The complete mitochondrial genome of Microtus fortis pelliceus (Arvicolinae, Rodentia) from China and its phylogenetic analysis

Abstract

The complete mitogenome sequence of Microtus fortis pelliceus was determined using long PCR. The genome was 16,310 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, 1 origin of L strand replication, and 1 control region. The overall base composition of the heavy strand is A (32.7%), C (27.7%), T (26.2%), and G (13.4%). The base compositions present clearly the A–T skew, which is most obviously in the control region and protein-coding genes. Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees. Results of phylogenetic analysis showed that Microtus had close relationship with Myodes, and had distant relationship with Eothenomys, Cricetulus, Dicrostonyx, Peromyscus, and other genera. This study verifies the evolutionary status of Microtus fortis in Microtus at the molecular level. The mitochondrial genome would be a significant supplement for the M. fortis genetic background. Results of phylogenetic analysis showed that M. f. pelliceus had close relationship with M. f. fortis in three subspecies of M.s fortis.

Keywords:

• Control region
• mitogenome
• phylogenetic trees
• Microtus fortis pelliceus

The Far Eastern vole (Microtus fortis corbet 1978) is a polytypical species widely distributed in Russia, Mongolia, Korea, and China (Corbet 1978). Microtus fortis is mainly distributed in more than 17 provinces and autonomous regions in China. Six subspecies forms were described within M. fortis, one of which, namely, M. f. pelliceus (Thomas, 1911) inhabits in the Heilongjiang Province, Jilin Province and Inner Mongolia Autonomous Region. Many studies found that M. fortis is resistant to Schistosome japonicum infection (He et al. 1999). The vole can become a laboratory animal model for the study on the mechanism of S. japonicum resistance.

In this paper, the complete mitochondrial genome of M. f. pelliceus was sequenced for the first time on ABI 3730XL using a primer walking strategy and the long and accurate PCR, with five pairs of long PCR primers and with 14 pairs of sub-PCR primers. A muscle sample was obtained from a female M. f. pelliceus captured from Mudanjiang region of Changbaishan Mountains in Heilongjiang Province, China (44°47′48″N, 129°04′52″E). The specimen is stored in Animal and Plant Herbarium of Mudanjiang Normal University. The voucher number is MD2018144.

The mitochondrial genome is a circular double-stranded DNA sequence that is 16,310 bp long including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, 1 origin of L strand replication, and 1 control region. The accurate annotated mitochondrial genome sequence was submitted to GenBank with an accession number MK805519. The arrangement of the multiple genes is in line with other Cricetidae species (Triant and DeWoody 2006; Fan et al. 2011; Hao et al. 2011; Bendová et al. 2016; Chen et al. 2016; Cong et al. 2016; Kang et al. 2016; Luo and Liao 2016; Park et al. 2017) and most mammals (Meganathan et al. 2012; Yoon et al. 2013; Xu et al. 2012, 2013; Jin et al. 2017; Liu, Tian, Jin, Dong, et al. 2017; Liu, Tian, Jin, Jin, et al. 2017; Liu, Wang, et al. 2017; Liu et al. 2018; Liu, Dang, et al. 2019; Liu, Qin, et al. 2019).

The control region of M. f. pelliceus mitochondrial genome was located between the tRNA-Pro and tRNA-Phe genes, and contains only promoters and regulatory sequences for replication and transcription, but no structural genes. Three domains were defined in M. f. pelliceus mitochondrial genome control region (Zhang et al. 2009): the extended termination-associated sequence (ETAS) domain, the central conserved domain (CD), and the conserved sequence block (CSB) domain.

The total length of the protein-coding gene sequences was 11,389 bp. Most protein-coding genes initiate with ATG except for ND1, ND2, ND3, and ND5, which began with ATA, ATT, or GTG. Nine protein-coding genes terminated with TAA. The incomplete stop codons (T– – or TA–) were used in ATP6, COX3, and ND4. A strong bias against A at the third codon position was observed in the protein-coding genes. The frequencies of CTA (Leu), ATT (Ile), TTA (Leu), and ATA (Met) were higher than those of other codons. The length of tRNA genes varied from 59 to 75 bp. Twenty-one of them could be folded into the typical cloverleaf secondary structure except the tRNA-Ser (AGY), whose complete dihydrouridine arm was lacking.

Most M. f. pelliceus mitochondrial genes were encoded on the H strand, except for the ND6 gene and eight tRNA genes, which were encoded on the L strand. Some reading frame intervals and overlaps were found. One of the most typical was between ATP8 and ATP6. The L-strand replication origin (OL) was located within the WANCY region containing five tRNA genes (tRNATrp, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr). This region was 32 bp long and had the potential to fold into a stable stem-loop secondary structure. The total base composition of M. f. pelliceus mitochondrial genome was A (32.7%), C (27.7%), T (26.2%), and G (13.4%). The base compositions clearly present the A–T skew, which was most obviously in the control region and proteincoding genes.

In order to explore the evolution of Cricetidae species which include 28 genera, especially the evolution of genus Microtus, here, we investigate the molecular phylogenetics of Chinese M. f. pelliceus using complete mitochondrial genome sequence of 54 species, and including three subspecies of M. fortis (M. f. pelliceus, M. f. Calamorum and M. f. fortis). All sequences generated in this study have been deposited in the GenBank (Figure 1).

Figure 1. Phylogenetic tree generated using the maximum parsimony method based on complete mitochondrial genomes. Akodon montensis (KF769456), Allocricetulus eversmanni (KP231506), Cricetulus kamensis (KJ680375), C. griseus (DQ390542), C. migratorius (KT918407), C. longicaudatus (KM067270), Cricetus cricetus (MF405145), Dicrostonyx torquatus (KX066190), D. groenlandicus (KX712239), D. hudsonius (KX683880), Eothenomys miletus (KX014874), E. chinensis (FJ483847), E. regulus (JN629046), E. inez (KU200225), Habromys ixtlani (KY707304), Isthmomys pirrensis (KY707312), Lasiopodomys mandarinus (KF819832), Meriones meridianus (KR013227), M. libycus (KR013226), M. tamariscinus (KT834971), Mesocricetus auratus (EU660218), Microtus rossiaemeridionalis (DQ015676), M. f. Pelliceus (MK805519), M. f. calamorum (JF261175), M. f. fortis (JF261174), M. levis (NC 008064), M. kikuchii (AF348082), M. ochrogaster (KT166982), M. arvalis (MG948434), M. agrestis (MH152570), Myodes glareolus (KF918859), M. rufocanus (KT725595), Neodon irene (HQ416908), N. sikimensis (KU891252), Neotoma mexicana (KY707300), N. magister (MG182016), Neotomodon alstoni (KY707310), Onychomys leucogaster (KU168563), Oligoryzomys stramineus (MF696155), Ondatra zibethicus (KX377613), Peromyscus maniculatus (MH260579), P. leucopus (MH256659), P. megalops (KY707305), P. crinitus (KY707308), P. melanophrys (KY707303), P. polionotus (KY707301), P. pectoralis (KY707309), P. aztecus (KY707306), P. attwateri (KY707299), Phodopus roborovskii (KU885975), Proedromys. liangshanensis (FJ463038), Podomys floridanus (KY707302), Reithrodontomys mexicanus (KY707307), Sigmodon hispidus (KY707311), Tscherskia triton (EU031048), Wiedomys cerradensis (KF769457). The out group is Apodemus peninsulae (JN546584).

Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees, indicating a close phylogenetic affinity of species. The phylogram obtained from Maximum Parsimony method is shown in Figure 1. Results of phylogenetic analysis showed that Microtus had close relationship with Myodes, and had distant relationship with Eothenomys, Cricetulus, Dicrostonyx, Peromyscus, and other genera. This study verifies the evolutionary status of M. fortis in Microtus at the molecular level. The mitochondrial genome would be a significant supplement for the M. fortis genetic background. Results of phylogenetic analysis showed that M. f. pelliceus had close relationship with M. f. fortis in three subspecies of M. fortis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by the Heilongjiang Provincial Department of Education filing project [1354ZD004], Project of Mudanjiang Normal University [PT2018007] and Heilongjiang Provincial Natural Funds [C2017065].