Differentiation of poplar and willow clones using RAPD fingerprints

The technique of random amplified polymorphic DNA (RAPD) fingerprinting was used to differentiate species and identify clones of 15 poplar and 15 willow clones (ramets of three poplar clones were also included to verify the stability of the results). Four random DNA primers (Deca-11, Chl-1, Chl-4 and Chl-10) and an M13 universal primer were used to determine DNA polymorphism among the clones. Based on the DNA banding pattern obtained with the four DNA primers by polymerase chain reaction (PCR) amplification we conclude that RAPD fingerprinting with properly selected primers can be used for clonal and species differentiation of poplar and willow. The technique is simple, accurate and inexpensive.     

Assessment of genetic diversity in <Emphasis Type="Italic">Dalbergia sissoo</Emphasis> clones through RAPD profiling

We studied the genetic polymorphism among 29 clones of shisham (Dalbergia sissoo Roxb) belonging to different geographic regions using random amplified polymorphic DNA (RAPD) markers. Out of 30 primers used, only 20 primers generated polymorphism in amplified product. In total 232 bands were amplified with 20 primers, of which 192 (82%) were polymorphic with an average of 9.6 bands/primer. The resolving power (Rp) ranged from 2.14 (Primer 5) to 11.93 (Primer 4). Primer 4 and Primer 3 possessed high Rp value. Polymorphism information content (PIC) ranged from 0.15 (Primer 5) to 0.37 (Primer 4). Primer 4 amplified total 18 bands in 29 genotypes with PIC value of 0.37 hence; this set of primer was most informative. The similarity coefficient analysis revealed two clusters. The first cluster comprised of only 10 clones and the second major cluster comprised of 19 clones. The genetic similarity among 29 clones ranged from 25.86% (clone 10 and 235) to 100% (clone 19 and 59), suggesting a wide genetic base in shisham clones.     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

23个油橄榄品种的RAPD分析

采用RAPD技术对23个引种的油橄榄品种进行分类和鉴定研究.从80个10 bp的随机引物中筛选出11个扩增效果较好的引物进行扩增,共产生127条带,其中78条为多态性带,占61.4%,平均每个引物扩增的DNA多态性带数为7.09条.4个品种具有特异的位点,可作为种质鉴定的依据.根据扩增结果构建反映品种间亲缘关系的UPGMA聚类图,23个品种可划分为2大类.     

用RAPD技术测定黑荆树种源遗传关系初报

黑荆树(Acacia mearnsii Willd)系含羞草科(Mimosaceae)金合欢属(Acacia)植物,是一多用途、速生树种。我国引种时间较短,种源混杂,但黑荆树象其它树种一样,表型的变化受环境影响极大,种内各品种之间的差异很小,很难用常规的方法加以识别和划分。而应用RAPD技术可以快速、简便、灵敏地检测DNA的多态性,将品种间的差异显著地反映出来。这已在医学上、农业上得到广泛的应用。RAPD在林业上也已有应用,例如,苏晓华用RAPD技术估测了柳树种及无性系的变异;李宽鈺利用RAPD分子标记对白杨派,青杨派之间的系统进     

Phylogenetic relationship and genetic differentiation of Populus caspica and Populus alba using cpDNA and ITS noncoding sequences

Populus caspica Bornm.(section Leuce and subsection Albida), one of the most endangered endemic tree species in the Hyrcanian Forest in Iran, has numerous morphological characteristics that are closely similar to Populus alba; to clarify their taxonomic relatedness and genetic differentiation and thus inform conservation strategies, we used the noncoding regions of chloroplast DNA(cpDNA; trnL-F and trnH-psbA) and the internal transcribed spacer(ITS). Leaf samples were collected from six populations across northern Iran. cpDNA and ITS fragments were amplified by universal primers using the PCR technique and directed sequencing. The results showed that P. caspica is genetically differentiated from P.alba, and two ITS variants were detected within some P.caspica individuals. Conflicts between topologies from ITS and plastid genomes were observed. High differentiation of P. caspica from the other Populus species shown in this study confirmed the diverging taxonomic status of this endangered species. We recommend in situ conservation measures(e.g., protected areas) for at least several populations of this species, especially in the plain regions of the Hyrcanian forest.     

Monitoring genetic stability inQuercus serrata Thunb. somatic embryogenesis using RAPD markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.     

杨山牡丹和牡丹种间杂交后代的DNA分子证据

以杨山牡丹作母本，分别以牡丹品种‘赵粉’(Paeonia suffruticosa Andr.cv.‘Zhao Fen’)和‘紫二乔’(P.suffruticosa cv.‘Zi Er Qiao’)作父本，进行人工杂交，获得了杂交后代。利用DNAISSR标记技术构建的亲子代DNA指纹图谱显示，在杂交后代中检测到了分别来自双亲的特征带。建立起来的专用于牡丹研究的ISSR标记技术方法可以用于牡丹杂交种的苗期快速鉴定。     

核桃早实基因的SCAR标记

根据与核桃早实性状连锁的RAPD标记OPB-08958序列,设计SEA-F/SEA-R和SEB-F/SEB-R2对SCAR引物。在亲本、杂交后代和栽培品种(系)中的检测结果表明:SEA-F/SEA-R引物对在早实亲本‘绿园’中能稳定扩增出SCAR-SEA958特异标记;在‘绿园’ב绿丰’杂交F1后代群体中,SCAR-SEA958标记与核桃早实性状共分离,与核桃早实性状间的遗传距离为1.99cM;用晚实优系‘T-12’和栽培品种‘元丰’及‘T-12’ב元丰’杂交F1后代,检测SCAR-SEA958标记的分离情况,SCAR-SEA958与核桃早实性状共分离;在9个核桃栽培品种(系)中,SCAR-SEA958在8个供试品种的7个早实品种上出现,在1个早实品种‘鲁光’和1个晚实优系‘T-12’上没有扩增出条带。SCAR-SEA958标记在不同遗传背景下有较高的稳定性。     

Morphological and molecular methods to identify butternut (Juglans cinerea) and butternut hybrids: relevance to butternut conservation

Butternut (Juglans cinerea L.) is a native, cold-tolerant, hard-mast species formerly valued for its nuts and wood, which is now endangered. The most immediate threat to butternut restoration is the spread of butternut canker disease, caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Other threats include the hybridization of butternut with the exotic Japanese walnut (Juglans ailantifolia Carr.) and poor regeneration. The hybrids, known as buartnuts, are vegetatively vigorous, highly fecund, more resistant than butternut to butternut canker disease and difficult to identify. We review the vegetative and reproductive morphological traits that distinguish butternut from hybrids and identify those that can be used by field biologists to separate the taxa. No single trait was sufficient to separate butternut from hybrids, but pith color, lenticel size, shape and abundance, and the presence or absence of a notch in the upper margin of leaf scars, can be used in combination with other traits to identify butternuts and exclude most hybrids. In at least one butternut population, reduced symptoms of butternut canker disease were significantly associated with a dark barked phenotype. We also describe two randomly amplified polymorphic DNA (RAPD) markers that differentiate butternuts from hybrids based on DNA polymorphism. Together, these results should assist in the identification and testing of non-hybrid butternut for breeding and reintroduction of the species to its former habitats.     

昆虫分子生物学分类技术及在植保上的应用前景

介绍了目前在昆虫分类鉴定中常用的分子生物学技术,包括分子杂交技术、PCR技术、RFLP技术、RAPD技术,SSCP和DSCP技术、DNA条形码。结合自身工作,对分子生物学昆虫鉴定方法在植保中的应用前景进行了阐述。     

杨属部分种及杂种的AFLP分析

利用13对AFLP引物对杨属4个派19个种及杂种的47个无性系进行分析,共检测到858个标记,其中多态性标记为771个,多态性位点的百分率为89.9%,每对引物产生的多态性位点的百分率在80.7%～98.1%之间,表明杨属种间及无性系间在DNA水平上存在广泛变异.根据AFLP标记结果计算杨属派间、派内种间、种内无性系间分子遗传距离,对它们的亲缘关系进行定量描述.聚类分析结果表明,派间聚类与经典形态分类完全一致,派内种间及种内无性系间聚类与形态分类基本相同.最后,探讨根据分子标记结果进行杂交亲本选配及杂种子代早期选择的可行性.     

小菊花色芽变品种的AFLP分析

采用AFLP(Amplified Fragment Length Polymorphism)技术对小菊花色芽变品种与原始品种的DNA进行差异性分析,旨在建立芽变品系形态学以外的分子水平鉴定技术。在引用13对引物中,每对引物平均扩增出13.5条多态性带,分子量在110～800bp之间。3对引物组合(第8对E-ACG/M-CAT、第10对E-AGG/M-CTC、第11对E-AGG/M-CTG)产生5条清晰特异性条带可以将二者区分,这些特异性片段可能包含引起花色芽变的基因序列,经计算,二者遗传相似系数为0.986,遗传物质多态性为2.81%。     

山茶属植物油茶组与金花茶组的分子分类

以山茶属5种油茶组植物和20种金花茶组植物的叶片为材料抽提DNA,利用筛选出的16种多态性随机引物进行PCR扩增,将扩增出来的多态型谱带转化为0-1数据,利用0-1型数据聚类软件进行RAPD聚类分析,将5种油茶组植物分为3大类,20种金花茶组植物分为4大类,分类结果与张宏达分类系统大致相同,并对差异很小的一些物种提出了合并建议.     

18份樱属材料亲缘关系的ISSR分析

对福建山樱花、11份日本樱花品种和6份选优材料进行了标记,通过筛选出来的12条引物进行聚合酶链式反应(PCR)扩增。结果显示,共获得144个位点,多态性位点135个,多态性占93.75%。选取引物UBC808和UBC835构建指纹图谱,简单重复序列间扩增(ISSR)分子标记能有效地对18份樱属材料进行区分和鉴定。供试材料间的遗传相似系数在0.4532~0.8333之间,平均0.6337;遗传距离在0.1823~0.7651之间。通过非加权组平均法(UPGMA)进行聚类分析,在遗传相似性系数为0.5899时,可将18份樱属材料划分为4大类,其中选优材料与福建山樱花或日本樱花亲缘关系较近,但在花色、花期等方面有一定差异,且能区分和鉴定供试材料的亲缘关系。研究结果能为国内樱花新品种鉴定、优选和商品化等提供参考依据。     

浙江马尾松毛虫不同地理种群间基因差异的ISSR分析

马尾松毛虫(Dendrolimus punctatus)是浙江省重要的森林害虫,也是我国南方林区松林主要食叶害虫之一,在浙江往往呈周期性发生并猖獗成灾,造成严重的经济损失和生态环境的破坏.     

6个复叶槭品种间亲缘关系的RAPD分析

采用RAPD法对复叶槭5个品种及1个芽变品种进行基因组DNA多态性分析,从300个引物中筛选出具有丰富多态性的20个10bp的随机引物,这20个引物共扩增出170个DNA片段,片段大小在200bp～2800bp之间,其中多态性谱带为112条,多态率为65.88%,表现出较为丰富的RAPD多态性;利用UPGMA进行遗传距离计算的结果表明,6个复叶槭品种间的亲缘关系和遗传距离均有较大差异.     

刺五加AFLP分子标记技术体系的建立

用改良的的试剂盒法提取刺五加叶片的基因组DNA,可以获得高质量的刺五加基因组DNA,DNA降解较少、污染低、电泳条带清晰,可以用于PCR扩增反应,可以被内切酶EcoRⅠ和MseI彻底消化。通过优化酶切反应、引物连接、预扩增、选择扩增,建立了优化的刺五加AFLP分析技术体系,从64对引物中筛选到5对高效扩增引物,并利用这5对引物在6个刺五加种源中获得了89个多态性AFLP分子标记。建立了利用红外荧光检测技术和Li-Cor4300DNA分析系统,进行刺五加AFLP分析的分子标记技术。     

小兴安岭红松原始林的遗传多样性研究

通过39个红松植株的DNA进行10个引物的ISSR实验,得出71个条带,其中特异性条带为55个,特异性位点百分率77.46%,平均每个引物产生5.5个特异性条带。可见,利用ISSR分子标记鉴别红松DNA多态性,多态性高,扩增的片段多,可用于红松种群间的亲缘关系分析研究。     

毛竹小RNA高通量测序及病毒分析

以毛竹叶片为材料,采用小RNA高通量测序结合生物信息学对小RNA数据库进行组装,进一步分析了毛竹中存在的病毒和类病毒,并采用RT-PCR和RACE进行验证。结果表明:在竹子样品中存在水稻东格鲁病毒(RTBV),覆盖率达到91.0%。在毛竹样品中扩增得到1 992 bp RTBV病毒类似序列,占其基因组的24.9%。RTBV病毒在多个毛竹样品中存在且不存在多态性。RTBV病毒可能是一个古老的植物病毒,在进化过程中禾本科植物将其序列整合到基因组中来防御RTBV病毒的浸染。