【作者】 袁菊红；

【导师】 夏冰； 孙视；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2007， 博士

【摘要】 石蒜属（Lycoris Herb.）是石蒜科中的一个重要的属,该属全世界约20种,中国种类最多,约15种,广泛分布华南、华中、华东各省区,华东地区为其多样性分布中心。该属植物具有很高的观赏价值,鳞茎富含生物碱,具有多种药理活性作用。石蒜属同种异名或异种同名的现象严重,种间易杂交、天然杂交种多,有些种内形态和染色体核型变异丰富,鉴别石蒜物种、确定属內种间亲缘关系和研究种內遗传多样性显得特别重要。石蒜（L.radiata（L’Hér.） Herb.）作为石蒜属中分布最广、资源最丰富、观赏性最强的种类之一,其系统研究尚未进行。因此,本研究利用ISSR分子标记、trn L-F序列分析、高效液相色谱（HPLC）技术以及基于形态特征的数量分类方法从不同角度、不同方面研究我国原产的石蒜属种间亲缘关系、物种的系统位置以及分类问题。同时运用多种分子标记技术和数量分类学方法对石蒜居群DNA和形态多样性进行研究,主要结果总结如下:1.利用基于形态特征的数量分类方法对中国石蒜属13种2变种的种间亲缘关系进行研究,以中国水仙（Narcissus tazetta L.var.chinensis Roem.）为外类群。Q聚类结果显示石蒜属物种聚为两类,第一类包括安徽石蒜（L.anhuiensis Y.Hsu & G.J.Fan）、长筒石蒜（L.longituba Y.Hsu & G.J.Fan）、黄长筒石蒜（L.Jongituba var.flavo Y.Hsu & X.L.Huang）、中国石蒜（L.chinensis Traub.）、忽地笑（L.aurea（L’Hér.）Herb.）、乳白石蒜（L.albiflora Koidz.）、香石蒜（L.incarnata Comes cx C.Sprenger）、鹿葱（L.squamigero Maxim）和换锦花（L.sprengeri Comes ex Baker） 9个种;第二类由矮小石蒜（L.radiata（L’ Hér.）Herb.var.pumila Grey）和石蒜（L.radiata（L’ Hér.）Herb.）、玫瑰石蒜（L.rosea Traub & Moldenke）、稻草石蒜（L.straminea Lindl.）、江苏石蒜（L.houdyshelii Traub.）、红蓝石蒜（L.haywardii Traub.）组成,皆为秋出叶物种。中国水仙单独成一类。与经典分类结果基本相符。研究结果支持鹿葱、乳白石蒜、江苏石蒜、稻草石蒜、玫瑰石蒜和红蓝石蒜的杂交起源观点。但不支持安徽石蒜作为分类学意义上的种,认为将其作为长筒石蒜的变种或长筒石蒜与中国石蒜的杂交种更为合适。R聚类和主成分分析表明雄蕊与花被片的位置关系和鳞茎形状是划分人类群的重要指标。2.利用高效液相色谱（HPLC）对我国石蒜属13种1变种及其近缘植物中国水仙生物碱进行检测,根据生物碱成分的相似性采用Q聚类,石蒜属物种聚成两大类,该属大多数种的归类与基于形态特征的数量分类结果一致,但红蓝石蒜和忽地笑的聚类发生了变化。中国水仙的HPLC图谱与石蒜属植物明显有别,单独成一类。外形相似的中国石蒜与忽地笑,石蒜与矮小石蒜的HPLC图谱差异也较大。3.采用PCR产物直接测序法,测定了我国石蒜属13种2变种的叶绿体DNAtrnL-F序列,以中国水仙为外类群。结果表明:所测物种的序列全长在905～1036bp之间,经排序后两端切平,得到886bp的整齐序列,其中trnL（UAA）內含子长度为529bp,trnL（UAA）3’外显子至trnF（GAA）之间的基因间区长度为357bp。所测物种的变异位点14个,信息位点10个,占序列总长度1.13%,序列的G+C含量为32.9%。序列中出现4个碱基“ATAT”缺失/插入和7个碱基的置换（颠换6个,转换1个）,用以区别石蒜属与其近缘植物水仙;trnL-F序列的177bp处和497bp处分别是香石蒜和矮小石蒜种特异性鉴别位点。基于trnL-F序列运用最大简约法获得供试样品的分子系统树:水仙属植物形成一个单系类群;石蒜属13种2变种可分为3类,多数种的归类与经典分类结果基本一致。结果还表明换锦花很可能作为红蓝石蒜、玫瑰石蒜的杂交母本;安徽石蒜可能是长筒石蒜的变种或以长筒石蒜为母本的杂交种。对石蒜13个居群和其变种矮小石蒜、近缘种玫瑰石蒜和红蓝石蒜的trnL-F序列也进行测定分析,结果表明:所测样品trnL-F序列存在一定变异,碱基突变以转换为主。石蒜种内不同居群（细叶型和宽叶型）trnL-F序列呈现一定差异,江苏江宁的石蒜居群trnL-F序列与玫瑰石蒜、红蓝石蒜的完全相同。4.采用简单重复序列间区（ISSR）分子标记,研究了中国原产的石蒜属13种1变种之间亲缘关系,以中国水仙为外类群。16个扩增清晰、多态性好的ISSR引物共扩增出253个位点,其中244个多态性位点,多态性位点百分率达96.44%。种间遗传距离以江苏石蒜与中国水仙的的最远,达0.8700,石蒜与矮小石蒜遗传距离最近,为0.2104。中国石蒜与忽地笑虽然花部性状极为相似,但DNA差异较大。UPGMA法将供试石蒜物种聚成2人类,除换锦花、鹿葱、香石蒜在人类土划分有所不同外,ISSR标记结果与传统形态分类、核型和RAPD标记结果基本相同;还与前文中基于形态特征的数量分类和HPLC聚类分析有较高相似处。5.将基于形态特征的数量分类、HPLC图谱聚类分析和ISSR分子标记中相同的一组供试样品的原始数据联合起来利用SPSS软件进行聚类分析,结果表明:（1）中国水仙与石蒜属植物亲缘关系远,在较高的聚合水平,中国水仙形成一个单系类群。（2）15份石蒜属植物被分成了两类,第一类除忽地笑和中国石蒜外,其它全为整齐花亚属物种;第二类是以石蒜和矮小石蒜为代表的石蒜亚属物种。（3）联合分析与单一分类方法获得的结果基本一致。即中国产石蒜属5个原种（长筒石蒜、中国石蒜、忽地笑、换锦花和矮小石蒜）的系统位置比较明确,玫瑰石蒜、鹿葱、红蓝石蒜和稻草石蒜等具有杂交起源的种其杂交种身份也得到证实,系统位置较为确定。然而,不支持安徽石蒜作为一个分类学意义上的种,它可能是长筒石蒜的变种或长筒石蒜与中国石蒜的杂交种。香石蒜和乳白石蒜的起源及系统位置还难以确定,有待进一步研究。6.用ISSR和RAPD标记分别对石蒜属4个种37份样品的基因组DNA的遗传多样性进行检测。16个ISSR和17个RAPD引物分别扩增出229和215条带,多态比率分别为85.59%和69.77%,ISSR检测出的多态性条带的能力优于RAPD;ISSR和RAPD标记检测同组样品的Nei’s遗传相似系数范围分别为0.5459～0.9345（平均0.6836）,0.6419～1.0000（平均0.7428）。两者相关系数r=0.8582,达极显著水平。ISSR和RAPD标记的分子聚类结果相近,两种标记均能准确地把不同来源的样品先按种聚类,石蒜种內的遗传多态性高于同一试验中忽地笑、中国石蒜、换锦花的种内多态性。种内不同采集地样品间存在一定的遗传差异。用POPGENE3.2分析的种间基因分化系数（Gst）与用AMOVA进行的遗传变异巢式方差分析所得结果基本一致。两种标记均可用于石蒜属植物居群的遗传多样性研究。7.石蒜有代表性的13个居群和其变种矮小石蒜、近缘种玫瑰石蒜和红蓝石蒜的数量分类研究结果与同一组植物样品基于trnL-F序列的聚类结果存在较高的相似性。石蒜居群之间外部形态、生长发育习性、次生代谢产物加兰他敏含量存在较大差异,这些差异与DNA差异之间有一定联系,石蒜具有较宽的遗传基础和丰富的遗传多样性。8.应用SRAP标记对中国13个省24个石蒜居群共89个样品进行检测,10对引物组合共获得218条带,其中173条为多态性条带,多态性百分比达79.36%。多态谱带比率以贵州3个居群的最高,达37.61%,其次是四川忠县居群（SC）,为36.70%,多态谱带比率最低的是安徽广德居群（AH1）,为25.69%。物种水平的总等位基因数（na）、有效等位基因数（ne）、基因多样性指数（h）、Shannon多样性指数（I）依次为1.7936、1.4131、0.2415和0.3664。AMOVA遗传变异巢式方差分析得出的居群间变异（96.05%）与物种水平遗传分化系数（Gst）0.9748、基因流（Nm）0.0129基本相符。居群间和样品间聚类结果皆显示,所有样品可分为两大类,第Ⅰ大类7居群中除连云港居群外,均来自西南或西北地区;第Ⅱ大类由分布华南、华中和华东地区的17个居群组成。SRAP遗传距离与经度、纬度、年均降雨量、年均温呈极显著正相关,相关系数依次为0.202^**0.248^**、0.194^**、0.171^**（r0.01=0.000～0.005,n=276）:但与海拔没有显著相关。SRAP技术不仅能检测到石蒜种内、居群间丰富的遗传多态性,还能检测到居群内样品间的遗传差异。本研究采用多种方法对石蒜居群的遗传多样性进行了分析,其结果均揭示出石蒜种内存在较为丰富的遗传变异,根据多种研究结果我们将石蒜分为细叶型、宽叶型和中间类型3种类型。更多还原

【Abstract】 The genus Lycoris(Amaryllidaceae) is comprised of about 20 species,and there are 15 species in China.They widely distribute in many provinces of China,and east China is the diversity center of them.The plants of Lycoris are endowed with medicinal and ornamental values.Alkoilds which are rich in bulbs of Lycoris,have pharmacological effects,for example,galanthamine have been used for the clinical treatment of Alzheimer’s disease(AD) in recent years.It is common that different species having the same name or the same species having different names in Lycoris,which cause some confusion in germplasm collection,preservation,and utilization.Since some species are easy to hybridize,there are many natural hybrids.The morphological feature,chromosome number, and karyotype of several species of Lycoris vary in wide range at interspecific and intraspecific level.However,L.radiata(L’ Hér.) Herb.as an ornamental and medicinal plant,its genetic diversity has not been studied.It is necessary to identify species with very similar outer characters,to research interspecific relationships of Lycoris and genetic diversity of L.radiata(L’ Hér.) Herb.Thus,interspecific relationships and taxonomy of Lycoris were investigated by means of numerical taxonomy which based on the morphology,high-performance liquid chromatography(HPLC),inter-simple sequence repeats(ISSR) markers and cp DNA trn L-F sequences analysis,and genetic diversity of L.radiata were also studied with numerical taxonomy and DNA molecular markers in this paper.The main results were as follows:1.Based on the field observation and the data collected from references,Q cluster analysis for 38 characters of 13 native species and 2 varieties of Lycoris in China and one Narcissus tazetta var.chinensis were conducted.The results of cluster analysis showed that N.tazetta var.chinensis was a monophyletic group and 15 Lycoris species were classified as two groups,group one included L.anhuiensis,L.longituba,L.longituba var.flava,L. chinensis,L.aurea,L.albiflora,L.incarnata,L.squamigera,and L.sprengeri,group two consisted of L.radiata var.pumila,L.radiata,L.rosea,L.straminea,L.houdyshelii,and L. haywardii,which agree with the traditional taxonomic studies.The results supported the viewpoint that hybrid origin of L.squamigera,L.albiflora,L.houdyshelii,L.straminea,L. rosea,and L.haywardii.While L.anhuiensis as an independent species was not supported, it should be recognized as a variety of L.longituba or a hybrid between L.longituba and L. chinensis.The principle component analysis and R cluster analysis revealed that bulb shape and the position relation of the stamen and perianth can be used as the characters of Lycoris for major group division.2.13 native species and 1 variety of Lycoris in China and one N.tazetta var.chinensis were studied with HPLC technique.The result of Q clustering obtained according to the similarity of alkaloids indicated that all Lycoris species were divided into two major groups. The clustering result was generally consistent with that of numerical taxonomy which based on the morphology except for L.haywardii and L.aurea.N.tazetta var.chinensis formed a independent group due to obvious difference of HPLC chromatograms exited between N. tazetta var.chinensis and genus of Lycoris.The differences of HPLC chromatograms also existed between L.aurea and L.chinenesis,L.radiata and L.radiata var.pumila,which indicated there is a far phylogenetic distance between them.3.The cpDNA trnL-F sequence of 17 taxa representing 13 species and 2 varieties of Lycoris and N.tazaetta var.chinensis as one outgroup were determined by using direct sequencing of PCR product,and they were analyzed by means of the software of CLUSTRAL and MEGA.The length of trnL-F of all taxa was 905～1036bp,When the gaps were always treated as missing,there were 14 variable sites,10 were parsim-info ones, and account for 1.13%of the total length.The(G+C) content was 32.9%.At the intergeneric level,4 nucleotides inserteions or deletions and 7 substitutions(6 transversions and 1 transition) occurred.Three substitution sites which located on 22bp, 214bp,and 636bp were used to identify some species of Lycoris.L.incarnata and L. radiata var.pumila had its species-specific SNPs.Maximum-parsimony tree of 17 accessions were constructed based on trn L-F sequence by bootstrap test.N.tazetta var. chinensis was a monophyletic clade,three infrageneric clades of Lycoris were resolved.The trees suggested that L.anhuiensis can not be taken as an independent species,while it may be a variety or a hybrid of L.longituba,and that the maternal parents of L.rosea and L. haywardii are L.sprengeri.The trnL-F sequence of 13 populations of L.radiata,L.radiata var.pumila,L.rosea, and L.haywardii were conducted by using direct sequencing of PCR product.The result showed that minor variation of trnL-F among these taxa occurred and nucleotide substitution was mainly due to transition.There some difference of trnL-F sequences between narrow-leaf type and width-leaf type of L.radiata,while the trnL-F sequences of L.rosea,L.haywardii and Jiangsu,Jiangni population of L.radiata were the same.4.Interspecific relationships of 13 species and 1 variety of Lycoris were evaluated using inter-simple sequence repeat(ISSR) markers,N.tazaetta var.chinensis as outgroup. 16 primers produced 253 discernible DNA fragments,out of which 244 were polymorphic, and the percentage of polymorphic bands(PPB) was 96.44%.The genetic distance between L.houdyshelii and N.tazaetta var.chinensis is farthest(0.8700),while the genetic distance between L.radiata and L.radiata var.pumila is the nearest(0.2104).The DNA differences is obvious between L.chinesis and L.aurea although both of them have similar outer characters.All species of Lycoris were recognized as 2 major groups by UPGMA cluster analysis,which were basically consistent with that of by morphology, karyotype,and RAPD analysis,and also consistent with that of numerical taxonomy which based on the morphology and HPLC chromatograms cluster analysis of Lycoris except for the major group division of L.sprengeri,L.squamigera,and L.incarnata.5.Interspecific relationships of 13 native species and 1 variety of Lycoris in China were discussed by combined the data of numerical taxonomy which based on the morphology,HPLC chromatograms cluster analysis,and ISSR markers,N.tazetta var. chinensis as outgroup.The result indicated(1) N.tazetta var.chinensis was a monophyletic group,which displayed that far relationship between N.tazetta var.chinensis and gnenus of Lycoris.(2) Lycoris was divided into two groups,species in the first group were attributed to Symmanthus subgenus except for L.chinesis and L.aurea,while,species in the second group such as L.radiata,L.radiata var.pumilar were attributed to Lycoris subgenus.(3) Our results confirmed that phylogenetics of 5 primary species of Lycoris in China(L.longituba,L.chinensis,L.aurea,L.sprengeri,and L.radiata var.pwnilar).The status and phylogenetics of hybrid origin species such as L.rosea,L.squamigera,L. haywardii,L.straminea,and L.houdyshelii were supported.However,L.anhuiensis can not be taken as a distinct species,it may be a variety of L.longituba or derived from hybridization between L.longituba and L.chinensis.The status and phylogenetics of L. albiflora and L.incarnata was not resolved in this study.In conclusion,the results obtained by combined analysis were basically accordance with that of by numerical taxonomy which based on the morphology,HPLC chromatograms cluster analysis,and ISSR markers respectively. 6.37 accessions of Lycoris collected from different location in China,including 18 L. radiata,10 L.chinensis,5 L.sprengeri and 4 L.aurea were investigated with the technique of RAPD and ISSR,respectively.The result showed that 229 and 215 DNA bands were amplified with 16 ISSR and 17 RAPD primers respectively,the percentage of polymorphic bands(PPB) in ISSR detection(85.59%) was higher than that in RAPD(69.77 %).The coefficient ranges ofinterspecific and intra-specific GS(genetic similarity) in ISSR and RAPD analysis with the same materials were 0.5459～0.9345(average 0.6836) and 0.6419～1.0000(average 0.7428),respectively.There was significant difference of correlation coefficient(P＜0.01) between ISSR and RAPD(r = 0.8582).The similar molecular cluster results were obtained from RAPD and ISSR analysis,and showed that the genetic diversity of intraspecific was high,and the percentage of polymorphic bands (PPB) of L.radiata was the highest.The coefficient of gene differentiation(Gst) amonginterspecific consists with the result of molecular variance analysis(AMOVA). ISSR and RAPD marker are both efficient methods in revealinginterspecific or intra-specific genetic difference and diversity.7.13 populations ofL.radiata,L.radiata var.pumila,L.rosea,and L.haywardii were studied with numerical taxonomy and cpDNA trnL-F sequence analysis.The result obtained from Q cluster analysis is very similar to that from trnL-F sequence analysis. There were some differences in outer characters,content of galanthimine,and growth and develop habit among populations of L.radiata,and these differences was relation to that variation of trnL-F sequence.The results denoted that L.radiata has abundant genetic diversity.8.Genetic diversity and population genetic structure of L.radiata were examined with the technique of sequence-related amplified polymorphism(SRAP) 218 loci were identified with 10 SRAP primer combinations,out of which 173 were polymorphic and accounted for 79.36%of total genetic diversity at species level.The observed average number of alleles(ha),the effective number of alleles(he),Nei’s gene diversity(h), and Shannon’s information index(I) at species level were 1.7936,1.4131,0.2415,and 0.3664,respectively.The percentage of polymorphic loci(PPB) was the highest (37.61%) in Guizhou three populations(GZ1,GZ2,GZ3),Sichuan Zhongxian population(SC) was the second(36.70%),the lowest(25.69%) was in Anhui Guangde population(AH1) among populations of L.radiata.A large proportion of genetic variation(96.05%) resided among populations,while only 3.95%resided among samples within populations by Analysis of molecular variance(AMOVA).The result consisted with that of coefficient of gene differentiation(Gst = 0.9748) and Gene flow (Nm = 0.0129).These 24 populations of L.radiata surveyed were classified into two major groups.Group one included 7 populations which came from southwest or northwest of China,except for Jiangsu,Lianyungang population(JS3).Group two included the other 17 populations which distributed in from south to east China.Correlation analysis detected significant correlation between genetic distance and longitude,latitude,annual rainfall, annual average temperature.The coefficients were 0.202,0.248,0.194,and 0.171, respectively.There was no significant correlation between genetic distance and altitude. SRAP marker can detected not only genetic differences among populations,but also among samples within population.The results obtained with molecular markers and numerical taxonomy among populations of L.radiata showed that abundant genetic diversity existed in L.radiata, which can be classified as narrow-leaf type,width-leaf type,and intermediate type.更多还原