Sequencing and analysis of the complete mitochondrial genome of the Eurasian least shrew (Sorex minutissimus) from China

Abstract

The complete mitogenome sequence of the Eurasian least shrew (Sorex minutissimus) was determined using long PCR. The genome was 17,061 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 (32.6%), C (24.7%), T (29.1%) and G (13.6%). The base compositions present clearly the A–T skew, which is most obviously in the control region and protein-coding genes. The extended termination-associated sequence domain, the central conserved domain and the conserved sequence block domain are defined in the mitochondrial genome control region of the Eurasian least shrew. Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees. The ten Sorex species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

Keywords:

• Control region
• mitogenome
• phylogenetic trees
• The Eurasian least shrew

In this paper, the complete mitochondrial genome of the Eurasian least shrew (Sorex minutissimus) 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 the Eurasian least shrew captured from Hulun Lake regions of Changbaishan Mountains in Inner Mongolia Province, China (48°37′20″N, 117°53′17″E). The muscle tissue was preserved in 95% ethanol and stored at −75 °C before use. The specimen and its DNA is stored in Animal and Plant Herbarium of Mudanjiang Normal University. The voucher number is NM201607.

The mitochondrial genome is a circular double-stranded DNA sequence that is 17,061 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 accession number MH823669. The arrangement of the multiple genes is in line with other Soricidae species (Nikaido et al. 2001; Fontanillas et al. 2005; Kim et al. 2013; Huang et al. 2014; Huang et al. 2016; Xu et al. 2016; Liu et al. 2016; Jin et al. 2017; Kim et al. 2017; Liu, Tian, Jin, Li, et al. 2017; Liu, Tian, Jin, Dong, et al. 2017; Liu, Wang, et al. 2017; Liu et al. 2018) and most mammals (Meganathan et al. 2012; Xu et al. 2012; Xu et al. 2013; Yoon et al. 2013).

The control region of the Eurasian least shrew 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 the Eurasian least shrew 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. Three CSBs were found in the CSB domain and they were located in positions 16,284–16,319, 16,714–16,748 and 16,764–16,802. Also only one repetitive sequence region (RS) was found, which was located between the CSB1 and CSB2, and was rich in A and C. The repetitive pattern of segments in the RS was 5′-TG-(TACACGTACACGTA)n-CA-3′ (n = 12) .

The total length of the protein-coding gene sequences was 11,475 bp. Most protein-coding genes initiate with ATG except for ND2, ND3, ND5 and ND6, which began with ATA, ATT or TAC. Six protein-coding genes terminated with TAA whereas the Cyt b gene terminated with AGG. The incomplete stop codons (T–– or TA–) were used in ND1, ND2, COX3, ND3 and ND6. 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 the Eurasian least shrew 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 37 bp long and had the potential to fold into a stable stem-loop secondary structure. The total base composition of the Eurasian least shrew mitochondrial genome was A (32.6%), C (24.7%), T (29.1%) and G (13.6%). 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 Insectivora shrews which include Soricidae and Talpidae, especially the evolution of genus Sorex from China, here, we investigate the molecular phylogenetics of Chinese the Eurasian least shrew using complete mitochondrial genome sequence of 31 species. 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. Crocidura lasiura (KR007669), Crocidura shantungensis (JX968507), Crocidura attenuata (KP120863), Crocidura russula (AY769264), Episoriculus macrurus (KU246040), Episoriculus caudatus (KM503097), Neomys fodiens (KM092492), Nectogale elegans (KC503902), Anourosorex squamipes (KJ545899), Blarinella quadraticauda (KJ131179), Suncus murinus (KJ920198), Soriculus fumidus (AF348081), Sorex araneus (KT210896), Sorex cylindricauda (KF696672), Sorex unguiculatus (AB061527), Sorex tundrensis (KM067275), Sorex caecutiens (MF374796), Sorex roboratus (KY930906), Sorex isodon (MG983792), Sorex gracillimus (MF426913), Sorex mirabilis (MF438265), Sorex minutissimus (MH823669), Talpa europaea (Y19192), Urotrichus talpoides (AB099483), Uropsilus soricipes (JQ658979), Uropsilus gracilis (KM379136), Mogera wogura (AB099482), Mogera robusta (KT934322), Condylura cristata (KU144678), Galemys pyrenaicus (AY833419), Scapanulus oweni (KM506754) and Erinaceus europaeus (NC002080).

Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees, indicating a close phylogenetic affinity of shrews. The phylogram obtained from Maximum Parsimony method is shown in Figure 1. It shows that two major phyletic lineages were present in Insectivora: Soricidae and Talpidae. Soricidae comprised Crocidura lasiura, Crocidura shantungensis, Crocidura attenuata, Crocidura russula, Episoriculus macrurus, Episoriculus caudatus, Neomys fodiens, Nectogale elegans, Anourosorex squamipes, Blarinella quadraticauda, Soriculus fumidus, Suncus murinus, Sorex araneus, Sorex tundrensis, Sorex caecutiens, Sorex roboratus, Sorex isodon, Sorex gracillimus, Sorex mirabilis, Sorex cylindricauda, Sorex unguiculatus and Sorex minutissimus was supported by bootstrap values of 100%. Talpidae comprised Talpa europaea, Urotrichus talpoides, Mogera wogura, Condylura cristata, Uropsilus soricipes, Mogera robusta, Galemys pyrenaicus, Uropsilus gracilis and Scapanulus oweni was supported by bootstrap values of 100%. The ten Sorex species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This research was supported by the Heilongjiang Provincial Department of Education Filing Project [1351MSYYB009], Heilongjiang Provincial Natural Funds [C2017065] and the Doctoral Scientific Research Foundation Project of Mudanjiang Normal University [MNUB201404].