Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

The complete mitogenome and phylogenetic analysis of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> strain Qingzhou

The complete nucleotide sequence of the mitogenome of Bombyx mandarina strain Qingzhou was determined. The circular genome is 15,717 bp long and has the typical gene organization and order of lepidopteran mitogenomes. All protein-coding sequences are initiated with a typical ATN codon, except the COI gene, which has a 4-bp TTAG putative initiator codon. Eleven of the 13 protein-coding gene have a complete termination codon (all TAA), but the remaining two genes terminate with incomplete codons. All transfer RNAs (tRNAs) have a clover-leaf structure typical of the mitochondrial tRNAs, and some of them have a mismatch in the four-stem-and-loop structure. The length of the A + T rich region of B. mandarina strain Qingzhou is 495 bp, shorter than that of B. mandarina strain Tsukuba (747 bp) but similar to that of Bombyx mori. Phylogenetic analysis based on the whole mitochondrial genome sequences of the available sequenced species (B. mori strains C-108, Aojuku, Backokjam, and Xiafang, B. mandarina strains Tsukuba, Ankang, and Qingzhou, and Antheraea pernyi) shows the origin of the domesticated silkmoth B. mori to be the Chinese B. mandarina. Nuclear mitochondrial pseudogene sequences were detected in the nuclear genome of B. mori with the MEGA BLAST search program. A phylogenetic analysis of these nuclear mitochondrial pseudogene sequences suggests that B. mori was domesticated independently in different areas and periods.     

Characterization of mitochondrial genome of Chinese wild mulberry silkworm, <Emphasis Type="Italic">Bomyx mandarina</Emphasis> (Lepidoptera: Bombycidae)

The complete mitochondrial genome of Chinese Bombyx mandarina (ChBm) was determined. The circular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement identical to that of Bombyx mori (B. mori) and Japanese Bombyx mandarina (JaBm) except for two additional tRNA-like structures: tRNA ^Ser(TGA)-like and tRN ^AIle(TAT)-like. All protein-coding sequences are initiated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon. Eleven of 13 protein-coding genes (PCGs) have a complete termination codon (all TAA), but the remaining two genes terminate with incomplete codons. All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNA ^Ser(TGA)-like, with a four stem-and-loop structure. The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm (747 bp) and B. mori (494–499 bp). Phylogenetic analysis among B. mori, ChBm, JaBm, and Antheraea pernyi (Anpe) showed that B. mori is more closely related to ChBm than JaBm. The earliest divergence time estimate for B. mori-ChBm and B. mori-JaBm is about 1.08±0.18–1.41±0.24 and 1.53±0.20–2.01±0.26 Mya, respectively. ChBm and JaBm diverged around 1.11±0.16–1.45±0.21 Mya.     

The mitochondrial genome of the quiet-calling katydids, <Emphasis Type="Italic">Xizicus fascipes</Emphasis> (Orthoptera: Tettigoniidae: Meconematinae)

To help determine whether the typical arthropod arrangement was a synapomorphy for the whole Tettigoniidae, we sequenced the mitochondrial genome (mitogenome) of the quiet-calling katydids, Xizicus fascipes (Orthoptera: Tettigoniidae: Meconematinae). The 16,166-bp nucleotide sequences of X. fascipes mitogenome contains the typical gene content, gene order, base composition, and codon usage found in arthropod mitogenomes. As a whole, the X. fascipes mitogenome contains a lower A+T content (70.2%) found in the complete orthopteran mitogenomes determined to date. All protein-coding genes started with a typical ATN codon. Ten of the 13 protein-coding genes have a complete termination codon, but the remaining three genes (COIII, ND5 and ND4) terminate with incomplete T. All tRNAs have the typical clover-leaf structure of mitogenome tRNA, except for tRNA(Ser(AGN)), in which lengthened anticodon stem (9 bp) with a bulged nuleotide in the middle, an unusual T-stem (6 bp in constrast to the normal 5 bp), a mini DHU arm (2 bp) and no connector nucleotides. In the A+T-rich region, two (TA)n conserved blocks that were previously described in Ensifera and two 150-bp tandem repeats plus a partial copy of the composed at 61 bp of the beginning were present. Phylogenetic analysis found: i) the monophyly of Conocephalinae was interrupted by Elimaea cheni from Phaneropterinae; and ii) Meconematinae was the most basal group among these five subfamilies.     

野桑蚕、蓖麻蚕及家蚕基因组的RFLP分析

以家蚕Bombyx mori丝素重链基因、丝胶基因1和胰凝乳蛋白酶抑制因子13基因为探针,对野桑蚕B.mandarina、蓖麻蚕Philosamia cynthia ricini和家蚕B.mori基因组DNA进行限制性片段长度多态性分析。结果发现,在野桑蚕、蓖麻蚕基因组中存在着家蚕丝素重链基因、丝胶基因1的同源序列,而在中日野桑蚕以及蓖麻蚕品种间存在着限制性酶切位点差异;丝胶基因1在中国野桑蚕基因组的EcoRⅠ酶切图谱较日本野桑蚕与家蚕更为一致,表明家蚕与中国野桑蚕亲缘关系更近。此外,在野桑蚕基因组中发现了家蚕胰凝乳蛋白酶抑制因子13基因的同源序列,并且在家蚕品种间以及中日野桑蚕之间也存在着多态性。这些结果表明不同绢丝昆虫在适应生存环境的进化过程中,基因组发生了结构改变。     

The Complete Mitochondrial Genome of the Beet Webworm,Spoladea recurvalis (Lepidoptera: Crambidae) and Its Phylogenetic Implications

The complete mitochondrial genome (mitogenome) of the beet webworm, Spoladea recurvalis has been sequenced. The circular genome is 15,273 bp in size, encoding 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes and containing a control region with gene order and orientation identical to that of other ditrysian lepidopteran mitogenomes. The nucleotide composition of the mitogenome shows a high A+T content of 80.9%, and the AT skewness is slightly negative (-0.023). All PCGs start with the typical ATN codons, except for COX1, which may start with the CGA codon. Nine of 13 PCGs have the common stop codon TAA; however, COX1, COX2 and ND5 utilize the T nucleotide and ND4 utilizes TA nucleotides as incomplete termination codons. All tRNAs genes are folded into the typical cloverleaf structure of mitochondrial tRNAs, except for the tRNA^Ser(AGY) gene, in which the DHU arm fails to form a stable stem-loop structure. A total of 157 bp intergenic spacers are scattered in 17 regions. The overlapping sequences are 42 bp in total and found in eight different locations. The 329 bp AT-rich region is comprised of non-repetitive sequences, including the motif ATAG, which is followed by a 14 bp poly-T stretch, a (AT₁₁ microsatellite-like repeat, which is adjacent to the motif ATTTA, and a 9 bp poly-A, which is immediately upstream from the tRNA^Met gene. Phylogenetic analysis, based on 13 PCGs and 13 PCGs+2 rRNAs using Bayesian inference and Maximum likelihood methods, show that the classification position of Pyraloidea is inconsistent with the traditional classification. Hesperioidea is placed within the Papilionoidea rather than as a sister group to it. The Pyraloidea is placed within the Macrolepidoptera with other superfamilies instead of the Papilionoidea.     

基于amy基因的中国野桑蚕遗传多样性及其与家蚕的系统发育关系

为探索中国野桑蚕Bombyx mandarina的遗传多样性及其与家蚕B. mori的系统发育关系, 采用PCR产物直接测序法（少数样本克隆测序）获得34个家蚕和野桑蚕样本淀粉酶基因amy序列片段（715 bp）。分析发现56个多态性位点, 鉴定出28种单倍型（haplotype）; 核苷酸多样性π=0.01390±0.00103, 单倍型多样度Hd=0.988±0.011。核苷酸不配对分析（mismatch analysis）和Fu’s Fs 检测表明中国野桑蚕曾发生过种群扩张。分子方差分析（AMOVA）表明, 遗野桑蚕传差异主要在种群内, 种群间和地理组群间差异不显著。聚类树上34个样本聚为3枝/3蔟, 野蚕和家蚕都不按地理区域或系统（类型）聚类, A枝由来自不同地区的野蚕和不同类型的家蚕混合构成, 并且进一步分成3个亚枝, 每一亚枝也同时包含家蚕和野蚕, B枝由3个家蚕和1个野蚕混合构成, C枝全部由来自不同地区的野蚕构成。网络分析没有发现“祖先单倍型”和优势单倍型。结果提示, 淀粉酶基因是一个多态性丰富的分子标记, 中国野桑蚕遗传多样性十分丰富, 据此推测家蚕起源于多种生态类型混杂的野桑蚕。     

The complete mitochondrial genome of the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae)

We determined the complete nucleotide sequence of the mitogenome from Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). The 15,566 bp circular genome contains atypical gene organization and order for lepidopteran mitogenomes. The mitogenome contains the lowest A+T content (80.16%) among the known lepidopteran mitogenome sequences. An unusual feature is the occurrence of more Ts than As, with a slightly negative AT skewness (−0.021), in the composition of the major genome strand. All protein-coding genes are initiated by ATN codons, except for cytochrome oxidase subunit I, which is proposed by the TTAG sequence as observed in other lepidopterans. All transfer RNAs (tRNAs) have a typical clover-leaf structure of mitochondrial tRNA, except for tRNA ^Ser (AGN) , the DHU arm of which could not form a stable stem-loop structure. Two aligned sequence blocks with a length of more than 50 bp and 90% of the sequence identity were identified in the A+T-rich region of the Saturniidae and Bombycoidae species.     

大紫蛱蝶线粒体基因组全序列分析

通过PCR步移法对大紫蛱蝶Sasakia charonda coreana线粒体基因组全序列进行了测定和分析。分析结果表明:大紫蛱蝶线粒体基因组全长15233bp,包括13个蛋白编码基因、22个tRNA基因、2个rRNA基因以及长度为381bp的非编码区。A、T、C、G碱基含量分别为39.7%、40.2%、12.2%、7.9%。9个蛋白编码基因和14个tRNA基因在J链编码,其余4个蛋白编码基因和8个tRNA基因在N链编码,基因排列顺序与其它已知鳞翅目昆虫相同。13个蛋白编码基因中除COⅠ以CGA作为起始密码外,其余蛋白质基因均以ATN作为起始密码子,终止密码子多数为典型的TAA、TAG,只有COⅡ和ND4以单独的T作为终止密码子。在所测得的22个tRNA基因中,除tRNA Ser(AGN)缺少DHU臂外,其余tRNA均能形成典型的三叶草结构。与其它多数鳞翅目昆虫一样,大紫蛱蝶的非编码区序列中散在着一些长短不一的串联重复单元,在与其近缘物种非编码区的比较当中并未发现共同的保守序列区。     

中华雏蝗（Chorthippus chinensis Tarb）线粒体基因组分析

采用Lon-PCR扩增线粒体全基因组和保守引物步移法结合克隆方法测定并拼接获得了中华雏蝗（Chorthippus chinensis Tarb）线粒体基因组全序列．序列的注释和分析结果表 明,中华雏蝗线粒体基因组序列全长15 599 bp,共有13个编码蛋白质基因、22个tRNA基因、2个rRNA基因和1个A+T富集区．基因顺序与非洲飞蝗（Locusta migratoria）相同,也发生了2个 tRNA Asp(D)和tRNALys(K)的倒置．13个编码蛋白质基因都使用了ATN作为起始密码子．除ND1以TAG和ND5的终止密码子为不完全的T外,其余11个编码蛋白质基因的终止密码子都为完整的TAA．6种直翅类昆虫13个蛋白质的氨基酸序列的联合数据集构建的系统树与形态分类系统一致,中华雏蝗与非洲飞蝗为姐妹群,并与东方蝼蛄构成一单系群．     

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS((AGY)) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem-loop structure and control region has several conserved sequence elements.     

黄毛纺蚋线粒体基因组全序列测定与分析

利用PCR步移法对黄毛纺蚋的线粒体基因组全序列进行了测定和分析。黄毛纺蚋线粒体基因组全长15904 bp(Gen Bank序列号KP793690),包括13个蛋白编码基因、22个tRNA基因、2个rRNA基因以及长度为939 bp的非编码区。A、T、C、G碱基含量分别为39.1%、35.8%、10.4%、14.7%。9个蛋白编码基因和14个tRNA基因在J链编码,其余4个蛋白编码基因和8个tRNA基因在N链编码,基因排列顺序与其它已知双翅目昆虫相同。13个蛋白编码基因中除COI以TTG作为起始密码外,其余蛋白质基因均以ATN作为起始密码子,终止密码子多数为典型的TAA、TAG,只有COI和ND4L以单独的T作为终止密码子。在所测得的22个tRNA基因中,除tRNASer(AGN)缺少DHU臂外,其余tRNA均能形成典型的三叶草结构。     

cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.     

The complete mitochondrial genome of Accipiter virgatus and evolutionary history of the pseudo-control regions in Falconiformes

The complete mitochondrial genome sequence of Accipiter virgatus was determined. This mt-genome was 17,952 bp in length and consisted of 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes, one control region (CR) and one pseudo-control region (CCR). Phylogenetic analyses of 14,644 bp of mitochondrial DNA (12 protein-coding genes, 2 rRNAs and 22 tRNAs) revealed the phylogenetic position of Cathartidae (Cathartes aura) was more closer to Ciconiidae (storks) than Accipitridae. To investigate the divergence times of the CCRs in Falconiformes, detailed analyses of the noncoding regions (CR and CCR) were performed. We found the recently reported novel gene order in Falconiformes had multiple independent origins and hence cannot be used to infer phylogenetic lineages. Indeed, the molecular clock suggested the CCR in Falconidae emerged about 65.4 million years (Mya), while that in Pandionidae–Accipitridae clade emerged about 19.16 Mya. The intra-genomic homology between the noncoding regions was detected in Spilornis cheela, which supporting the duplication hypothesis. Furthermore, the structure of CCR should be featured by a region containing tandem repeats as two definitely separated clusters of tandem repeats were found. The findings presented here should be considered in future phylogenetic and evolutionary studies targeting the pseudo-control regions of all Falconiformes species.     

The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.     

Complete mitochondrial genome of Locusta migratoria migratoria (Orthoptera: Oedipodidae): three tRNA-like sequences on the N-strand

The complete 16053 bp mitochondrial genome (mitogenome) sequence of Locusta migratoria migratoria has been determined. This mitogenome contains the base compositional biases and codon usage typical of metazoans, and the RSCU values indicate a negative correlation with the C and G contents in codon. The orientation and gene order of the L. migratoria migratoria is identical to Locusta migratoria migratoiodes. An unusual feature of the L. migratoria migratoria mitogenome is the presence of three tRNA-like structures on the N-strand: one tRNA(Ile)-like and two tRNA(Leu(CUN))-like sequences. The tRNA-like sequences have proper folding structures and anticodons sequences. Two repeated DNA sequences, Rpt I and Rpt II, were found in the A+T-rich region of the L. migratoria migratoria mitogenome. Both repeated sequences have various features. In the 5' region of Rpt I, a 51 bp fragment is localized in the srRNA gene; and there are two tandemly sub-repeated DNA sequences (sub-Rpts), Rpt 1-4, within Rpt I and Rpt II. One stem-loop structure on the N-strand that may be involved in the N-strand replication initiation was found in the A+T-rich region.     

麦穗鱼线粒体基因组序列测定及分析

利用麦穗鱼Pseudorasbora parva和相关鱼类的部分线粒体基因序列,设计出2对长批引物和30对短批引物,采用基于长PCR的2次PCR扩增法测定并注释麦穗鱼线粒体基因组全序列。结果表明,麦穗鱼线粒体基因组长16600bp,A+T含量为58.9%,37个基因位置及组成与其它硬骨鱼一致,均由13个蛋白编码基因、22个tRNA、2个rRNA基因和1个控制区(D-loop)组成。其中L链仅含8个tRNA(Pro、T yr、Ser、Ala、Asn、Cys、Glu、Gln)及ND6基因,其余基因皆由H链编码。基因排列紧密,间隔序列共计13处64bp,长度从1～32bp不等;基因重叠区7处23bp,重叠碱基数在1～7bp之间。13个蛋白编码基因中,除COI起始密码子为GTG外,其余均以ATG为起始密码子;有8个基因(ND1、ND2、COI、ATP6、ATP8、ND4L、ND5、ND6)3’端有完全的TAA或TAG终止密码子,其它5个基因终止密码子为不完整的TA(ND3和ND4)或T(COⅡ,COⅢ,Cyt b)。除tRNASer(AGY)外,其余21个tRNA基因的二级结构均为典型的三叶草结构。预测的lrRNA二级结构共有6个结构域,53个茎环结构,srRNA二级结构包含43个茎环结构。控制区(D-loop)存在3个结构区:终止序列区(TAS)、中央保守区(CSB-F、CSB-D)和保守序列区(CSB-1、CSB-2、CSB-3),其中TAS与DNA复制终止相关,出现茎环结构。