亚口鱼科鱼类核DNA 18S-ITS1-5.8S序列比较分析

目前,对亚口鱼科鱼类的研究主要是在线粒体DNA 层次,如以线粒体DNA 的控制区和DN5/ 6 基因为分子标记,调查胭脂鱼种群间、种群内的遗传结构和遗传分化以阐明其多样性衰退的程度,以线粒体DNA 细胞色素b 基因以及16S 和18S 基因为分子标记探讨亚口鱼科鱼类的系统发育关系等。然而,线粒体DNA 只占鱼类遗传信息的极少部分,越来越多的学者希望以鱼类核DNA 为分子标记来检测遗传变异情况,其中,rRNA 基因受到普遍的关注。真核生物的rRNA 基因以串联重复方式存在,其中18S、5.8S、28S 组成一个转录元,其前体是由18S-ITS1-5.8S-ITS2-28S 剪切而来。本文以中国胭脂鱼为中心,比较了亚口鱼科6 属7 种在18S-ITS1-5.8S 序列变异情况,并以此序列为分子标记,构建了亚鱼科鱼类系统发育树。18S-ITS1-5.8S 既含有编码序列18S和518S,又含有非编码序列转录间隔区1(ITS1),适合用来分析比较亚口鱼科鱼类在核DNA 编码与非编码区域的遗传变异情况。       

Phylogenetic analysis of isolated biofuel yeasts based on 5.8S-ITS rDNA and D1/D2 26S rDNA sequences

The utilization of agro-industrial wastes such as whey as raw materials for the production of bio-ethanol is gaining importance as a result of the attractiveness of renewable fuel alternatives due to exhaustion of fossil fuel sources coupled with the positive impact to the environment. Here, we report the isolation of two Kluyveromyces spp. designated as BM4 and P41, able to produce ethanol as main fermentation product from fermenting whey. Three different molecular biological approaches including, the RFLP analysis of the 5.8S-ITS rDNA, the sequence of the 5.8S-ITS rDNA region and the sequence of the D1/D2 domain of the 26S rRNA gene were applied for accurate identification. While RFLP analysis of 5.8S-ITS region failed to accurate the differentiation between the two species, sequencing of this region and D1/D2 region of the 26S rRNA gene verified the identification. PCR amplification and sequence analysis of 5.8S-ITS rRNA and D1/D2 domain of the 26S rRNA genes revealed that the isolates BM4 and P41 were highly related to Kluyveromyces marxianus and Kluyveromyces lactis with homology of 99% for both. In addition, phylogenetic analysis indicated that both BM4 and P41 shared a cluster with K. marxianus and K. lactis, respectively. The fermentative performance of both strains on cheese whey to produce ethanol was evaluated at different parameters such as incubation temperature, initial pH, whey sugar concentrations, and yeast concentrations. Results show that the maximum ethanol productions achieved at pH 4.5 and 35 °C were 5.52% and 5.05% for K. marxianus and K. lactis, respectively. Our results demonstrated that K. marxianus and K. Lactis could be recommended for cheese whey bioremediation in the environment and produce renewable biofuel.     

The placement of South African strains of Beauveria in a phylogeny inferred from rDNA ITS1-5.8S-ITS2 sequences

This study aimed to confirm the identity of three strains of the entomopathogenic fungus Beauveria bassiana from South African soils and to investigate their phylogenetic relationship with non-indigenous strains from other geographic regions. Sequences of the rDNA ITS1-5.8S-ITS2 region of 23 strains were compared with the Genbank reference sequences of 20 other cosmopolitan strains. Fitch parsimony and neighbor-joining analyses of the ITS1-5.8S-ITS2 regions resolved the strains into two distinct clades and matched them to four species groups/lineages: Beauveria bassiana, B. cf. bassiana (pseudobassiana), B. brongniartii and B. caledonica. Two of the South African strains initially identified as B. bassiana grouped with B. caledonica, whereas the third strain was confirmed as B. bassiana. Because of the paucity of Genbank references for B. caledonica, we have designated the two South African B. caledonica strains as B. sp. aff. caledonica. Other reassignments included two strains from Norway, originally classified as B. bassiana, being grouped with B. brongniartii, and three of the B. brongniartii reference taxa from Brazil which were clearly placed in the B. bassiana clade. The study provides a first report of the presence of the B. caledonica lineage in Africa and confirms current Beauveria phylogenies inferred from molecular data.     

Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species.

The physical locations of the 18S-5.8S-26S rDNA sequences were examined in three sorghum species by fluorescence in situ hybridization (FISH) using biotin-labeled heterologous 18S-5.8S-26S rDNA probe (pTa71). Each 18S-5.8S-26S rDNA locus occurred at two sites on the chromosomes in Sorghum bicolor (2n = 20) and S. versicolor (2n = 10), but at four sites on the chromosomes of S. halepense (2n = 40) and the tetraploid S. versicolor (2n = 20). Positions of the rDNA loci varied from the interstitial to terminal position among the four accessions of the three sorghum species. The rDNA data are useful for investigation of chromosome evolution and phylogeny. This study excluded S. versicolor as the possible progenitor of S. bicolor.     

Isolation of culturable yeasts from market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes

AIMS: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. METHODS AND RESULTS: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. CONCLUSIONS: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.     

Molecular and cytological characterization of 5S rDNA in Oryza species: genomic organization and phylogenetic implications.

We investigated the molecular characteristics and chromosomal organization of 5S rDNA in the genus Oryza, including diploid and tetraploid species. A phylogenetic tree of Oryza species was constructed based on the non-transcribed spacer sequences of 5S rDNA, and some novel relationships were discovered. Specifically, comparative sequence analysis of 5S rDNA in several wild rice species showed unique characteristics inconsistent with the model of concerted evolution: (1) multiple distinct 5S rDNA types were detected within a species, leading to intraspecific divergence of 5S rDNA; (2) multiple identical 5S rDNA types were shared among species, resulting in interspecies clustering of 5S rDNA types; and (3) intraspecific nucleotide diversity was detected within a 5S rDNA class. Our results obtained by fluorescence in situ hybridization revealed that each rice species studied contained only one 5S rDNA locus with two hybridization sites, which were located on either chromosome 7 or chromosome 11. These results suggest that different 5S rDNA classes within the rice genome were arranged together and that one pair of 5S rDNA loci from a diploid progenitor of the tetraploid species might have been lost during evolution. Taken together, our data show that 5S rDNA in rice species is more informative at the gene level than at the chromosome level.     

Relationships amongOmphalotus species (Paxillaceae) based on restriction sites in the ribosomal ITS1-5.8S-ITS2 region

Genetic relationships for several species of the fungusOmphalotus were estimated by comparing the presence or absence of restriction sites in the ITS1-5.8S-ITS2 region of nuclear ribosomal DNA. Results placeO. olearius, O. subilludens andO. olivascens in a single clade.Omphalotus illudens, usually thought to be related toO. subilludens, was placed in a second clade, more closely related toO. nidiformis. Omphalotus mexicana is distinct from all other examinedOmphalotus species.     

5.8S rDNA variability in Allium species belonging to the third evolutionary group

Sequence analysis of 5.8S rDNA in 67 accessions of the subgenus Allium and six other subgenera belonging to the third evolutionary group of Allium genus (Friesen et al., 2006) was performed. Nucleotide substitutions in 5.8S rDNA sequences of Allium accessions were identified and studied for the first time. The probable secondary structure of 5.8S rRNA was constructed. It was shown that mutations in 5.8S rDNA do not involve conserved motifs, and they did not significantly affect the secondary structure of the RNA molecule in Allium accessions.     

绒泡菌目黏菌的ITS1-5.8S-ITS2二级结构的比较分析

为探讨核糖体DNA转录间隔区(r DNA ITS)的RNA二级结构在黏菌系统发育研究中的作用,以黏菌ITS通用引物PHYS4和PHYS5对绒泡菌目5属8种黏菌的r DNA ITS进行扩增和测序,利用RNA structure构建了ITS区的RNA二级结构模型。结果表明:ITS1在绒泡菌目黏菌中不能形成一个紧实的结构,但大部分物种都具有一段稳定的螺旋结构,可能对r RNA的成熟具有作用;5.8S r RNA的二级结构相似,由4个螺旋组成,主要为两种类型;基于5.8S r RNA和28S r RNA相互作用构建的ITS2的二级结构模型显示,它由一个封闭的多分支环和至少4个主要的螺旋组成,其中螺旋IV结构相对比较保守。由于ITS区的二级结构相比核苷酸序列更加保守,因此深入地分析其二级结构有助于认识其结构与进化的关系。     

Intraspecific phylogeography of Carchesium polypinum (Peritrichia, Ciliophora) from China, inferred from 18S-ITS1-5.8S ribosomal DNA

Based on the variation of site 34, 46, 241, 305 and 322 in the 18S-ITS1 rDNA sequence, 19 Carchesium polypinum populations collected from eight provinces of China were separated into northern and southern population along the delineation between the Yangtze River and the Pearl River. This geographic distribution pattern of Carchesium polypinum maybe results from two factors: the vicariance resulting from the formation of the delineation between the Pearl River and the Yangtze River accompanied with the uplift of Qinghai-Xizang Plateau, and the different dispersal paths of C. polypinum affected by the climate.     

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.     

Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East

The present study examined the molecular organisation and sequence variation in the nuclear ribosomal DNA (rDNA) region, including the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the Clonorchis sinensis from the Russian Far East. The relevant sequences from other parts of this species' area were downloaded from GenBank. The results showed 100% identity for all investigated 5.8S-ITS2 rDNA sequences. In contrast, two levels of intraspecific variations were revealed in the complete ITS1 sequences. The intra-genomic variation resulted from a C/T polymorphism in a single position. The inter-individual differences between the ITS1 sequences were both due to nucleotide and size polymorphisms resulting from a varying number of five-nucleotide repeats and followed by two ITS1 length variants. These variant frequencies correlate with the clonorchiasis level in some geographical localities. ITS1 differences, both in the mutation profile and mutation localisation, were revealed between northern and southern geographical samples. The presence of GC boxes that are identical to known regulatory motifs in eukaryotes was detected within the ITS1 sub-repeats. The predicted secondary structures for ITS1 consist of two large branches, one of which was invariable, while another depended on ITS1 length. The predicted secondary structure for ITS2 includes four helices around the core. The main differences between C. sinensis and other opisthorchids were localised on the tops of helices 2, 3, and 4. A phylogenetic MST reconstruction subdivided all ITS1 sequences into two well differentiated clusters, each with the major widespread ribotype, and showed that ribotype diversity in both Russia and Korea is much lower than in China. The results obtained demonstrate the feasibility of complete ITS1 sequences in C. sinensis population genetics and can be considered as a basis for further studies of the parasite infection because they may help to elucidate the molecular mechanisms of pathogen evolution and adaptation.     

Evolutionary insights inferred by molecular analysis of the ITS1-5.8S-ITS2 and IGS Avena sp. sequences

In an attempt to clarify phylogenetic and genome relationships among 35 diploid (A and C genomes), 13 tetraploid (AB and AC genomes) and 6 hexaploid (ACD genome) Avena taxa, 71 clones of the ITS1-5.8S-ITS2 fragment were sequenced, aligned and a network was constructed. In addition, the intergenic spacer (IGS) fragment was fingerprinted by means of a RFLP analysis using three different restriction enzymes. Both approaches led to comparable results. Clustering among the 54 Avena sp. entries was according to karyotype. Major genic divergence between the A and C genomes was revealed, while distinction among the A and B/D genomes was not possible. High affinity among the AB genome tetraploids and the A(s) genome diploid A. lusitanica was found, while AC genome tetraploids and ACD hexaploids were highly affiliated with the A(l) genome diploid A. longiglumis. The possible role of A. longiglumis in Avena sp. evolution is discussed.     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceol     

Concerted evolution of 18–5.8–26S rDNA repeats in Nicotiana allotetraploids

We review and extend data showing concerted evolution of parental 18–5.8–26S nuclear ribosomal DNA (18–26S rDNA) gene families in three natural Nicotiana allotetraploids ( N. tabacum , N. rustica and N. arentsii , each 2 n  = 4 x  = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2 n  = 24) × ♂ N. tomentosiformis (2 n  = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18–26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially ( N. tabacum, N. rustica ) or completely ( N. arentsii ) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris- derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 615–625.     

Phylogenetic position of yeast-like symbiotes from three rice planthoppers (Homoptera: Delphacidae ) based on 18S rDNA and ITS-5.8S rDNA sequences

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌( yeast-like symbiotes,YLS)的种属地位及与寄主的进化关系,测定了其体内YIS的18SrDNA及ITS-5.8S rDNA的全长序列.基于3种稻飞虱体内YLS的18S rDNA序列比对表明,褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLs的高(褐飞虱YLS和白背飞虱YLS为98.91％,灰飞虱YLS和褐飞虱YLS为95.74％,灰飞虱YLS和白背飞虱YLS为96.02％),而基于ITS-5.8S rDNA序列比对,灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高(白背飞虱YLS和灰飞虱YLS为99.57％,灰飞虱YLS和褐飞虱YIS为91.91％,白背飞虱YLS和褐飞虱YLS为90.46％).基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明,3种稻飞虱体内YLS与其他已知真菌进化关系较远.本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系,从而形成了不同于已知真菌的分类地位.     

三种稻飞虱体内类酵母共生菌18S rDNA和 ITS 5.8S rDNA序列克隆及进化分析

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、 褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌（yeast-like symbiotes, YLS）的种属地位及与寄主的进化关系, 测定了其体内YLS的18S rDNA及ITS-5.8S rDNA的全长序列。基于3种稻飞虱体内YLS的18S rDNA序列比对表明, 褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLS的高（褐飞虱YLS和白背飞虱YLS为98.91%, 灰飞虱YLS和褐飞虱YLS为95.74%, 灰飞虱YLS和白背飞虱YLS为96.02%）, 而基于ITS-5.8S rDNA序列比对, 灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高（白背飞虱YLS和灰飞虱YLS为99.57%, 灰飞虱YLS和褐飞虱YLS为91.91%, 白背飞虱YLS和褐飞虱YLS为90.46%）。基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明, 3种稻飞虱体内YLS与其他已知真菌进化关系较远。本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系, 从而形成了不同于已知真菌的分类地位。     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

血卵涡鞭虫核糖体18SrDNA和ITS1-5.8SrDNA-ITS2序列测定与系统发育分析

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