高等植物DNA条形码最新研究进展及其应用

植物DNA条形码的筛选工作主要集中于叶绿体基因上,包括位于编码区的mat K、rbc L、UPA、rpo B、rpo C1、acc D、ndh J和YCF5等以及间隔区的trn H-psb A、trn L-trn F、atp F-atp H、trn D-trn Y和psb K-psb I等,其中mat K、rbc L和trn H-psb A研究最为成熟,可作为核心条形码序列。由于每个基因进化速率不同以及植物种间杂交较为频繁等诸多原因,不同类群植物的最适条形码序列有很大差别。本文综述了DNA条形码的最新研究进展以及条形码技术在物种鉴定、分类学问题修订、新种隐种的发现、植物药材真伪鉴定、食品质量控制和安全、出入境植物检验检疫等方面的应用,提出了cp DNA条形码在应用中的不足之处以及解决措施等,并对DNA条形码未来的发展进行了展望,以期为高等植物的应用提供强有力的分子依据。     

6个地方鸡种线粒体COⅠ基因的DNA条形码

以我国6个地方鸡(Gallus gallus)品种为研究对象,利用DNA测序技术测定了线粒体细胞色素C氧化酶Ⅰ(cytochrome coxidaseⅠ,COⅠ)基因的2段序列,研究COⅠ这一特定基因的特定区段作为DNA条形码在识别地方鸡种方面的可行性和有效性。研究结果表明,所选择的2段序列中,经过多态性分析筛选,以Bar1序列(712～1359位)突变位点较多,为24个,为24种单倍型,其中22种单倍型为6个鸡品种所特有;品种间平均多态性3.860%,6个鸡品种均有其特异位点;6个品种的NJ聚类分析结果显示,6个鸡品种基本被聚为不同的类别,与形态学分类基本一致,COⅠ基因的Bar1序列用于这些品种鉴定是可行的。而对Bar2序列(1800～2314位)进行序列多态性分析,只发现5个突变位点,为5种单倍型,且大多品种没有其特异位点。由于其多态性低,且NJ聚类分析6个品种的聚类结果与形态学分类不一致,Bar2序列不利于品种鉴定。研究结果表明,COⅠ基因的Bar1序列更适合地方鸡品种鉴定条形码。     

基于DNA条形码ITS2的高分辨率熔解曲线鉴定市售核桃乳真伪

市场上核桃乳掺杂使假现象层出,急需建立一种快捷、准确、高效的真伪鉴定方法.本研究选取核桃基因组间隔区ITS2、叶绿体基因组编码区rbcL以及非编码区psbA-trnH作为候选DNA条形码基因.综合扩增、测序、比对成功率以及生物信息学结果,筛选得到ITS2是区分核桃、花生等不同坚果的最适DNA条形码.利用高分辨率熔解曲线...     

DNA条形码技术在地方猪种质资源保护中的应用

本文先概述了动物DNA条形码技术的研究现状,统计分析了DNA条形码研究中所选用的基因序列和研究对象等,发现目前并没有DNA条形码技术应用于地方猪的研究报道;随后,分析我国地方猪种的系统发育研究现状表明,当前中国地方猪的分类体系在分子上的证据还很匮乏,研究所涉及的品种较少,分子标记数量有限,分子系统发育学的研究才刚起步;最后,阐述和讨论了DNA条形码技术应用于中国地方猪种保护中的重要意义及其可行性.     

基于物种特异性单一DNA标记扩增检测的食品真实性定性鉴别技术研究进展

脱氧核糖核酸(DNA)分析技术已经成为食品真实性鉴别中物种源性成分鉴定最主要也是最有效的技术,其中基于物种特异性单一DNA标记扩增检测的食品真实性定性鉴别技术是最准确可靠,也是目前最成熟、应用最广的技术。本文综述了该技术物种特异性单一DNA标记主要类型及目前常用的扩增检测技术优缺点,并探讨了该技术的未来发展趋势。     

基于cpDNA条形码鉴定铁皮石斛种质资源

为选择适合鉴定铁皮石斛的条形码,以5份铁皮石斛种质资源和1份串珠石斛(外类群)为试验材料,采用cpDNA条形码技术进行分子鉴定。结果表明,5个条形码序列(matK、rbcL、trnH-psbA、petApsbJ和rpl32-trnL)种间遗传距离均远高于种内遗传距离,符合作为条形码的基本要求,其中petA-psbJ变异位点最多(8.03%),说明其进化速率亦最快,petA-psbJ可作为石斛属候选条形码之一;单个条形码鉴定率均较低(60%),核心条形码matK+rbcL鉴定率亦较低(68.75%),而在核心条形码基础上结合其它序列(≥3)却可以显著提高鉴定率(≥81.25%),matK+rbcL+petA-psbJ鉴定率达到100%,可作为铁皮石斛分子鉴定的最适条形码组合。综上,利用cpDNA条形码技术,可将不同种质资源的铁皮石斛区分开,基本上可将不同采集地的铁皮石斛聚类;同时,建议matK+rbcL+petA-psbJ作为铁皮石斛种质资源鉴定的DNA条形码。本研究结果为铁皮石斛种质资源的快速鉴定提供了一定的参考。     

武夷红樱分类地位的修订

为了重新确定武夷红樱的分类地位,以16个野生樱属种为材料,利用SSR分子标记技术进行聚类分析,结合形态观测和DNA条形码分子鉴定技术进行辅助分类学研究。为了获得3个种间的亲缘关系,利用4个叶绿体基因序列(trnH-psbA、rpl32-trnL、petA-psbJ和trnL-trnF)进行DNA条形码分析,结果表明,武夷红樱(PcW1和PcW2)与华中樱4个基因序列差异明显,小于与钟花樱之间的差异。其中,武夷红樱(PcW2)的trnH-psbA和rpl32-trnL序列与华中樱完全一致,尤其是trnL-trnF序列,武夷红樱与华中樱仅有3个位点差异,而与钟花樱有75(77)个位点差异。基于trnH-psbA+rpl32-trnL+petA-psbJ+trnL-trnF构建的进化树表明,武夷红樱与华中樱聚在一起,而与钟花樱关系较远。结合SSR和形态观测结果,确定武夷红樱为华中樱与钟花樱的杂交种。由于叶绿体基因呈母系遗传,可确定华中樱为母本,钟花樱为父本。本试验利用分子生物学、遗传学结合形态观测的方法,确立了武夷红樱新的分类地位,武夷红樱形态上虽与钟花樱较为相似,但基因水平上却与华中樱更为接近,因此,建议将武夷红樱由钟花樱下的一个变种升级为一个独立的种。此外,本试验方法也可为其他樱属植物的分类学修订、新种的发现以及为新品种的选育提供重要的参考。     

基于核糖体DNA第二内部转录间隔区(ITS2)序列鉴定四季青及其近缘种和混伪品

中药材四季青(Ilicis Chinensis Folium)的基原植物冬青(Ilex chinensis)是重要的园林观赏物种。利用DNA条形码技术可以快速、准确鉴定四季青及其近缘种和混伪品。本研究以冬青、铁冬青(Ilex rotunda)、秤星树(Ilex asprella)、枸骨(Ilex cornuta)和女贞(Ligustrum lucium)等37份植物样本为实验材料,提取全基因组DNA,扩增核糖体DNA第二内部转录间隔区(internal transcribed spacer 2,ITS2)序列并进行双向测序。测序结果利用Codon Code Aligner 4.2.7进行序列拼接,获得高质量ITS2序列。同时从Gen Bank中获得28条冬青的同属近缘种以及混伪品女贞、四川山矾(Symplocos setchuensis)的ITS2序列。基于隐马尔科夫模型(hidden Markov model,HMM)注释5.8S和28S,获得完整的ITS2区域,进而应用MEGA6.0(molecular evolutionary genetics analysis)进行冬青种间和种内序列分析,计算种内种间K2P(Kimura 2-parameter)遗传距离,构建邻接树(neighbor-joining tree,NJ tree)。结果表明,琼脂糖凝胶电泳得到清晰明亮的均一条带,说明利用通用的ITS2引物可以成功扩增到用于测序的PCR产物。通过PCR产物测序、数据处理和序列分析可知,冬青种内ITS2长度经过比对后为239 bp,全部样品的种间ITS2比对后长度为269 bp。冬青种内有4个变异位点以及6个单倍型,种间有143个变异位点,远远多于其种内变异位点。MEGA6.0分析结果表明,种间最小K2P遗传距离(0.094)大于种内最大K2P遗传距离(0.013)。邻接树显示,女贞和四川山矾分别以100%的自展支持率分别聚类,表现出良好的单系性,冬青属物种聚为一大支。冬青在冬青属下可以单独聚为一支,可以进行区分。冬青属其他物种以同一亚属聚为一支。研究结果表明,作为一种快速、准确、简便的分子生物学鉴定方法,ITS2可以应用于冬青及其近缘种和混伪品的鉴定,同时对于准确鉴定冬青属物种亦有巨大潜力,可为冬青属植物在临床上的用药安全和开发应用提供科学依据。     

DNA芯片技术应用研究进展

DNA芯片技术是近年迅速发展的一门生物高新技术 ,其突出特点在于高度并行性、多样性 ,即能一次性对生物遗传信息进行大规模的快速、同步分析。DNA芯片技术目前已用于基因重复测序、基因表达分析、新基因的发现、基因单核苷酸多态性 ( SNPs)研究、基因诊断、药物筛选等领域 ,而且其应用范围还在不断扩展。本室运用这一新技术 ,检测水稻在受水稻黄单胞菌水稻致病变种及该菌株的 rpf C基因缺失突变体感染时基因表达的差异 ,发现一些基因可能与水稻抗白叶枯病相关     

用ITS序列识别牧草种质资源

DNA条形码技术能够快速、准确地识别物种,对于开展基础性的分类学研究和应用性的生物多样性研究极为重要。本文以广西畜牧研究所牧草种质资源圃的8个物种共22个品种为材料,进行植物条形码ITS测序。PCR结果显示ITS的扩增成功率达到100%,同源性的分析表明各物种中的ITS序列变异显著,共找到99个可以用来区分物种的变异位点,系统进化树的结果表明利用ITS序列进行物种水平鉴定成功率达100%,能够作为DNA条形码识别植物物种。     

DNA barcoding for conservation and management of Amazonian commercial fish

DNA barcoding is used to assign a biological specimen to a species. DNA-based procedure has become the preferred forensic tool for criminal prosecution in cases involving the sale of incorrectly identified food. The aim of this work was to develop a DNA-based marker for allowing an accurate and reliable identification of Amazonian fish species of commercial interest. For this purpose, we extracted DNA from fish directly purchased in local markets and identified de visu by local experts. We PCR amplified the mitochondrial 12S rRNA and cytochrome oxidase I (COI) genes. Twenty-nine commercial species accounting for most commercial landings in the River Amazon markets were unambiguously identified based on their DNA for the first time. Phylogenetic trees reconstructed based on the sequences of the two mitochondrial genes clustered species in concordance with their taxonomic classification. We illustrated the utility of DNA barcoding demonstrating that the group of fish generically sold as “Acará” includes seven different species, which are being exploited together as a single species, thus estimation of exploitation rates was not possible until now. Application of genetic markers for species authentication in markets and control of commercial landings will contribute to recognition of the real fishing targets and to the conservation of fish resources in the Amazon basin.     

Identifying earthworms through DNA barcodes

With almost 3000 species, earthworms provide important model systems for studying soil fauna. However, species identification of earthworms is difficult and therefore limiting. The use of DNA barcodes, which are short sequences from standardized regions of the genome, has been regarded as a promising approach to resolve this taxonomic dilemma. We evaluated sequence diversity in the mitochondrial cytochrome-c oxidase I (COI) gene as a tool for resolving differences among species of Chinese earthworms. Members of six genera and 28 species were examined, and species were successfully discriminated in all cases. Sequence divergence within species was generally less than 1%, whereas divergence between species was greater than 15% in all cases. Divergence among individuals of Eisenia fetida were much higher (up to 7.8%); however, this may represent the presence of unrecognized sibling species or subspecies. We conclude that although it cannot completely replace taxonomy, the DNA barcode is a powerful tool for identifying species of earthworms and provides a useful complement to traditional morphological taxonomy.     

DNA sequences identify invasive alien Cardamine at Lake Constance

The shores of Lake Constance in southwest Germany inhabit an endemic and highly endangered lake-shore community. In spring 2004, a so far unknown Cardamine (Brassicaceae) was detected at the lake-shores. In the subsequent years, this taxon has spread rapidly, 95 locations have been recorded until spring 2007. We tested the utility of DNA sequences to distinguish between two alternative hypotheses regarding the appearance of this new invasive taxon: a local formation via hybridization between native species versus an introduction of a non-native taxon. The relative DNA contents was analysed as an additional independent character. DNA sequences provided substantial evidence that the unknown Cardamine taxon, recently discovered at Lake Constance in southwest Germany, is a non-native species introduced from abroad. Sequences of the internal transcribed spacer of the large subunits of nuclear ribosomal DNA and two noncoding regions of chloroplast DNA (trnL intron, trnL/F spacer) were distant from sequences of all native Cardamine species providing evidence against a hybridization hypothesis. In contrast, DNA sequences of the unknown Cardamine were identical to one accession of Cardamine flexuosa auct. non With. (Asian C. flexuosa) from Japan. The introduction history of Asian C. flexuosa at Lake Constance and the potential threat to the native lake-shore vegetation is discussed. Our study highlights the potential of DNA sequences to identify invasive genotypes and source regions.     

DNA条形码与实时荧光定量PCR技术在铁皮石斛鉴定中的应用

为建立铁皮石斛准确、高效的鉴定体系,本研究以101份石斛属和蝴蝶兰属植物样品为试验材料,通过对比ITS、psbA-trnH、matK及rbcL基因在石斛属植物中的鉴定能力,筛选出ITS作为本研究最理想的DNA条形码。以ITS序列作为靶基因,设计铁皮石斛特异引物和特异探针,以及石斛属通用引物和探针,利用实时荧光定量PCR(TaqMan)技术,建立铁皮石斛多重实时荧光PCR检测新体系,通过特异性、灵敏度和实际样本验证,发现参试样品中的25份铁皮石斛均可被有效鉴定,与其他鉴定方法相比,该方法具有特异性强、灵敏度高(高出普通PCR 100倍)、重复性好且高效经济的优势。本研究结果对铁皮石斛的资源保护与利用起到了积极作用。     

Results of molecular analysis of an archaeological hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.) DNA sample from North West China

Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C. sativa sample showed that C. sativa races might have been introduced into China from the European–Siberian center of diversity.