Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Complete Sequence and Gene Organization of the Mitochondrial Genome of Tokay (Gekko gecko)

Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mt genome of Gekko is similar to most of the vertebrates in gene components, order, orientation, tRNA structures, low percentage of guanine and high percentage of thymine, and skews of base GC and AT. Base A was preferred at third codon positions for protein genes is similar to amphibians and fishes rather than amnion vertebrates. The standard stop codes (TAA) present only in three protein genes, less than those of most vertebrates. Transfer RNA genes range in length from 63 to 76 nt, their planar structure present characteristic clover leaf, except for tRNA-Cys and tRNA-Ser (AGY) because of lacking the D arm.

DGGE Analysis on Mitochondrial Cyt b Gene of Eriocheir sinensis and Eriocheir hepuensis

[Objective] The aim was to investigate the possibility to analyze the genetic diversity of Eriocheir sinensis and Eriocheir hepuensis by using the technique denaturing gradient gel electrophoresis（DGGE）.[Method] Mitochondrial cyt b gene fragment was amplified from 180 individuals of five populations of E.sinensis and a population of E.hepuensis and then analyzed by using DGGE.[Result] All PCR products showed two kinds of electrophoretic mobility on DGGE.The PCR products of all individuals from E.hepuensis showed the same mobility with that of the individuals from 46.7% of Jiangdu population,23.3% of Yizheng population and 20.0% of Wenzhou population of E.sinensis,while the rest of the individuals from the three populations of E.sinensis mentioned above as well as all the individuals of Nanjing and Panjin populations showed the same mobility,which was higher compared with that of E.hepuensis.The results indicated that there was the same genetic marker in E.sinensis populations as that of E.hepuensis population,which was consistent with previous studies.[Conclusion] DGGE technique could be used to analyze the genetic diversity of Chinese mitten crab.

Cloning,Sequencing and Molecular Phylogenetic Analysis of the Mitochondrial Cytochrome Oxidase Ⅰ Gene of Chilo suppressalis

[Objective] The research aimed at cloning and analyzing mitochondrial cytochrome oxidase I gene（cox 1）of C.suppressalis.[Method] The mitochondrial cox 1 gene of C.suppressalis was cloned with PCR method and sequenced.Then,cox1 sequences of other 21 Lepidopteran species were obtained by blasting the GenBank with cox 1 gene sequence of C.suppressalis.Finally,homology comparison and molecular phylogenitic analysis among the 22 Lepidopteran species were conducted.[Result] The open reading frame of cox 1 gene from C.suppressalis contained 1 531 nucleotides encoding a putative protein of 510 amino acids.The cox1 gene used a start codon CGA,and an incomplete termination codon composed of only T.Based on the amino acid sequences of cox 1,the molecular phylogenetic tree of Lepidoptera was reconstructed using the maximum likelihood（ML）method.The molecular phylogenetic tree was similar to the morphological phylogenetic tree mainly,but also showed some differences.[Conclusion] The result will provide reference for further research on expression and application of cox 1 gene.

Mitochondrial COI Gene Sequence Variation and Taxonomic Status of Three Macrobrachium Species

Freshwater prawns （Decapoda： Caridea： Macrobrachium） play an important role in domestic fishery resources. Culturing M. rosenbergii and M. nipponense brings great economic benefits, as the two species were widely farmed in China. M. qilianensis, a native species with natural distribution limited in Gansu province, was classified into genus Macrobrachium based on external morphological characters. In order to understand the molecular genetic differences among the three species of Macrobrachium, i.e., M. rosenbergii, M. nipponense, and M. qilianensis, we analyzed sequences of mitochondrial cytochrome oxidase subunit I （COI） of them. It would provide theoretical basis of exploiting and utilizing Macrobrachium resources rationally. A total of 30 individuals （10 individuals of each species） were collected from Gansu and Zhejiang province. Samples of M. qilianensis were wild, however, that of M. rosenbergii and M. nipponense were cultured. Their mitochondrial COI gene segment sequences were obtained by using the method of PCR amplification and sequencing. After alignment, 649 bp consensus sequences of COI were obtained. One hundred and sixty-nine variable sites were detected in all 30 individuals, accounting for 26.04% of total sequence. A total of seven haplotypes were also detected. Nucleotide diversity was 0.411% within M. rosenbergii, 0.092% within M. nipponense, and 0.031% within M. qilianensis. Genetic diversity of wild M. qilianensis was much lower than that of cultured M. rosenbergii and M. nipponense. Genetic distances between different haplotypes of the three prawns ranged from 19.87% to 23.84%. It suggested that the three species were valid species, because genetic distances among them were quite great. To further determine the taxonomic status of the three prawns in family Palaemonoidae, we downloaded the corresponding COI sequences of Palaemonoidae prawns from Genbank and analyzed the phylogenetic relationships of them. Phylogenetic tree （NJ） showed that M. nipponense, M. rosenbergii and other Macrobrachium species constituted one monophyletic group. However, M. qilianensis, Exopalaemon carinicauda, and Palaemon debilis formed the other clade. Thus, results of COI sequences did not support that M. qilianensis belonged to genus Macrobrachium. The taxonomic status ofM. qilianensis should be reevaluated with more comprehensive evidences.

Genetic Variation Among the Geographic Population of the Grain Aphid, Sitobion avenae (Hemiptera: Aphididae) in China Inferred from Mitochondrial COI Gene Sequence

In order to characterize the genetic relationship of the geographic populations of Sitobion avenae （Hemiptera： Aphididae） in China, a 588 bp region of the mitochondrial cytochrome oxidase subunit I （mtDNA-COI） gene was sequenced and analyzed among the different geographic populations. 269 individuals were collected from 17 localities in different wheat-growing areas in China that covered most of the range reported for this species. Within the sequence among these geographic populations, 15 polymorphic sites defined 16 distinct haplotypes, ranging in sequence divergence from 0.2% （one nucleotide） to 1.7% （10 nucleotides）. Of the 15 variable sites, 12 were transitional substitutions, 2 were transversional substitutions and 1 was transitional and transversional substitution. Phylogenetic analysis showed that all haplotypes were highly interconnected with each other, in absence of phylogeographic structing. Each of 8 haplotypes was found only at one locality, and the other haplotypes were the widespread distributed in the different localities. The higher genetic diversity was found in the northern China populations than that in the southern China populations. The low genetic differentiation （FST=-0.06945-0.69857） and high migration rate （Nm=0.21575-infinite） of Chinese populations suggest that dispersal over long distance is a major factor in the demography of S. avenae.

Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome

Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly chosen individuals, and evenamong siblings. A mtDNA pseudogene in the Ychromosome was observed in a YAC clone carrying onlyrepetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showedthat there were at least 5.7×105 hp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part ofthe human nuclear genome is discussed.

The cotton mitochondrial chimeric gene orf610a causes male sterility by disturbing the dynamic balance of ATP synthesis and ROS burst

Plant cytoplasmic male sterility(CMS)is maternally inherited and often manifested as aborted pollen development,but the molecular basis of abortion remains to be identified.To facilitate an investigation of CMS in cotton,the complete sequence of cotton mitochondrial(mt)genome for CMS-D2 line ZBA was determined.The mt genome was assembled as a single circular molecule with 634,036 bp in length.A total of 194 ORFs,36 protein-coding genes,six r RNAs,and 24 t RNAs were identified.Several chimeric genes encoding hypothetical proteins with transmembrane domains were identified.Among them,a previously unknown chimeric gene,orf610a,which is composed of atp1 and a 485-bp downstream sequence of unknown nature,was identified.RT-PCR and q RT-PCR validation indicated that orf610a was expressed specifically in a sterile line.Ectopic expression of orf610a in yeast resulted in excessive accumulation of reactive oxygen species and reduction in ATP content,in addition to inhibition of cellular growth.Transgenic A.thaliana overexpressing orf610a fused with a mitochondrial targeting peptide displayed partial male sterility.Interaction between ORF610a and the nuclear-encoded protein RD22 indicated an association between ORF610a and pollen abortion.Positive feedback during transcriptional regulation between nuclear regulatory factors and the mt CMS gene may account for the male sterility of ZBA.

Functional analysis of the novel mitochondrial tRNA^(Trp)and tRNA^(Ser(AGY))variants associated with type 2 diabetes mellitus

BACKGROUND Mutations in mitochondrial tRNA(mt-tRNA)genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus(T2DM).We previously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel mt-tRNA^(Trp)A5514G and tRNA^(Ser(AGY))C12237T variants,however,the effects of these mt-tRNA variants on T2DM progression are largely unknown.AIM To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic,molecular,and biochemical levels.METHODS Cytoplasmic hybrid(cybrid)cells carrying both m.A5514G and m.C12237T variants,and healthy control cells without these mitochondrial DNA(mtDNA)variants were generated using trans-mitochondrial technology.Mitochondrial features,including mt-tRNA steady-state level,levels of adenosine triphosphate(ATP),mitochondrial membrane potential(MMP),reactive oxygen species(ROS),mtDNA copy number,nicotinamide adenine dinucleotide(NAD+)/NADH ratio,enzymatic activities of respiratory chain complexes(RCCs),8-hydroxy-deoxyguanine(8-OhdG),malondialdehyde(MDA),and superoxide dismutase(SOD)were examined in cell lines with and without these mt-tRNA variants.RESULTS Compared with control cells,the m.A5514G variant caused an approximately 35%reduction in the steady-state level of mt-tRNA^(Trp)(P<0.0001);however,the m.C12237T variant did not affect the mt-tRNA^(Ser(AGY))steady-state level(P=0.5849).Biochemical analysis revealed that cells with both m.A5514G and m.C12237T variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells:ATP,MMP,NAD+/NADH ratio,enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells(P<0.05 for all measures).By contrast,the levels of ROS,8-OhdG and MDA were significantly increased(P<0.05 for all measures),but mtDNA copy number was not affected by m.A5514G and m.C12237T variants(P=0.5942).CONCLUSION The m.A5514G variant impaired mt-tRNA^(Trp)metabolism,which subsequently caused mitochondrial dysfunction.The m.C12237T variant did not alter the steady-state level of mt-tRNA^(Ser(AGY)),indicating that it may be a modifier of the m.A5514G variant.The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.

Single and Mitochondrial Gene Inheritance Disorder Prediction Using Machine Learning

One of the most difficult jobs in the post-genomic age is identifying a genetic disease from a massive amount of genetic data.Furthermore,the complicated genetic disease has a very diverse genotype,making it challenging to find genetic markers.This is a challenging process since it must be completed effectively and efficiently.This research article focuses largely on which patients are more likely to have a genetic disorder based on numerous medical parameters.Using the patient’s medical history,we used a genetic disease prediction algorithm that predicts if the patient is likely to be diagnosed with a genetic disorder.To predict and categorize the patient with a genetic disease,we utilize several deep and machine learning techniques such as Artificial neural network(ANN),K-nearest neighbors(KNN),and Support vector machine(SVM).To enhance the accuracy of predicting the genetic disease in any patient,a highly efficient approach was utilized to control how the model can be used.To predict genetic disease,deep and machine learning approaches are performed.The most productive tool model provides more precise efficiency.The simulation results demonstrate that by using the proposed model with the ANN,we achieve the highest model performance of 85.7%,84.9%,84.3%accuracy of training,testing and validation respectively.This approach will undoubtedly transform genetic disorder prediction and give a real competitive strategy to save patients’lives.

Three New Ranidae Mitogenomes and the Evolution of Mitochondrial Gene Rearrangements among Ranidae Species

Various types of gene rearrangements have been discovered in the mitogenoes of the frog family Ranidae. In this study, we determined the complete mitogenome sequence of three Rana frogs. By combining the available mitogenomic data sets from GenBank, we evaluated the phylogenetic relationships of Ranidae at the mitogenome level and analyzed mitogenome rearrangement cases within Ranidae. The three frogs shared an identical mitogenome organization that was extremely similar to the typical Neobatrachian-type arrangement. Except for the genus Babina, the monophyly of each genus was well supported. The genus Amnirana occupied the most basal position among the Ranidae. The [Lithobates ＋ Rana] was the closest sister group of Odorrana. The diversity of mitochondrial gene arrangements in ranid species was unexpectedly high, with 47 mitogenomes from 40 ranids being classified into 10 different gene rearrangement types. Some taxa owned their unique gene rearrangement characteristics, which had significant implication for their phylogeny analysis. All rearrangement events discovered in the Ranidae mitogenomes can be explained by the duplication and random loss model.

Mitochondrial DNA Markers for PCR-Based Phylogenetic Analysis of Ark Shells

Arcidae species are commercially important bivalves in Japan and are commonly referred to as bloody ark due to their red blood. They have thick shells with distinct radiating ribs, and the numbers of these ribs are important morphological features for species discrimination. However, some Arcidae species are morphologically indistinguishable, with a similar number of the ribs in adults and deficient rib formation, particularly among juveniles. Thus, we developed a reliable molecular marker to genetically discriminate between 7 Arcidae species belonging to Scapharca, Anadara, and Tegillarca based on species-specific polymorphic segments of mitochondrial DNA. PCR amplification of partial COI, 16S rRNA, 12S rRNA, and Cyt b genes was performed on 7 species using 8 primer sets. Only the set of Scapharca-specific forward primer and universal reverse primer for the partial COI gene successfully yielded single PCR products from all 7 species examined. Thus, nucleotide sequences of 481 bp portion of these PCR products were determined, and the degrees of nucleotide substitutions ranged from 0.4% between S. broughtonii and T. granosa to 20.2% between S. satowi and A. antiquata. In addition, a phylogenetic tree showed significant differences between 7 species, with higher bootstrap support than 69.

Frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among children with sensorineural deafness in China

Objective： To investigate the frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among Chinese with sensorineural deafness. Methods： Blood samples from 78 sporadic cases with sensorineural deafness were obtained and DNA was extracted from the leukocytes, then the mitochondrial DNA target fragments were amplified by polymerase chain reaction（PCR）. The 1555G mutations were detected by BsmA 1 restriction endonuclease digestion, every fragment was analyzed by sequencing; All the 961 insC mutation were detected by direct sequencing. Results： The percent age of A1555G mutation and mt961C insertion were 6.4% and 2.6% in the hearing-impaired Chinese subjects respectively. Conclusion： A1555G and 961insC mutations in mitochondrial DNA 12S rRNA gene regions may play a role in the pathogenesis of hearing loss in the sporadic cases.

Mitochondrial genome of Chthamalus challengeri(Crustacea:Sessilia): gene order comparison within Chthamalidae and phylogenetic consideration within Balanomorpha

Acorn barnacles are important model species in researches on intertidal ecology, larval development and biofouling. At present, with the development of mitochondrial genomics, it is helpful to understand the phylogenetic relationship from the mitogenomic level. The complete mitochondrial genome of Chthamalus challengeri was presented. The genome is a circular molecule of 15 358 bp. Compared with other species in Balanomorpha, the non-coding region is longer, while the length of the genes is similar to the other species. The overall A+T content of the mitochondrial genome of C. challengeri is 70.5%. There are variations of initiation and stop codons in the known Balanomorpha mitochondrial genomes. The C. challengeri and C. antennatus within the same genus share the identical gene arrangement. However, the gene arrangement of different genera in Chthamalidae is different, as there is a translocation between two tRNA genes and an inversion involving a large gene block. In particular, both srRNA and lrRNA of the two species in Chthamalus are encoded in the heavy strand, differing from the former Balanomorpha species. The topology and gene arrangement in Chthamalidae support each other. Phylogenetic analysis indicates that the Chthamalidae is monophyletic, while the Balanidae and Archaeobalanidae are polyphyletic.

Genomic Analysis of Mitochondrial Carrier Genes in the Bombyx mori

This study is performed to investigate the mitochondrial carrier gene family in silkworm genome. In total, 30 genes are identified and claded into eight well-conserved groups. Gene duplication contributes to the expansion and complexity of this family. Diverse expression patterns suggest their functional differentiation. Analyses of the sitespecific profiles reveal critical amino acid residues for functional divergence. This study highlights the molecular evolution of the mitochondrial carrier gene family in silkworm and may provide a starting point for further experimental verification.

Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

RNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins.This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other.DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10,wild abortive(WA)cytoplasm based male sterile(A line Pusa 6A),maintainer(B line Pusa 6B)and restorer(R line PRR78)lines.Pusa 6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines.The genomic and cDNA amplicons for eight mitochondrial genes(18SrRNA,atp6,atp9,cobII,coxI,coxIII,nadI and rps3)were sequenced and compared.Differences in genomic and cDNA sequences were considered as editing.Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in 18SrRNA(5.74%)and the lowest in coxI(0.60%).The highest editing sites were observed in fertile maintainer Pusa 6B followed by PRR78 and Pusa 6A,of which random five editing sites in five different rice mitochondrial transcripts namely atp9,cobII,coxIII,rps3 and 18SrRNA were chosen and validated through cleaved amplified polymorphism sequence(CAPS)analysis and found to be partially edited in four genes.The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.

Genetic Differences of Mitten Crabs Based on RFLP Analysis on Mitochondrial Cytochrome Oxidase Subunit I(COI) Gene

The genetic differences of 15 mitten crab populations from 6 river systems in China's Mainland and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I （CO I）. The results showed that Tas I-RFLP pattern could be used as a genetic marker to distinguish Eriocheir hepuensis from Eriocheir sinensis, Eriocheirjaponica and Eriocheir leptognathus; genetic distances among 13 populations of Eriocheir sinensis range from 0 to 0.015, indicating that they were different geographic strains; the subspecies status of Eriocheir sinensis and Eriocheir hepuensis （population from Nanliujiang） were considered owning to their genetic distances of 0.02-0.044, indicating that genetic divergence between them was low; Eriocheir leptognathus （population from Nanpaihe, Tianjin） was the most distant taxon with genetic distances value of 0.147-0.195, which could be defined as genetic distances between species in genus Eriocheir.

Complete mitochondrial genomes of two deep-sea pandalid shrimps,Heterocarpus ensifer and Bitias brevis:insights into the phylogenetic position of Pandalidae(Decapoda:Caridea)

The mitochondrial genome(mitogenome)analysis is a significant tool for investigating the evolutionary history of metazoan animals.The family Pandalidae is a diverse caridean group containing mainly deep-sea species.Until May 302019,only two complete mitogenomes are available in GenBank.Here we present the complete mitogenome sequences of two deep-sea pandalid shrimps,Heterocarpus ensifer and Bitias brevis through Illumina sequencing.The mitochondrial genomes were determined to be 15939 bp and 15891 bp long,and both consist of 13 protein-coding genes(PCGs),23 transfer-RNA genes(tRNAs),two ribosomal-RNA genes(rRNAs),and one control region.The nucleotide composition is biased toward adenine and thymine.Overall,the gene contents and arrangements are consistent with the pancrustacean ground pattern.The alignment of the control regions of four pandalids reveals a conserved sequence block(CSB)(104 bp in length,average GC%=29.47%and 69.23%similarity).A phylogenetic analysis based on 51 Caridea complete mitogenomes revealed that the deep-sea pandalid shrimps are situated an intermediate lineage,with a tendency to originated from those living in shallow sea area.

A phylogeny of the Passerida(Aves: Passeriformes)based on mitochondrial 12S ribosomal RNA gene

Background: Passerida is the largest avian radiation within the order Passeriformes. Current understanding of the high-level relationships within Passerida is based on DNA–DNA hybridizations; however, the phylogenetic relationships within this assemblage have been the subject of many debates.Methods: We analyzed the 12 S ribosomal RNA gene from 49 species of Passerida, representing 14 currently recognized families, to outline the phylogenetic relationships within this group.Results: Our results identified the monophyly of the three superfamilies in Passerida: Sylvioidea, Muscicapoidea and Passeroidea. However, current delimitation of some species is at variance with our phylogeny estimate. First, the Parus major, which had been placed as a distinct clade sister to Sylvioidea was identified as a member of the super family;second, the genus Regulus was united with the Sturnidae and nested in the Muscicapoidea clade instead of being a clade of Passerida.Conclusion: Our results were consistent with Johansson's study of the three superfamilies except for the al ocation of two families, Paridae and Regulidae.

STUDY ON THE RELATIONSHIP BETWEEN MITOCHONDRIAL GENE MUTATION AND LATENT AUTOIMMUNE DIABETES MELLITUS IN ADULTS

Objective.To identify the A3243G mutation of mi tochondrial(mt )DNA in patients with latent autoimmu ne diabetes mellitus in adults(LADA)of Han nationality in the northeast a rea of China.Methods.Seventy-nine diabetics of Han natio nality,whose families have resided in the northeast area of China for more than 3generations,we re divided into 3groups:Group 1(22cases of type 2diabetes with mater-nal inheritance history),Group 2(34cases of LADA),Group 3(23cases of type 1diabetes in adolesc ents).The A3243G of mt DNA was detected in th ese 79subjects with the method of PCR-RFLP.Results.None of the 79diabetics studied was p ositively identified for the A3243G mutation of mt DNA.Conclusion.The A3243G mutation of mt DNA might not be related to the onset of LADA in dia betic popula-tion of Han nationality in northeast area of China and there might not be close relationship between A3243G mut a-tion of mt DNA and autoimmunity.