Generic allocation of Indian and Sri Lankan Philautus (Anura: Rhacophoridae) inferred from 12S and 16S rRNA genes

The generic allocation of Indian and Sri Lankan Philautus needs further examination. In this study, a comprehensive understanding of the phylogeny of Indian and Sri Lankan Philautus is obtained based on 125 and 16S rRNA genes. All phylogenetic analyses indicate that Indian-Sri Lankan Philautus, Philautus menglaensis, Philautus longchuanensis, and Philautus gryllus form a well supported clade, separate from Philautus of Sunda Islands that form another well supported clade representing true Philautus. This result supports the designation of the genus Pseudophilautus to accommodate the Indian and Sri Lankan species. Pseudophilautus consists of two major lineages, one comprises the majority of Indian species, Chinese species, and Southeast Asian species, and one comprises all Sri Lankan species and a few Indian species. Pseudophilautus may have originated in South Asia and dispersed into Southeast Asia and China. Based on the results, we further suggest that Philautus cf. gryllus (MNHN1997.5460) belongs to the genus Kurixalus. (C) 2010 Published by Elsevier Ltd.

Reevaluation of the phylogenetic relationship between Mobilid and Sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

木根麦冬rRNA基因进化的研究

木根麦冬（Ophiopogon xylorrhizus Wang et Dai）属于铃兰科（Convallariaceae）或广义百合科（Liliaceae s.l.）沿阶草族（Ophiopogoneae）沿阶草属（Ophiopogon Ker-Gawl.），属于典型的濒危植物。前人已从细胞学、种群生态学、生殖生物学和遗传结构与多样性等方面对木根麦冬进行了研究，但在分子进化和分子细胞遗传学水平上的研究近为空白。本文运用染色体的荧光原位杂交(FISH)、PCR扩增和克隆、DNA测序、系统发育重建等方法，对18S rDNA作了染色体原位定位，研究了木根麦冬的Ss rRNA基因结构特点，并重建了该基因的系统发育树，探讨了5S rRNA多基因家族的分子进化模式和木根麦冬的濒危机制。主要结果如下： 1．对木根麦冬三个居群七个个体、及其最近姐妹种林生麦冬（Ophiopogon svlvicola Wang et Tang）一个个体的5S rRNA基因进行了PCR扩增和TA克隆，在两个种中共得到1085个具有插入片段的阳性克隆。 2．对木根麦冬三个居群六个个体的294个SS rRNA基因克隆，及林生麦冬一个个体的45个克隆，总计339个克隆进行了DNA序列测定，这是目前已完成的最大的单个物种的5S rRNA数据。结果表明：两个种的序列高度多样化，在339个拷贝中仅仅有13对(3.8%)是相同的，序列长度变化在307bp-548bp之间，长度变异主要发生在间隔区，单个碱基的插入和缺失(indel)频率很高，5bp以上片段的插入，缺失有11个，插入的序列通常是其两侧序列的重复和倒位。术根麦冬序列的分化指数（sequence differentiation index，SDI）是0.078，林生麦冬是0.032，两个物种间是0.149，木根麦冬的序列之间的分化明显大于林生麦冬。 3．以PAUP程序对339个5S rRNA基因拷贝的DNA序列（包括编码区和间隔区）作了系统发育分析，结果如下：在得到一个唯一的最俭约树中，所有木根麦冬的拷贝被聚成一支，而林生麦冬的则被聚到另一支，统计支持率(bootstrap)达到lOO%，表明这两个物种所有的的5S rRNA基因拷贝分别来自各自的一个祖先拷贝（建立者拷贝），而其共同祖先的其它拷贝则在物种形成中或之后丢失：在多基因家族中如此长期而单一的拷贝偏选( sorting)过程尚未有前人报道;由此基因系统发育树可以看出，在这两个物种形成之后，“建立者拷贝”经历了多次扩增过程而形成了一个直系的（orthologous）多基因家族。 4．在木根麦冬分支中，很少有亚分支是全部由一个居群或一个个体的拷贝组成的，不同居群、不同个体的拷贝混合在同一个亚分支中;对基因系统发育树、序列多样性和序列分化指数分析表明，5S rRNA基因家族内一致化（homogeruzation）过程很弱，不同拷贝是独立进化的，这在串联．重复的多拷贝基因家族中是不寻常的;由上述分析我们推测，在术根麦冬的进化历史上，居群间的基因交流远远比今天频繁，可能是某些外在因素在近期发生变化，导致自交和自交衰退，并进而导致濒危。 5．利用荧光原位杂交技术，成功地将18S rRNA基因定位在木根麦冬减数分裂期的染色体上，两对强信号和一对弱信号分别位于三对二价体染色体上。

松属植物rRNA基因的变异模式及其进化生物学意义

被子植物的rRNA基因已经得到深入研究。二倍体被子植物一般拥有1-4对18S-5.8S-26S rDNA位点和1-2对5S rDNA位点。作为特殊的多基因家族成员，rDNA会受均一化力 (homogenizing forces) 的作用，通过基因转换、不等交换等机制，形成基因的致同进化 (concerted evolution)。长期以来，我们一直认为动植物rDNA致同进化水平很高，各种拷贝的序列几乎完全一致，因此可以直接应用PCR测序的方法进行分子系统学研究。但是在裸子植物中由于研究资料的匮乏，使我们对裸子植物rDNA的变异模式了解甚少。松属植物作为裸子植物的最大类群，它的rDNA变异和进化有何特点、与被子植物是否相同，是这个重要类群的进化研究中目前尚未解决的问题。本文的研究内容从三个方面进行： (1)rDNA的染色体定位 目前，松属的18S-5.8S-26S rDNA的染色体定位研究只包括5种植物，其中的3种同时涉及到5S rDNA定位。这些研究结果表明，不同种存在相异的rDNA位点数目，甚至不同的个体的rDNA位点均有变化。其共同点是，18S-5.8S-26S rDNA位点数平均较被子植物多，5S rDNA除Pinus radiata外，在其它种里则与被子植物相似。这种现象是松属或裸子植物的共同特征，亦或是特例呢？有限的研究限制了对裸子植物rDNA的了解。本研究的目的之一就是研究松属植物rDNA的染色体空间分布特征，希望借此了解松属植物间的关系，比较裸子植物和被子植物rDNA在染色体组水平的差异。 (2)5S rDNA的分子进化 5S rDNA的序列水平的进化研究在松属中尚属空白。5S rDNA在染色体数目上没有显示裸子植物与被子植物的差异，是否意味着松属乃至裸子植物的5S rDNA也同被子植物一样——致同进化完全，序列高度一致呢？利用克隆测序方法对松属植物5S rDNA的研究无疑是有开创性的工作，可以探讨裸子植物的5S rDNA的进化机制和种间关系。 (3)杂种基因组研究 杂交物种的起源演化是当前生物学研究的热点，通过杂种基因组的研究，可以了解杂种的的基因组构成，组织方式和进化历史，探讨杂交事件对成种过程的影响及意义。这项研究涉及到高山松、云南松和油松。之所以采用这三种植物，因为等位酶、cpDNA和mtDNA证据证明高山松为油松和云南松的自然杂交种。但这些证据不足以反映杂种核基因组的重组特征和构成及其进化规律。我们利用rDNA-FISH、5S rDNA和基因组原位杂交分析三种松树间的基因组关系，为揭示高山松的进化机制和历史提供新的依据。 本项研究得到以下结果： 一. rDNA荧光原位杂交 (FISH) 通过对华山松和白皮松两种单维管束亚属植物及油松、云南松、高山松、马尾松和南亚松等五种双维管束亚属植物的18S rDNA与5S rDNA的荧光原位杂交，结果表明： ⑴ 裸子植物的18S rDNA位点数目明显多于二倍体被子植物。其中主要位点数目，油松有7对，高山松5对，云南松8对，马尾松10对，南亚松6对，白皮松3对，华山松10对，平均在7对;另外，部分松树还存在弱位点。无论强弱位点都有部分存在于染色体的着丝粒区，除了赤松 (Pinus densiflora)，在其它松科植物中并没有发现这种现象。究竟是基因转移的结果或该位点是18S rDNA的原始起源位置还有待确证。 ⑵ 5S rDNA位点相对变异较小，与被子植物相当。除了华山松5S rDNA有4对位点，马尾松只有1对位点外，其它松树的5S rDNA位点数目均为2对，并且在双维管束亚属植物中有一对属于弱位点。 ⑶ 两种rDNA存在不同连锁模式。双维管束亚属植物中，5S与18S rDNA连锁在同一染色体的同一臂或两条臂上。在同一染色体臂时，18S rDNA在臂的远端。单维管束亚属植物的5S与18S rDNA或连锁于同一染色体的同一臂上，或分别处于不同染色体。前一情况，5S rDNA位于臂的远端。据此可以说明两个亚属的rDNA结构在染色体组水平的很大分化。 ⑷ 松属植物的关系及高山松核型特征。由于5S与18S rDNA连锁关系的不同，可以将单维管束亚属和双维管束亚属分开。各亚属的不同物种可以依据杂交位点的多少、位置、信号强弱构成的核型图加以区分，并且构成一定的系统关系。杂交起源的高山松在染色体组上，表现出对油松和云南松两亲本不同染色体特征的分别继承与重组，并产生独有的特征。其II同源染色体之一18S rDNA位点的缺失，可能是染色体重组的痕迹。 二. 5S rDNA的序列变异与分子进化 利用分子克隆和DNA测序分析了油松、云南松、马尾松、白皮松和不同遗传背景的高山松居群的5S rRNA基因序列变异及基因进化规律，得到以下主要结果： ⑴ 5S rDNA的结构特征。双维管束亚属植物长度在658-728 bp，白皮松则为499-521 bp。长度差异体现在基因间隔区，而基因区极端保守，基本为120 bp。基因转录区内部存在着转录控制区，决定了5S rRNA的转录起始与转录效率。5S rRNA基因能够折叠成正常的二级结构，其中，相对于干区来说，环区要保守，但环E却表现出异乎寻常的变异，转换/颠换比值高达7.1，这种突变可能是假基因的产物。基因间隔区存在一定的保守单元，其中一些与转录的起始和终止调控相关，有些是裸子植物未知功能的特异保守区。 ⑵ 松属植物5S rDNA存在着基因组内与种间的异质性。基因组内的各个克隆中有超过80%的特异的，彼此不相同。整个5S rDNA分化距离为0.042 - 0.051，其中，间隔区的分化比基因区高，其速度约是基因区的3-7倍。比较种间5S rDNA序列发现：在122个克隆中，基因区只有50个特异的序列。基因组间的序列变异度与基因组内 (个体内) 没有明显差别。白皮松的间隔区与双维管束亚属松树的5S rDNA间隔区差异极大，几乎不能排序，而四种双维管束亚属植物的5S rDNA间隔区种间种内差异不大。 ⑶ 松属植物5S rDNA进化。PAUP分析建立的5S rRNA基因树显示，5S rRNA基因在基因组内是多系的 (polyphyletic)，表明成种事件以前，祖先种就已经存在序列的分化。观测到的5S rRNA基因序列变异状况，并非完全是致同进化或独立进化的单一因素造成的，而是二者的相互作用的结果。致同进化确实存在，只是速度较慢而已。 ⑷ 高山松5S rDNA 组成。高山松拥有最高的基因组内的序列多样性，高山松的5S rDNA拷贝既有亲本类型，又有重组类型，并且不同地理及遗传来源的高山松显示一定的分化趋势，有更多的拷贝来自母系亲本。 三. 基因组原位杂交 以油松和云南松总DNA作为探针，相互进行基因组原位杂交，结果显示云南松和油松的染色体组可以完全被对方探针标记，在现有基因组原位杂交的分辨率下不能将两个基因组区分开。说明云南松和油松基因组之间存在高比例的同源序列，两种松树的基因组组成十分相似。利用油松和云南松总DNA作为探针，对高山松的染色体组进行双探针基因组原位杂交。结果表明，高山松全部基因组都能与两亲本探针完全杂交，说明三者间有着异乎寻常的亲缘关系。但在PH失调影响下，高山松只有部分基因组被杂交，并且两种探针的杂交信号有轻微差异。这可能是高度重复序列优先杂交的结果。这些情况表明，高山松虽然在基因组构成上与两个亲本基本一致，但基因在染色体组的空间排布上是存在差异的，这一点可以从rDNA-FISH中证明。

Identification of Sarcocystis hominis-like (Protozoa : Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences

DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single

Phylogenetic analysis of the family Thermaceae with an emphasis on signature position and secondary structure of 16S rRNA

The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis. The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17). The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept. Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the alpha-, gamma- and delta-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13A degrees N.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Chromosomal mapping of the major ribosomal RNA genes in the dwarf surfclam (Mulinia lateralis Say)

Chromosomal location of the major ribosomal RNA genes (rRNA) were studied in the dwarf surfclam (Mulinia lateralis, Say) using fluorescence in situ hybridization (FISH). FISH probes for the rRNA genes were made by polymerase chain reaction (PCR), labeled with digoxigenin-11-dUTP and detected with fluorescein-labeled antidigoxigenin antibodies. Mulinia lateralis had a diploid number of 38 chromosomes and all chromosomes were telocentric. FISH with the rRNA probe produced positive and consistent signals on two pairs of chromosomes: Chromosome 15 with a relative length of 4.6% and Chromosome 19, the shortest chromosome. Both loci were telomeric. The rRNA location provides the first physical landmark of the M. lateralis genome.

Phylogenetic relationships among Rhacophorinae (Rhacophoridae, Anura, Amphibia), with an emphasis on the Chinese species

The partial nucleotide sequence of mitochondrial 12S and 16S rRNA genes was determined for 23 Chinese species of Rhaeophoridae (Amphibia: Anura), representing four of the eight recognized genera. Using Buergeriinae as the outgroup, phylogenetic analyses (

PCR-DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were characterized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communities, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eukaryotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE-patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the concentration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR-DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations.