HER2基因组DNA标准物质互通性研究

采用实验室建立的数字PCR方法 (ddPCR)和荧光定量PCR (qPCR)方法研究HER2基因组DNA标准物质的互通性,评价HER2基因组DNA标准物质与临床样本的互通性,为临床实验室检测提供具有互通性的可溯源标准物质.将研制的5个水平标准物质随机穿插于29例临床样本间,使用ddPCR方法与qPCR方法同时进行检测.参考美国临床实验室标准化协会(CLSI)指南文件EP30和中华人民共和国卫生行业标准基质效应与互通性评估指南WS/T356-2011的推荐,采用Deming回归法评价标准物质的互通性.结果显示,5种标准物质与临床样本之间具有良好的互通性,可以用于临床实验室对HER2基因拷贝数变异检测方法的方法验证和质量控制.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

用RAPD技术检测野生鲫鱼的四个金鱼代表品种的基因组DNA多态性

利用随机扩增多态DNA技术检测了两个不同地区的野生鲫鱼(Carassius a uratus L.)和4个金鱼(Carassius auratus Var.)代表品种的基因组DNA的多态性。用26个随机引物对各品种实验鱼的基因组DNA进行扩增,平均每个品种观察到约134个标记,单个引物获得的标记在1-16个之间。实验结果经统计学分析表明,金鱼和鲫鱼的随机扩增多态DNA共亨度高,进一步证实了金鱼由野生鲫鱼演化而来,聚类结果表明,草金鱼形成后,首先演化为文种鱼,然后由文种再形成龙种和蛋种两个品系。 Abstract:Genomic DNA polymorphisms in the wild crucian from rwo different areas and in four representative varieties of goldfish were detected by using random amplified polymorphic DNA(RAPD)technique.The genomic DNA of each variety of fish was amplified with 26 primers.On average,about 134 RAPD markers were observed by each variety.The markers obtained by a single primer varied from 1 to 16.The statistical analysis of the experimental results indicated that random amplified polymorphic DNA of the goldfish and wild crucian have a high proportion of random amplified polymorphic DNA fragment shared.This further verified that goldfish is evolved from wild crucian.The cluster analysis suggested that after emerging,grass goldfish is evolved to wen goldfish,then through wen goldfish evolved to dragoneye goldfish and oval goldfish.     

Mitochondrial DNA Polymorphisms Revealed by RAPD Assays Distinguish the Male-sterile and Male-fertile Cytoplasms in Pearl Millet

Diversification of cytoplamic male sterile (CMS) sources is of considerable significance in pearl millet, considering that almost all the commercially cultivated hybrids, particularly in India, are based on a single CMS source, A₁. We analyzed the mitochondrial DNA (mtDNA) polymorphisms among five pearl millet CMS sources (A₁ to A₅) and a male-fertile maintainer (B) line, all in an isonuclear background. Analysis using 21 random primers led to identification of polymorphic bands specific to the A₁, A₂, A3 and A₅ CMS lines. Two RAPD primers, OP-G12 and OP-G19, in combination were able to distinguish all the male-sterile and male-fertile cytoplasms. Highly effective for this purpose also were four RAPD markers, OP-B7, OP-D8, OP-F10 and OP-G12. Cluster analysis, followed by bootstrap analysis, of the mtDNA dataset revealed two distinct clusters: cluster-I comprising the A₁, A₂, A₃ CMS lines and the male-fertile line, and cluster-II comprising the A₄ and A₅ CMS lines.     

紫斑牡丹遗传多样性的ISSR分析

利用ISSR标记对一个居群内的16个紫斑牡丹品种材料进行基因组多态性分析,从100条引物中筛选出15条用于紫斑牡丹的ISSR扩增。共扩增出134条带,其中多态性条带96条,多态性百分率为71.6%。根据ISSR扩增结果,利用NTSYSpc 2.10e软件进行Jaccard相似性系数分析,16个紫斑牡丹品种的遗传相似系数为0.45～0.93。UPGMA聚类分析结果显示,在遗传相似系数0.534处,16个紫斑牡丹品种材料可分为4类。第Ⅰ类中的6个紫斑牡丹品种材料中有5个为皇冠型品种,1个单瓣型,其他类中也有皇冠花型品种但未聚在Ⅰ类中;第Ⅱ类仅有1个‘粉盘托桂’,它是所选材料中唯一的托桂型品种,单独聚为一类;第Ⅲ类由不同花色、花型品种聚为一类,可见亲缘关系较近的紫斑牡丹品种性状变异性也很大;第Ⅳ类中A组品种材料均为白色,花型有单瓣型、荷花型和绣球型;B组仅有一个‘银线女’,它是所选材料中唯一的红色绣球型材料。研究表明,不同相似系数的遗传聚类划分与花色、花型之间并非完全具有相关性。     

中籼杂交水稻亲本多态性的AFLP分析

对15个籼型杂交水稻亲本进行AFLP分析,结果表明：亲本间遗传距离小,在0.0589~0.3305之间,平均为0.2033。15个亲本按类平均法可聚为两类,Ⅰ类为不育系,Ⅱ类为恢复系。其中Ⅱ类又分为两个亚类,Ⅱ-1不含明恢63血缘、Ⅱ-2全部含明恢63血缘。Ⅰ/Ⅱ-1与Ⅰ/Ⅱ-2间的遗传距离无明显差异,揭示恢复系的遗传基础较一致,这可能是当前的品种不能超过汕优63的重要原因之一。要提高水稻的杂种优势,需丰富亲本的遗传基础,扩大其遗传差异。 Abstract:The polymorphisms were analyzed in 15 semilate indica parental materials with AFLPs.The genetic distances between parental materials were small with an average of 0.2033,ranged from 0.0589 to 0.3913.Fifteen parental materials were classified into two groups,cytoplasmic male-sterile lineⅠand restorer line Ⅱ.The latter were classified into two subgroups,Ⅱ-1 and Ⅱ-2.SubgroupⅡ-2 was consanguinity with Minghui63.The genetic distances between male-sterile line and two restorer line subgroups(Ⅱ-1 and Ⅱ-2) did not show significant difference,indicating that the genetic bases of restorer lines were similar,which maybe one of major reasons for the yield still not surpassing Shanyou63 in indica hybrid rice presently.To increase the heterosis of hybrid rice,we must enrich the genetic diversity and expand the genetic differences between parental lines.     

Genomic islands in Photorhabdus

Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.     

Genomic Variants Revealed by Invariably Missing Genotypes in Nelore Cattle

High density genotyping panels have been used in a wide range of applications. From population genetics to genome-wide association studies, this technology still offers the lowest cost and the most consistent solution for generating SNP data. However, in spite of the application, part of the generated data is always discarded from final datasets based on quality control criteria used to remove unreliable markers. Some discarded data consists of markers that failed to generate genotypes, labeled as missing genotypes. A subset of missing genotypes that occur in the whole population under study may be caused by technical issues but can also be explained by the presence of genomic variations that are in the vicinity of the assayed SNP and that prevent genotyping probes from annealing. The latter case may contain relevant information because these missing genotypes might be used to identify population-specific genomic variants. In order to assess which case is more prevalent, we used Illumina HD Bovine chip genotypes from 1,709 Nelore (Bos indicus) samples. We found 3,200 missing genotypes among the whole population. NGS re-sequencing data from 8 sires were used to verify the presence of genomic variations within their flanking regions in 81.56% of these missing genotypes. Furthermore, we discovered 3,300 novel SNPs/Indels, 31% of which are located in genes that may affect traits of importance for the genetic improvement of cattle production.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

Palaeosymbiosis Revealed by Genomic Fossils of Wolbachia in a Strongyloidean Nematode

Wolbachia are common endosymbionts of terrestrial arthropods, and are also found in nematodes: the animal-parasitic filaria, and the plant-parasite Radopholus similis. Lateral transfer of Wolbachia DNA to the host genome is common. We generated a draft genome sequence for the strongyloidean nematode parasite Dictyocaulus viviparus, the cattle lungworm. In the assembly, we identified nearly 1 Mb of sequence with similarity to Wolbachia. The fragments were unlikely to derive from a live Wolbachia infection: most were short, and the genes were disabled through inactivating mutations. Many fragments were co-assembled with definitively nematode-derived sequence. We found limited evidence of expression of the Wolbachia-derived genes. The D. viviparus Wolbachia genes were most similar to filarial strains and strains from the host-promiscuous clade F. We conclude that D. viviparus was infected by Wolbachia in the past, and that clade F-like symbionts may have been the source of filarial Wolbachia infections.     

宝兴百合与匍茎百合遗传多样性的ISSR分析

利用5条ISSR引物对宝兴百合(Lilium duchartrei)9个居群和匍茎百合(Lilium lankongense)13个居群的遗传多样性进行了初步检测。结果表明:(1)宝兴百合在物种水平上多态位百分率(PPB)为97.26%,Nei’s基因多样度(H)为0.309 8,Shannon’s多样性信息指数(Hsp)为0.469 4;匍茎百合在物种水平上多态位百分率(PPB)为100%,Nei’s基因多样度(H)为0.3390,Shannon’s多样性信息指数(Hsp)为0.503 0,均略高于宝兴百合。(2)宝兴百合与匍茎百合的遗传多样性在居群水平上相对较低;宝兴百合和匍茎百合居群间遗传分化系数(Gst)分别为0.642 5和0.563 7,表明2个物种居群间的遗传分化大于居群内的遗传分化。(3)经Mantel检测,两种居群间的遗传距离与地理距离均不存在显著的相关性(宝兴百合r=0.263 7,P=0.844 0;匍茎百合r=0.104 2,P=0.695 0);宝兴百合与匍茎百合的遗传多样性及遗传分化现状可能是两者的生活史特性、地理隔离等作用的结果。(4)UPGMA聚类结果显示,宝兴百合与匍茎百合在分子水平上出现了明显分化,支持二者是独立的物种。     

云杉矮槲寄生遗传多样性的ISSR分析

为探究云杉矮槲寄生(Arceuthobium sichuanense)的遗传多样性及其与不同寄主选择压力和地理分布的关系,采用ISSR分子标记方法,对青海、甘肃、四川等地区5种寄主上的100份云杉矮槲寄生样本进行遗传多样性和遗传结构分析。结果表明:(1)10条引物共扩增出130个条带,其中多态性条带129条,多态位点百分率(PPB)为99.23%。(2)物种水平上的Nei’s遗传多样性指数(He)和Shannon信息指数(I)分别为0.313 9和0.476 5,表明云杉矮槲寄生物种水平的遗传多样性较高,但群体间的基因流(Nm=0.528 7)较弱,可能会加速群体间的遗传分化(Gst=0.486)。(3)UPGMA聚类结果显示,来自甘肃、青海的样本聚为一组,表现出较高的相似性,而四川的样本独立聚类;不同寄主来源的聚类结果显示,寄生于鳞皮云杉(Picea asperata)与川西云杉(P.likiangensis var.balfouriana)的样本聚为一类,而寄生于青海云杉(P.crassifolia)、青杄(P.wilsonii)和紫果云杉(P.purpurea)的样本聚为一类,表明地理隔离和寄主选择压力对云杉矮槲寄生的遗传分化起到了重要作用。     

流式细胞术揭示出枯草芽孢杆菌多态异质性

新近的研究发现,微生物群体异质性现象普遍存在,与微生物群体许多关键功能密切相关.微生物群体中的多种异质性状态需要单细胞水平的分析技术才能被揭示,流式细胞术是获取异质性状态精确分布的重要工具.但微生物细胞尺寸微小、生物分子含量少、常常缺乏特异性试剂等都限制着传统流式细胞技术在微生物研究领域的应用.本论文采用新型的低背景、高灵敏度和高分辨率流式细胞仪,以增强的前向散射光、侧向散射光以及紫外光激发的细菌自发荧光水平这三个无需任何荧光标记就可以检测的信号为参数,首次揭示出不同生长状态的枯草芽孢杆菌具有复杂、动态的异质性状态分布.这一方法鉴定出的枯草芽孢杆菌多种状态及其与生理功能相关的、高度关联的变化,可能对该菌的生理变化规律及其分子机理的认识提供新的机遇.本论文也讨论了这一采用新型高灵敏度、高分辨率流式细胞仪测量非标记细胞参数的方法对于广泛开展各种微生物多态性研究具有巨大潜力.     

A Link among DNA Replication, Recombination, and Gene Expression Revealed by Genetic and Genomic Analysis of TEBICHI Gene of Arabidopsis thaliana

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.     

Epigenetic Regulation of Programmed Genomic Rearrangements in Paramecium aurelia1

ABSTRACT In ciliates, development of the polyploid somatic macronucleus after sexual events involves extensive and reproducible rearrangements of the germ-line genome, including chromosome fragmentation and precise excision of numerous internal sequence elements. In Paramecium aurelia, alternative macronuclear versions of the same germ-line genome can be maternally inherited across sexual generations, showing that rearrangement patterns are not strictly determined by the germ-line sequence. Homology-dependent maternal effects can be evidenced by transformation of the vegetative macronucleus with cloned macronuclear sequences: new fragmentation patterns or internal deletions are specifically induced during differentiation of a new macronucleus, in sexual progeny of transformed clones. Furthermore, transformation of the maternal macronucleus with germ-line sequences containing internal eliminated sequences (short single-copy elements) can result in a specific inhibition of the excision of the same elements in the zygotic macronucleus. These experiments show that the processing of many germ-line sequences in the developing macronucleus is sensitive to the structure and copy number of homologous sequences in the maternal macronucleus. The generality and sequence specificity of this trans-nuclear, epigenetic regulation of rearrangements suggest that it is mediated by pairing interactions between germ-line sequences and sequences imported from the maternal macronucleus.     

Dead-End Hybridization in Walnut Trees Revealed by Large-Scale Genomic Sequence Data

Although hybridization plays a large role in speciation, some unknown fraction of hybrid individuals never reproduces, instead remaining as genetic dead-ends. We investigated a morphologically distinct and culturally important Chinese walnut, Juglans hopeiensis, suspected to have arisen from hybridization of Persian walnut (J. regia) with Asian butternuts (J. cathayensis, J. mandshurica, and hybrids between J. cathayensis and J. mandshurica). Based on 151 whole-genome sequences of the relevant taxa, we discovered that all J. hopeiensis individuals are first-generation hybrids, with the time for the onset of gene flow estimated as 370,000 years, implying both strong postzygotic barriers and the presence of J. regia in China by that time. Six inversion regions enriched for genes associated with pollen germination and pollen tube growth may be involved in the postzygotic barriers that prevent sexual reproduction in the hybrids. Despite its long-recurrent origination and distinct traits, J. hopeiensis does not appear on the way to speciation.     

鲍氏层孔菌基因组DNA提取及RAPD扩增条件优化

鲍氏层孔菌Phellinus baumii Pilát在系统分类学上属于担子菌门(Basidiomycota),非褶菌目(Aphyllophorales)锈革孔菌科(Hymenochaetaceae),是大型珍稀药用真菌(戴玉成,2003)。现代药理学研究证明鲍氏层孔菌有抑制肿瘤、保护肝脏、提高免疫力等多方面药用功能。层孔菌属的子实体多呈蹄形,宏观上区分有困难,目前对鲍氏层孔菌的分类和命名有很多争议(戴玉成,2003),有必要从DNA分子水平上对鲍氏层孔菌种质资源进行研究,探索属内各种间的遗传差异。本文简要报道鲍氏层孔菌基因组DNA的提取及RAPD扩增条件优化,为采用RAPD技术进行鲍氏层孔菌种质资源的分类鉴定、亲缘关系和遗传多样性的研究提供依据。