A Report on the Multivariable Sites of the Mitochondrial Genome of a Bamboo Rat, Rhizomys pruinosus from Wenzhou, China

Yijiang Liu1, Kun Li2, Hui Zhang2, Khalid Mehmood2,3, Muhammad Shahzad3, Houqiang Luo1,*, Muhammad Asif Yaseen4 and Xiong Jiang2,5,*

1College of Animal Science, Wenzhou Vocational College of Science and Technology, Wenzhou 325006, People’s Republic of China

2College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China

3University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan

4Barani Livestock Production Research Institute, Kherimurat, Attock, Pakistan

5College of Agriculture, Hubei Three Gorges Polytechnic, Yichang 443000, People’s Republic of China

Yijiang Liu and Kun Li contributed equally to this study.

ABSTRACT

The sequencing of the complete mitochondrial (mt) genome of the pet bamboo rat Rhizomys pruinosus, which had a problem in standing and walking in a Bamboo farm in Wenzhou, China was carried out, using its tibia. The mt genome (16,579 bp) is composed of 13 protein coding genes, 22 tRNA genes and 2 rRNA genes. The ratio of the bases of the mt genome of R. pruinosus from Wenzhou are A (32.31%), T (31.04%), C (24.77%), G (11.88%), A+T (63.35%), respectively. The multivariable sites in rRNA were 1.98% and 5.53% in 12S rRNA and 16S rRNA, respectively. The multivariable sites in protein coding gene were ranged from 0 to 4.83%, while the multivariable sites in amino acids were ranged from 0 to 5.22%. The current results contributes to the perform prevention of leg problems in the bamboo rat industry.

Article Information

Received 30 November 2017

Revised 01 March 2018

Accepted 06 March 2018

Available online 11 May 2018

Authors’ Contributions

HL, KL and XJ planned the study. YL and KL designed the study. KL, YL, HZ, KM, MS and XJ performed the trial, KL and MAY analyzed the data. KL wrote the manuscript.

Key words

Rhizomys pruinosus, Mitochondrial genome, Sequence, Multivariable sites, Phylogenetic analysis.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.sc4

* Corresponding author: [email protected]; [email protected]

0030-9923/2018/0004-1545 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Bamboo rats (Rhizomys sinensis, Rhizomys pruinosus, Rhizomys sumatrensis and Cannomys badius) are extremely popular in China for their medicinal value, delicious and nutritious meat, thick and soft fur. Because of successful breeding in captivity, bamboo rats industry was born in the 1990s (Liu et al., 2011). It was estimated that there were over 30 million farmed bamboo rats in 2011 (Liu et al., 2011; Tang et al., 2017).

The mitochondrial (mt) DNA have been widely utilized as molecular markers in the study of taxonomy, population genetics, phylogenetic and evolutionary analyses due to its maternal inheritance (Li et al., 2008, 2016a, b, 2017a, b; Yan et al., 2018). The mt DNA was reported to have a higher mutation rate than that of nuclear DNA (Zhao et al., 2014), and it was reported to be highly related to various diseases due to the alteration of mt DNA content (Zong et al., 2016; Chen et al., 2016). However, scare information is available about the mt characteristics of Rhizomys pruinosus (R. pruinosus) from Wenzhou, China, especially ailing bamboo rat. Therefore we carried out this research to sequnce the mt of Rhizomys pruinosus with a leg problem in this area to reveal whether it has relationship with the mitochondrial genome.

Materials and methods

Samples were collected after permission from the relevant institutions. All procedures were performed under the instructions and approval of Laboratory Animals Research Centre of Zhejiang province in P.R. China.

A 3 months old male, pet R. pruinosus with heavy leg problem (can not stand up) was obtained from the hospital of College of Animal Science, Wenzhou Vocational College of Science and Technology. This rat was fed on fresh bamboos. Under euthanasia tibia of this pet was removed and stored at -70 °C.

For sequencing mt DNA was isolated from an improved extraction method previously reported by Sorensen et al. (2006). After purification of mt DNA, short-insert libraries (insert size 350 bp) were constructed according to the manufacturer’s instructions (Illumina). The short library sequences were sequenced by using a commercial Illumina Hiseq 4000 sequencing system at total genomics solution (TGS) Institute in Shenzhen, China.

For variation analysis the current mt genome was multiple aligned with previously reported 12S rRNA of mt sequences by MEGA (6.0). The reference sequences were Rhizomys pruinosus (AJ250358.1), Rhizomys pruinosus (KC789518.1) and Rhizomys sinensis (NC_026124.1). The phylogenetic analysis was performed to determine Rhizomy species using MEGA version 6 by pilotting methods of the neighbor-joining algorithm, and the distances were computed using the Tajima-Nei method. The stability of branches was assessed after bootstrapping with 1000 replicates (Li et al., 2016b). The reference mt genes are as follows: R. pruinosus mt (KC789518.1), Rhizomys sinensis mt (KM434232.1), Eospalax rothschildi mt (JN544420.1), Eospalax baileyi mt (JN540033.1), Myospalax psilurus mt (JX014234.1), Eospalax cansus mt (KC514112.1), Rattus exulans mt (KY814718.1), and Myodes rufocanus mt (KT725595.1).

Results and discussion

The sequence of the present mt genome of R. pruinosus was submitted to NCBI database with the Genebank accession number of MG193909. The mt genome (16,579 bp) is composed of 13 protein coding genes, 22 tRNA genes and 2 rRNA genes, which is in line with previous of KC789518 (Zhao et al., 2014) (Table I, Fig. 1). The ratio of the bases of the mt genome of R. pruinosus from Wenzhou are A (32.31%), T (31.04%), C (24.77%), G (11.88%), A+T (63.35%), which present a little difference with previous reported in southern China (KC789518) (Fig. 2). Interestingly, a lot of multivariable sites of the mitochondrial genome of R. pruinosus from Wenzhou, China were found when compared with KC789518. The multivariable sites in rRNA were 1.98% and 5.53% in 12S rRNA and 16S rRNA, respectively (Table I). The multivariable sites in protein coding gene ranged from 0 to 4.83%, while the multivariable sites in amino acids ranged from 0 to 5.22% (Table I).

Table I.- Comparison of the mt genome of R. pruinosus from Wenzhou (WZ) with previously reported from South China (SC) KC789518.

Though, a low multivariable sites in protein coding gene were found, the NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and phylogenetic analysis revealed that the mt genome of R. pruinosus from Wenzhou is 99% in homology with KC789518 by pilotting Neighbor Joining method with available mt sequences of R. pruinosus mt (KC789518.1), Rhizomys sinensis mt (KM434232.1), Eospalax rothschildi mt (JN544420.1), Eospalax baileyi mt (JN540033.1), Myospalax psilurus mt (JX014234.1), Eospalax cansus mt (KC514112.1), Rattus exulans mt (KY814718.1 ), and Myodes rufocanus mt (KT725595.1) (Fig. 3).

We also performed the phylogenetic analysis of 12S rRNA by employing Neighbor Joining method with available sequences of R. pruinosus (AJ250358.1), R. pruinosus (KC789518.1) and R. sinensis (NC_026124.1), which also demonstrated the high homology 99.99% and 99.987% of R. pruinosus from Wenzhou with KC789518.1 and AJ250358.1, respectively (Fig. 4).

Conclusion

Though the R. pruinosus from Wenzhou shows a homology with previous reported one, low multivariable sites in protein coding gene were found with the leg problem rat, which may contribution to the perform prevention of leg problems in the bamboo rat industry.

Acknowledgements

This study was supported by the General Project of Education of the Zhejiang province in 2017 (Y201737824), and the Startup Project for Doctoral Scientific Research of Wenzhou Vocational College of Science and Technology in 2016 (No. 201604).

Statement of conflict of interest

The authors state that there are no competing interests.

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