6种中国特有闭壳龟的人工驯养繁育种群状况

2003年1月～2006年6月期间,调查了6种中国特有闭壳龟的人工驯养繁育种群状况。结果显示,三线闭壳龟（含越南三线闭壳龟和中国三线闭壳龟）的人工驯养繁育种群数量最多,种龟8000～10000,其中30％～40％是中国三线闭壳龟;金头闭壳龟134只,潘氏闭壳龟49只;百色闭壳龟39～44只,周氏闭壳龟29只;云南闭壳龟最少,仅2只。6种中国特有闭壳龟中,除中国三线闭壳龟外,其他种类人工驯养繁育种群数量均不超过50～150只;没有形成稳定的人工种群数量。     

基于线粒体Cyt b基因的全长序列探讨闭壳龟类的系统进化

采用PCR技术对淡水龟科具闭壳结构的黄缘盒龟、黄额盒龟、金头闭壳龟、潘氏闭壳龟、锯缘龟和白腹摄龟的线粒体Cytb基因的全长序列进行了PCR扩增和序列测定,并结合GenBank中16种淡水龟科物种的同源序列,进行了序列变异和系统发生分析。经C lustalX1.8软件对位排列后共有1154个位点,其中可变位点413个,简约信息位点301个;A＋T的平均含量（56.5%）高于G＋C（43.5%）。在氨基酸密码子中,第一位富含A,第二位富含T,第三位富含C;碱基转换/颠换率为5.97,碱基替换多发生在密码子第三位。以中华鳖和马来鳖为外群,通过最大简约法,最大似然法和贝叶斯法重建了分子系统树,均具有一致的拓扑结构,结果表明：金头闭壳龟和潘氏闭壳龟最先聚成一支,再和三线闭壳龟聚成一组,说明形态上相似的三种闭壳龟亲缘关系最近;闭壳龟属、盒龟属和单种锯缘龟属聚成一个单系的闭壳龟群,建议合并为闭壳龟属;齿缘龟属和果龟属聚为一支,它们与新的闭壳龟属关系较远,揭示闭壳结构的形成不是由一个共同祖先分化而来;乌龟属、花龟属和拟水龟属三属为并系起源,建议三属可以合并为一属。     

越南三线闭壳龟、中国三线闭壳龟和金头闭壳龟的新亚种（英文）

最近,遗传学研究证明了越南三线闭壳龟Cuoro cyclornata的有效性,并表明这个种内还存在第3个尚未描述的亚种,这个亚种也能从体色和形态量度上与以前认定的2个亚种相区分。同时,也证明中国三线闭壳龟C.trifasciata有一个来自中国海南岛在遗传学上存在歧化的种群。此现象也存在于金头闭壳龟C.aurocapitata,很久以来就发现该种有一个在体色量度上明显不同的种群,并得到了微卫星标记和形态学证据的支持。以上两种方法都显示了中国三线闭壳龟、越南三线闭壳龟和尚未描述的亚种之间的区别。为此,本文将这3个种群分别描述为新亚种C.cyclornata annamitica,C.trifasciata luteocephala和C.aurocapitata dabieshani。     

真菌通用引物Its1和Its4在丝状真菌鉴定中的价值评价

目的 对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.方法 收集中山大学附属第一医院2012年1月至2012年8月间分离的丝状真菌11株,使用真菌通用引物Its1和Its4采用PCR法扩增核糖体基因,对PCR产物进行序列测定,将测序结果与GenBank中已知标准或临床菌株DNA序列比对,确定丝状真菌的菌种;同时与传统形态学鉴定方法进行比较,从而对真菌通用引物Its1和Its4在丝状真菌鉴定中的价值进行评价.结果 从DNA提取到序列测定结束,在2个工作日内即可完成.所有菌株均测序成功,测序结果与GenBank中的序列比对,烟曲霉、杂色曲霉、橘青霉、溜曲霉可以鉴定到种;黄曲霉和米曲霉、阿姆斯特丹散囊菌和冠突散囊菌由于高度同源性无法区分鉴定到种.结论 利用真菌通用引物Its1和Its4结合PCR技术和测序技术,可快速、准确将大部分丝状真菌鉴定到种.     

周氏闭壳龟的人工繁殖

笔者作为龟类养殖爱好者,在北京家中饲养闭壳龟属龟类十余年.于2000年12月经出售人自广西购得成体周氏闭壳龟Coura zhoui 1对.购得时公龟有严重残疾,两后肢均无后脚掌,母龟正常.几年来与三线闭壳龟、潘氏闭壳龟以及金头闭壳龟混养,养殖池内设有产卵场,平时投喂小鱼、小虾、猪内脏、蟋蟀和香蕉,每周1次,以吃饱为准.     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件...     

中药杜仲原植物的分子鉴定

杜仲是我国特有贵重中药材,市场供不应求,各地出现许多伪品,为克服目前仅依据形态、显微特征及理化性状进行鉴别的不足,本研究旨在运用分子标记技术鉴定中药杜仲Eucommia ulmoides Oliv的真伪。采用PCR产物直接测序法测定杜仲原植物matK基因序列,通过Clustal软件将其与GenBank中同源序列进行排序比较,分析杜仲序列特征。结果:测定的杜仲matK序列长度为1140bp,同源序列比较分析表明,杜仲matK序列中存在32个特异性位点,其中特异性A、T、C、G位点分别为8,2,11,11,序列中有一个GAC插入为杜仲所独有。matK基因是良好的分子标记,能够为杜仲原植物鉴定提供足够的特异性变异位点,matK基因序列的测序分析可成为杜仲正品鉴定的有效手段。     

新一代DNA测序技术给农业育种带来革命

利用新一代DNA测序技术,可以高效、廉价地测定所有重要农业生物的基因组序列,测定农业核心种质的基因组序列,全面了解农作物遗传变异情况,进而大规模地鉴定、克隆功能基因.基于新一代DNA测序技术的种质基因组学,将实现农业育种由经验依赖型向知识决定型的转变,给农业发展带来革命.我国应该把握这个稍纵即逝的历史机遇,树立我国农业育种强国的地位.     

纳米孔测序技术在实蝇类害虫检疫鉴定中的应用

【目的】纳米孔测序技术是单分子实时测序技术的一种,正在被广泛应用于临床快速诊断及微生物检测等领域。本研究以实蝇这一类重要的检疫性有害生物为例,探究该技术在昆虫检疫鉴定中应用的可行性,为昆虫检疫鉴定提供新方法。【方法】分别采用一代测序技术和纳米孔测序技术,对14种经形态学鉴定的实蝇成虫进行DNA条形码测序,通过BOLD和NCBI数据库对测序结果进行比对分析,并比较2种测序技术所得序列结果准确性的差异。【结果】通过纳米孔测序,14个实蝇样品在44 min内获得181 Mb bases,每个样品平均得到11280条reads,单个reads的准确度为92.10%~94.53%;经一致性序列校正,所有实蝇样品均可得到与一代测序结果完全一致的序列,序列分析结果与形态学鉴定结果完全一致。【结论】采用本研究的实验流程和数据分析方法,纳米孔测序技术可以应用于实蝇类害虫的检疫鉴定,测序结果准确、高效;本研究提供的实验方案同样适用于基于扩增子测序的物种鉴定,满足大规模样品的高通量精准鉴定需求。     

前沟藻18S rDNA序列克隆和分子鉴定分析

旨在扩增一株未知微藻18S rDNA,并进行序列分析和物种鉴定分析.提取该微藻总DNA,用PCR技术扩增其18S rDNA,对序列进行测序,在GenBank进行序列比对分析,用ClustalX软件构建进化树;用光学显微镜进行形态学分析.经测序其序列长度为1 736 bp,G+C为47％;同源性分析表明,与GenBank中多个强壮前沟藻种的18S rDNA基因序列同源性迭99％;与前沟藻属的其它藻种同源性均迭95％以上.而与裸甲藻属和环藻属的藻株同源性为85％左右.经光学显微镜观察,该藻具有典型的强壮前沟藻形态.结合该藻18S rDNA序列分析和粗略的形态学分析,推断该藻可能为前沟藻属的强壮前沟藻.微藻形体微小,结构简单,需借助电子显微镜才可准确地从形态学上签定其种属,鉴定工作繁琐费时;而利用分子技术,则可能使鉴定工作变得简单快捷.     

三线闭壳龟的人工保育

三线闭壳龟为集药用、观赏和食用于一身的珍稀动物,其自然资源日益枯竭,开展人工保育具有重要意义。人工保育措施主要包括：（１）提供良好的自然生态环境;（２）采用人工孵卵;（３）加强饲养管理;（４）做好敌害与病害的防治工作。     

Molecular phylogeny of the critically endangered Indochinese box turtle (Cuora galbinifrons)

The Indochinese box turtle Cuora galbinifrons is a polytypic, critically endangered species from Vietnam, Laos, and Hainan Island, China. We analyze up to 1790bp of mitochondrial DNA under maximum parsimony and maximum likelihood criteria to test if the five historically recognized subspecies represent evolutionary lineages, and to elucidate the relationship of C. galbinifrons to other Cuora. C. galbinifrons is composed of three major mitochondrial DNA clades corresponding to the three subspecies galbinifrons, bourreti, and picturata. These three lineages are also morphologically diagnosable, and consequently we recommend elevating each to full species. Cuora galbinifrons hainanensis nests within the galbinifrons clade, and we retain it as a synonym of galbinifrons, as supported by morphology. Cuora "serrata" is known to be a hybrid of male Cuora mouhotii and female C. galbinifrons, and our findings show that C. "serrata" originates from both female galbinifrons and bourreti. Little or no mitochondrial DNA variation was found among the morphologically distinct species Cuora aurocapitata, Cuora pani, and Cuora trifasciata, for which hypotheses are proposed. Recognizing galbinifrons, bourreti, and picturata as separate species has consequences for ongoing ex situ captive breeding programs and prioritization of in situ conservation activities, particularly in Vietnam.     

黄喉拟水龟（♀）与三线闭壳龟（♂）杂交后代的形态特征及其与父母本的比较研究

本研究通过黄喉拟水龟Mauremys mutica（♀）与三线闭壳龟Cuora trifasciata（♂）进行杂交,成功获得了杂种龟。这说明黄喉拟水龟和三线闭壳龟是可以进行属间远缘杂交的,但杂交组合的受精率及孵化成功率均低于黄喉拟水龟的同种组合。杂种稚龟与黄喉拟水龟稚龟在背甲纹路、腹甲黑斑、四肢和尾腹面的皮肤颜色、喉盾前端形状及起点位置、喉盾宽/喉盾缝长、喉盾缝长/肱盾缝长、股盾缝长/肛盾缝长存在差异。1龄前,杂种龟生长快于黄喉拟水龟。形态特征上,杂种龟头顶部淡棕黄色,头侧眼后有两条黑色纵纹,颈腹部淡黄色;背甲棕色,腹甲浅黄色,每一盾片中间都有边缘呈放射状的黑斑;四肢、尾腹面及裸露皮肤部分为黄褐色。形态可量数据分析显示杂种龟在形态上接近黄喉拟水龟。建立了三种龟的形态判别公式,判别的准确率为100%（p<0.01）,判别分析中贡献最大的4个变量分别是腹甲后半部长/背甲长、喉盾宽/背甲长、肱盾缝长/背甲长、股盾缝长/背甲长,可见三线闭壳龟、黄喉拟水龟与杂交种腹部的腹甲后半部长、喉盾宽、肱盾缝长、股盾缝长等参数可作为鉴别三者的直接依据。本实验的结果对杂种龟的鉴定、龟类的杂交育种及养殖生产实践都有一定的借鉴作用。     

Identification and differentiation of Cryptosporidium species by capillary electrophoresis single-strand conformation polymorphism

Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening.     

Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses

The taxonomy of Volvocales (Chlorophyceae, Chlorophyta) was traditionally based solely on morphological characteristics. However, because recent molecular phylogeny largely contradicts the traditional subordinal and familial classifications, no classification system has yet been established that describes the subdivision of Volvocales in a manner consistent with the phylogenetic relationships. Towards development of a natural classification system at and above the generic level, identification and sorting of hundreds of sequences based on subjective phylogenetic definitions is a significant step. We constructed an 18S rRNA gene phylogeny based on 449 volvocalean sequences collected using exhaustive BLAST searches of the GenBank database. Many chimeric sequences, which can cause fallacious phylogenetic trees, were detected and excluded during data collection. The results revealed 21 strongly supported primary clades within phylogenetically redefined Volvocales. Phylogenetic classification following PhyloCode was proposed based on the presented 18S rRNA gene phylogeny along with the results of previous combined 18S and 26S rRNA and chloroplast multigene analyses.     

Unusual intraindividual variation of the nuclear 18S rRNA gene is widespread within the Acipenseridae

Significant intraindividual variation in the sequence of the 18S rRNA gene is unusual in animal genomes. In a previous study, multiple 18S rRNA gene sequences were observed within individuals of eight species of sturgeon from North America but not in the North American paddlefish, Polyodon spathula, in two species of Polypterus (Polypterus delhezi and Polypterus senegalus), in other primitive fishes (Erpetoichthys calabaricus, Lepisosteus osseus, Amia calva) or in a lungfish (Protopterus sp.). These observations led to the hypothesis that this unusual genetic characteristic arose within the Acipenseriformes after the presumed divergence of the sturgeon and paddlefish families. In the present study, a survey of nearly all Eurasian acipenseriform species was conducted to examine 18S rDNA variation. Intraindividual variation was not found in the polyodontid species, the Chinese paddlefish, Psephurus gladius, but variation was detected in all Eurasian acipenserid species. The comparison of sequences from two major segments of the 18S rRNA gene and identification of sites where insertion/deletion events have occurred are placed in the context of evolutionary relationships within the Acipenseriformes and the evolution of rDNA variation in this group.     

Phylogenetic relationships of the Asian box turtles of the genus Cuora sensu lato (Reptilia: Bataguridae) inferred from mitochondrial DNA sequences

Phylogenetic relationships of the genus Cuora sensu lato (Cuora sensu stricto and Cistoclemmys) and other testudinoid genera were inferred from variations in 882 base positions of mitochondrial 12S and 16S rRNA genes. Results yielded a robust support to the monophyly of a group (Cuora group) consisting of Cuora sensu lato and the monotypic Pyxidea. Within the Cuora group, the continental Cuora (sensu stricto) and the two subspecies of Ci. flavomarginata constituted two well-supported monophyletic groups. Distinctly small interspecific genetic distances in the former groups suggested that in the continent speciations in Cuora took place much later than the primary divergences in the Cuora group, or speciations in other related genera, such as Mauremys. Our analyses failed to provide a substantial support to the monophyly of any other combinations of taxa within the Cuora group, including Cuora in broad and strict senses, and Cistoclemmys as consisting of Ci. galbinifrons and Ci. flavomarginata. Besides these, our results also suggested the non-monophyly for the Batagurinae and the Geoemydinae, and sister relationships of the Bataguridae with Testudinidae rather than with the Emydidae.     

Fungal communities on decaying leaves in streams: a comparison of two leaf species

Decomposition of leaf litter is a microbial mediated process that helps to transfer energy and nutrients from leaves to higher trophic levels in woodland streams. Generally, aquatic hyphomycetes are viewed as the major fungal group responsible for leaf litter decomposition. In this study, traditional microscopic examination (based on identification of released conidia) and phylogenetic analysis of 18S rRNA genes from cultivated fungi were used to compare fungal community composition on decomposing leaves of two species (sugar maple and white oak) from a NE Ohio stream. No significant differences were found in sporulation rates between maple and oak leaves and both had similar species diversity. From the 18S rRNA gene sequence data, identification was achieved for 12 isolates and taxonomic affiliation of 12 of the remaining 14 isolates could be obtained. A neighbor-joining tree (with bootstrap values) was constructed to examine the taxonomic distribution of the isolates relative to sequences of known operational taxonomic units (OTUs). Surprisingly, only 2 of the isolates obtained were aquatic hyphomycetes based on phylogenetic analysis. Overall, there were no differences between the two leaf types and a higher diversity was observed via culturing and subsequent 18S rRNA gene sequencing than by conidia staining. These differences resulted from the fact that traditional microscopy provides estimates of aquatic hyphomycete diversity while the other approach revealed the presence of both aquatic hyphomycete and non-aquatic hyphomycete taxa. The presence of this broad array of taxa suggests that the role of aquatic hyphomycetes relative to other fungi be re-evaluated. Even though the functional role of these non-aquatic hyphomycetes taxa is unknown, their presence and diversity demonstrates the need to delve further into fungal community structure on decomposing leaves.     

Phylogeny of Intestinal Ciliates,Including Charonina ventriculi,and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.     

Taxonomic Resolutions Based on 18S rRNA Genes: A Case Study of Subclass Copepoda

Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1–9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.