中国葡萄属植物野生种多样性分析

中国是葡萄属植物的主要起源地之一,也是世界葡萄属植物种类最多、遗传资源最丰富的国家之一,起源于中国的葡萄属植物共有40种、1亚种、13变种。本研究根据《葡萄种质资源描述规范和数据标准》,选择多项性状指标作为形态鉴定参数,对起源于中国的葡萄属植物23个种的生物学性状、植物学特征、农艺性状等进行鉴定,分析其遗传多样性特点。结果表明,中国葡萄属植物的主要物候期,嫩梢新梢的绒毛、颜色,叶片的形状、颜色、锯齿,果实大小、颜色、香型和花器官等形态性状都有丰富的变化,表现出丰富的多样性。上述研究结果将为葡萄属植物的分类鉴定提供依据,也将为葡萄属植物演化研究和育种利用提供参考。根据本研究结果,建议对目前使用的《葡萄种质资源描述规范和数据标准》作进一步修订和完善,以适应我国野生葡萄资源多样性评价研究。     

一株海洋藻类的分子鉴定及分类

海洋藻类种类繁多,由于形态的易变性和生活史的复杂性,传统的形态分类往往十分困难.分子标记技术的发展,使DNA分子成为区分物种的有效手段,成功地解决了许多疑难藻种的分类鉴定问题.本研究测定和比较了一株海洋藻类ST3的18srDNA基因部分核苷酸序列,序列长度为1733bp,通过与GenBank中序列数据进行比较分析,对其进行了分子鉴定和分类.研究结果表明藻株ST3属于硅藻门海链藻目,与骨条藻属的亲缘关系较海链藻属为近,但与骨条藻属遗传距离远大于骨条藻属种间差异,说明ST3可能是硅藻门海链藻目新的藻属.18srDNA可用于海洋藻类分子分类与鉴定.     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株, 利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究, 探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布。共鉴定出13属26种, 其中优势属为汉逊酵母属Hanseniaspora(5种), 假丝酵母属Candida(4种), 毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种)。对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较, 以探讨不同地理起源地酵母种内序列稳定性及其变异。     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株,利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究,探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布.共鉴定出13属26种,其中优势属为汉逊酵母属Hanseniaspora(5种),假丝酵母属Candida(4种),毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种).对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较,以探讨不同地理起源地酵母种内序列稳定性及其变异.     

桃叶珊瑚属植物的叶绿体基因组结构特征及系统发育分析

为确定桃叶珊瑚属(Aucuba)植物叶绿体基因组的结构及其序列变异,揭示其属下种间亲缘关系,该研究对桃叶珊瑚(A. chinensis)、花叶青木(A. japonica var. variegata)等6种桃叶珊瑚属植物和丝缨花属植物黄杨叶丝缨花(Garrya buxifolia)进行二代测序,利用生物信息学软件对其叶绿体基因组序列进行组装和注释,并进行基本特征分析、序列比较以及系统发育分析。结果表明:(1)桃叶珊瑚属植物叶绿体基因组具典型的环状四分体结构,6条序列全长157 891～158 325 bp,均编码114个基因,包括80个蛋白质编码基因、30个tRNA基因和4个rRNA基因。(2)6种植物叶绿体基因组高频密码子数均为29个,偏好以A/U结尾,确定了这6条序列的最优密码子共100个,包含12个共有的最优密码子。(3)6条叶绿体基因组序列共检测到270条散在重复序列,133条串联重复序列以及412个SSR位点。(4)比较基因组学分析结果表明,该属植物叶绿体基因组序列高度保守。(5)从叶绿体基因组中筛选出10个高变片段。(6)系统发育分析结果显示支持桃叶珊瑚属为一个支持率较高的单系,与丝缨花属关系较近。该研究中的5种桃叶珊瑚属植物以及1种丝缨花属植物的叶绿体基因组均为首次测序组装,揭示了桃叶珊瑚属及其属下种间的系统发育关系,为桃叶珊瑚属植物的分类鉴定和系统发育提供了参考资料。     

三种荆州半夏的ITS序列分析与种属鉴定

通过采集自湖北荆州地区的三种不同表现型的半夏属植物的叶片组织中提取基因组,纯化后经直接PCR扩增得到其对应的ITS序列并测序。运用NCBI Gen Bank数据库进行序列比对与进化树分析。根据比对与分析结果,结合植物形态学特征显示,HBB-01为Pinellia ternata,为《中国药典》中规定的半夏类药材品种;HBD-02为P.tripartita,为药用半夏的混淆品;HBF-03的结果较为复杂,其不仅与芋属(Colocasia)天南星属(Arisaema)、半夏属(Pinellia)等植物ITS序列的同源性高达80%以上,而且在进化关系上,它们也有一定关联。所以推测HBF-03可能为天南星科不同属但亲缘关系较近的植物产生的变种,具体种属来源有待进一步论证。通过三种半夏种属来源及其亲缘关系的分析,鉴定了荆州地区的半夏人工种植的主要品种,对其当地药农种植符合《中国药典》规范的半夏药材具有指导意义,为后续优质半夏资源的保护与选育提供了参考依据。     

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。     

吸引人的葡萄科

葡萄科植物有12属约700种,中国有7属100多种。本科的分类和应用都吸引人。 葡萄的知识面 葡萄(Vitis vinifera)是大家习知的檀物,但主要是由于它的果实为著名水果,而对于它的植物形态能认识深入些的却并不多(对一般人而言)。有的人知道是藤本植物,搭架栽培的,结葡萄串……。如果问葡萄的花怎样,怕是说不清了。从植物分类上说认识葡萄最好先弄清葡萄属的特征。对此,还应与接近的属——     

利用rbcL-a序列模拟检测小鼠食性

动物食性是研究动物生活习性中非常重要的一环,利用DNA条形码进行检测变得非常快捷和方便。利用这项技术,模拟生态环境研究动物食性,来检测小白鼠的食物组成。首先,给小白鼠喂食了6种植物(白菜、玉米、胡萝卜、小麦、生菜、花椰菜)并收集其粪便,然后对这些样本进行DNA提取、PCR扩增与克隆,最后测序并获得来自质体的rbcL基因(ribulose-1,5-bisphosphate carboxylase large subunit gene)的部分序列(rbcL-a)。粪便样本中得到的序列分别与GenBank数据库和实验中得到的作为食物的5种植物的rbcL-a序列(玉米中未发现)进行比对,确定其分类单元。利用GenBank数据库,共鉴定出了3科和1属。与实验中的5种植物rbcL-a序列比对能鉴定出3个植物物种。表明实验用的rbcL-a序列在检测粪便中物种方面具有很大的潜力,对野生动物的食性分析及分子生态学的研究具有重要的意义。     

基于4个叶绿体基因识别蓑藓属(Macromitrium)植物的可行性研究

我国的蓑藓属植物形态变异式样复杂,分类问题多.DNA条形码技术是一种新的物种鉴定技术.本研究以采自浙江、福建、云南、广西、四川等省的蓑藓属(Macromitrium)7个物种及其外类群直叶藓Macrocoma tenue subsp. sullivantii和火藓Schlotheimia grevilleana的38份标本为对象,获得了它们的叶绿体基因trnL、trnG、psbT和rps4序列,基于这些基因的不同组合构建了15棵贝叶斯系统发育树,获得了相应的蓑藓属植物的物种识别率、种内和种间的遗传距离.发现基于trnL-rps4、trnL-trnG-rps4、trnL-psbT-rps4、trnG-psbT-rps4和trnL-trnG-rps4-psbT等5个组合能够较好地识别本研究中蓑藓属植物,均得到了100%的物种识别率.考虑到扩增和测序的成功率和得到的7种蓑藓属植物的系统发育关系,建议将trnL-trnG-psbT组合用于蓑藓属植物的系统发育分析和物种识别的DNA条形码.     

Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification

Guignardia bidwellii is the etiological agent of grape black rot, a disease affecting Vitis and other Vitaceae that can cause heavy crop losses in vineyards. Its identification is based mainly on morphological characters and the symptoms on plants but, due to their variability, they may be difficult to interpret to reliably distinguish the pathogen to species. To date, despite the economic importance of G. bidwellii, no molecular investigations have been carried out on Vitis isolates and few sequence data are available for cultures derived from ornamental host plants. We analyzed samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins by morphological, molecular and proteomic techniques, including ITS1-ITS2 regions and calmodulin gene sequencing, as well as matrix-assisted laser desorption/ionization analysis by time-of-flight mass spectrometry (MALDI-TOF MS). This polyphasic approach allowed assessing the phylogenetic relationships among the different isolates and suggested the existence of two distinct species. The advantages of a polyphasic approach for the identification of G. bidwellii are highlighted.     

Testing reticulate evolution of four Vitis species from East Asia using restriction‐site associated DNA sequencing

Reticulate evolution is an important driving force of angiosperm evolution. It has been proposed as an important evolutionary process in Vitis L. subgenus Vitis. Events of natural hybridization and introgression of several taxa native to North America have been hypothesized and discussed. However, there is no convincing evidence of reticulate evolution reported for closely related Vitis taxa from East Asia. We aim to explore natural hybridization and introgression among four closely related Vitis taxa from East Asia (V. amurensis Ruprecht, V. romanetii Romanet du Caillaud, V. shenxiensis C. L. Li, and V. piasezkii Maximowicz) with the restriction‐site associated DNA sequencing technique. A total of 46 accessions, covering the potential morphological and geographic variation of each species, are sequenced. Our results show a complex evolutionary pattern of the four Vitis species. The phylogenetic inference suggests that V. amurensis is monophyletic, however, V. romanetii, V. shenxiensis, and V. piasezkii do not appear to be monophyletic. Significant signals of introgression in some accessions have been detected by population structure analyses. D‐statistics analysis and population structure analyses support the presence of introgression between V. shenxiensis/V. piasezkii and V. romanetii in sympatric populations, but a strong signal of admixture has not been recognized between distantly located populations. Our results provide strong evidence of reticulate evolution among V. romanetii, V. shenxiensis, and V. piasezkii.     

刺葡萄种内遗传多样性研究进展

刺萄萄主要分布在我国长江中下游及以南地区,是中国野生萄萄中果粒最大的一个种,其果实高抗炭疽病和白腐病,在湖南、江西、福建、贵州等省的部分地区一直作为鲜食和酿酒品种栽培利用。刺葡萄种内遗传多样性较为丰富,为了充分挖掘利用刺葡萄资源,提高刺葡萄资源的利用效率,本文从刺葡萄的物候学、花器类型、形态性状、果实理化性状、花粉形态、DNA遗传水平和抗性等方面进行种内多样性的研究和综述,以期为刺葡萄种质资源的保护、鉴定评价和发掘利用奠定基础。     

Molecular phylogeny of the genus Vitis (Vitaceae) based on plastid markers

? Premise of the study: This work represents the first molecular phylogeny of the economically important genus Vitis, an important genetic resource for breeding in grapevine, Vitis vinifera. ? Methods: A molecular phylogeny of Vitis using a combined data set of three noncoding regions of the plastid DNA genome was constructed from 47 accessions covering 30 species of Vitis. The data for the trnL-F marker were combined with previously published data across the Vitaceae. ? Key results: The molecular phylogeny demonstrated monophyly of the genus Vitis. Based on the combined analysis of three genes, Vitis is split into three clades that mirror the continental distribution of these accessions. The diversity is highest in the Asian clade, but the general genetic distances across taxa from different continents are relatively small. ? Conclusions: The findings support a relatively recent and intense gene flow between East Asia and North America and the possible impact of hybridization on the evolution of the genus Vitis. Taxon identity in important stock collections should be screened carefully because roughly 10% of the accessions analyzed in the present study had been misidentified.     

A detailed study of leaf micromorphology and anatomy of New World Vitis L. subgenus Vitis within a phylogenetic and ecological framework reveals evolutionary convergence

We investigated leaf anatomy and micromorphology in the New World Vitis using light and scanning electron microscopy to understand the correlation of these traits to molecular phylogenetic relationships and environmental affinity. We observed traits known to differ among species of Vitis with importance in traditional taxonomy of Vitis: trichome type, stomata morphology, mesophyll organization, and midrib vascularization. We found that traits associated with water conductance and photosynthesis comprised the highest loadings of axis one of a principal components analysis (PCA) while traits related to gas exchange (i.e., the stomatal apparatus) had high loadings on axis two. Using the PCA, we identified seven clusters of species, which showed little correlation to recently reported molecular phylogenetic relationships. Moreover, analyses using Bayes Traits and Bayesian Binary Method revealed little to no phylogenetic signal in trait evolution. PCA axes one and two separated species occurring in dry southwestern North American habitats from those in mesic places. For example, a cluster of V. monticola and V. arizonica occurred adjacent to a cluster of V. californica and V. girdiana in ordination space, and the latter three species share key leaf anatomical traits. Nevertheless, among these, only V. arizonica and V. girdiana are closely related according to molecular phylogeny. Thus, the leaf micromorphological/anatomical traits of Vitis observed in this study are highly correlated with environment, but not phylogenetic relationships. We expect that trait similarities among distantly related species may result from evolutionary convergences, especially within xeric habitats of western North America.     

PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism

We report two polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar species based on amplification of a variable region of the 28S gene of ribosomal DNA. The four species we investigated are mosquitoes of the Anopheles minimus group: An. aconitus, An. varuna and An. minimus species A and C. The formally named species are vectors of human malaria parasites in south-east Asia but are difficult to distinguish with certainty on the basis of morphology. Allele-specific amplification was used to differentiate An. minimus A from An. minimus C. This technique has been widely used for the diagnosis of species. Single-strand conformation polymorphisms (SSCPs) were used to separate all four species. This technique, which has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplification; it is ideally suited for the detection of heterozygotes; it utilizes more of the information in the PCR product than allele-specific amplification; it distinguishes all four species considered here and could easily be extended to other species; previously unknown intraspecific variation and additional species are likely to be detected. Thus, SSCPs provide valuable population genetic information which allele-specific amplification does not.     

A study on pollen morphology of the genus Vitis L.

The pollen grains of 32 species, 1 subspecies and 4 varieties of Vitis and 1 species of Muscadinia were scanned with Hitachi S-800 scanning electron microscope and described in the present paper. In Vitis, the pollen morphology is found to be valuable for the identification of species, though it is of little significance in supraspecific classification and systematic consideration. The ornamentation of Vitis could be divided into four types: ( 1 ) The pollen grains are oblate or rarely spheroidal (P/E =0.97 ～ 1.08) in shape, with colpi wide enough to show dense granulae inside. The pollen grains of V. coignetiae Pull. Ex Planch. belong to this type. (2) The pollen grains are prolate to subprolate (P/E = 1.25 ～ 1.76 ), with sparse granulae within the colpi. This type includes l0 species, 1 subspecies and 3 varieties, such as V. hui Cheng etc. (3) The pollen grains of V. romanetii Roman du Caill. ex Planch. and V. davidii (Roman du Caill. ) Foex. var. ferruginea Merr. et Chun are prolate (P/E= 1.87 ～ 2.1), with colpi narrow and linear-shaped, but rhombic in the equational region. (4) The pollen grains are prolate or perprolate (P/E= 1.30 ～ 2. 0), with narrow and linear-shaped colpi. The species with this type of pollen grains are Muscadiniarotundifolia (Michx.) Small and 20 species of Vitis.     

SSR分子标记鉴定山葡萄和河岸葡萄种间杂种

利用SSR分子标记技术,对山葡萄和河岸葡萄种间杂交后代的真伪性进行鉴别。从12对多态性SSR引物中筛选出能扩增出父本特异性条带的7对引物,作为杂种鉴定的标记。用这7对引物对239株山葡萄和河岸葡萄的杂交后代进行鉴定。结果表明,有161株后代具有父本的特异性条带,结合田间形态学分析,确认为真杂种。另外,后代中还出现了新的条带,表明杂交后代产生了丰富的变异。因此,SSR标记可以有效地对葡萄属种间杂交后代进行真实性鉴定,可作为葡萄种质创新的有效辅助手段。     

Phylogeny and diversity of Japanese truffles (Tuber spp.) inferred from sequences of four nuclear loci

The genus Tuber, which includes some highly valued truffles, comprises ascomycetous ectomycorrhizal fungi associated with ecologically important tree species. Although the genus is distributed over northern temperate regions, we know little about the phylogeny and diversity of Tuber species in Japan. We have collected 186 new Tuber ascoma samples in Japan over a 10 y period. The identities and phylogenies of the samples were analyzed with sequences of four nuclear loci (i.e. internal transcribed spacer [ITS] and large subunit [LSU] regions of rDNA, elongation factor 1 alpha [EF1-α], and RNA polymerase II large subunit [rpb2] genes). Based on the species delimitation of 95% sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified 20 Tuber species. The number of observed species did not reach an asymptote with our maximum sampling localities in a species accumulation curve. The Chao2 species richness estimator indicated that at least 40 Tuber species should be present in Japan. Molecular phylogenetic analyses revealed that Japanese Tuber species belong to five major phylogroups, including Macrosporum, which had not been reported previously in Asia. Two Japanese species were morphologically and phylogenetically distinct from other known phylogroups, and here we propose a new Tuber phylogroup, Japonicum. In addition most of the other Japanese species formed separate clades within individual major phylogroups and deserve to be proposed as new species. Detailed molecular phylogeny within individual phylogroups revealed the existence of phylogeographic structures at both continental and within-Asia scales, indicating that migration and allopatric speciation have occurred even between the mainland and islands in Asia. Although our findings substantially advance current understanding of Tuber diversity and phylogeny, comparable richness estimation and multilocus phylogeny in other geographic regions are necessary to unequivocally address global patterns of Tuber diversity and biogeography.     

Molecular phylogeny and biogeography of Picea (Pinaceae): implications for phylogeographical studies using cytoplasmic haplotypes

The center of diversity is not necessarily the place of origin, as has been established by many plant molecular phylogenies. Picea is a complicated but very important genus in coniferous forests of the Northern Hemisphere, with a high species diversity in Asia. Its phylogeny and biogeography were investigated here using sequence analysis of the paternally inherited chloroplast trnC-trnD and trnT-trnF regions and the maternally inherited mitochondrial nad5 intron 1. We found that the North American P. breweriana and P. sitchensis were basal to the other spruces that were further divided into three clades in the cpDNA phylogeny, and that the New World species harbored four of five mitotypes detected, including two ancestral ones and three endemics. These results, combined with biogeographic analyses using DIVA and MacClade and fossil evidence, suggest that Picea originated in North America, and that its present distribution could stem from two times of dispersal from North America to Asia by the Beringian land bridge, and then from Asia to Europe. Most of the northeastern Asian species and the European P. abies could arise from a recent radiation given the very low interspecific genetic differentiation and pure mitotype of them. Considering that the ancestral mtDNA polymorphism can be preserved in many descendant species, even distantly related ones, we suggest that more species, at least the closely related ones, should be sampled in the phylogeographical study using cytoplasmic haplotypes if possible. In addition, we also discussed the evolution and phylogenetic utility of morphological characters in Picea.