DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

Serologic Response to SARS-CoV-2 in COVID-19 Patients with Different Severity

Virologica Sinica - The immense patient number caused by coronavirus disease 2019 (COVID-19) global pandemic brings the urge for more knowledge about its immunological features, including the...     

天蚕素AD在毕赤酵母中的多拷贝整合表达及表达条件优化

【背景】天蚕素抗菌肽是目前研究最清楚、效果最显著的抗菌肽,实现工业化生产为其在农业、养殖业中的应用奠定了基础。【目的】获得一株高效生产天蚕素AD的基因工程菌株。【方法】构建重组载体pGAPZαA-CAD通过电击转化至PichiapastorisX33中,表达天蚕素AD基因并获得X33/GCAD菌株;构建重组载体pUCGAP-CAD导入至X33/GCAD菌株中。pGAPZαA-CAD是以博来霉素为抗性筛选标签被整合到P. pastoris X33的GAPDH启动子区域,pUCGAP-CAD是以遗传霉素为抗性筛选标签被整合到P. pastoris X33的非翻译rDNA区域,最终获得一株高效表达天蚕素AD的酵母菌株X33/GUCAD。【结果】通过质谱分析鉴定X33/GUCAD表达的抑菌物质为天蚕素AD,通过发酵条件的优化,表明X33/GUCAD菌株在以甘油为碳源和以蛋白胨、酵母提取物为有机氮源的情况下具有较强表达天蚕素AD的能力。【结论】较高的拷贝数更有利于提高天蚕素AD的产量,此工程菌株在后期发酵过程中稳定性较好,适于工业化生产。     

馆藏植物标本与iFlora

构建准确、有效的物种遗传信息库并运用其作为主要的物种鉴定检索数据库,是新一代植物志（iFlora）与传统植物志的本质区别。采集并准确鉴定数以万计的植物样品用于建立遗传信息库存在一定困难,随着遗传信息获取技术的快速发展,使馆藏植物标本成为获取遗传信息的重要补充,并具有提升遗传信息库建设速度和可靠性的较大潜能。本文结合已有的研究和实验室多年工作实践,论述了植物标本作为iFlora物种遗传信息提取材料的可行性和不可替代性;总结了标本遗传信息提取过程中的主要困难和存在问题,并提出一些解决方法;阐述了使用馆藏标本特别是利用模式标本材料构建遗传信息标准库对iFlora构建的重要作用和意义。     

Comparative study of four rice cultivars with different levels of cadmium tolerance

Cadmium (Cd) has been identified as a significant pollutant due to its high solubility in water and soil and high toxicity to plants and animals. Rice, as one of the most important food crops, is grown in soils with variable levels of Cd and therefore, is important to discriminate the Cd tolerance of different rice cultivars to determine their suitability for cultivation in Cd-contaminated soils. This study investigates the primary mechanisms employed by four rice cultivars in attaining Cd tolerance. HA63 cultivar reduces Cd uptake by increasing Fe absorption through activation of phytosiderophores. T3028 cultivar accumulates the highest level of Cd in leaves while also activating its reactive oxygen species (ROS) scavenging system, including antioxidant enzymes and phytochelatins. In some rice cultivars (such as HA63), a cyanide-resistant respiration mechanism, important in Cd detoxification, was also promoted under the Cd stress. In conclusion, different rice cultivars may adopt different biochemical strategies and respond with different efficiency to Cd stress.     

Comparative analysis of complete plastid genome reveals powerful barcode regions for identifying wood of Dalbergia odorifera and D. tonkinensis (Leguminosae)

Dalbergia odorifera T. C. Chen (Leguminosae), a rare and endangered tree species endemic to Hainan Island of China, produces the most expensive and rarest wood in China. The wood characteristics of D. odorifera are remarkably similar to those of D. tonkinensis (a much less sought-after species from Vietnam), and the DNA from wood is often highly degraded, making it very difficult to identify the two species using anatomical features or DNA barcoding based on regular DNA markers. To solve the confusion of identifying wood reliably from the two species, we built and analyzed the plastome library of 26 samples from 18 Dalbergia species, of which 12 samples from eight closely related species of D. odorifera are newly sequenced in this study. Phylogenomic analysis suggested that the relationships among the 26 samples are mostly well resolved, and conspecific individuals from different populations of D. odorifera and D. tonkinensis clustered together. Between the plastid genomes of the two species, we identified 129 indels and 114 single nucleotide polymorphisms. By assessing a subset of 20 nucleotide polymorphisms and 10 indels using 37 population-level samples (20 samples of D. odorifera and 17 samples of D. tonkinensis), we recovered eight species-specific barcode regions that could be suitable for identifying the wood D. odorifera and D. tonkinensis. To examine their utility in wood identification, we amplified the eight DNA barcodes using six wood samples and recovered an amplification success rate of 83.3%, demonstrating a reliable method for precise wood identification of the two species.     

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron

Standard plant DNA barcodes based on 2–3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3–4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.     

Natural hybridization origin of <Emphasis Type="Italic">Rhododendron agastum </Emphasis>(Ericaceae) in Yunnan,China: inferred from morphological and molecular evidence

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. The assumed parents, Rhododendron delavayi Franch. and R. decorum Franch., are morphologically distinct, and the putative hybrid species, R. agastum Balf. f. et W. W. Smith, has an intermediate morphology. We used the main morphological characters, sequences of the nuclear ribosomal DNA ITS region, and the chloroplast DNA trnL-F intronspacer to analyze the three species, and compared these morphological and molecular data with an artificial hybrid between R. decorum (♀) × R. delavayi (♂). From the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum.     

四个DNA片段在山茶属分子系统学研究中的应用

选取山茶属14个组中的10组21种植物对目前常用于属内种间的4个DNA片段（ITS、waxy、trnL-F、rpL16）进行序列测定。结果表明：（1）来自叶绿体基因组的2个片段（trnL-F、rpL16）其PCR扩增和测序都很容易,但两者的进化速率都非常慢,序列矩阵只有很少信息位点（trnL-F含9个,rpL16为20个）,不能提供必要的系统发育信息。（2）来自核基因组的ITS片段其PCR产物比较容易获得,但其序列的测定存在较多问题。（3）waxy是来自核基因组的另一个片段,其PCR扩增因受模板DNA的数量和质量的影响很大而有一定难度,但其进化速率较快,序列矩阵具有较多信息位点（92个）,并且在山茶属是单拷贝,这对于解决山茶属这类具有许多近缘物种的类群的系统关系有重要价值。基于tsnL-F、rpL16和waxy三组数据所建分子系统树支持山茶属为一单系,但属下系统由于取样等原因有待进一步研究。