ITS作为粒毛盘菌属DNA条形码的探索

以种内与种间差异以及PCR扩增和测序成功率为评价DNA条形码的重要指标,探索ITS序列作为粒毛盘菌属DNA条形码的可行性。结果表明,ITS成功区分了研究涉及的22个种,其PCR扩增和测序成功率为100%,该片段有望成为该属区分物种的DNA条形码。     

木霉属补充DNA条形码筛选

木霉属真菌是一类重要的生物资源,在工农业、环境保护等方面具有较高经济价值,对其进行快速、准确的物种鉴定兼具理论意义和应用前景。以木霉属35个概念清晰的种为材料,选择ITS、rpb2和 tef1作为候选基因序列,利用TaxonGap对231个序列片段进行分析,将种内与种间序列差异以及序列获取难易程度作为评价指标,筛选该属的补充条形码片段。结果表明,rpb2具有适宜的种内与种间序列差异,其最小的种间差异（2.48%）,大于最大种内差异（1.8%）,种内、种间遗传距离存在明显的间隔区,并且该基因序列具有较高的PCR扩增与测序成功率（94.4%）;ITS和tef1基因序列的种内与种间序列差异之间存在交叉重叠。因此建议rpb2作为木霉属的补充DNA条形码序列。     

用于灵芝属10个物种快速鉴定的DNA条形码筛选与验证

灵芝栽培品种常出现同名异物或同物异名的现象,依赖于灵芝的形态学特征鉴定物种的传统分类方法不足以鉴定物种的种间和种内差异。为开发简便而准确的分子水平灵芝属物种鉴定方法,以常用于种属鉴定的核糖体内转录间隔区（ITS）、微管蛋白基因（β-tubulin）片段、大亚基核糖体DNA(LSU)片段和基因编码RNA聚合酶Ⅱ第二大亚基（RPB2）片段4个DNA条形码,对灵芝属10个物种的44份菌株进行PCR扩增和测序,比较4个条形码的扩增成功率和变异率,根据种内、种间遗传距离差异分析筛选特异性的DNA条形码间隔（DBG）,并利用单独及联合条形码构建系统发育树验证筛选的DBG鉴定灵芝属物种的准确性。研究结果表明：β-tubulin和LSU片段的PCR扩增成功率均达100%,ITS和RPB2片段分别为95.35%和90.91%;对比ITS和LSU片段,β-tubulin和RPB2片段的种内最大遗传距离分别小于各自的种间最小遗传距离,存在明显的DBG,有利于准确对物种进行种间鉴定;利用ITS、β-tubulin和RPB2片段分别构建的系统发育树均能使灵芝属10个物种的所有个体聚为1个单系分支,而LSU片段不...     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序, 比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图, 使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力, 进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明, ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高, 其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势, 且纳入60个属316个种共1 228个样品的网上数据后, 其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高, 其鉴定成功率为63.2%, 并能鉴别一些ITS序列无法鉴别的种。实验结果表明, ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序,比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图,使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力,进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明,ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高,其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势,且纳入60个属316个种共1228个样品的网上数据后,其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高,其鉴定成功率为63.2%,并能鉴别一些ITS序列无法鉴别的种。实验结果表明,ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

亚热带森林乔木树种DNA条形码研究——以哀牢山自然保护区为例

作为新一代植物志iFlora的重要组成部分,DNA条形码已经成为物种鉴定中重要且有效的方法。本研究以亚热带森林的乔木树种为研究对象,开展了DNA条形码的尝试性工作。为评估DNA条形码对鉴定亚热带森林树种的有效性,收集并研究了来自哀牢山自然保护区内5l科111属中204个树种的525个乔木个体。结果显示,所选4个DNA片段（rbcL,matK,trnH-psbA和ITS）的PCR扩增成功率都超过90％;测序成功率rbcL和matK最高,分别为90．7％和90．5％,trnH-psbA次之（83．6％）,ITS最低（73．5％）,表明4个片段在亚热带森林乔木中都具有较好的通用性。应用BLAST与NJ Tree两种方法,对物种和属水平的鉴别成功率进行统计,发现单片段中ITS最高,分别为68．4％-81．3％和99．0％～100％,核心条码rkL和matK组合的成功率是52．8％～60．2％和86．7％～90．5％,再与补充条码trnH-psbA和ITS联合,可以成功鉴别74．7％～79．6％哀牢山自然保护区亚热带森林中的乔木物种。由于ITS片段在亚热带森林部分重要树种类群（樟科和壳斗科等）中的测序成功率较差,所以对这些植物类群采用trnH-psbA作为DNA条形码是一个更好的选择。     

基于芸香科的植物通用DNA条形码研究

植物DNA条形码研究是近10年来进展最迅速的学科之一,其通用序列的筛选一直是该领域研究的热点问题.2009年,生命条形码联盟植物工作组推荐rbcL+matK组成复合序列作为植物通用条形码,但其研究对象中近缘属、种较少,且物种水平鉴定成功率仅为72%,所以仍在进行验证和新序列的研究工作.本研究选取nrDNAITS2序列,利用其具有Ⅱ级结构的特性、通过不同物种类型模型判定全长,将其和目前热点候选序列(matK,rbcL,psbA-trnH,rpoC1,ycf5)及nrDNAITS序列针对芸香科72属192种300个样本进行比较,试图在同一科属下更多近缘种存在时,真实判定候选序列的鉴定能力.结果表明,自行设计引物的ITS2序列具有较好的PCR扩增和测序成功率,在所考察的候选序列中具有最大的种间变异和较小的种内变异,且两者存在极显著差异,同时物种鉴定成功率最高,各评价指标均优于其他候选序列.证实ITS2序列在单一科属内的"高效性",故推荐其作为植物DNA条形码通用序列之一.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

中国秋海棠属 (秋海棠科) 植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限;ITS/ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100%/96%,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS/ITS2纳入种子植物DNA条形码核心片段中的观点。     

基于形态特征和ITS序列对7个鹅膏菌属菌株的分类鉴定

以采自浙江省丽水地区的7个鹅膏菌属菌株作为研究材料,在基于形态特征进行初步鉴定的基础上,对7种鹅膏菌的rDNAITS区段进行克隆测序和序列特征比较分析。进一步对ITS序列进行核酸序列数据库GenBank同源性检索比对,将从GenBank检索获得的9个最相似物种的ITS序列连同7种鹅膏菌的ITS序列一起作系统发育分析。结果表明:基于ITS序列对f6、f9和f493个菌株的分子鉴定支持了基于形态特征的鉴定结果,对f5的分子鉴定不支持形态鉴定的结果,f8为鹅膏菌属内某种,f66为鹅膏菌属内某种,并与Amanitafulva,A.atrofusca,A.orientifulva3种鹅膏菌的亲缘关系较近,f7与另外6种鹅膏菌的亲缘关系相差甚远。研究结果提示基于分子水平上的ITS序列分析不能单方面作为大型真菌分类鉴定的可靠依据,可以作为基于传统形态学分类鉴定的辅助参考依据。     

DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China

Some species of the genus Amanita are economically important gourmet mushrooms, while others cause dramatic poisonings or even deaths every year in China and in many other countries. A DNA barcode is a short segment or a combination of short segments of DNA sequences that can distinguish species rapidly and accurately. To establish a standard DNA barcode for poisonous species of Amanita in China, three candidate markers, the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1 alpha (tef1 α) were tested using the eukaryotic general primers for their feasibility as barcodes to identify seven species of lethal fungi and two species of edible ones which can easily be confused with the lethal ones known from China. In addition, A.phalloides—a European and North American species closely related to one of the seven taxa, A.subjunquillea was also included. PCR amplification and sequencing success rate, intra and inter specific variation and rate of species identification were considered as main criteria for evaluation of the candidate DNA barcodes. Although the nLSU had high PCR and sequencing success rates (100% and 100% respectively), occasional overlapping occurred between the intra and inter specific variations. The PCR amplification and sequencing success rates of ITS were 100% and 85.7% respectively. ITS showed high sequence variation among species group and low variation within a given species. There was a relatively high PCR amplification and sequencing success rate for tef1 α (85.7% and 100% respectively), and its intra and inter specific variation was higher than that of ITS or nLSU. All three candidate markers showed hight species resolution. ITS and tef1 α had a more clearly defined barcode gap than nLSU. Our study showed that the tef1 α and nLSU can be proposed as supplementary barcodes for the genus Amanita, while ITS can be used as a primary barcode marker considering that the ITS region may become a universal barcode marker for the fungal kingdom.     

我国云南食用牛肝菌的DNA条形码研究

iFlora是结合传统分类学与DNA测序和信息技术,通过系列关键技术进行集成,构建便捷、准确识别物种和掌握相关数字化信息的新一代智能植物志。iFlora研发中首要和迫切的任务之一就是寻找适合于大多数植物和经济蘑菇的标准DNA条形码序列。为筛选适合大型经济蘑菇的DNA条形码,本研究以云南食用牛肝菌为例,选取野生食用菌市场上常见的、被当地人认为的4“种”牛肝菌为研究对象,利用核糖体大亚基（nrLSU）、翻译延长因子1-α（tefl-α）、RNA聚合酶II大亚基（rpbl）和RNA聚合酶II的第二个大亚基（rpb2）四个DNA序列,使用真核生物通用引物进行扩增、测序和测试。研究发现这4“种”样品实际上代表了12个独立的物种,进一步研究表明4个候选片段的扩增和测序成功率均为100％,且不存在种问和种内变异的重叠。4个片段的物种分辨率均较高,但与nrLSU相比,rpbl、tefl-α和rpb2具有更为明显的条形码间隔。鉴于rpbl比tefl-α和rpb2具有更高的种间变异和较低的种内变异,建议将rpbl作为牛肝菌属的核心条形码,tefl-α和rpb2可作为该属的辅助条形码。     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.     

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intra- and inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra- and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intra- and inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.     

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.