Plastid phylogenomics provides new insights into the systematics,diversification,and biogeography of Cymbidium(Orchidaceae)

Cymbidium(Orchidaceae:Epidendroideae),with around 60 species,is widely-distributed across Southeast Asia,providing a nice system for studying the processes that underlie patterns of biodiversity in the region.However,phylogenetic relationships of Cymbidium have not been well resolved,hampering investigations of species diversification and the biogeographical history of this genus.In this study,we construct a plastome phylogeny of 56 Cymbidium species,with four well-resolved major clades,which provides a framework for biogeographical and diversification rate analyses.Molecular dating and biogeographical analyses show that Cymbidium likely originated in the region spanning northern IndoBurma to the eastern Himalayas during the early Miocene(~21.10 Ma).It then rapidly diversified into four major clades in East Asia within approximately a million years during the middle Miocene.Cymbidium spp.migration to the adjacent regions(Borneo,Philippines,and Sulawesi)primarily occurred during the Pliocene-Pleistocene period.Our analyses indicate that the net diversification rate of Cymbidium has decreased since its origin,and is positively associated with changes in temperature and monsoon intensity.Favorable hydrothermal conditions brought by monsoon intensification in the early Miocene possibly contributed to the initial rapid diversification,after which the net diversification rate was reduced with the cooling climate after the middle Miocene.The transition from epiphytic to terrestrial habits may have enabled adaptation to cooler environments and colonization of northern niches,yet without a significant effect on diversification rates.This study provides new insights into how monsoon activity and temperature changes affected the diversification dynamics of plants in Southeast Asia.

Diversity of Orchids in Terms of Their Distribution, Uses and Conservation in Annapurna Conservation Area of Nepal

The Annapurna Conservation Area (ACA), the first conservation area and the largest protected area (PA) in Nepal, is incredibly rich in biodiversity. Notwithstanding this, orchids in the ACA have not been explored enough yet thus making the need for ambitious research to be carried out. Previous study only included 81 species of orchids within ACA. This study aims to update the record of species and genera richness in the ACA. In total 198 species of orchids, belonging to 67 genera (40% and 62% of the total recorded orchid species and genera in Nepal) has been recorded in ACA. This represents an increase of 144% in species and 56% in genera over the previous data. Out of the 198 species, 99 were epiphytes, 6 were holomycotrophic and 93 were terrestrial. Among the 67 genera, Bulbophyllum (17) species were dominant, followed by Dendrobium (16), Herminium (10), Coelogyne, Plantanthera (9 each), Eria, Habenaria, Oberonia (8 each), Calanthe (7), and Liparis (6). Fifty-six species were found to be ornamentally significant and 85 species medicinally significant.

Progress in systematics and biogeography of Orchidaceae

Orchidaceae are one of the largest families of angiosperms in terms of species richness.In the last decade,numerous studies have delved into reconstructing the phylogenetic framework of Orchidaceae,leveraging data from plastid,mitochondrial and nuclear sources.These studies have provided new insights into the systematics,diversification and biogeography of Orchidaceae,establishing a robust foundation for future research.Nevertheless,pronounced controversies persist regarding the precise placement of certain lineages within these phylogenetic frameworks.To address these discrepancies and deepen our understanding of the phylogenetic structure of Orchidaceae,we provide a comprehensive overview and analysis of phylogenetic studies focusing on contentious groups within Orchidaceae since 2015,delving into discussions on the underlying reasons for observed topological conflicts.We also provide a novel phylogenetic framework at the subtribal level.Furthermore,we examine the tempo and mode underlying orchid species diversity from the perspective of historical biogeography,highlighting factors contributing to extensive speciation.Ultimately,we delineate avenues for future research aimed at enhancing our understanding of Orchidaceae phylogeny and diversity.

Metabolomic Analysis of the Anthocyanins Associated with Different Colors of Cymbidium goeringii in Guizhou, China

Cymbidium goeringii is an economically important ornamental plant,and flower color is one of the main features of C.goeringii that contributes to its high economic value.To clarify the molecular mechanisms underlying the role of anthocyanins in mediating differences in color among varieties,liquid chromatography–tandem mass spectrometry was used to perform anthocyanin-targeted metabolomics of seven C.goeringii varieties,including‘Jin Qian Yuan’(JQY),‘Jin Xiu Qian Yuan’(JXQY),‘Miao Jiang Su Die’(MJSD),‘Qian Ming Su’(QMS),‘Shi Chan’(SC),and‘Yang Ming Su’(YMS),as well as the C.goeringii.We detected 64 anthocyanins,including cyanidins,delphinidins,malvidins,pelargonidins,peonidins,petunidins,procyanidins,and flavonoids.We identified six shared differentially accumulated metabolites(DAMs),including cyanidin-3-O-rutinoside,delphinidin-3-Osophoroside,pelargonidin-3-O-rutinoside,peonidin-3-O-(6-O-malonyl-beta-D-glucoside),peonidin-3-Osophoroside,and chalcone.Most DAMs were enriched in the anthocyanin biosynthesis pathway.Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed metabolites were significantly enriched in the anthocyanin biosynthesis pathway.Analysis of the content of differentially expressed metabolites indicated that peonidin-3-O-(6-O-malonyl-beta-D-glucoside)was the key metabolite underlying color differences among C.goeringii varieties.Procyanidin B2,pelargonidin-3-O-galactoside,and naringenin might also affect the color formation of JQY and QMS,SC,and MJSD,respectively.The results of this study shed light on the metabolic mechanism underlying flower color differences in C.goeringii at the molecular level.Our findings will aid future studies of the mechanism of flower color regulation in C.goeringii and have implications for the breeding of new varieties.

Transcriptome Analysis Reveals Clues into leaf-like flower mutant in Chinese orchid Cymbidium ensifolium

The floral morphology of Cymbidium ensifolium,a well-known orchid in China,has increasingly attracted horticultural and commercial attention.However,the molecular mechanisms that regulate flower development defects in C.ensifolium mutants are poorly understood.In this work,we examined a domesticated variety of C.ensifolium named‘CuiYuMuDan',or leaf-like flower mutant,which lacks typical characteristics of orchid floral organs but continues to produce sepal-to leaf-like structures along the inflorescence.We used comparative transcriptome analysis to identify 6234 genes that are differentially expressed between mutant and wild-type flowers.The majority of these differentially expre ssed genes are involved in membrane-building,anabolism regulation,and plant hormone signal transduction,implying that in the leaf-like mutant these processes play roles in the development of flower defects.In addition,we identified 152 differentially expre ssed transcription factors,including the bHLH,MYB,MIKC,and WRKY gene families.Moreover,we found 20 differentially expressed genes that are commonly involved in flower development,including MADS-box genes,CLAVATA3(CLV3),WUSCHEL(WUS),and PERIANTHIA(PAN).Among them,floral homeotic genes were further investigated by phylogenetic analysis and expression validation,which displayed distinctive spatial expression patterns and significant changes between the wild type and the mutant.This is the first report on the C.ensifolium leaf-like flower mutant transcriptome.Our results shed light on the molecular regulation of orchid flower development,and may improve our understanding of floral patterning regulation and advance molecular breeding of Chinese orchids.

Phylogenetic incongruence in Cymbidium orchids

Cymbidium,which includes approximately 80 species,is one of the most ornamental and cultivated orchid genera.However,a lack of markers and sparse sampling have posed great challenges to resolving the phylogenetic relationships within the genus.In the present study,we reconstructed the phylogenetic relationships by utilizing one nuclear DNA(nrITS)and seven plastid genes(rbcL,trnS,trnG,matK,trnL,psbA,and atpI)from 70 species(varieties)in Cymbidium.We also examined the occurrence of phylogenetic conflict between nuclear(nrITS)and plastid loci and investigated how phylogenetic conflict bears on taxonomic classification within the genus.We found that phylogenetic conflict and low support values may be explained by hybridization and a lack of informative characteristics.Our results do not support previous classification of the subgenera and sections within Cymbidium.Discordance between gene trees and network analysis indicate that reticulate evolution occurred in the genus Cymbidium.Overall,our study indicates that Cymbidium has undergone a complex evolution.

In vitro germination and propagation of a threatened medicinal orchid,Cymbidium aloifolium(L.) Sw.through artificial seed

Objective:To study the in vitro germination and plantlet regeneration from artificial seeds of Cymbidium aloifolium(C.aloifolium),a highly threatened medicinal orchid of Nepal.Methods:Artificial seeds were produced in vitro by encapsulation of protocorms with 4%sodium alginate and 0.2 mol/L calcium chloride solution.In vitro germination and plantlet regeneration of the artificial seeds were tested by culturing them on different strength of Murashige and Skoog(MS) liquid media(0.25,0.5 and 1.0) and MS liquid medium supplemented with 0.5 mg/L benzyl amino purine and 0.5 mg/L naphthalene acetic acid.Freshly produced artificial seeds were stored up to 28 d at 4 ℃.In order to check the viability,storage artificial seeds were treated with five different sterilization techniques(T_1 T_2,T_3 T_4,T_5) and inoculated on full strength(1.0) of MS liquid medium after each 7 d of interval upto28 th days.Results:The highest percentage of germination(100%) of artificial seed was obtained on quarter(0.25),half(0.5) and full(1.0) strength of MS liquid medium.Experimentally,full strength of MS liquid medium was more effective for earlier seedling development of C.aloifolium.Artificial seeds were successfully stored at 4 ℃ till 28 th days.Treatments T_1 and T_2showed 97.5%viability of storage artificial seeds and hence considered as the most effective sterilization techniques to recover the plant from storage artificial seeds.Plantlets developed from artificial seeds were successfully acclimatized in potting mixture containing cocopeat,litter and sphagnum moss with 85%survival rate.Conclusions:The present study revealed that artificial seeds are the good alternative explants for in vitro mass propagation and short term conservation of C.aloifolium.

Orchid diversity and distribution pattern in karst forests in eastern Yunnan Province,China

Global biodiversity loss and mass extinction of species have raised serious global concerns,especially in fragile ecosystems.Karst forest,a well-known fragile ecosystem,is served as a natural laboratory for biogeographical,ecological,evolutionary,and taxonomic research.However,species richness and distribution patterns in karst forests largely remain unclear.Orchidaceae has been regarded as the“flagship”group of plant diversity.In this study,we use Wanfengshan Nature Reserve in southeast Yunnan,China,as a model to determine the conservation status of orchids in the karst forest by conducting systematic field surveys in the past three years from 2019 to 2022.In total,we identified 78 orchid species in 35 genera in Wanfengshan Nature Reserve.The dominant species were Bulbophyllum andersonii,Eria coronaria,Nervilia mackinnonii and Paphiopedilum micranthum.Multiple life forms were also observed,including epiphyte(14.29%),facultative epiphyte(18.18%),lithophyte(23.38%),and terrestrial(44.15%).Orchid species richness showed a bimodal pattern varied with altitude,the higher richness was recorded at 1,500-1,600 and 1,200-1,300 m,while the minimum was below 1,100 m.Each independent hill is rich in orchid species including‘endemic’species.A total of 25(33.8%)orchid species were considered threatened by the Redlist of Chinese Vascular Plants(Qin et al.,2017),including 3 CR species,5 EN species and 17 VU species.We call for more research addressing the mechanisms of ecological adaption,mycorrhizal interactions,and pollination of orchids in karst forests,and effective ways for recovery and conservation of orchids.

烯效唑对杂交兰苗期植株的矮化效应研究

为明确烯效唑(S_(3307))对杂交兰苗期植株生长的影响,以自育新品系GZ1-11为试材,采用不同浓度的S_(3307)进行灌根处理。结果表明,适宜浓度的S_(3307)可以有效抑制新苗株高、叶长和根长,增加叶宽和根粗,但不影响假鳞茎的纵径和与横径。随着S_(3307)施用浓度的增加,植株的矮化效应显著增强,其中30 mL 10.00 mg/L S_(3307)的矮化效果最强,但综合杂交兰植株高度、叶片形态等表型特征及成本,生产上施用30 mL 2.50 mg/L的S_(3307)为宜。

兰属植物珍珠矮试管开花技术研究

为了保护和开发利用兰花新种珍珠矮,加快育种进程,本研究通过根状茎直接和再生植株间接的方式来诱导试管开花。结果表明,在含有1 mg/L 6-BA,30 g/L蔗糖以及2 g/L蛋白胨的调整MS培养基(165 mg/L NH_(4)NO_(3)、380 mg/L KNO_(3)和850 mg/L KH_(2)PO_(4))上直接诱导根状茎获得的正常试管花数量最高,平均为2.6个。含有3 mg/L 6-BA、1 mg/L NAA和100 mL/L椰汁的MS或1/2 MS培养基最适宜珍珠矮根状茎再生;在含有3 mg/L 6-BA和20 g/L蔗糖的MS培养基间接诱导再生植株获得试管花数量最高,平均为0.8个。

大花蕙兰(Cymbidium hybridum)的研究动向

系统论述了大花蕙兰(Cym bidium hybridum)的概况和发展简史,综述了国内外对于大花蕙兰在组织培养、栽培技术及育种技术等方面的最新研究动向,提出了我国大花蕙兰生产中存在的问题以及对我国兰花产业发展的展望。

TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒

为建立一种同步定量检测建兰花叶病毒(cymbidium mosaic virus,CymMV)和齿兰环斑病毒(odontoglossum ringspot virus,ORSV)的高效检测方法,本研究根据CymMV和ORSV CP基因高度保守区分别设计引物和探针并筛选,获得156 bp和148 bp的靶标序列及对应的最优特异性引物探针组合,建立了基于TaqMan探针的双重实时荧光定量PCR方法。该方法最低检出限为1拷贝或6.2×10^(-3)fg,最低稳定检出限为10拷贝或6.2×10^(-2)fg,是RT-PCR的10~100倍;构建的标准曲线线性关系良好,扩增效率分别为97.7%和100.2%,相关系数R2分别为1.000和0.999;对其他5种常见病毒均无扩增曲线,检测特异性强;批组内与批组间重复性试验Ct值变异系数≤0.60%,重复性和稳定性好。利用该方法和基因芯片法分别对4个种属的66个兰花样品进行方法验证,该方法相较于基因芯片法检出率提高了53.85%(CymMV)和162.5%(ORSV)。综上所述,本方法可同时高通量检测CymMV和ORSV 2种病毒靶基因,结果可靠,应用前景广阔,可为开展病毒精准鉴定、科学防控以及从源头遏制病毒传播提供技术支撑。

大花蕙兰(Cymbidium hybridium)离体快速繁殖技术

以大花蕙兰茎尖和茎节段作外植体进行了离体快速繁殖技术研究。结果显示:茎尖诱导芽的效果优于茎节段;适宜诱导培养基为MS+BA4.0mg·L-1+NAA0.2mg·L-1+AC0.6g·L-1,茎尖在该培养基上芽的诱导率为71.4%;丛芽增殖适宜培养基为MS+BA2.0mg·L-1+NAA0.2mg·L-1+AC03.g·L-1,增殖率为3.79;原球茎增殖、分化的适宜培养基为MS+BA1.0mg·L-1+NAA0.2mg·L-1+AC0.3g·L-1,增殖率为6.1,分化率为71.1%;生根和壮苗的适宜培养基为MS+NAA0.5mg·L-1+AC0.3g·L-1,试管苗生根率达100%。

利用RAPD技术分析兰属(Cymbidium)品种间的亲缘关系

本文利用RAPD技术来检测兰属 13个品种间的亲缘关系 .通过对 5 5种 10个碱基随机引物的筛选 ,其中 4种引物能得到重复性、稳定性较高扩增产物 ,四种引物共扩增出 93条多态性带 ,多态率为 10 0 % .根据RAPD标记进行邻体聚类分析 ,表明兰属各种间的亲缘关系与形态学分类结果不完全一致 .实验结果认为RAPD技术可用于兰属植物的系统学研究 .图 2表 3参

兰属Cymbidium植物ISSR遗传多样性分析

将简单重复序列区间扩增多态标记(inter-simple sequence repeats,ISSR)技术应用于16种兰属的遗传多样性分析,15个引物共扩增出836条带,其中有227条多态带,多态百分比为27.2%。UPGMA聚类结果显示:春兰与春剑的亲缘关系最近,而兔耳兰与其他15种兰属植物的距离最远。ISSR聚类结果与传统分类结果基本相似,表明该技术能在分子水平上对传统兰属分类进行必要补充。

5种国兰(Cymbidium)的光合特性

通过气体交换和叶绿素荧光分析等方法研究了5种国兰(Cymbidium)的光合特性。其光饱和 点、光补偿点、CO2补偿点和CO2饱和点表明它们具有C3植物特征;光合速率在3.0～5.9μmol· m-2·s-1之间,在低于2000μmol·mol-1CO2浓度下,光合速率受CO2浓度影响较大;5种国兰的光合能 力、表观量子效率、羧化效率等都有所差异;光饱和点在350～650μmol·m-2·s-1之间,属于喜阴植物, 其中春兰、建兰耐阴性相对较低,墨兰、春剑耐阴性相对较高,蕙兰居中。从暗处暴露到强光下,5种国 兰的光化学反应启动差异较大,启动后的光化学效率差异也较大,这可能与国兰不同种起源地的生态环境, 适应性及进化有关。

基于ISSR标记的96份兰属种质资源遗传多样性分析及指纹图谱构建

为加强我国兰属种质资源的保护利用,本研究通过ISSR分子标记对96份兰属种质进行多样性分析和指纹图谱构建。结果显示,共筛选出11条可扩增清晰条带的多样性引物,在96份材料共检测67条多态性条带,平均多态性条带比例为73.63%;等位基因数(Na)为1.925,有效等位基因数(Ne)为1.450,Nei′s遗传多样性指数(H)为0.277,Shannon多样性指数(I)为0.427,多态性位点百分比(PPL,percentage of polymorphic loci)为92.54%;种群内基因多样性(Hs)为0.1934,基因分化度(Gst)为0.3009,总遗传多样性指数(Ht)为0.2767,种群间的平均基因流(Nm)为1.1619,种群间的两两遗传分化固定指数值范围为0.002~0.527,平均值为0.325。系统聚类结果表明,兰属种群间遗传分化程度高,8个种群可分为3类,春兰和墨兰为一大类,寒兰、春剑、蕙兰、莲瓣兰、建兰为第二类,杂交种独为一类,与其他两类种群之间的遗传距离较大。主坐标分析表明,莲瓣兰和春兰表现出较远的亲缘关系。本研究筛选出6对引物构建了96个品种的指纹图谱二维码。本研究结果可为今后兰属新品种选育及品种鉴定提供重要依据。

51个春兰(Cymbidium goeringii)品种的AFLP遗传多样性分析

为了揭示春兰品种的遗传多样性和亲缘关系,为春兰种质资源的有效利用和开发提供依据,采用AFLP技术对51个春兰品种进行了遗传多样性分析,经筛选得到了8对条带清晰、多态性高的引物,共扩增出1315条DNA片段,其中多态性条带为1217条,平均1对引物扩增条带164条,多态性带152条,多态性位点频率为92.5%,表明春兰品种具有丰富的遗传多态性。49个品种含有特有带。51个品种间遗传相似系数变化范围为0.501-0.716,聚类分析表明,51个春兰品种共分为5个类群,来自同一地区的品种并没有聚在一起,表明春兰品种的遗传背景混乱。AFLP分子标记技术能有效地分析春兰品种的遗传多样性和亲缘关系。

利用同工酶和SDS-PAGE技术对一些兰属(Cymbidium)品种的分析(简报)

利用同工酶和ＳＤＳ－ＰＡＧＥ技术对兰属（Ｃｙｍｂｉｄｉｕｍ）５个种和２个变种的１１个品种进行了分类研究，酯酶和细胞色素氧化酶的同工酶共出现２４条带，其中２３条具有多态性；ＳＤＳ－ＰＡＧＥ有３０条带，其中２５条带为多态性。根据同工酶和ＳＤＳ－蛋白质标记进行聚类分析，生化水平与传统的形态学分类结果基本一致，但某些兰属品种的分类地位与传统的分类有一些差异。

中国兰花(Chinese Cymbidium)部分品种的叶片同工酶分析

利用聚丙烯酰胺凝胶电泳和淀粉胶凝胶电泳,对中国兰花的分属于六个种一个变种二十个品种的叶片过氧化物酶、酯酶、酸性磷酸酯酸、多酚氧化酶的同工酶进行了分析,共获得98条酶带,其中的95条具多态性。各个种的品种之间,有基本相似的特征酶谱。而各种间的酶谱差异较大。四种同功酶的聚类分析结果与传统分类学的划分比较接近,为中国兰花品种的鉴定和分类提供了生物化学依据。