Nuclear Mitochondrial DNA Pseudogenes in the Genome of the Silkworm, Bombyx Mori

Journal of Computational Biology and Bioinformatics Research Vol. 3(7), pp. 103-119, December 2011 Available online at http://www.academicjournals.org/JCBBR ISSN-2141-2227 ©2011 Academic Journals Full Length Research Paper Nuclear mitochondrial DNA pseudogenes in the genome of the silkworm, Bombyx mori Guangli Cao1,2, Renyu Xue1,2, Yuexiong Zhu1, Yuhong Wei1 and Chengliang Gong1,2* 1School of Biology and Basic Medical Science, Medical college of Soochow University, Suzhou, 215123, China. 2National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, 215123, China. Accepted 26 October, 2011 To understand the types of mitochondrial deoxyribonucleic acid (mtDNA) pseudogenes in the genome of the silkworm Bombyx mori L. (Lepidoptera: Bombycidae) and to determine the origin of the B. mori, the mtDNA of the wild silkworm, Bombyx mandarina Morre was sequenced. Several fragments from the B. mori genome database were obtained with varying degrees of homology to B. mandarina mtDNA by sequence alignment between the B. mori genome and B. mandarina mtDNAs. The results showed that in the B. mori genome database there are not only mtDNA (Dazao strain) sequence but also pseudogenes derived from B. mandarina mtDNA. There is a potential reverse repeated sequence at the terminal sequence of the pseudogenes, and there are random point mutations and insertion mutations in the pseudogenes sequences. One B. mandarina mtDNA could be repeatedly inserted into different positions within the nuclear genome. Evolutionary analysis indicates that the transfer of mtDNA pseudogenes might have occurred at different points during evolution, some sequences might be transferred after Lepidoptera had formed, and possibly before the families of Lepidoptera were formed. B. mori probably originated from B. mandarina of China. Key words: Bombyx mori, Bombyx mandarina, nDNA, mtDNA, nuclear mitochondrial DNA pseudogenes. INTRODUCTION The mitochondria of the eukaryotic cells are important (Zhang and Hewitt, 1996; Bensasson et al., 2001a; Song organelles. The genome of mitochondrial et al., 2008). Experiments show that mtDNA was reverse- deoxyribonucleic acid (mtDNA) contains mitochondrial transcribed from the mitochondrial RNA in the cytoplasm, tRNA genes, rRNA genes and cytochrome genes and through the nuclear pore of the nuclear membrane and thus is able to synthesize the specific protein of itself, but randomly integrated into the nDNA by DNA ligase the mitochondrial genome is regulated by the nuclear (Woischnik and Moraes, 2002; Nugent and Palmer, genes, including mitochondrial genome transcription and 1991). These numts copies may be very long, but their translation process, especially for the greater impact on richness varied in different species and they and their the regulation of transcriptional level. The evidence has corresponding mtDNA have strong homology. mtDNA and been found that there are similar mitochondrial genes similarities in the sequence of the nuclear genome shows sequences in nuclear Deoxyribonucleic acid (nDNA). there has been an exchange of genetic material between These sequences that exist in the pseudogene form were nucleus and cytoplasm during the evolution of the cell. named nuclear mitochondrial pseudogenes (numt, The high mutation rates of mtDNA can produce nuclear mitochondrial DNA segments). The numts are intraspecific polymorphism and deep interspecific nonfunctional copies of mtDNA in the nucleus that have divergence in relatively short evolutionary times (Avise et been found in major clades of eukaryotic organisms al., 1987). This makes mtDNA particularly informative for the determination of genetic population structure and inference of population history within species as well as for deducing phylogenetic relationships between closely *Corresponding author. E-mail: [email protected]. Tel: +86- related species (Hlaing et al., 2009). The use of mtDNA 521-65880183. Fax: +86-521-65880183. sequences has been proposed for several DNA 104 J. Comput. Biol. Bioinform. Res. barcoding initiatives for taxonomic identification and the Shandong Province of China. Mitochondrial DNA was extracted biodiversity assessment (Song et al., 2008; Hebert et al., from larval insects using a standard phenol/chloroform method for 2003). However, such studies can generate misleading genomic DNA extraction (Sambrook and Russell, 2001). Restriction enzymes PstI (use for identification of TA clone), PCR results if the species concerned contain numts as these reagents, T-vector, the T4 DNA ligase and the gel DNA purification may amplify in addition to, or even instead of, the kit were purchased from the Sangon (Shanghai Sangon Biological authentic target mtDNA. The evolutionary rate of the Engineering Technology and Services Co., Ltd, Shanghai, China) numt sequences is very low, numts result from the and TaKaRa (Dalian, China) or Bio Flux (Hangzhou, China). The full translocation of mitochondrial sequences from the mitogenome of B. mandarina strain Qingzhou was amplified in 15 overlapping fragments by PCR using insect-specific designed mitochondrial genome into the nuclear genome and, once primers (Hu et al., 2009). The mtDNA fragments were amplified integrated, these non-functional sequences accumulate using a standard PCR method. After purification with the gel DNA mutations freely (Hlaing et al., 2009). Because the numt purification kit, each fragment was cloned into the T-vector and still can represent the original form of mtDNA sequence sequenced (Sangon, Shanghai, China). The mitochondrial genome to a certain extent, it is possible to determine the early sequence of B. mandarina strain Qingzhou was deposited in the evolutionary relationships between species (Zhang and GenBank database under the accession no. FJ384796. Hewitt, 1996; Sorenson and Quinn, 1998; Perna and Kocher, 1996). Sequence analysis There are seven complete mtDNA genome of Bombycidae strains (or varieties) have been sequenced, Sequence analysis was performed as follows. Initially, the mtDNA including B. mori variety C-108 and Xiafang (Chinese), sequence and numt sequence were identified using the NCBI Aojuku (Japanese), Backokjam (South Korea), B. internet blast (Altschul et al., 1990) search function. The probe sequence was used complete mtDNA sequences, including four B. mandarina strain Ankang and Qingzhou (Chinese), and mori strains (Xiafang, AY048187; Aojuku, AB083339; C-108, Tsukuba (Japanese). These data provides the basis for AB070264; Backokjam, AF149768), three B. mandarina strains an evolutionary analysis. Bombyx mori is used as a (Tsukuba, AB070263; Ankang, AY301620; Qingzhou, FJ384796), model organism for Lepidopteran insects (Goldsmith, and other insects (Drosophila melanogaster, AJ400907; 1995; Goldsmith et al., 2005), and the draft sequence for Adoxophyes honmai, DQ073916; Locusta migratoria, NC_011119; the genome of B. mori provided a good platform for the Tribolium castaneum, AJ312413), respectively. After selected blast analysis the “highly similar sequences, megablast” and “somewhat study of the biological information of Lepidoptera (Xia et similar sequences, blastn” were chosen for comparison with the B. al., 2004; Mita et al., 2004). mori genome (Dazao strain) database “wgs” (whole – genome There is a lot of evidence to support the origin of the B. shotgun reads). Suspected mtDNA sequence of the Dazao strain, mori in China. There are many theories for the and suspected numt sequences (Bmnumt) were obtained. The geographical origin of silkworms and differentiation of sequences were aligned with Clustal X (Thompson et al., 1997). voltinism. The “multi centers” and “mono center” hypotheses of B. mori origin are examples. In recent Phylogenetic analysis years, molecular biology research provided strong evidence at the molecular level for the origin of silkworms The numt sequences and homologous regions in different B. mori in China, and the evidence from molecular biology also mitogenome sequences were aligned with Clustal X (Thompson et support the “multi-center” hypotheses of silkworm origin al., 1997). A phylogenetic analysis was performed based on the concatenated nucleotide sequences of the Bmnumt using the (Zhang and Lü, 2005). The origin and differentiation of B. neighbor joining (NJ), minimum evolution (ME), maximum mori is related to the major issues, such as silkworm parsimony, and UPGMA programs of the MEGA3 software package genetics, breeding, character formation, and other (Kumar et al., 2004; Pan et al., 2008), with a bootstrap of 500 subjects, therefore also the need for more direct or replicates. Pairwise genetic distances were calculated with MEGA3 indirect evidence to understand the problem. There are using the Kimura two-parameter model for nucleotide substitution (Kumar et al., 2004). numerous reports of the analysis of molecular evolution Based on the supposition that the different strains of B. of silkworms using mtDNA protein coding genes or rRNA mandarina arose through geographic isolation and the generally genes (Chen et al., 2007), but so far there are no reports accepted belief that the Japanese islands were separated from the of the analysis of silkworm evolution using the numt of Asian continent 0.02 Mya, the MEGA3 program was used to set the nDNA sequence. This paper uses sequence searching to divergence time to understand the divergence time during origin of compare and analyze the B. mori genome numt (Bombyx B. mori. The molecular clock was based on a substitution rate of (7.8 to 10.2) × 10-9 per site per year (Zakharov

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