【作者】 郑玉红；

【导师】 夏冰； 杭悦宇；

【作者基本信息】 南京农业大学 ， 植物学， 2006， 博士

【摘要】 周生翅组(Sect．Enantiophyllum)隶属薯蓣科(Dioscoreaceae)薯蓣属(Dioscorea L．)，全世界约120种，我国仅14种5变种，广泛分布于长江以南的广大省区。周生翅组茎右旋，单叶，全缘；雄花序为穗状花序，单生或簇生于叶腋，或排列成圆锥状；雌花序为穗状花序，单一或分支，1-3个着生于叶腋；蒴果不反折，种翅周生；是薯蓣属中一个进化的自然类群。本研究运用形态学、解剖学和分子生物学的方法对周生翅组进行了研究，得到了以下结论：1．借助于SEM(Scanning Electron Microscopy，扫描电子显微镜)观察了周生翅组10种1变种植物叶片下表皮细胞、表皮毛和气孔的超微结构。结果表明：薯蓣属周生翅组主脉，二级脉和叶肉上均存在表皮毛，且主脉和二级脉的密度大于叶肉。表皮毛的类型有两种，即单细胞不分枝的非腺毛和多细胞头、单细胞柄的腺毛。表皮细胞大多呈不规则形，表皮细胞蜡质有粉末状、屑状、颗粒状、片状，或稀疏或密集。气孔器以不定型为主，三胞型也占有一定的比例。气孔内缘壁光滑或呈波浪形。副卫细胞平周壁角质层具颗粒状、颗粒状-条纹状及条纹状3种纹饰，气孔器保卫细胞垂周壁呈拱形弯曲，气孔周围纹饰有弯曲条纹、环绕后扩散形条纹和放射形条纹3种。本研究结果还表明表皮细胞、表皮毛和气孔器的超微结构均在周生翅组系统演化和种类鉴别中均具有十分重要的意义。2．利用LM(Light microscopy，光学显微镜)，对薯蓣属周生翅组10种1变种植物脉序进行观察。周生翅组植物均为掌状弧形脉序，基出脉5-9枚，弧形，聚顶。最高脉次5-6，二级脉均有分支；二级脉与一级脉夹角大多大于65°，二级间脉为复合型；网眼发育不完全，大多呈类方形、多角形；网眼内盲脉大多简单，不分枝，边缘末次脉大多发育不完全。虽然周生翅组植物脉序的变异程度不大，但在薯蓣属中单复叶类群的脉序还是呈现出明显的演化趋势，脉序特征在薯蓣属及周生翅组系统演化研究中的地位不容忽视。3．在总结了前人研究结果的基础上，对5种周生翅组植物的退化雄蕊的形态特征进行了观察。除丽叶薯蓣和多毛叶薯蓣雄花中3枚雄蕊具花药，3枚雄蕊退化外，周生翅组其余种雄花中均具6枚雄蕊，雌花中大多具有6枚退化雄蕊，一般柱头所对的退化雄蕊较大，花药的颜色较深，花丝几乎透明，除无翅参薯退化雄蕊具少量花粉外，其余均没有花粉。周生翅组植物雌花中具有退化雄蕊的现象无疑是一种进化的性状。4．利用LM对周生翅组10种1变种地下储藏器官—块茎的组织结构进行了观察。周生翅组块茎由外到内是由周皮、基本组织和散生在基本组织内的维管束组成。周皮是最外层的褐色、黄褐色或棕褐色的保护组织，它是由原来表皮下方的基本薄壁组织细胞次生分化而产生的，包括多层栓质化(或带木质化)的木栓层，1～2层扁平细小由切向分裂能力的木栓形成层和由其衍生的栓内层薄壁细胞。外侧基本组织为薄壁细胞组成，比较菲薄，组织中有粘液细胞(内含草酸钙针晶束)、树脂、鞣质细胞(统称树脂细胞)。内侧基本组织占广大部位，由基本薄壁细胞组成，维管束散生，外韧型，无形成层，后生木质部中有大形管胞或导管，韧皮部中有大形筛管，有些种类有树脂细胞和粘液细胞。5．利用LM对薯蓣属周生翅组11种1变种植物的淀粉粒形态特征进行了观察。周生翅组植物的淀粉粒有单粒和复粒之分。单粒淀粉粒主要以类圆形为主，兼有有贝壳形、三角形和长条形等形状；脐点多为点状，飞鸟状和裂缝状也比较常见。复粒淀粉粒有两种类型，A型由2-3小粒构成，形状以卵圆形、三角形为主，脐点点状，大多不明显，少数种层纹清晰；B型由10个以上的淀粉粒构成，形状以圆形为主，脐点不明显，无层纹。按照颗粒的大小，周生翅组淀粉粒可以分为3种类型：小粒类型(长径小于14μm)、中粒类型(长径在15-40μm之间)和大粒类型(长径大于41μm)。淀粉粒脐点和层纹的有无与其大小有关，小粒类型淀粉粒的脐点和层纹都不明显，大粒类型绝大多数都很清晰，中粒类型部分清晰，部分难以辨认。6．测定了周生翅组共10种1变种的trnL-F、rbcL和matK序列。序列分析结果表明：周生翅组trnL-F序列长689-834bp；加上外类群，当空位始终作缺失处理时，有变异位点67个，其中信息位点11个，占序列总长度1.52％；种间碱基差异百分率为2.2％；序列的(G+C)含量为32.5％。周生翅组rbcL序列长1096-1160bp；加上外类群，存在变异位点42个，其中信息位点10个，占序列总长度的0.93％；种间碱基差异百分率为0.9％；序列(G+C)含量为44.1％。周生翅组mark序列长1032-1165bp，排序后，两端切平，序列长1031bp；加上外类群，序列中存在变异位点67个，其中信息位点8个，占序列总长度的0.78％；种间碱基差异百分率为1.3％；序列的(G+C)含量达32.4％。基于trnL-F、rbcL和matK序列分别重建了薯蓣薯周生翅组的系统发育树。三个系统树的结构基本一致。同时，本研究也证实叶绿体基因组序列是周生翅组植物及药材鉴定快速、可靠、有效的方法。7．采用简单重复序列间区(ISSR)标记，研究了薯蓣属周生翅组9种1变种植物的遗传特点，共使用了11条简单的重复序列引物，扩增出92个位点，其中88个多态性位点，多态百分率为95.67％。根据ISSR分子标记数据用POPGENE软件计算的周生翅组植物的遗传距离和遗传相似系数。其中褐苞薯蓣和薯莨的遗传距离最远，达0.7151，薯蓣与光叶薯蓣和多毛叶薯蓣之间的遗传距离最近，均为0.2733。种间Nei’s基因多样性为(h)0.3156，有效等位基因数为(Nei)1.6198，Shannon多样性指数(I)为0.5276。聚类分析将周生翅组植物分为3个类群。本研究结果还表明ISSR能快速把形态相似的周生翅组植物区分开来。8．基于45个广义的形态学性状，运用spss13.0软件，UPGMA聚类分析，构建了周生翅组表征树系图。聚类结果将周生翅植物分成3个类群。基于广义形态学性状，去掉淀粉粒、脉序等演化特征不明显的9个指标，运用PAUP软件，采用最大简约法(MP)构建薯蓣属周生翅组系统发育树。采用启发式搜索共搜索到6棵最佳树。从种系发生上看，丽叶薯蓣和多毛叶薯蓣属较原始的种类，薯莨、异块茎薯莨和光叶薯蓣为最进化的种类。表征聚类分析和分支分析及分子系统学分析对周生翅组植物种间亲缘关系的分析基本一致。但基于ISSR遗传相似系数的聚类树与表征聚类树和分支树的差异都比较大。9．采用随机扩增多态性DNA(RAPD)标记对周生翅组重要药用植物薯蓣的遗传多样性进行了研究，共使用了11条寡聚核苷酸引物，扩增出82个位点，其中65个多态性位点，多态百分率为79.27％。居群间Nei’s基因多样性为(h)0.3159，有效等位基因数为(Ne)1.5552，Shannon多样性指数(I)为0.4626。聚类分析将8个薯蓣居群分为两类。遗传多样性分析结果表明，薯蓣的野生种群的基因库已经萎缩，其分子水平的遗传多样性正在下降，有必要采取相应的措施，加快薯蓣的新品种培育进程，这将有助于更加合理、充分、有效的利用这种重要的资源。更多还原

【Abstract】 Sect. Enantiophyllum belongs to Dioscorea L. Dioscoreaceae. There are about 120species in the section in the world, but only 14 species, 5 varieties in China. They widelydistribute in many provinces south of Yangtse Rive. Stem twining to right. Maleinflorescences spikes, solitary or clustered, or grouped into axillary or terminal panicles.Flowers solitary, sessile; Female spike sometimes branched. Seeds inserted near middle ofcapsule, winged all round. This section is an evolutive natural group in Dioscorea L.Researches on Sect. Enantiophyllum were conducted by micromorphological, anatomical andmolecular biological means. The results were stated as follows:1 The ultrastructure of leaf epidermis cells, epidermis hair and stomatal of 10 species and1 variety in Sect. Enantiophyllum were observed by SEM (Scanning Electron Microscopy)There are epidermis hairs both on nervure and mesophyll. And there exsit 2 types ofepidermis hair: single cell nonglandular hairs and multicell glandular hairs. Leaf epidermiscells of most species are irregular. Appurtenances of leaf epidermis are grain, bits or pieces,which are sparseness or denseness. Most stomatal are ambiquity. The inner margin of stomatalis smooth or lumpy. The appurtenances of periclinal wall is grain, grain-stripe, stripe or cluster.The style of anticlinal wall is arciform. The decorations around stomatal are curving stripe,diffusing stripe after circle or stripe diffusing in 4 directions. This research proved thatultrastructure of leaf epidermis cells, epidermis hair and stomatal are of significance inphylogeny of Sect. Enantiophyllum.2 Using LM (Light microscopy), leaf venation was studied. The vein type of Sect.Enantiophyllum is actinodromous; the number of primary vein is 5-9; the grade of primaryvein is 5-6; the secondary veins’ angle is more than 65°; the intersecondaries is complex;areola develops unconsummate, which is square or polygon; bland vein is not ramified; mostmarginal vein ateliosis. Venation characters should not be neglected in the phylogeny of Sect.Enantiophyllum. 3 Based on the former results, the conditions of staminodes of 5 species wereobserved. There are 6 staminodes in the female flowers except for D.aspersa and D.decipiens.Generally, the staminodes against chapiters are bigger. The colour of anthers is darker thanthat of the chapiters. Staminodes have no pollen except that of D.alata. Staminodes in femaleflowers may be one of the evolutive characters in Sect. Enantiophyllum.4 The tuber of Sect. Enantiophyllum is built up by periderm, ground tissue and vascularbundle. Outside the periderm, there is brown defence tissue, which is differentiation fromground weak-wall tissue under epidermis, including phellem, phellem cambium andendophellem weak-wall cells. The outer ground tissue is made up with ground weak-wallcells, thinner, which contains mucous cells (raphides), resiniferous cells. The inner groundtissue is also made up of ground weak-wall cells, vascilar bundle scattering, without anycambium, tracheid or vessel in the center of metaxylem; sieve tube in the center of phloem.There are mucous cells and resiniferous cells in inner ground tissue in some species.5 The micromorphology of starch granules of Sect. Enantiophyllum were described withthe help of LM. There have two forms of starch granules:single and compound. Most singlegranules are similar to round, and a few of them are like shells, triangle or stip. The hilumsseem like dot, flying bird or slit. There are 2 types of compound granules. Type A is consistof 2-3 single granules, which mainly are ovum and trangle and the hilums are like dot whichcan’t be seen clearly, but only annular striation of a few species are clear. Type B is consist ofmore than 10 granules, which are round and have illegible hilum and no annular striation.According to the size, starch granules can be devided into 3 types:tiny (long diameterrange, LR＜14μm), Middle (40μm＞LR＞14μm),and Big (LR＞41μm).The hilum & annularstriation and size relate to one another. Specifically, tiny granule has illegible hilum & annularstriation while Big grain has clear hilum & annular striation. Some of the middle sizedgranules have clear hilum & annular striation and some do not.6 Total DNA was isolated by CTAB technique and the PCR products were applied tosequence directly. We analyzed cpDNA trnL-F, rbcL and matK of 11 species of Sect. Enantiophyllum. The results were as follows.: The length of trnL-F is 689-834bp. When thegaps were always treated as missing, there were 67 variable sites, of which 11 wereporsim-info, account for 1.52% of the total length. The (G+C) content was 32.5%. Thepairwise distance between species was 2.2%, of which 0.8% was transitions and 1.4% wastransversions. The total length of rbrL was 1096-1160bp. When the gaps were always treatedas missing, there wa 42 variable sites, of which 10 were parsim-info ones, and account for0.93% of the total length. The pairwise distance between species was 0.9%, of which 0.6%was the transitions and 0.3% was the transversions. The (G+C) content was 44.1%. The totallength of matK was 1032-1165bp. When the gaps were always treated as missing, there were67 variable sites, of which 8 were parsim-info ones, and account for 0.78% of the total length.The pairwise distance between species was 1.3%, of which 0.8% was transitions and 0.5%was the transversions. The (G+C) content was 32.4%. Based on the 3 sequences, thephylogeny trees were constructed by bootstrap test. The molecular trees held the same pointwith the classification of the morphological and anatomical characters. Besides, it is provedthat the cpDNA sequecing is the rapid, efficient, credible way to distinguish RhizomaDioscoreae and its related species.7 Genetic character from 10 species in Sect. Enantiophyllum of Dioscorea L. was studied byISSR markers.92 loci were identified with 11 primers, out of which 88 were polymorphic andaccount for 95.67% of total genetic diversity above species level, shannons indices of diversity(I) was 0.5236 at the section among species level, and nei’s gene diversity was 0.3516 and theeffective number of alleles was 1.6068. The genetic distance between D.persimilis andD.cirrhosa is farthest to 0.7051, while the genetic distance between D.polystachya andD.glabra or D.decipiens is nesearest to 0.2733.Cluster analysis grouped all the 10 speciesinto 3 groups. Besides, the results proved that the ISSR marker can be successfully used todistinguish the plants in Sect. Enantiophyllum that have similar exterior.8 Based on 46 morphological characters, using SPSS13.0, phenetic tree was constructedby UPGMA. The tree grouped 10 species and 1 variety into 3 groups. Throwing awaycharacters without plesiomorphy or apomorphy (e.g., the characer of starch and venation), using PAUP, the cladistic tree was constructed by MP. 6 trees were obtained. According to thephylogeny, D.aspersa and D.deipiens are original species, while D.cirrhosa and D.cirrhosavar.cylindrica are more evolutive ones. Phenetic cluster held the same points on the relative ofSect. Enantiophyllumwith cladistic analysis. But they didn’t agree with the tree base oncoefficients of ISSR.9 Genetic diversity from 8 populations of D. polystachya Turcz. was studied by RAPDmarkers. 82 loci were identified with 11 primers, out of which 65 were polymorphic andaccounted for 79.27% of total genetic diversity among species level. Shannons indices ofdiversity (I) was 0.4626 among species level, while nei’s gene diversity was 0.3159 and theeffective number of alleles was 1.5552. Cluster analysis grouped all the 8 population into 2groups. The results of genetic diversity analysis showed that the gene library of wildpopulation of D.polystachya Turcz. is shrunk and the genetic diversity on molecular leveal isdropping. It is necessary to quick the breeding programe, and this will be helpful to use theimportant medical resource reasonably, sufficiently and efficitively.更多还原