Institutional Research Project for this study. Financial assistance in the form of DBT Overseas Associateship for the first author is gratefully acknowledged. IL and MM were supported by a DST and DBT funded grant, Govt. Of India, respectively. Assistance of DBT Biotech Hub in carrying out a part of wet lab work is thankfully acknowledged.
Funding
This work was carried out under an Institutional research project funded by ICAR-NRC on Mithun, Nagaland, India. Financial support in the form of DBT Overseas Associateship for the first author played a significant role to carry out some of the bioinformatic analyses and their interpretations at Aarhus University, Denmark.
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SM, AM and CR conceived the study. SM, ZC and AM run analysis and wrote the manuscript consulting other authors. IL, MM, KV, KK collected samples and run wet lab work. ABM, BG, MS, and GS critically read and revised the manuscript. All authors read and approved the final manuscript.
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All the animal experiments and sample collections procedures were carried out by qualified Veterinarians of the Institute. The ethics approval for animal experimentation was obtained from the Institute Ethics Approval Committee under the mandatory regulations of CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals) guidelines, prescribed by the Indian Council of Agricultural Research (ICAR), Ministry of Agriculture and Farmers Welfare, Government of India. Since no specimen was used, no permissions were necessary to collect the specimens in our present study.
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Additional files
Additional file 1:
NC3Rs ARRIVE Guidelines Checklist. (PDF 160 kb)
Additional file 2:
Table S1. Genes found in mithun genome with at least one domain hit. (XLSX 2390 kb)
Additional file 3:
Table S2. Genes found in mithun genome with GO analysis. (XLSX 5227 kb)
Additional file 4:
Table S3. Genes found in mithun genome with KEGG entries. (XLSX 510 kb)
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Mukherjee, S., Cai, Z., Mukherjee, A. et al. Whole genome sequence and de novo assembly revealed genomic architecture of Indian Mithun (Bos frontalis). BMC Genomics 20, 617 (2019). https://doi.org/10.1186/s12864-019-5980-y
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DOI: https://doi.org/10.1186/s12864-019-5980-y