DNA条形码技术在林业科学研究中的应用

DNA条形码是利用生物体内普遍存在的一个或几个、较短的且标准化的基因片段作为通用片段,通过碱基序列差异来实现物种水平准确鉴定的工具。相对于传统的分类学,DNA条形码技术具有不依赖形态特征和发育阶段、鉴定数字化、快速准确、操作简单规范等优势。已成功应用在生物多样性监控、海关、中药材真伪鉴定、法医鉴定、动植物检疫、生物入侵、食品和药物市场监督等诸多领域。经过15年时间发展,DNA条形码研究已基本确定针对不同生物类群使用不同的通用片段,同时构建了标准的全球生命条形码数字化数据库。目前DNA条形码研究主要集中在如何提高近缘类群物种分辨率和构建区域条形码数据库两个方向。在林业科学研究中,DNA条形码在木材识别、森林群落生态和生物多样性监控与评估等领域发展迅猛,但同时也面临一些问题,如木材DNA降解、整条片段的扩增等,需要在条形码片段选择、数据库构建、数据库与高通量测序技术结合、分析方法改进等进一步深入研究。未来DNA条形码将在林业资源评价、保护和可持续利用等方向具有广阔的应用前景。     

木材分子考古研究进展

木材考古学研究是推动木质文物自然和历史信息挖掘、保护和修复的重要基础。近年来,随着古DNA捕获和测序技术的快速发展,从木质遗存中可获取古DNA信息,在木材解剖学基础上,创新开展以古DNA为核心的木材分子考古研究已成为木材考古学的前沿热点。本文首先对木材分子考古研究进行概述,从古DNA的保存和降解、获取以及数据处理和序列分析3方面归纳木材古DNA的研究进展,并指出古DNA因高度降解、含量极低和化学损伤特征导致其难于提取和信息解译的难题。然后总结木材分子考古在解读先民认知与利用森林资源方式、复原历史时期地域性森林植被类型和物种多样性以及重建古代树木应对气候和生境变化的微进化反应等方面的主要应用。最后提出该研究领域未来应优先开展的工作：1）建立考古木材标本库及其DNA信息数据库;2）研究不同时空维度下木材古DNA损伤及变化规律;3）构建稳定高效的木材古DNA提取及序列信息解译技术体系。通过进一步加强木材分子考古等多学科交叉研究,推动新理论、新方法、新技术在木材学和考古学领域的应用,为木质文物的用材树种识别、保护利用以及重建历史时期森林植被、环境气候与人类活动的耦合关系提供重要科学依据。     

DNA条形码在龟鳖类物种鉴定中的应用

研究中获得了龟鳖类18个个体长度为660bp的COI基因序列。利用MEGA软件采用P-距离法计算龟鳖类种间及种内遗传距离并构建序列间的UPGMA和邻接系统发生树。结果显示,龟鳖类的种间遗传距离显著大于种内遗传距离。在系统树中,龟鳖类每一物种的个体分别形成各自独立的分支。基于COI基因的DNA条形码在识别龟鳖类物种方面和传统形态学基本一致,而且该基因可以探讨龟鳖类种间的系统发育关系,以COI基因作为DNA条形码,对龟鳖类进行物种鉴定具有一定的可行性。     

降香黄檀木材DNA提取及rDNA-ITS序列条形码分子鉴定

以不同产地人工林中采取的降香黄檀(Dalbergia odorifera T.Chen)木材为研究对象,提取并扩增不同温度处理后的降香黄檀心、边材DNA和ITS片段,分析不同温度处理对降香黄檀木材DNA提取和PCR扩增的影响;对不同产地的降香黄檀木材及其近缘种多裂黄檀(Dalbergia rimosa Roxb)木材的rDNA-ITS序列进行测定和差异分析。结果表明,25℃和65℃热处理后的木材DNA呈不同程度的弥散分布,105℃热处理后的木材DNA降解成250bp以下的小片段;不同温度处理后的降香黄檀木材,仅有25℃处理后的木材ITS片段能够被成功地扩增。降香黄檀和多裂黄檀ITS序列共存在6个变异位点且ITS2区变异大于ITS1区,聚类分析结果可以将降香黄檀及其近缘种多裂黄檀木材区分开来,为利用ITS条形码序列鉴定降香黄檀木材及其常见混伪品提供理论依据。     

我国首次依据木材DNA出具树种鉴定报告

<正>日前,受国内某检验认证机构委托,中国林科院木材工业研究所对近期我国海关部门截获的一批进口木材开展了鉴定工作。木材鉴定团队在形态解剖学基础上,通过提取木材样品DNA,采用DNA条形码技术进行分析,准确鉴定出送检木材样品为豆科紫檀属的刺猬紫檀(Pterocarpus erinaceus Poir.,2016年已被列入《濒     

DNA技术在木材来源鉴定中的应用

DNA技术在木材树种鉴别和产地鉴定方面有较高的准确性,可用于提高木材合法性检验的可信度和木制品产销链跟踪,具有国际通用性和可行性.然而由于DNA技术特点和木材特性,其实际应用尚有一定难度.未来还需加大投资和研发力度,尽快建立公共数据库,发挥其在合法木材贸易中的重要作用.     

白条天牛DNA条形码鉴定技术

白条天牛属(鞘翅目:天牛科)昆虫是口岸植物检疫工作中多次截获的类群,为探讨线粒体细胞色素C氧化酶亚基I(mt DNA COI)基因的特定区段作为DNA条形码鉴定白条天牛种类的可行性,尝试应用该技术对国内外13种白条天牛进行分子鉴定,测定各种类mt DNA COI基因序列(其中10种检疫性白条天牛COI基因序列为国内外首次测序)并进行比对,采用MEGA 5.05构建系统进化树。结果表明:基于mt DNA COI基因的DNA条形码技术可实现对白条天牛种类快速、准确的鉴定。     

猴头菇DNA条形码的构建及验证

为构建猴头菇品种专属的DNA条形码。应用分子生物学方法、选定β-tubulin基因作为条形码序列的目标基因,依据NCBI中猴头菇β-tubulin基因序列的全长,设计了一对用于扩增目标基因的引物:Betu-F1:ATGCGTGAAATCGTCCACC;Betu-R2:GAAGACGGGGGAAAGGAAC。应用上述引物对现有的19个猴头菇的品种进行PCR鉴定。测序后的序列结合BLAST、Clustal、MEGA、BioEdit等软件分析、比对得到多态性位点,分析多态性位点设计出构建条形码序列的特异性引物。经验证表明,猴头菇RT9品种的3个特有的条形码成功获得。试验结果表明,实验成功获得的DNA条形码序列可以将猴头菇RT9品种与猴头菇的其它品种区分鉴定。     

木材树种识别技术现状、发展与展望

介绍了木材树种宏观及其与微观特征结合的识别技术、木材识别特征的术语和定义、木材识别辅助工具和软件以及命名依据;阐述了DNA标记、稳定同位素、近红外光谱分析等木材树种识别新技术的发展,及对木材树种和产地鉴定的应用前景.     

茶油总DNA提取技术及扩增适用性

近年来茶油掺假问题日益突出,传统理化方法难以进行有效鉴定,开发基于特征DNA的茶油掺假的高效鉴定技术十分必要。本研究探索不同方法提取茶油总DNA的提取效果,通过优化提取过程,确立了茶油总DNA最优的改良SDS提取方法。该法可从40 m L茶油中提取出足量用于PCR实验的茶油总DNA。进一步将获得的茶油总DNA作为模板进行特征DNA的PCR扩增验证,经DNA测序分析表明其PCR扩增适用性较好。     

Identification and species-typing of wood rotting fungi using melting curve analysis

A method for identification and typing of wood rotting fungi using the melting temperature [T(m)] of DNA fragments coding rRNA (rDNA) was examined. The T(m)s of four DNA fragments, inter transcribed spacer (ITS) I, ITS II, and two partial fragments of 28S rDNA from each of 20 species of wood rotting fungi, were measured by melting curve analysis. The T(m) variation was large enough between species to enable identification based on the T(m) values. A pair of T(m)s of the ITS I region (between the primers ITS1 and ITS2) and the ITS II region (between the primers ITS3 and ITS4) had the highest resolution for identifying wood rotting fungi. To assess about the diversity of the T(m), intraspecific diversity of these DNA fragment sequences was evaluated using test strain sequences and data from the GenBank/EMBL/DDBJ biological database. The intraspecific diversity of T(m) was considered to be small because the nucleotide diversity of each fragment was small within the species.     

A database for poplar gene co-expression analysis for systematic understanding of biological processes,including stress responses

Reforestation in the humid tropics and arid zones, where trees are often subject to stresses, is an effective approach for mitigating global warming. Forestation with Populus species that are tolerant to the stresses in such regions has been conducted. The selection of poplar trees with higher stress tolerance leads to more efficient reforestation. The genome-wide bioinformatics approaches of gene function have been used for revealing the mechanisms of biological processes, including such stress tolerance. The decoding of the poplar genome has been followed by the genome-wide identification of genes and then the inference of gene function for systematic understanding of biological processes. To predict gene function in poplar, we analyzed poplar gene expression data using DNA microarray datasets obtained from the Gene Expression Omnibus database and developed a database for poplar gene co-expression analysis. Using the database, we illustrate the steps to retrieve two groups of co-expressed genes that are specifically expressed in experiments of hypoxic stress response in gray poplar, a flooding-tolerant tree species. Our database allows users to extract genes involved in biological processes, such as stress reaction, and then is useful for understanding such mechanisms in tree species.     

Identification of market adulterants in East Indian sandalwood using DNA barcoding

Context

East Indian sandalwood (Santalum album L.) in commercial markets is highly prone to adulteration. A number of cases were registered with regard to the adulteration of East Indian sandalwood, but the lack of technical tools for the precise species identification of the source wood stalled most of the court cases.

Aims

The standard DNA barcode regions, the rbcL, matK and trnH-psbA chloroplast genomic sequences recommended by the Consortium of Barcode of Life (COBOL) were analysed to distinguish wood adulterants of East Indian sandalwood.

Methods

Standard polymerase chain reactions with COBOL recommended primers were performed for all three barcode loci. The PCR products after gel elution were sequenced and alignments were performed using CLUSTALX.

Results

Single nucleotide polymorphisms (SNPs) identified with rbcL and trnH-psbA sequences of Erythroxylum monogynum Roxb. as well as with matK sequences of Osyris wightiana Wall ex. Wight could be efficiently utilized for the detection/monitoring of East Indian sandalwood adulterants. Among the two common adulterants O. wightiana and E. monogynum, the former was more similar to S. album and grouped together in the dendrogram.

Conclusion

The study recommends the exploitation of DNA barcoding technique using standard barcodes to trace sandalwood timber adulterants.     

Validation of DNA barcoding as an efficient tool for taxon identification and detection of species diversity in Italian conifers

DNA barcoding may be particularly important in impacting the current trends of world’s biodiversity crisis, public economic issues and science. Forests play a crucial role in self-sustaining natural ecosystems and human environment at both local and regional scales. Over 39% of the world’s forests are formed by conifers; most species are highly exposed to threats and exploited at high frequency. Conifers have an urgent need to be identified through DNA barcodes, but received little attention up to date. The application of standard barcoding protocols appears to be hampered by a lack of consensus on the selection of markers and low rates of successful species identification. We examined whether four marker regions (trnh-psba, rbcL, rpoc1 and matK) matched conifer species taxonomy in a practical biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co-occurrence of the species. Twenty-five conifer species were included in a floristic study based on regional samplings. Genetic distance, similarity and phylogeny approaches were used to assess identification of taxa and species monophyly. We obtained 100% total discrimination success, with rbcL?+?trnH-psbA as the suggested two-locus barcode in terms of universality and efficacy. Consistent barcoding gaps, supported monophyly and regional patterns of diversity, were evidenced. Species requiring further investigation to be fully assessed at a broader context are indicated. We conclude that the time seems now ripe for coherent barcoding campaigns of entire forest tree regional floras.     

基于DNA的木材识别新技术发展与应用

介绍了基于DNA方法的木材识别新技术在国内外的研究进展,在分析现存技术问题的基础上,提出完善DNA方法在木材识别领域应用的建议.为开辟木材识别的新途径,保护木材资源、推进木材合法贸易提供技术支撑.     

Sequencing and quantifying plastid DNA fragments stored in sapwood and heartwood of <Emphasis Type="Italic">Torreya nucifera</Emphasis>

The selection of wood species and the styles of sculpture play key roles in the characterization of Buddhist statues. After Jianzhen, a Chinese Buddhist monk, visited Japan in the mid-eighth century, wood of the genus Torreya had been frequently used to produce single-bole statues. Establishing measures for the accurate identification of wood in the genus Torreya is effective for investigating the drastic change in the production of statues during this period. Analyzing the plastid deoxyribonucleic acid (DNA) fragments extracted from wood is considered helpful in the identification of species in the same genus. This study analyzed the sequences and residual amounts of plastid DNA fragments in the wood of Torreya nucifera. Nucleotide substitutions in the plastid DNA were clearly identified between T. nucifera and the species distributed in China, indicating that the wood of Torreya sp. can be discriminated based on the plastid DNA sequences. DNA polymorphism analyses revealed sequence diversity for the intergenic spacers on the T. nucifera plastid DNA. A series of polymerase chain reaction (PCR) analyses demonstrated that the plastid DNA fragments with a length of approximately 100 bp could be amplified from the residual DNA extracted from the T. nucifera sapwood with longer elapsed years after cutting. Therefore, an identification of wood species in the genus Torreya based on their plastid DNA is considered to be one of the most effective measures taken in the study regarding the historical changes of Buddhist statues.     

植物DNA条形码研究进展

DNA条形码技术是利用标准的、具有足够变异的、易扩增且相对较短的DNA片段在物种内的特异性和种间的多样性而创建的一种新的生物身份识别系统,从而实现对物种的快速自动鉴定。本文从植物DNA条形码的开发、应用、国内研究现状、植物DNA条形码面临的挑战以及发展前景等进行了综合分析,以期推动我国植物DNA条形码和分类学研究的发展。     

Geographical traceability of an important tropical timber (Neobalanocarpus heimii) inferred from chloroplast DNA

The inbuilt unique properties of DNA within the timber could serve as tracking and monitoring tools to verify the legality of a suspected timber in the context of illegal logging, forest certification and chain of custody certification. By using Neobalanocarpus heimii (Dipterocarpaceae) as an example, a population identification database and haplotype distribution map in Peninsular Malaysia were generated for authenticity testing based on four chloroplast DNA markers (trnL intron, trnG intron, trnK intron and psbK-trnS spacer). Twenty one haplotypes were identified from 10 significant intraspecific variable sites. The results clearly revealed that only northern and southern regions of Peninsular Malaysia were distinguishable. Thus, this database could only be used to determine the wood lot of unknown origin at the regional level. Statistical procedure based on the composition of wood lot was used to test whether a suspected timber conforms to a given regional origin. Overall, the observed type I and II errors of the database showed good concordance with the predicted 5% threshold, which might indicate that the database is useful to reveal provenance and establish conformity of wood lot from northern and southern regions of Peninsular Malaysia. Applications of this database for timber tracking are discussed.     

DNA芯片技术在细胞和分子生物学研究上的应用

基因组序列信息和DNA芯片技术给研究者同时分析大规模的基因表达创造了条件,这一技术对细胞和分子生物学研究有极大的促进作用,可以同时分析数百万基因表达的DNA芯片技术将有利于科学家们对重要生物学过程的研究和探索。本文综述了有关生物芯片技术的关键技术及这些技术在基因组和基因功能分析、基因表达研究、生物信号和防御系统、细胞周期调控、转录条控机制、蛋白质组学和食品组成分析上的应用。图3参59