Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

An innovative protein expression system using RNA polymerase I for large-scale screening of high-nucleic-acid content Saccharomyces cerevisiae strains

Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high-nucleic-acid content yeasts more efficiently, a cap-independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I-recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo-dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap-dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high-nucleic-acid content yeasts.     

基于CRISPR/Cas9的基因分析方法研究进展

自2012年首次证明了CRISPR/Cas9可以在体外进行DNA切割试验以来,CRISPR技术逐渐在基因编辑研究中获得了迅速的发展,除了应用于基因编辑领域之外,它在基因表达调控、基因成像、基因分析等方面也展现出了巨大的应用潜力。尤其在基因分析领域,CRISPR技术由于其精确的基因识别、室温的反应条件、易设计性和操作性等特色,使得一系列新型的基因检测技术得以发展,并取得了超越常规技术的一些检测参数。本文以Cas9蛋白为对象,综述了近些年来在该领域取得的研究进展。主要论述Cas9蛋白的功能、改造、引导RNA(sgRNA)的设计及其在基因分析方法上的应用。     

Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development

Fertilization Independent Endosperm (FIE) is an essential member of Polycomb Repressive Complex 2 (PRC2) that plays important roles in the developmental regulation of plants. OsFIE1 and OsFIE2 are two FIE homologs in the rice genome. Here, we showed that OsFIE1 probably duplicated from OsFIE2 after the origin of the tribe Oryzeae, but has a specific expression pattern and methylation landscape. During evolution, OsFIE1 underwent a less intensive purifying selection than did OsFIE2. The mutant osfie1 produced smaller seeds and displayed reduced dormancy, indicating that OsFIE1 predominantly functions in late seed development. Ectopic expression of OsFIE1, but not OsFIE2, was deleterious to vegetative growth in a dose‐dependent manner. The newly evolved N‐terminal tail of OsFIE1 was probably not the cause of the adverse effects on vegetative growth. The CRISPR/Cas9‐derived mutant osfie2 exhibited impaired cellularization of the endosperm, which suggested that OsFIE2 is indispensable for early seed development as a positive regulator of cellularization. Autonomous endosperm was observed in both OsFIE2^+? and osfie1/OsFIE2^+? but at a very low frequency. Although OsFIE1‐PRC2 exhibited H3K27me3 methyltransferase ability in plants, OsFIE1‐PRC2 is likely to be less important for development in rice than is OsFIE2‐PRC2. Our findings revealed the functional divergence of OsFIE1 and OsFIE2 and shed light on their distinct evolution following duplication.     

中国缺齿藓科新记录种——冻原丝瓜藓(新拟,Pohlia tundrae A. J. Shaw)

冻原丝瓜藓(Pohlia tundrae)在北美被首次描述,随后在欧洲中部、俄罗斯远东地区和西伯利亚西北部也相继报道。最近,作者在西藏林芝也发现了冻原丝瓜藓的分布。该文详细描述了冻原丝瓜藓的形态结构特征,提供了显微形态学照片,并对其生境、地理分布以及与相似种的形态学进行了比较讨论。凭证标本存放于中国农业大学标本馆(BAU)。     

青藏高原高寒草甸土壤环境对线虫功能多样性的影响

土壤线虫是地下食物网的重要组成部分, 在生态系统能量流动和物质循环中起着至关重要的作用。大量研究报道了肥力等土壤环境对土壤线虫物种多样性及各功能群多度的影响, 而我们对土壤线虫功能多样性如何响应土壤环境变化依然知之甚少。本研究以群落水平个体大小和个体大小多样性表征土壤线虫功能多样性。在青藏高原高寒草甸选择3个研究点, 调查和分析了不同生境(沟底平地、阴坡、阳坡和山顶)土壤线虫物种多样性、各功能群多度和功能多样性及其与土壤理化因子和植物多样性的关系。结果表明: (1)土壤线虫个体多度和物种多样性在阳坡最高, 随土壤pH值和土壤总磷增加而升高; 而基于个体大小的土壤线虫功能多样性主要受土壤养分影响, 随土壤总氮和有机质增加而增加, 随土壤总磷含量增加而减少; (2)食细菌和食真菌线虫多度在沟底最高, 植食与捕食杂食线虫多度在山顶最低; 除捕食杂食线虫外, 各功能群多度也主要随土壤磷增加而升高; 除食真菌线虫外, 各功能群多度随植物物种丰富度的增加而减少。本研究强调了土壤线虫物种和功能多样性受不同土壤环境因子的影响, 丰富了土壤线虫多样性研究的内容, 为理解高寒草甸土壤动物多样性形成、维持和变化提供了更广阔的 视角。     

Overexpression of the chitinase gene CmCH1 from Coniothyrium minitans renders enhanced resistance to Sclerotinia sclerotiorum in soybean

Transgenic Research - Pathogenic fungi represent one of the major biotic stresses for soybean production across the world. Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a...     

青藏高原多年冻土区不同水分条件的高寒草甸根系功能性状对增温的响应

在青藏高原多年冻土广泛分布的风火山地区,选择小嵩草（Kobresia pygmea）草甸和藏嵩草（Kobresia tibetica）沼泽化草甸为研究对象,采用开顶增温室（Open top chambers, OTCs）模拟气候变暖,探讨模拟增温对土壤水分差异的两种草甸地下生物量及根系功能性状的影响。结果显示,（1）增温显著增加小嵩草草甸0—20 cm根系生物量,主要是由于表层（0—10 cm）根系生物量显著增加,而对藏嵩草沼泽化草甸根系生物量无影响。（2）增温显著增加了小嵩草草甸根组织密度,同时提高了藏嵩草沼泽化草甸10—20 cm的比根长和比根面积（3）增温降低了小嵩草草甸的根系碳含量及10—20 cm根系氮含量,增加了藏嵩草沼泽化草甸的碳含量及10—20 cm根系氮含量,显著提高了小嵩草草甸和藏嵩草沼泽化草甸深层（10—20 cm）根系碳氮比。这些结果预示着增温使得土壤水分较低的小嵩草草甸朝着资源保守的慢速生长型发展,以适应暖干化的环境;土壤水分较高的藏嵩草沼泽化草甸朝着资源获取的快速生长型发展,加速利用土壤中的养分满足植物生长需要。可见,土壤水分可以调节高寒草甸对气候变暖的演变趋势,强调了水分的重要性。