See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/316316844 Mitochondrial genome of Abraxas suspecta (Lepidoptera: Geometridae) and comparative analysis with other Lepidopterans Article in Zootaxa · April 2017 DOI: 10.11646/zootaxa.4254.5.1 CITATIONS READS 7 192 10 authors, including: Yu Sun Muhammad Nadeem Abbas Bengbu Medical College Southwest University in Chongqing 47 PUBLICATIONS 356 CITATIONS 93 PUBLICATIONS 814 CITATIONS SEE PROFILE SEE PROFILE Cen Qian Guoqing Wei Anhui Agricultural University (AHAU) 54 PUBLICATIONS 437 CITATIONS 123 PUBLICATIONS 1,388 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: microbiolgy View project Silkworm/Mulberry/Silk and Health View project All content following this page was uploaded by Yu Sun on 30 March 2018. The user has requested enhancement of the downloaded file. Zootaxa 0000 (0): 000–000 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.0000.0.0 http://zoobank.org/urn:lsid:zoobank.org:pub:00000000-0000-0000-0000-00000000000 Mitochondrial genome of Abraxas suspecta (Lepidoptera: Geometridae) and comparative analysis with other Lepidopterans YU SUN1, JIAWEI ZHANG1, QINGQING LI1, DAN LIANG1, MUHAMMAD NADEEM ABBAS1, CEN QIAN1, LEI WANG1, GUOQING WEI1, BAO-JIAN ZHU1,2 & CHAO-LIANG LIU1,2 1College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China 2Corresponding authors. E-mail: [email protected] (B.-J. Zhu); [email protected] (C.-L. Liu) Abstract In this study, a complete mitochondrial genome (mitogenome) sequence of Abraxas suspecta (Lepidoptera: Geometridae) is isolated and characterized. The complete DNA is 15,547 bp length and contains 2 ribosomal RNA genes, 23 putative transfer RNA (tRNA) genes including an extra tRNAAsn (AUU), 13 protein-coding genes and an adenine (A) + thymine (T)-rich region. The nucleotide composition and gene organization are identical to those of other lepidopteran, except for the presence of an extra copy of trnN (AUU). Of the 38 genes, twenty-five genes (9 PCGs and 16 tRNAs) are encoded by heavy strand (H-strand), while thirteen are encoded by light strand (L-strand). Among the 13 PCGs, 12 PCGs employ ATN as initiation codon, while cytochrome c oxidase subunit 1 (cox1) utilizes CGA as initiation codon. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. All tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNAs, except for the tRNASer (AGN) gene, in which the DHU arm fails to form a stable stem-loop structure. The A+T-rich region is 532 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17bp poly-T stretch, a microsatellite-like element (AT)8(AAT)3 and also a poly-A element. A short Phylogenetic analysis based on 13 PCGs using maximum likelihood (ML) and Bayesian infer- ence (BI) revealed that A. suspecta resides in the Geometridae family. We present the method and approach to use moths as model organisms for further genetic and evolutionary biology studies. Key words: mitogenome, geometrid moth, phylogenetic analysis, evolution Introduction The complete mitogenome DNA of an insect is a compact, double stranded and closed circular molecule that ranges almost 14–16 kbp in length. The gene arrangement and organization of mitogenome in insects and especially in Lepidoptera is fairly conserved. With a few exceptions, e.g. Ctenoptilum vasava (Lepidoptera) and Bombus ignites (Hymenoptera), an insect mitogenome contains a variable number of tRNA genes (Cha et al. 2007; Hao et al. 2012) and usually encodes 37 genes including 13 protein-coding genes (PCGs), two ribosomal-RNA- coding genes (rRNAs), 22 transfer-RNA-coding genes (tRNAs), and an A + T rich displacement loop (D-loop) control region (Cameron 2014; Liu et al. 2016). Because of their maternal inheritance, compact structure, lack of genetic recombination, and relatively fast evolutionary rate, mitogenomes have been extensively used in molecular, phylogenetics and evolutionary studies (Cameron 2014). Abraxas suspecta (Warren, 1894) (Lepidoptera: Geometridae: Ennominae), is considered a most damaging pest of the medicinally important plant Euonymus japonicas Thunb. The larvae of this species extensively feed on young leaves and cause death of small branches (Yingqi 1999). The A. suspecta is widely distributed in the northern parts of China. It belongs to Geometridae, the second largest family of Lepidoptera that includes approximately 23,000 described species worldwide (Scoble & Hausmann 2007). The management of this devastating species is extremely important, despite this importance, only a few studies are available on this species (Yang et al. 2009, 2013; Liu et al. 2014; Chen et al. 2016). Hence, to improve the management of A. suspecta, it is enormously important to know more about this pest, particularly its genetic characteristics and phylogenetic position. Accepted by J.De Prins: 13 Mar. 2017; published: ?? Month 2017 1 Here, we analyzed the genomic organization, gene arrangement, codon usage of A. suspecta, and the complete mitogenome of A. suspecta was compared its sequence to selected Lepidoptera species, particularly Geometridae. The use of molecular phylogenetic studies based on the mitochondrial genome can provide a strong support to the taxonomic study and make it more accurate. Moreover, a reconstructed phylogeny of the superfamily Geometroidea based on the concatenated nucleotide sequences of 13 PCGs of moth mitogenome enables to characterize A. suspecta among its relatives within the family Geometridae. Materials and methods Sampling and DNA extraction. The A. suspecta specimens were collected from Anhui Agricultural University, Anhui Province, China. The collected specimens were identified as A. suspecta based on the morphological characters by the taxonomist of the Department of Entomology, Anhui Agricultural University, Hefei, China (AHAU). After a careful examination of the morphological characters and the comparison of voucher specimens to the referenced publications of Chinese moths published by the Institute of Zoology, Chinese Academy of Sciences we are confident that the species under our study is identified correctly. The samples were preserved in 100% ethanol and stored at -80°C. A single specimen was used to extract total genomic DNA using the Genomic DNA Extraction Kit, according to the manufacturer's instructions (Aidlab Co., Beijing, China). The quality of extracted DNA was determined by 1% agarose gel electrophoresis (w/v) and used to amplify the complete mitogenome of A. suspecta. The Abraxas suspecta specimens were collected on the Buxus megistophylla H.Lév. (Buxaceae) plants in the campus of Anhui Agricultural University, Hefei city, China. The owner of the land gave permission to conduct the study on this site. Our field studies did not involve endangered or protected species. The voucher specimens are preserved at the collection of the Department of Entomology, Anhui Agricultural University, Hefei, China (AHAU). Design of primers, PCR amplification and sequencing. To determine the mitogenome of A. suspecta (Liu et al. 2013; Dai et al. 2014), we designed twelve pairs of primers from the conserved nucleotide sequences of known mitogenome of several lepidopteran species and then synthesized them following the methods presented in Dai et al. (2015) and Sun et al. (2016) (Sangon Biotech Co., hanghai, China) (Table 2). All amplifications were performed on an Eppendorf Mastercycler and Mastercycler gradient in 50 µL reaction volumes, which contained 35 µL sterilized distilled water, 5 µL 10 Taq buffer (Mg2+ plus), 4 µL dNTP (25 mM), 1.5 µL extracted DNA as a template, forward and reverse primers 2 µL each (10 µM) and 0.5 µL (1 unit) Taq DNA polymerase (Takara Co., Dalian, China). The PCR amplification conditions were as follows: an initial denaturation one cycle at 94°C for 4 min followed by 38 cycles, one cycle at 94°C for 30s, one cycle at 48–59 °C for 1–3 min (depending on the putative length of the fragments), and a final extension one cycle at 72°C for 10 min. The PCR products were resolved by 1% agarose gel electrophoresis (w/v), and were purified using a DNA gel extraction kit (Transgen Co., Beijing, China), and directly sequenced with PCR primers. Sequence assembly and gene annotation. Sequence annotation was performed using blast tools available from NCBI (http://blast.ncbi.nlm.nih.gov/Blast), and SeqMan II program from the Lasergene software package (DNASTAR Inc.; Madison, USA). The protein-coding sequences were translated into putative proteins on the basis of the Invertebrate Mitochondrial Genetic Code. The skewness was measured by the method given by Junqueira et al. (2004), and the base composition of nucleotide sequences were described as: AT skew = [A−T]/[A+T], GC skew = [G−C]/[G+C]. The relative synonymous codon usage (RSCU) values were calculated using MEGA version 5.1 program (Tamura et al. 2011). The tRNA genes were determined using the tRNAscan-SE software (http://lowelab.ucsc.edu/tRNAscan-SE/) (Lowe & Eddy 1997), or predicted by sequence features of being capable of folding into the typical clover-leaf secondary structure with legitimate anticodon. The tandem repeats in the A+T-rich region were determined by the tandem repeats finder program (http://tandem.bu.edu/trf/trf.html) (Benson 1999). Phylogenetic analysis. To reconstruct the phylogenetic relationship among Lepidopterans 36 complete mitogenome sequences were downloaded from the GenBank database (Table 1). Further, the mitogenomes of Drosophila melanogaster (U37541.1) (Lewis et al. 1995) and Anopheles gambiae (L20934.1) (Beard et al. 1993) were used as outgroup. The multiple alignments of the 13 PCGs concatenated nucleotide sequences were 2 · Zootaxa 0000 (0) © 2017 Magnolia Press SUN ET AL. conducted using ClustalX version 2.0. (Thompson et al. 1997), and then was used for phylogenetic analyses. Two analytical approaches, Maximum Likelihood (ML) with the MEGA version 5.1 program (Tamura et al.