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1. Seed dormancy type ofManglietia grandis seeds, The seeds of Manglietia grandis had good water-absorption and the embryos were underdeveloped. Fresh seeds only germinated to 2% at 25 ℃, so the seeds were morphophysiological dormant. The seeds germinated to 82% at 25/15 ℃,the embryos do not need 0~10 ℃ stratification to break the morphophysiological dormancy and only grew obviously when they were nearly germinated. So we can say that the seeds of Manglietia grandis have non-deep morphophysiological simple dormancy, and the cold stratification can break the physical dormancy component of there complex dormancies. The 50 days germination rate of Manglietia grandis seeds was higher than the 30 day germination rate at 30/20℃,25/15℃,20/10℃, and the GA3 had broken the seeds physical dormancy effectively, so the Manglietia grandis seeds were conditional dormant. After different time cold stratifications, we found that the 60 days stratification was the most effective way to break the dormancy. The others’ stratifications made a long time and moist environment, so the seeds would have a secondary dormancy, entering into the dormancy circle. 2. The way to break the dormancy of Manglietia grandis seeds effectively and to improve the germination, 1000 mg·L-1 GA3 could break the dormancy of Manglietia grandis seeds at 20~30℃ somehow. 6-BA and KNO3 couldn’t improve germination effectively, but these two chemicals could make a considerably high germination at 20/10℃ no matter with the concentration. And the differences of each other were not remarkable. The dormancy of 60 days cold stratification Manglietia grandis seeds almost could been broken. But at 20/10℃ the highest germination happened at the 40 days stratification, the 100 days stratification’ s germination also high to 82%. 3. The storage behavior of Manglietia grandis seeds, After the desiccation tolerance test, we can know generally that the storage behavior of Manglietia grandis seeds are intermediate type. In the further experiment, when the seeds were dehydrated to 10~12% water content, the viability was above 90%. Go on the dehydration to 5% water content, the seeds viability was 87%,when the water content was 3.7%, there still were considerable live seeds. Then the seeds were air tightly stored at -20℃ for 3 months, the viability was only about 30%(comprehend the viability of 5% and 4.7% water content together), most seeds lost their viability. So the Manglietia grandis seeds were intermediate seeds. 4. The storage condition of Manglietia grandis seeds, The Manglietia grandis seeds can endure 4℃ low temperature, and keep the viability for quite a long time, but can’t endure the sub-zero low temperature. At the 10℃ above temperature, the seeds viability went down as the water content lost. So we can airtightly store the Manglietia grandis seeds directly at 4℃ or make proper dehydration to about 10% water content then store at -20℃. After low temperature moist storage 60 days and 120 days, the seeds viability were all above 90%, we can concluded that the low temperature moist storage was more effective than the dry storage, and easy to manipulate. 5. The in vitro culture of Manglietia grandis seeds, The in vitro culture experiment used seedlings of Manglietia grandis as the explants. Put the seedlings which only have the cotyledons into the culture medium of H+6-BA0.25+NAA0.125+KT0.1+IAA0.05 mg·L-1(the fallowing units are as the same), several adventitious buds had be induced out. Put all these adventitious buds into the medium of H+6-BA0.5+ KT0.1, the multiplication coefficient is 3.5, and all of the buds grew well. It’s not hard to induce out the adventitious buds and make proliferations, the problem was that during the process there had be browning effects on the seedlings and buds, the problem was settled by cutting and using the antioxidants 1 1ncarvillea zhongdianensis is one of alpine plants with horticultural and medicinal values distributes in the Hengduan Mountains. It is necessary to understand the environmental adaptation for the effective acclimation and cultivation of this species. Many years’ observation in the process of cultivation showed that temperature and light are important factors in its growth and acclimation. In this research, the photosynthesis and leaf traits are analysed to understand the adaptive mechanism of 1. zhongdianensis and develope effective strategy for its domestication and cultivation.The results are summarized as follows: 1. For the teatment of 20℃ , the responses of light intensity, concentration of CO2, chlorophyll fluorescence were better than the teatment of 30℃ in varing degrees; for the traits of photosynthesis, except the apparent quantum efficiency (AQE) was lower under 20℃, other parameters were better than those under 30℃. 2. Contents of chloropyll a,b decreased , content of leaf nitrogen (LNCa) increased with the increasing temperature; the teatment of 30℃ had maximal length of compound leaf and number of folioles; the teatment of 10℃ had maximal ratio of root to shoot (R/S), the relative growth rate (RGR)of 20℃ and 30℃ treatments were better than other treatments. 3. The responses of light intensity, concentration of CO2, chlorophyll fluorescence of three treatments in Shangri-La decreased with the decreasing light intensity; for the traits of photosynthesis, except the apparent quantum efficiency (AQE) was steady,other parameters decreased with the decreasing light intensity. 4. Specific leaf area (SLA), contents of chloropyll b and leaf nitrogen (LNCa) increased with the decreasing light intensity; length of compound leaf and number of folioles of three treatments in Shangri-La increased with the decreasing light intensity, but the relative growth rate (RGR) was opposite, teatment of X100 had maximal ratio of root to shoot (R/S). 5. After transplanted from Shangri-La to Kunming, the photosynthetic rate(Pn)decreased 10% under the full light intensity condition,Vcmax decreased 13%, Jmax decreased 22%, both chloropyll(Chla+b)and carotenoids(Car)were higher than in Shangri-La from June to August, a little drop in September, Chla/bwas higher than Shangri-La from June to September, but LNCa was opposite, RGR rised 30%. Most parameters of the leaf traits of lncarvillea zhongdianensis subject to full light intensity in Kunming were between Shangri-La full and 40% light intensity. 1 Aconitum L. (Ranunculaceae) is a large genus of about 300 species distributed in the temperate regions of the Northern Hemisphere. The southwestern China, particularly its Hengduan Mountains region, is the most important center of diversity and speciation of the genus. The C18-, C19- and C20-diterpenoid alkaloids had been reported as major constituents from the genus Aconitum and also had a high toxicity. During the course of our searching bioactive chemicals from natural sources, A. hemsleyanum and A. hemsleyanum were phytochemically investigated to afford the isolation and identification of 76 compounds. Among them, 14 new compounds including two novel skeleton compound, were elucidated based on spectroscopic methods. 1
Aconitum carmichaeli Debx., a traditional Chinese medicinal herb, which has been cultivated for more than one thousand years, and has different morphological and cytological characters. Until now, the questions about its original and so on are still unknown. In this study, molecular phylogeny and phylogeography have been adopted to tracing the original plant(s) of Chuanwu, distinguished it from A. kusnezoffii and other medicinal Aconitum plants. With the help of nrDNA and cpDNA sequencing, the members of the A. carmichaeli polyploid complex were reclassified, and the relationships between the different clusters were also studied. Finally, based on these data, phylogeographic studies were carried out for the complex and Chuanwu, the origin of theChuanwu and the formation of the authentic characters were proposed. Main conclusions are summarized as follows: 1. Aconitum carmichaeli var. carmichaeli and A. carmichaeli var. pubescens were most likely the botanical original plants of Chuanwu, and the former was the principal source. 2. A 56 bp sequence in the psbA-trnH intergenic spacer could distinguish the A. carmichaeli and A. kusnezoffii from the other Aconitum medicinal species. 3. Aconitum carmichaeli polyploid complex includes the following taxa: A. carmichaeli, A. kusnezoffii, A. jaluense and A. paniculigerum var. wulingense. A. carmichaeli var. carmichaeli probablycame from the hybridization between A. carmichaeli var. hwangshanicum and A. carmichaeli var. tripartitum, and A. carmichaeli var. pubescens is likely ahybridized product of Northeastern region taxa (A. kusnezoffii, A. paniculigerum var. wulingense) and Central region taxon (A. carmichaeli var. hwangshanicum), while A. carmichaeli var. hwangshanicum, A. carmichaeli var. tripartitum and A. paniculigerum var. wulingense might all contribute to the speciation of A. carmichaeli var. truppelianum. 4. Based on ndhA and psbA-trnH intergenic spacer sequence data in 45 populations of 525 individuals of A. carmichaeli polyploid complex 32 haplotypes were revealed. High genetic diversity at the complex level and relatively low genetic diversity at the population level. Some refugia may be located in Mts. Dabieshan (JZ), Huangshan (HS) and Changbaishan (QS), and these populations may all expand or immigrate to southward or(and)northward via Mts. Taihangshan and Dabieshan-Tongbaishan during the intreglacial/glacial cycles. Mts. Qinling and Nanling have important effects on the haplotype’s pattern of A. carmichaeli var. carmichaeli.  5. The results indicated that the Chuanwu had very low genetic diversity and genetic differentiation. Chuanwu could have originated from at least two places twice. Range expansion of the original plants was likely the main genetic differentiation model for the formation of genuine character of Chuanwu. 1 Astilbe Buch.-Ham. ex D.Don, Rodgersia A.Gray together with Astilboides Engler. blong to the tribe Astilbeae in Saxifragaceae. Rodgersia is a genus of tall, statuesque herb with unique compound leaves. It consists of six or five species, ranging from Japan to east Himalaya through Qinling-Daba mountain areas and southwest China. Astilbe is a well defined genus of Saxifragaceae, with a disjunct distribution between eastern Asia and eastern North America extending westward to northern India and southward to Philippines, Java Island, and New Guinea.Two separate molecular phylogeny studies using multiple DNA sequences were conducted on Astilbe and Rodgersia as well as their allies. 1. Molecular phylogeny of eastern Asian-eastern North American disjunct Astilbe,Phylogenetic analyses were conducted for the eastern Asian-eastern North American disjunct genus Astilbe (Saxifragaceae) and allies using sequences of plastid matK, trnL-trnF, psbA-trnH, and nuclear ribosomal ITS region. A worldwide 65 collections of Astilbe species and allies were sampled from Mainland China, Taiwan, Korea, Japan, Nepal, United States and New Guinea. The monophyly of Astilbe was supported based on analyses of both ITS and plastid data sets. Astilbe is distinct from Rodgersia and Astilboides, but close related to western North American Saxifragopsis. The combination of Astilbe and Saxifragopsis suggested by Soltis with the name of Astilbe Group was supported by our molecular result, as well as morphological and cytological traits. The plastid and the ITS trees differed primarily on the placement of eastern North American Astilbe biternata. Hybridization and subsequent chloroplast capture may best explain the topological incongruence between neulcear and plastid data. Our results supported a hybrid origin of allotetraploid A. biternata with two Asian dimorphism diploid progenitors. Astilbe rivularis var. myriantha (= A. myriantha) was suggested to be elevated to the species level. Without being allied to A. rivularis, A. myriantha was related to taxa with perfect petals. Astilbe rubra was not monopyletic, “A. rubra” recorded from Korea and Japan is grouped together with A. chinensis and A. koreana, while two collections of A. rubra sampled from of western Yunnan of China grouped with A. longicarpa, A. macroflora from Taiwan, and A. philippinensis from Papua of Indonesia. In present analyses, the northestern Asian Astilbe koreana was not supported as synonym of A. grandis, but being closely allied with A. chinensis and “A. rubra”.Astilbe simplicifolia occupied a derived position other than being a primitive position. Molecular result supported regarding it as a new neotenous derivative of A. thunbergii. High genetic variations were first found in Astilbe rivularis, our molecular deriviations were corresponded with biogeographic distributions of A. rivularis complex. Astilbe rivularis might have an origin in Hengduan Mountains and with a development westward to Himalayas and eastward to eastern Hengduan mountains. Dating analysis based on the Bayesian approach with BEAST estimated the age of the crown Astilbe to be 11.96 mya, the divergent time for intercontinental disjunction in Astilbe was inferred to be not earlier than in middle Miocene. Astilbe originated from northeastern Asia with its allies in western North America. The Bering land bridge might be the most likely migration route for origin of the new world Astilbe. Based on present molecular results, as well as original description papers and Type specimen, 16 species and four variaties were recognized in Astilbe. For the other three species: Astilbe indica (recorded from Java, Indonesia), Astilbe khasiana (recorded from Khasia, India), and Astilbe stoliczkai (recoded from northwest Himalaya), the taxonomical status were still questionable. According to morphological and molecular evidences, Astilbe rivularis var. angustifoliolata was raised to sepecies level with the name of Astilbe angustifoliolata (H.Hara) W. D. Zhu, H. Sun et J. Wen stat. nov.2. Molecular phlogeny of Rodgersia and allies,Rodgersia is a well defined genus native to eastern Asia. Molecular phylogenetic analyses were conducted for Rodgersia, as well as its alies Astilboides, Darmera, Oresitrophe, Bergenia, Mukdenia. The sequences of matK, trnL-trnF and nuclear ITS regions were used. The monophyly of Rodgersia was well supported. Rodgersia was closely related to eastern Asian endemic Astilboides and western North American Darmera; the combination of Rodgersia, Astilboides, Darmera, Oresitrophe, Bergenia, and Mukdenia by Soltis with the name of Darmera group was supported. The key taxonomic traits of leave arrangement and pubescence were not suppoted by molecular result, especially for taxa from Hengduan Mountains and Himalayas. Multiple sampled Rodgersia aesculifolia was not monophyly, samples from Hengduan Mountains (R. henrici = R. aesculifolia var. henrici) were nested with R. pinnata and R. sambucifolia, while samples from southeast Tibet (R. henrici = R. aesculifolia var. henrici) form a clade sister to the former taxa. Samples of R. aesculifolia from Qingling and Daba mountains (R. aesculifolia var. aesculifolia = Triditional R. asculifolia) are distinct with all the above. R. aesculifolia var. henrici is distinct from A. aesculifolia var. aesculifolia and is suggested be raised to spcies level again as Rosgersia henrici Franchet. Populations of R. henrici from western Yunnan are grouping with R. pinnata, natural hybridization are supposed to occur. Rodgersia podophylla from Korea and Japan is sister to Chinese Rodgersia. The furthermore study of infraspecific taxonomy of R. aesculifolia is suggested.The relict Rodgersia nepalensis from eastern Nepal branched first in the combined ITS and plastid tree, which is different from evidences of the traditional morphology and cytology. This might due to its narrow distribution disjuct from other species of Rodgersia, low level of gene flow and subsequent conserved genetic system. It may evolved by polyploidy, the spcecialized morphological character of R. nepalensis may be a strategy for ecological tolerance and self-protection. Our molecular phylogeny of Rodgersia is accordant with the former morphological and cytological evidences. Hybridization and polyploidy may play an important role in evolution and speciation in Rodgersia. Rodgersia may origin from northestern Asia and migrated into Hengduan mountains and Himalayas through Qingling and Daba mountains. Based on present molecular results, as well as original description papers and Type specimen, six species and two variaties were recognized in Rodgersia. Rodgersia henrici was recognized in our study, and was supported to be raised to species level again 1 Bambusoideae belongs to BEP clade of Poaceae. Bambusoideae in general, and Arundinarieae in particular, has long been considered a complex and taxonomically difficult group. In this study, six bamboo chloroplast genomes were sequenced. One of the six bamboos belongs to Bamabuseae (Bambusa emeiensis) and the other five belong to Arundinarieae (Ferrocalamus rimosivaginus, Acidosasa purpurea, Indocalamus longiauritus, Phyllostachys edulis and Phyllostachys nigra var. henonis). It was a first attempt to address phylogenetic issue in Bambusoideae using phylogenomics. The aim of the thesis is to resolve the relationships of five species in Arundinarieae and to evaluate the efficiency of phylogenomic strategy in bamboo phylogenetic study. Through comparing eight bamboo chloroplast genomes, we were able to identify fast-involving fragments for further phylogenetic studies in Bambusoideae. The main results were summarized as follows: 1. The complete sequences of six bamboo chloroplast genomes have verified the feasibility and reliability of using the next-generation platform to sequencing the chloroplast genomes isolated through improved method of high ionic strength in combination with low pH buffer. Comparative studies indicated that eight chloroplast genomes of Bambusoideae which encoded an identical set of genes were rather conserved and perfectly syntenic. We identified repeat sequences of the six newly sequenced bamboo chloroplast genomes and coded these repeat sequences as whole genome features according to their distributions in six chloroplast genomes. The coded matrix was used for construction of phylogenetic tree using maximum parsimony analysis. The result provided strong support for the position of F. rimosivaginus as the earliest diverging species of the five bamboos in Arundinarieae, followed by A. purpurea, I. longiauritus and a clade consisted of P. edulis and P. nigra var. henonis. 2. Phylogenetic studies based on twenty-four complete chloroplast genomes of Poaceae provided a strong support for the sister relationship between Bambusoideae and Pooideae within BEP clade. Within Arundinarieae, I. longiauritus, P. edulis and P. nigra var. henonis were proved to be a monophyletic clade (referred as Phyllostachys clade) in which P. edulis and P. nigra var. henonis had a closer relationship. A. purpurea was siter to Phyllostachys clade. However, this relationship was only supported by Bayesian analysis. This result was considered reasonable while combining with the phylogenetic result based on whole genome features. Phylogenetic analyses were performed based on the following data partitions: (1) the complete chloroplast genome sequences, (2) a set of 74 common protein-coding genes, (3) the large single copy region, and (4) the small single copy region. The result from the complete chloroplast DNA sequence was proved to be better than results from other three data partitions. Unambiguously possibly informative exon indels were identified and mapped to chloroplast genome-based phylogenetic tree. Of the forty-five indels, twenty-five mapped to monophyletic groups, therefore they may be synapomorphies. The remainder of these events may be homoplasies resulted from parallel evolution or back mutation. Forty-five indels were coded and added to the protein-coding gene matrix. Then the matrix was used for phylogenetic analysis and the result indicated that these coded indels did not change the tree topology, while the bootstrap values of some nodes were increased. In conclusion, it was proved to be efficient to reconstruct bamboo phylogeny using chloroplast phylogenomics. 3. The number and distribution pattern of variable characters in each homologous region were rather different in three subfamilies. Therefore, it is more reasonable to select fast-evolving regions from bamboo chloroplast genomes. The average proportion of variability in noncoding regions was as twice as in the coding regions. Therefore, we identified twenty fast-involving markers from noncoding regions. 1
Begonia L. belongs to the family Begoniaceae. Begonia species in China are mostly distributed S of Yangtze River, particularly concentrated in SE Yunnan and SW Guangxi, with only a few extended into N China. The thesis includes investigation and introduction of the genus Begonia from SE Yunnan and SW Guangxi. Preliminary studies on seed germination, seed storage characteristics, DNA content analysis of in vitro conservation Begonia species, seed morphology and pollen morphology were conducted. The main results were summarized as follows:1. Morphology,⑴ Seed morphological study, Seeds of three species of the Chinese Begonia Sect. Coelocentrum and ten species of Sect. Platycentrum were studied. The result showed that there was higher consistency in Sect. Coelocentrum and the seeds morphology had certain value at sectional level. In addition, the Chinese Begonia Sect. Platycentrum had high diversities in seed micromorphology especially in the aspect of the shape of operculum and the size of seed. The seed morphology of the same species showed steady characters.⑵ Pollen morphological study, Pollen of 21 Chinese Begonia species was examined. The results showed that striate was the most commonplace exine sculpture pattern among Begonia as well as punctuate, rarely rugose. Although the pollen morphology did not show its importance to the taxonomy at sectional level of the genus Begonia, it showed some value to some specific treatment. Our study also suggested that the species with middle pollens in Begonia could be more primitive, while the species with smaller or larger ones might be much advanced.2. Conservation of germplasm resources,⑴ Conservation status, There are only three populations of B. purpureofolia and two populations of B. pseudodryadis according to investigation. The total mature individuals ofthe two species are less than 2500 and no subpopulation estimated to contain more than 250 mature individuals. Based on the IUCN Red List Categories and Criteria (Version 3.1), the conservation status of B. purpureofolia and B. pseudodryadis is ranged as Endangered. ⑵ Introduction and cultivation, The species were collected mainly from SE Yunnan and SW Guangxi. Living environment, growing conditions, physical and chemical characters of soil samples of B. purpureofolia and B. pseudodryadis were studied. Then the suitable condition was obtained to cultivate the two species.⑶ Seed germination and storage characteristics, The effect of temperature on seed germination of thirteen Begonia species were studied. The results of the test were as follows: temperature had no significant effect on seed germination percentage on most of the studied Begonia species but had a significant effect on mean germination time (MGT). Storage characteristics of three species of Begonia were studied by setting different storage temperatures (ambient temperature, 4℃ and -20℃) and storage periods (30d, 60d, 90d and 180d). The results showed that the seeds storage in ambient temperature and 4℃ had higher germination percentage than -20℃. ⑷ Assessment of genetic stability, Flow Cytometry (FCM) analysis was used to assess the genetic stability in three Begonia species which were maintained in vitro for a period of over 10 years. The results showed that no significant variation in the nuclear DNA contents of B. cathayana and B. pseudodryadis. However, long-term in vitro maintenance induced aneuploid change in nuclear DNA contents in B. hemsleyana. 1 Begonia sect. Coelocentrum Irmsch. is a strict calciphilous plant. It consists of about 50 species, and is exclusively distributed in limestone areas of Guangxi, SE Yunnan, S Guizhou of China, and N Vietnam. Based on morphology of ovary placentae, embryology, micromorphology of leaf epidermis, literature collections, specimens examinations and field works, the taxonomy on Begonia. sect. Coelocentrum is studied. The main results are summarized as follows: 1. The Morphology of placentae of 26 species is observed through paraffin sections, results are summarized as follows: 1) Morphological characteristics of placentae are stable in sections of Begonia,can be taken as a primary evidence between sections; 2) In Begonia. sect. Coelocentrum, different species placentae have different development and considerably varied degrees of concrescence; 3) The types of placentae in other sections of Begonia could all be found in Begonia. sect. Coelocentrum. In this section, the development processes of placentae have implied the phylogeny of other sections. Based on the placentae types of every sections, the evolution trends in Begonia probably can be suggested: from groups with parietal placentae to groups with axial placentae, from 2- (or more) lobates to 1-lobate placentae, and so B. sect. Coelocentrum. is the base group.2. Embryological features of B. liuyanii C.-I Peng, S. M. Ku et W. C. Leong are reported for the first time. Ovules are numerous. The mature ovules are anatropous, bitegminous, and crassinucellate, usually linear tetrad (rarely T-shaped and inverted T-shaped).The embryo sac is formed by the chalazal megaspore in the tetrad. It is the polygonum type. The anther of each stamen is tetrasporangiate; mature anther wall falls into the basic type of development, including 4-layered (a epidermis, a fibrous endothecium, a ephemeral middle layer and a secretory anther tapetum), mature pollen grains with 3-grooves. Additionally, the ovule has a unique structure that the cells of outer layer of integument are significantly larger with radial extension, fewer inclusions, individual cells filled with tannin substances, At present, this unique structure has not yet been reported in other plants. 3. Leaf epidermal micromorphorlogy characters of 42 species and 3 varieties in B. sect. Coelocentrum and related sections, are examined under scanning electron microscope (SEM), results are showed that: Characteristics of leaf epidermal micromorphology have poor consistency in sections, but can provide important evidences for the division of species and varieties in Begonia. sect. Coelocentrum. By compared observations of leaf epidermis, some species are reduced to synonymy or degraded from species to variations. Moreover, the epidermal morphology of some species in Begonia. sect. Coelocentrum showed high consistency, t may be a relatively natural taxonomic character and can be used as the proofs for classification under the sections.4. Based on field works, literatures, specimen examinations, and morphology of placentae, micromorphology of leaf epidermis, Begonia. sect. Coelocentrum is revised. There are 46 species and four varieties are recognized in the world, the morphological characters and distributions are described and a key for the identification is also given, some taxonomic problems are clarified. 1) According to characters of placentae, habitats and habits, B. cavaleriei H. Lév.,B. jingxiensis D. Fang & Y. G. Wei,and B. wangii T. T. Yü are suggested to be transferred from B. sect. Diploclinium into B. sect. Coelocentrum; 2) On the basis of Leaf epidermal micromorphorlogy characters, B. bamaensis Yan Liu & C.-I Peng is treated as a synonymy of B. bonii, B. debaoensis C.-I Peng, Yan Liu & S. M. Ku is degraded from species to a variation and treated as a synonymy of B. bonii Gagnep. var. remotisetulosa Y. M. Shui & W. H. Chen, B. arachnoidea is also degraded to a variation and treated as a synonymy of B. umbraculifolia Y. Wan & B. N. Chang var. flocculosa Y. M. Shui & W. H. Chen 1 Below-ground ectomycorrhizae (ECM) community and diversity study is one of the intensively explored area in mycological ecology. Such study, however, was insufficient in China. Ectomycorrhizal fungus diversity and ectomycorrhizal morphology of Pinus yunanensis seedlings in central Yunnan province, were studied by combining morphology and the analysis of rDNA ITS and rDNA LSU sequences from ECM.114 rDNA ITS sequences and 15 rDNA LSU sequences were obtained from roots of 86 Pinus yunanensis seedlings. Totally, 37 species of 16 genera, 15 families of Basidiomycetes and 8 species of 6 genera, 5 families of Ascomycetes were recognized, among of which Thelephoraceae and Russulaceae are dominant. Tomentella and Russula hold the highest species diversity, both represented by 8 species. The most frequent species were Pseudotomentella tristis and T. subclavigera. Contact exploration and short-distance exploration were main exploration types of the ECM on Yunnan pine seedlings.Species diversity was higher on Yunnan pine than on other seedlings. Russula, a group that doesn’t prevail in young stage of other seedlings, was found to be dominant on Yunnan pine seedlings. Tuber and Suillus, common early-stage ECMF on other seedlings, were not found in this work. Pseudotomentella, a genus rarely occurring on other seedlings, shows relatively high frequency on Yunnan pine seedlings.Morphological characteristics of Tomentella, Russula and some other genera were given. The practice in this work demonstrated that integrating ECM morphotyping and sporocarps surveys with DNA analysis of ECMF is an effective approach for documenting the ECMF community. 1
C. sinensis var. assamica is an important member of section Thea, belonging to the genus Camellia. It becomes more and more attractive because of its medicine and health maintenance efficacy. The chloroplast genome sequence is now widely used to resolve the phylogeny at different levels of taxonomic categories, because of its small genome size and different evolutionary rates of coding and noncoding gene regions. Futhermore with the progression in chloroplast genetic engineering, we can improve the quantity and quality of crops efficiently. However, the isolation of high yield and purified chloroplast DNA of Camellia becomes very difficult due to special leaf structure and complicated metabolic products. Unfortunately there are not any efficient methods available to isolate the Camellia cpDNA. In this study, we established an efficient method to isolate the Camellia cpDNA on basis of its leaf structure and complicate compounds. With the purified and sufficient cpDNAs, we completed sequencing the first chloroplast genome in the Genus Camellia, even the first one in the order Ericales. Besides, we used the information of genomic rearrangement of chloroplast to reconstruction the phylogenetic relationship of the three basal algal groups to investigate the feasibility of using genomic rearrangement for phylogeny reconstruction. Results are as below: 1. Isolation of cpDNAs of Section Thea of the genus Camellia, Based on special leaf structure and components of Section Thea of the genus Camellia, we adjusted experimental conditions and established a suitable protocol for its chloroplast isolation. They included short time and high speed homogenate, combined with a low pH extraction buffer for cell breakage, differential centrifugation for isolating the chloroplasts, high-salt wash buffers for the elimination of nuclear DNA contamination and phenol-chloroform treatment. As a result, sufficiently purified and successfully digested cpDNAs was abtained. They can be directly sequenced by Solexa. 2. Complete chloroplast genome sequence of C. sinensis var. assamica, With the second generation sequencing technology of Solexa, we completed the first chloroplast genome sequencing of the genus Camellia, also the first one of order Ericales. The genome size is 157,140bp, and the lengths of LSC, SSC, IR are 86,600bp, 18,272bp, 26,134bp, respectively. The genome contains 115 genes, including 30 tRNA genes and 4 rRNA genes. Futhermore, with the complete cpDNA of C. sinensis var.assamica and other completely sequenced cpDNAs of Asterids, we compared shade-loving and sun plants, the chloroplast structures of the two kinds of plants are very similar, but we found that nucleotide substitution rates of shade-loving plants are lower than sun plants. A likely explanation is that more variations accumulated by more chloroplasts in cells of sun plants. 3. Feasibility to use genomic rearrangement for the phylogeny reconstruction: a case study on the phylogeny reconstruction of basal alagal groups, Phylogeny of three basal algal groups, glaucophyta, chlorophyta and rhodophyta, was first reconstructed by means of rearrangement information from the chloroplast genomes. It includes common genes content, conserved gene clusters and phylogenetic trees produced by gene order data. We found that glaucophyta and rhodophyta share more common genes, and the conserved gene clusters between them have fewer arrangements. Furthermore phylogenetic trees also showed closer relationships between glaucophyta and rhodophyta. Thus our results supported that chlorophyta are sister to glaucophyta plus rhodophyta. 1 Camellia taliensis (W. W. Smith) Melchior, a member of Camellia sect. Thea, is an indigenous species in local natural forest and has a long cultivative history in western Yunnan and its neighborhood, where the domestications of this species in different historical periods and in different ways can be found. C. taliensis is an important contributor to the formations of tea landraces by hybridization and introgression. In the present study, 14 microsatellite loci screened from 37 loci were used to explore the genetic diversity about this species with 579 samples from 25 populations (16 wild populations, 4 semi-cultivated populations and 5 cultivated populations). At the same time, the potential hybrid speciation of C. grandibracteata, was investigated using 39 individuals from 2 populations, along with C. taliensis and C. sinensis var. assamica (83 individuals from 4 populations) by the same microsatellite markers. C. taliensis had a moderate high level of genetic diversity (A = 14.3, Ne= 5.7, HE = 0.666, I = 1.753, AR = 7.2, PPB = 100%). This may result from several factors including K-strategy, genetic background, gene flow between populations, hybridization and introgression among species. Between wild populations of C. taliensis, the gene flow was moderate high (Nm = 1.197), and genetic variation was less than 20% (GST = 0.147, FST = 0.173), which was similar to other research results of long-lived woody plants, and reflected the genetic structure of its ancestry to same extent. There was a high significant correlation between geographic distance and Nei’s genetic distance (r = 0.372, P = 0.001) of populations, which accorded with isolation by distance model. Inferring from Bayesian clustering of genotypes, all individuals of C. taliensis were divided into two groups, conflicting with the result based on Nei’s genetic distance and real geographic distribution, which suggested there were heavy and non-random influences by human practices. According to allelic richness, there were no significant differences (P > 0.05) between wild (AR = 4.651), semi-cultivated (AR = 5.091) and cultivated (AR = 5.132) populations of C. taliensis, which suggested that the genetic background of long-lived woody plant was not easy to be changed, and there were moderate high gene flow between populations. However, there was a significant difference (P < 0.05) between wild (AR = 5.9) and cultivated (AR = 7.1) populations distributed in the same place in Yun county, Yunnan province, which may result from the hybridization and introgression of species in the tea garden and anthropogenic damages to the wild population. The hypothesis of hybrid origin of C. grandibracteata was tested by morphological and microsatellites analyses. Compared with other species, the locules in ovary of C. grandibracteata are variable, which showed a morphological intermediate and mosaic. Except one private allele, Ninety-nine percent alleles of C. grandibracteata were shared with these of C. taliensis and C. sinensis var. assamica. And C. grandibracteata was nested in the cluster of C. taliensis in the UPGMA tree. Conclusively, our results supported the hypothesis of hybrid origin of C. grandibracteata partly. The speciation of C. grandibracteata was derived from hybridization and asymmetrical introgression potentially. It is possible that C. taliensis was one of its parents, but it still needs more evidences to prove that C. sinensis var. assamica was another parent. 1 Chemical constituents and bioactivities research on Cimicifuga species have been became an international hotspot. There are abundant plant resources of this genus in China, however the step of exploitation is far fall behind the western world. In an attempt to fully utilize our domestic resources, our research group has been studying on the chemical constituents and their bioactivities, specially 9,19-cyclolanostanetriterpenes of this genus, since 2003. As a result, several cycloartane triterpenoids with potential anti-tumor activities were obtained for the first time in China. For further searching anti-tumor lead compounds with novel structures and more potent activities, in this thesis,the roots of C. yunnanensis and the aerial parts of C. foetida, which collected from Shangrila County in Yunnan Province, the roots of C. foetida, which collected from Heishitou Town in Guizhou Province and the roots of C. dahurica and C. heracleifolia, which collected from Qingyuan County in Liaoning Province were used as experimental materials and total 105 compounds were isolated and identified by different spectroscopic and chemical methods. Among these 105 compounds, there are 94 9,19-cyclolanostanetriterpenes,including 44 new constituents. All these new compounds can be categorized as Cimigenol-type, Shengmanol-type, Hydroxyshengmanol-type, Acteol-type, Dahurinol-type, Cimicidol-type, and 3,4-Seco-type, 15,16-Seco-type, which were very novel. The left 11 compounds are normal Chromones, sitosterols, oleanane triterpenes and a new Ergostane-type steoid. The isolated 9,19-cyclolanostane compounds were tested for their cytotoxicities against 15 human carcinoma cell lines, which included human hepatoma cell line HepG2, human breast carcinoma cell line MCF-7, human colorectal carcinoma cell line HT-19 and so on. 40 compounds showed activities and three of them, which isolated from C. yunnanensis exhibited as potent activities as positive control Taxol. Mechanism research indicated that these three compounds can increase the expression of p53 and bax, which led to the loss of mitochondrial potential and then resulted in the activation of caspase-7 and induced apoptosis of MCF-7. In addition, a series of compounds which isolated from the roots of C. foetida showed selective significant cytotoxicity (potent than positive controlcisplatin) against the human HepG2 cell line and a domestic patent was applied for these compounds. A conclusion of the structure-activity relationships of Cimigenol-type compounds were proposed: one of the key point for anti-tumor activities is at least one side of the compound has hydrophobic groups such as carbonyl, double bond substitute hydroxyl groups. At the end of this thesis, a comprehensive review about the distribution and utilization of the Cimicifuga genus worldwide, the structures of 9,19-cyclolanostanetriterpenes and their anti-climacteric syndrome, anti-osteoporotic and anti-tumor activities were gave. 1
Chemical investigation of twelve macrofungi and one lichen including Coriolopsis gallica, Conocybe siliginea, Albatrellus confluens, Scutellinia ascoboloides, Lactarius deliciosus, Thelephora terrestris, Collybia acervata, Shiraia bambusicola, Cortinarius alboviolaceus, Mycena galericulata, Polyporus elegans, Trogia sp., and Sulcaria virens were comprehensively reported in this dissertation.113 different compounds have been isolated and elucidated by varied chromatographic methods and extensive spectroscopic analysis, among which 26 compounds were new ones. The classes of these new compounds include acetylenic acids, tremulane-type sesquiterpenes, cleistanthane-type diterpenes, isocoumarin, norleucine-type non-protein amino acid, etc. The highlight of this dissertation is the phytochemical investigation of Trogia sp. leading to two norleucine-type non-protein amino acids, 2R-amino-4S-hydroxy-5-hexynoic acid (104) and 2R-amino-5-hexynoic acid (105), which are responsible for the toxicity of this fungus. Cellulose microcrystalline column chromatography with n-BuOH-EtOH-AcOH-H2O (4:1:1:2, v/v/v) eluting system was extensively used for isolation of amino acids in this study. Comparing to traditional n-BuOH-AcOH-H2O (BAW) solvent system, a time-saving and efficient TLC solvent system OWF (organin layer-water-formic acid) for amino acids detection was developed. TLC solvent system OWF was prepared as below: solvent OW was composed of CHCl3-CH3COCH3-MeOH (10:4:4, v/v/v), which was saturated with water; 1 to 4 drops of formic acid was added to 1 ml solvent OW when used.In the last chapter, the chemical, biological and mycological literature dealing with the isolation, structure elucidation, biological activities, and synthesis of pigments produced by those fungi that produce conspicuous fruiting bodies (macromycetes) was reviewed. 198 references between June 2003 to December 2009 are cited. 1 Cold stress is one of the major environmental factors that adversely influence plants growth. Cold stress not only limits plants geographic distribution, but also reduces plants yield by shortening growing season, which brought billions of dollars economic losses for global crop. In nature, responses of overwintering plants to low temperature can be divided into three distinct phases: cold acclimation (CA), freezing, and post-freezing recovery (PFR). Until now, plenty intensive study about molecular mechanism of cold stress mainly focused on the above-zero low temperature phase. However, the studies on the freezing phase below zero and the following PFR phase with temperature going up to above-zero were rare. The previous research form our lab hinted that the responses of plants to freezing and PFR were complex and important. Except for passive reflection, there were also crucial active responses during this process. Several special rules were presented at the different levels including gene expression, signal transduction and membrane lipids changes, and fully understanding these rules would be helpful for us to explore the responses of plants to low temperature and then proceed to improve the freezing resistance of plants. In the present study, the mechanisms of respond to freezing and PFR of model plant Arabidopsis thaliana and its close relative Thellungiella halophlia that with extreme tolerance to abiotic stresses were carried out, including regulation of gene expression, signal transduction pathway and membrane lipids changes three levels which were essential for the freezing resistance of plants. Ground on these work, we obtained results from the following five aspects. First, the complete picture of A. thaliana responding to freezing and PFR at transcriptome level was elaborated and three functional genes closely related to the phases were identified. Second, the cis-elements with high frequent presence in differentially expressed genes were elucidated, and the practical binding of one elements among them was experimental verified during freezing and PFR. Moreover, we predicted the new elements which would respond to freezing and PFR. Third, the regulation of freezing stress by microRNA in A. thaliana was preliminarily investigated and 36 functional genes possibly regulated by miRNA during freezing and PFR were gained. Fourth, the negative effect of phytohormone Auxin on A. thaliana subjected to freezing stress was identified. Fifth, for the freezing-resistant plant T. halophlia, the rules of membrane lipids composition changes under freezing stress were uncovered. 1 Craigia yunnanensis W. W. Smith & W. E. Evans (Tiliaceae) is an endangered deciduous tree species which has high scientific and economic value. C. yunnanensis is seriously threatened and has been pushed to the verge of extinction due to vegetation destruction in China and consequent contraction of its distribution. Hence, it was listed as a nationally rare and endangered plant in 1999 and has also been proposed as a second-ranked plant for national protection in China and included in IUCN red list. As a scientifically important and valued tree species with endangered status, the wild populations of C. yunnanensis therefore represent is a genetic resource that must be conserved. To provide basic information for its conservation, the population dynamics and population size structures, pollination biology and breeding system, eleven fitness-related characters and the genetic variability based on AFLP were comprehensively studied. The main results are summarized as follows: A total of six wild populations of C. yunnanensis were found in two disjunct regions of Yunnan, i.e. WenShan (SE Yunnan) and DeHong (SW Yunnan), from 2005 to 2007. Additionally, in all but one of the populations we detected, mature trees were felled between 2005 and 2007, so destruction of most of these populations is ongoing. Across the six populations of extant C. yunnanensis found during our study, the total number of mature (reproductive) individuals detected was 584 in 2007,plus larger numbers of seedling and resprouts from cut trunks. The result of surveying Population structure showed that there are two regeneration types which are seedlings and sprouts. Seedlings occurred abundantly in gaps or open areas and the size class frequency distributions were often discontinuous, and the same general pattern occurred in all the investigated populations for juveniles and adults. The numbers of seed-origin individuals did however decline sharply with increasing size, indicating a high mortality rate going from seedling to sapling stage may be a problem for this species. Additionally, the cash crop cultivation and logging seriously threaten the survival of the species. We conducted field observations and artificial pollination experiments on the floral biology, pollination process and breeding system of Craigia yunnanensis in Fadou, Xichou county of Yunnan province. The lifespan of a single hermaphrodite flower is approximately 3-4 days. A cyme has 2-9 flowered. The flowering period of an inflorescence is usually 5-14 days. The flowers of C. yunnanensis were protandrous. The stamens were within petal-like staminodes in the opening flowers until the flower withered. Without touchment, the bractlike staminodes can’t open. Self-pollination was partially avoided by temporal and spatial isolation of male and female organs within the same flower. However, autogamous and geitonogamous pollination is unavoidable because of the large number of flowers on a single tree and the action of pollinators. The values of both OCI (≥4) and P/O (1381) and the results of bagging tests indicated there was no apomixes in C. yunnanensis and the breeding system of the species was outcrossing with partial self-compatibility and the pollinators were required during the pollination process. The most frequent effective floral visitor was only beautiful fly (Chrysomyia megacephala). Fruit set and seed set in natural condition were 56.67±3.85% and 6.26±0.75%, respectively. Therefore, lack of pollinators, low pollination efficiency, unavoidable geitonogamous pollination and partial self-compatibility and inbreeding in small populations may account for the low fruit set, especially seed set.Variations in seed traits, seed germination, and seedling growth characters among six Craigia yunnanensis populations were evaluated. All seed and seedling traits exhibited significant differences among populations (P < 0.05). The fitness of seed as assessed by seed size, seed germination and seedling trait was independent of population size, except for the number of seeds per capsule (r = 0.93,P < 0.01). Correlations between geo-climatic variables of seed origin and seed and seedling related characters were insignificant (P > 0.05). For some populations, germination capacity in 12-h photoperiod was significantly higher than that in completed darkness(W-FD: P < 0.01, W-JD: P < 0.05).Genetic variation within and among six populations was assessed using AFLP markers. Genetic diversity was higher at species level (PPL = 69.19%, HE = 0.221) than at population level (PPL = 26.22%, HE = 0.095, Is =0.140), and populations in southeast Yunnan were strongly differentiated from those in southwest Yunnan (Nei’s GST = 0.575; FST = 0.655). UPGMA analysis demonstrated a clear genetic division between the two populations from DeHong (SW Yunnan; D-JD and D-HG) and the four from WenShan (SE Yunnan; W-FD, W-LH, W-ML, and W-MG). Within-population genetic variation was significantly correlated with population isolation (r(PPL) = -0.94, P = 0.006; r(HE) = -0.85, P = 0.032; r(Is) = -0.87, P = 0.025), but not with population size (r(PPL) = 0.63, P = 0.178; r(HE) = 0.54, P = 0.268; r(Is) = 0.56, P = 0.249). 1
Cyatheaceae species, usually called tree ferns, are considered as relicts of a time when dinosaurs were common. In recent several decades, the number of Cyatheaceae plants decreases dramatically. In order to find the reasons and provide directions for protecting these endangered plants, the biological characteristics of Cyatheaceae were surveyed. Using AFLP and cpDNA sequence variations, the genetic diversity and phylogeography of Sphaeropteris brunoniana were also analyzed. Based on these findings, implications for conservation strategies were discussed for this relict tree fern. Main results of the dissertation were summarized as follows, (1) Cyatheaceae plants have extensive distribution in Yunnan, China, and most of them distribute in southeast of Yunnan. In southeast, they usually inhabit margins of evergreen broad-leaved forests or secondary coniferous forests; however, the population update is very different and the age structure is unscientific. The spore of Cyatheaceae is trilete, radially symmetrical, and perinous. The spores of Alsophila species feature a ridged perine and a granular, verrucate or smooth exine. The spores of S. brunoniana are characterized by an incipient granular outermost layer and a verrucate exine. The metaphase chromosome numbers of gametophytes in the three examined species, viz. A. podophylla, A. gigantea and A. austro-yunnanensis, are 69, indicating that they are diploid and do not display variety in chromosome number. The chemical constituents of S. brunoniana are main simple and familiar compounds, such as saccharides, fatty acids and alcohols, and stigmasterols. (2) An unexpectedly high level of nDNA genetic diversity and low cpDNA diversity were detected in S. brunoniana. (3) This study showed that the genetic differentiation among populations within regions was low and between regions was significant. (4) There were several refugia of S. brunoniana in Yunnan during glacial periods. The Hainan populations were likely new colonizations and originated from Southeast Asia. (5) To retain existing genetic diversity, whether in situ or ex situ conservation or collection of germplasm is used, the populations of the two regions should be considered equally. Furthermore, ex situ conservation of this species should be preferably conducted on large populations. 1 Cycas micholitzii complex is composed of 5 species: C. micholitzii Dyer, C. bifida (Dyer) K. D. Hill,C. longipetiolula D. Y. Wang, C. debaoensis Y. C. Zhong et C J. Chen, C. multipinnata C J. Chen et S. Y. Yang,and distributed from southwest China to central Vietnam and eastern Laos. Based on sequence data from two maternally inherited cpDNA and one biparentally nuclear DNA fragments, our study revealed the population genetic structure of C. micholitzii complex and explored the potential causes. The evolutionary and demographic histories were investigated. The genetic relationship among species in the complex was also clarified.The results were summarized as follows: 1. Phylogeographic analysis based on chloroplast sequences,We examined chloroplast sequence variation of the atpB-rbcLand psbA-trnHintergenic spacers in 27 populations of C. micholitzii complex, recovering 26 haplotypes. The average within-population diversity (HS = 0.140) was low while total diversity (HT = 0.911) was high. Population differentiation was also high(GST = 0.846, NST = 0.919), indicating significant phylogeographical structure (NST > GST,p < 0.001) and low levels of seed-based gene flow. C. debaoensis (Cycadaceae) is an endangered species restricted to the border of Guangxi and Yunnan province in southwest China. This species has been classified into two types: sand and karst, according to the soil matrix they grow on. We examined chloroplast sequence variation of the cpDNA sequences from 11 populations of this species. Significant population genetic differentiation was detected (GST= 0.684 and FST = 0.74160). There was marked genetic differentiation between populations in the sand and karst regions and no expansion was detected. Climate changes during glacial periods have had significant effects on the current distribution of cycads. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggest that C. debaoensis experienced range contraction during glacial periods, and that the current populations are still confined to the original refugia in southwest China which have favorable habitats in glacial period. These results imply that small refugia were maintained in both sand and karst regions during the LGM (last glacial maximum). This species had no postglacial recolonization and only stayed in these refugia up to now. The low within-population diversity of C. debaoensis suggests that there were strong bottleneck events or founder effects within each separate region during the Quaternary climatic oscillations. Relatively high genetic and haplotype diversities were detected in the newly discovered populations, which located at intermediate locality of sand regions and had morphological variation; this is probably the consequence of the admixture of different haplotypes colonizing the area from separate sources. C. micholitzii occurs in the Annan Highlands in central Vietnam near the Laos border. C. bifida occurs in North Vietnam; its distribution extends across the border into adjacent localities in Guangxi and Yunnan in China. For the comparability between them,theywere considered as the same species C. micholitzii by many academicians. The cpDNA sequences from 11 populations showed that these very controversial species, C. micholitzii and C. bifida, is paraphyletic and should belong to the same species C. micholitzii. AMOVA analysis showed that the component of among-population within region/species (76.46%) was unexpectedly larger than the among-species/region component (14.97%), which also indicates that there is no justification for recognizing two species as C. micholitzii and C. bifida. This hypothesis was also supported by the geological data, especially the neotectonic history of the indo-china block, which started to move south since Oligocene and cause the geographic isolation of these two groups. Therefore, the most likely explanation to the phenotypic similarities between these two groups may be the retention of ancestral polymorphisms in the paraphyletic group due to incomplete lineage sorting. Furthermore, the similarities may also be ascribed to pollen-mediated gene flow among geographically proximate populations and/or phenotypic convergence under similar selection schemes in the same region. C.micholitzi had the higest genetic diversity (HT = 0.980,) and genetic differentiation (GST = 0.830, NST = 0.915) among the C. micholitzii complex. The high genetic diversity might be attributed to its long evolutionary history, highly diverse habitats. The ineffective mode of seed dispersal and dramatic neotectonic movement in the distribution range of this species could result in the high genetic differentiation. 2. Phylogeographic analysis based on nuclear ribosomal sequences, We sequenced the nrDNA ITS in all 27 populations sampled, 7 haplotypes were identified, among which C. micholitzii had 6, while C. multipinnata, C. longipetiolula and C. debaoensis shared the remaining one. Compared to chloroplast genes, nuclear genes had higher correlation between genetic and geographical distance, but lower interspecies differentiation (54.42% vs 25.24%). Phylogeographical structure of C. micholitzii and C.bifida based on ITS Variation was consistent with the morphology differentiation. This similar in nuclear gene should be ascribed to pollen-mediated gene flow among geographically proximate populations.Long-distance gene flow over the two groups was clearly interrupted, which brought on the nrDNA genetic differenciation between the geographically isolated groups, to a certain extent affected the morphological variation. 3. Interspecies relationships among Cycas micholitzii complex, We analysed chloroplast sequence variation of the atpB-rbcL and psbA-trnH intergenic spacers in 27 populations sampled of C. micholitzii complex, AMOVA analysis showed that the component of among-species/region component (59.21%). However, phylogenic analysis showed that the haplotypes of C. micholitzii complex couldn`t grouped into four clusters closely corresponding to the narrowly defined C. micholitzi, C. multipinnata, C. debaoensis and C. longipetiolula. We concluded that the conflict may result from several factors: firstly incomplete lineage sorting of C. micholitzii; secondly hybridization/introgression of sympatrically cycads, which would be supported by evidence base on nrDNA ITS sequences; thirdly intramolecular recombination in cpDNA of cycads; eventually the neotectonic movement in the distribution range of this species. 1 Cytology study can reveal important biological features of plants and answers to a certain degree in phylogeny and distribution of genetic materials and so forth. By hard working of cytologists, chromosome data of plants have been increased to a great abundance, but yet disorderly distributed in different magazines, which made researches based on the whole chromosome data of one taxon rarely launched. Scientific databases have become increasingly indispensable as researching data growing daily. As Cytological studies are booming in China, in order to fill the absence of digital and statistical data of plant chromosome researches and chromosome atlas, we started to develop a Chinese Seed Plants Chromosome Database, aiming to construct a database and start to record published chromosome data of Chinese seed plants. Based on this database, we chose the part of gymnosperms and gave a discussion to the features of its chromosomes’ evolution and variation. Cytological experiments have been applied to some important phyto-groups for phylogeny research and germplasm identification.Part I: The Chinese Seed Plants Chromosome Database and Discussion on the features of Gymnosperms chromosomes,1  The Chinese Seed Plants Chromosome Database,The frame of database was constructed by Microsoft Access 2003. 19 items of data were included in, they are: Chinese and Latin names of family, genus and species; plant pictures, mitosis metaphase and karyotype figures; morphological characteristics and distributions of the plant; chromosome numbers and basic numbers; karyotype formula; karyotype description; origin of the plant material; literature and the source of photos. In this database, data can be checked and shared easily by extracted out in species sorted interface or family sorted interface. 120 species in 29 genera and 10 families of Gymnospers have been collected and input to the database. In Angiosperms, 61 species in 10 genera of family Magnoliaceae and 80 species in 3 genera of family Theaceae have been collected and input to the database.2 Discussion on the features of evolution and variation of Gymnosperms chromosomes,By data collection of the database, we analyzed chromosome features of the group Gymnosperm. Plants of Gymnosperm had been through a long historical evolution on earth, fossil records of which originated from the late Devonian period. Once an authoritative and major classification level in the plant kingdom, most Gymnosperms have been extinct unless conifers, cycads, Ginkgo and Getales. Three main features of Gymnosperm chromosomes are: relatively large chromosome, which can be recognized from figures in the database; constant chromosome numbers, in most families of Gymnosperm the basic chromosome number keeps a certain value; comparatively low variation, karyotype under family level differs a little. The variation of chromosomes in Gymnosperm is dominated by Robertsonian changes. Contrary to common variation type in Angiosperms, the variation from high unsymmetric karyotype to low unsymmetric karyotype was found in existence in Gymnosperm.Part II: cytology research on some important phyto-groups,3 Karyomorphology of three species in the order Huerteales and their phylogenetic implications,The karyomorphology of three species in Dipentodon (Dipentodontaceae), Perrottetia (Celastraceae), and Tapiscia (Tapisciaceae), namely Dipentodon sinicus, Perrottetia racemosa, and Tapiscia sinensis, was investigated in the study. Recent molecular research has discovered close relationships among these three genera, which has led to the establishment of the order Huerteales with Perrottetia being placed in Dipentodontaceae. Herein we report the chromosome numbers of D. sinicus and P. racemosa for the first time, and present their karyotype formulas as 2n = 34 = 22sm + 12st (D. sinicus), 2n = 20 = 11m + 9sm (P. racemosa), and 2n = 30 = 22m(2SAT) + 8sm (T. sinensis). Asymmetry of their karyotypes is categorized to be Type 3B in D. sinicus, Type 2A in P. racemosa, and Type 2A in T. sinensis. Each of the species shows special cytological features. Compared with Perrottetia, Dipentodon has a different basic chromosome number, a higher karyotype asymmetry, and different karyomorphology of its interphase nuclei, mitotic prophase, and metaphase. Thus, on the basis of these results, we have reservations regarding the suggestion of placing Dipentodon and Perrottetia together in the family Dipentodontaceae.4 Genomic analyses of intergeneric hybrids between Michelia crassipes and M. calcicola by GISH,Genomic in situ hybridization (GISH) is becoming the method of choice for identifying parental chromosomes in interspecific hybrids. Interspecific F1 hybrid between Michelia crassipes and M. calcicola, tow highly ornamental species in Michelia of Magnolicaceae, has been analized by double-colored GISH with its parents’ genome as the probe. Research gave the results that the chromosome number of the F1 hybrid is 2n=38 as the same of species in Michelia and other genera in Magnoliaceae, the basic chromosome is x=19, the karyotype formula is 2n=38=32m+6sm, and the asymmetry of karyotype is 1B type. Based on chromosome data of Michelia in our database, the karyotype of this genus is featured mostly by metacentric chromosomes and submetacentric chromosomes. In Mechelia, the variation range of submetacentric chromosomes is 4 to 18 and of the karyotype asymmetry is 1A to 2B type. Both the karyotype and karyotype asymmetry type of F1 hybrid is among the variation range of Michelia. The figure of GISH showed that all the 38 chromosomes of F1 hybrid have crossing parental signals, and signal on the no.1 and no.7 chromosome showed differences, which proved that both the parental genome have been transmitted to and recombinated in F1 hybrid. 1
Dendrobium officinale is a valuable medicinal plants,mainly distributed in Yunnan, Guangxi and Anhui. It is necessary to understand the environmental adaptation for the effective acclimation and cultivation of this species. Up till now, there is little information on the ecophysiological adaptation of D. officinale, especially on the photosynthetic response to temperature. This paper investigated the response of photosynthesis and growth of D. officinale to temperature, and the stem polysaccharide content of D. officinale at different temperatures, in order to understand how growth temperature affect the growth and development of D. officinale and to determine the suitable temperature ranges and day-night temperature differences for the growth and development of D. officinale. The result are summarized as follows: 1. Temperature has a significant effect on the photosynthetic rate (Pn) of D. officinale, The light saturated photosynthesis at ambient CO2 concentration (Pmax) of the plants were highest at T-30/20. High photosynthetic rate at T-30/20 were related to a larger leaf area (LA) and the more balance between the maximum rate of electron transport and maximum rate of RuBP-mediated carboxylation. 2. Temperature also has a significant effect on the growth and polysaccharide content of D. officinale’s stem. The polysaccharide content of D. officinale at T-20/10 was significantly higher than at the other temperatures, but the stem length, stem node number, stem fresh weight and stem dry weight was the highest at T-30/20. 3. The utilization of solar energy were highest at T-30/15 temperature difference between day and night, it also has the highest content of chlorophyll, and respiration rate was lower, resulting in higher dry matter accumulation and accumulation of relatively higher polysaccharide content. 4. The polysaccharide content of D. officinale T-30/20 temperature difference between day and night was significantly higher than at the other temperatures, but the leaf area was smaller and chlorophyll content, stem length, node number, the average stem length, stem fresh weight and stem dry weight and other indicators are relatively low. 5. My thesis illuminated how temperature affect the growth and development of D. officinale. The suitable temperature ranges and day-night temperature differences for the growth of D. officinale are recommended as below: day temperature is 25℃ ~ 30 ℃, night temperature is 15℃ ~ 20℃, and day-night temperature difference should be maintained at 10℃ ~ 15℃. 1 During a field trip at a brule in Shangri-La, a mixed population of Ligularia Cass. was found, which including L. subspicata (Bur. et Franch.) Hand.-Mazz., L. nelumbifolia (Bur. et Franch.) Hand.-Mazz., L. tongolensis (Franch.) Hand.-Mazz., L. cymbulifera (W.W.Smith) Hand.-Mazz., L. lingiana S.W.Liu, and also some individuals morphologically intermediate between L. subspicata and L. nelumbifolia. Hence, these intermediate individuals were preliminarily assumed as natural hybrids of the two Ligularia. According to their morphology, they’re assumed to form hybrids A and B. Through careful comparison of specimens in herbarium and those we collected, the inflorescence of putative hybrid A is close to L. nelumbifolia, but the shape of laminae are intergradation of L. subspicata and L. nelumbifolia; overall morphology of putative hybrids B is similar to L. nelumbifolia, but inflorescence color is as same as L. subspicata. Compared to L. nelumbifolia (39%) and L. subspicata (36.8%), the germination rate of putative hybrid B (45.7%) slightly higher than the two; but that of hybrid A is extraordinarily low (0.3%). One possible interpretation of the low rate is hybridization. 60 individuals were collected, including putative parents, other 4 species of Ligularia nearby, putative hybrid A and B. They were all direct sequenced of four cpDNA fragments, and direct sequenced or cloning sequenced of nrDNA ITS4-5. The results support that L. nelumbifolia and L. subspicata are parents of putative hybrid A, and the majority female parent is L. subspicata, L. vellerea may also be involved in the hybridization in some degree; the nuclear sequences of putative hybrid B have no superposition, and its chloroplast DNA sequences are identical with L. nelumbifolia, so putative hybrid B could not be hybrid; and there are backcross individuals exist among the putative parent L. subspicata. NewHybrids analysis of ISSR markers indicated that, the individuals of putative hybrid A are almost L. nelumbifolia and L. subspicata F1 hybrid generation (10/11), only 1/11 possibly backcross or other forms; all individuals of hybrid B are L. nelumbifolia; except one individual of L. subspicata as backcrossed, the other parent individuals are 100% reliable. This study focused on molecular evidence, complemented by ecological, reproductive and other characteristics, we demonstrated that the morphologically intermediate individuals’ origin, and the probability of belonging to each parental or hybrid class. And concluded that L. nelumbifolia and L. subspicata are the parents of putative hybrid A, L. vellerea may also be involved in the hybridization in some degree, hybrids mainly are the first generation, a few individuals may be involved in backcross, and most probably backcross with L. subspicata according to the anthesis, while the assumption of hybrid B is not supported. 1