红莲型细胞质雄性不育水稻线粒体DNA的AP-PCR分析

为了研究红莲型细胞质雄性不育与线粒体基因组的关系。以水稻红莲型粤泰细胞质雄性不育系A和保持系B及杂种一代F1为材料。应用AP-PCR分析,用10个单引物对其线粒体DNA进行扩增。实验结果表明,不同的引物在3种材料间均有不同程度的差异。为红莲型细胞质雄性不育分子机理的研究提供了线索;此外,在引物6F1的扩增图谱中找到一条在YTA和F1中特异的带TAF6F2,Sounthern分析TAF6F2不育胞质的特异性,可能与红莲型水稻细胞质雄性 不育性状的形成有关。     

BT型细胞质雄性不育水稻及其三系的线粒体DNA研究

用RAPD技术对BT型水稻胞质雄性不育系秀A及其保持系秀B、恢复系湘晴以及杂种F1代的线粒体DNA进行了比较分析。结果表明不育系与其保持系间存在显著差异;不育系与其F1之间mtDNA也存在差异。在引物OPJ－08的扩增产物中,秀A扩增出一条分子量为800bp的多态性片段,在引物OPK－10的扩增产物中,杂种F1扩增出一条分子量为900bp的片段。把这两片段回收、克隆并制备探针,OPJ－08800的Southern杂交结果显示不育系与其F1杂交图谱存在多态性;OPK－10900的Suthern杂交结果显示不育系与其保持系同存在差异。推测这两片段与育性可能有一定的联系。     

Mitochondrial DNA-like sequence in the nuclear genome of an akodontine rodent.

Initial amplification and sequencing of a 366-bp fragment of the cytochrome b gene by a conserved primer pair (MVZ 03 and MVZ 04) revealed a nonfunctional copy of the gene with two deletions (one of which is 17 bp in length and the other of which is 3 bp in length) in Chroeomys jelskii, a South American akodontine rodent. By means of an alternative primer to MVZ 03--namely, MVZ 05--from the region of the tRNA for glutamic acid, a functional copy of cytochrome b was subsequently amplified. Both primer pairs amplify functional sequence when applied to purified mitochondrial DNA (mtDNA). Restriction-endonuclease digestion of purified mtDNA from C. jelskii did not reveal any additional sets of bands that would suggest heteroplasmy in the mitochondrial genome. When probed with both functional and nonfunctional gene fragments, MboI restriction digests revealed the same pattern, providing further evidence that the nonfunctional copy must be located in the nucleus. Observed differences in the mitochondrial and nuclear sequences from two populations are consistent with a faster rate of change in mtDNA than in nuclear DNA.     

RAPD标记鉴定水稻3种不同细胞质雄性不育类型的杂交种及其亲本

利用RAPD引物对3种不同细胞质雄性不育类型的杂交水稻组合及其亲本共21个材料进行了DNA多态性分析。从264个随机引物中筛选出具有非常明显多态性的引物25个, 对25个引物在3种不同细胞质不育类型的杂交组合及其亲本间的DNA扩增多态性差异进行比较,最终选出具有不同类型间特异性扩增带的引物7个,利用这些特异性扩增带能有效地区分和鉴定目前在生产上大面积种植和推广,或者是具有应用潜力的3种不同细胞质雄性不育类型—野败型(WA)、红莲型(HL)和包台型(BT))—的6种杂交水稻组合及其亲本。     

红莲型杂交稻(红莲2号)及其骨干亲本的RAPD分析与鉴定

利用RAPD技术,从248个随机寡核苷酸引物（10－mer）中筛出18个引物对红莲型杂交稻组合红莲2号及其亲本（T－07A、T－07B、YD6－05）,另6个红莲型胞质不育系的骨干恢复和汕优63及其亲本共14份水稻材料进行分析。共检测到173个多态性标记。聚类分析结果表明：不育系与保持系间因核背景相似,遗传差异很小;杂种（F1）的基因型更倾向于恢复系;恢复系与保持系间遗传距离的相对较大,但各恢复系之间的遗传距离较小。利用这些标记能有效地地区交组合中不育系,保持系、恢复系和杂种（F1）。     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.     

Multiple populations of deleted mitochondrial DNA detected by a novel gene amplification method

A gene amplification method for detecting small populations of deleted mitochondrial DNA was used in analysis of skeletal muscle from a patient with ocular myopathy. Multiple populations of differently deleted mtDNA were detected in the patient muscle. The presence of deleted mtDNAs was further confirmed by comparison of the shift in the sizes of the amplified fragments with the shift in the positions of the primers used for the amplification, (the primer shift PCR method). Other methods, namely Southern blotting, enzymic activity measurement, and Western blotting, were inefficient at detecting the mitochondrial abnormality. These findings suggest that the primer shift PCR method could be valuable for accurate diagnosis of ocular myopathy associated with mtDNA deletion.     

A set of 35 consensus primer pairs amplifying genes and introns of plant mitochondrial DNA

Being generally uniparentally inherited, the plant mitochondrial genome is a source of original markers potentially useful for studies of phylogeny and population genetics. We designed 24 new pairs of plant mitochondrial DNA primers that allow amplification of either introns, intergenic regions or genes. They have been defined for consensus over angiosperms and were tested along with 11 previously described mitochondrial primer pairs on 28 plant species representing 19 families of higher plants. The total set allows amplification of 40 kb (～11%) of the mtDNA genome of Arabidopsis thaliana. The amplification rate ranged between 76% and 100% depending on the species.     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

Unusual mitochondrial genome structure of the freshwater goby Odontobutis platycephala: rearrangement of tRNAs and an additional non-coding region

Mitochondrial DNA was isolated from the Korean freshwater gobioid fish Odontobutis platycephala by long-polymerase chain reaction with conserved primers and this mtDNA was sequenced by primer walking using flanking sequences as sequencing primers. The resultant O. platycephala mtDNA sequence was found to be 17 588 bp in size with a mostly conserved structural organization when compared with that of other teleost fish. Rearrangements of tRNAs (tRNA-Ser, tRNA-Leu, tRNA-His) and an additional non-coding region (533 bp) were present between the ND4 and ND5 genes. In the present paper, the basic characteristics of the O. platycephala mitochondrial genome is reported, including its structural organization, base composition of rRNAs, tRNAs and protein-encoding genes, characteristics of mitochondrial tRNAs and the peculiar rearrangement features of some parts of the mtDNA. Phylogenetic analysis performed using the cytochrome b gene sequences of 16 Korean freshwater fishes (15 gobioids) with the Bayesian algorithm showed that O. platycephala forms a clade (1·00 of posterior probability) with other species of Odontobutis . This suggests that the observed rearrangement between the ND4 and ND5 genes in the O. platycephala mitogenome reflects independent events.     

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome. Received: 10 September 1998 / Accepted: 24 February 1999     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

暗纹东方鲀线粒体基因组核苷酸全序列测定与分析

以暗纹东方鲀(Takifugu fasciatus)肝的线粒体DNA为模板,参照红鳍东方鲀(T.rubripes)等近源鱼类的线粒体基因组DNA序列,设计合成14对特异引物,进行PCR扩增并测序,首次获得了暗纹东方鲀线粒体基因组全序列。结果表明,暗纹东方鲀线粒体基因组序列全长16 444 bp(GenBank登录号为GQ409967),A+T含量为55.8%,其mtDNA结构与其他脊椎动物相似,由22个tRNA基因、2个rRNA基因、13个蛋白质编码基因和1段819 bp非编码的控制区(D-loop)所组成。蛋白质基因除COⅠ和ND6的起始密码子为GTG、CCT以外,均为典型的起始密码子ATG。ND1、ATPase8、COⅢ、ND4L、ND5、Cyt b使用典型的终止密码子TAA,其他的使用不完全终止密码子。除ND6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测定的鲀类一致,这显示了鲀类线粒体基因排列顺序上的保守性。tRNA基因核苷酸长度为64～73nt,预测了22个tRNA基因的二级结构,均呈较为典型的三叶草状。基于19种鲀类mtDNA全序列构建的进化树表明,暗纹东方鲀与红鳍东方鲀、中华东方鲀(T.chinensis)聚成一个姊妹群。结果还支持东方鲀属鱼类为一单系类群。     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.     

Primer design for Whole Genome Amplification using genetic algorithms

Whole Genome Amplification (WGA) is an important process to increase limiting amounts of genomic DNA prior to genomic analyses. Current amplification methods based on primer extension or strand displacement principles employ primers of partially or totally random sequence. In this paper, we present a method using Genetic Algorithms to optimize a single primer design to be used in a primer extension reaction to achieve unbiased WGA. Computational simulation and prediction of a suitable primer proposed two candidates NYP6-1 (ATCTCA) and NYP6-2 (TGAGAT). NYP6-1 amplified to a maximum length of 2537 base pairs (bp), had genome coverage of approximately 45.62%, with an average of 493 and variance of 163 amplicons per 1 megabasepairs (Mb). NYP6-2 amplified to a maximum length of 2926 bp and covered 54.35% of the genome with an average of 579 and a variance of 191 amplicons per Mb. In contrast, the original primer used in Degenerate Oligonucleotide-Primed PCR (DOP-PCR) had coverage of 20.93%, an average of 74 and variance of 188 amplicons per Mb when extended up to a length of 2000 bp. Successful WGA of miniscule amounts of genomic DNA requires the amplification method used to resolve issues on efficiency, accurate representation of the whole genome and ability to degraded DNA. The sequence NYP6-2 discovered using our method can be confidently used in a primer extension based protocol to perform quantitatively unbiased WGA.     

青花菜细胞质雄性不育系线粒体DNA的提取与RAPD分析

利用分子生物学方法提取青花菜不育系及其保持系线粒体的DNA,经琼脂糖凝胶电泳表明所提取的线粒体DNA纯度较高。通过40个引物进行随机引物多态性扩增,结果表明,由引物S66扩增出一条1 000 bp左右的特异条带BMS1000,该条带是不育系m tDNA所特有的,初步推断该特异条带可能与不育性相关。     

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish,Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.     

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.