【作者】 唐建民；

【导师】 周志钦；

【作者基本信息】 西南大学 ， 果树学， 2009， 博士

【摘要】 变叶海棠(Malus toringoides(Rehd.)Hughes)属于蔷薇科(Rosaceae)苹果属(Malus Miller)陇东海棠系(Series Kansuenses Rehd.)。变叶海棠属于兼性无融合生殖种。作苹果砧木利用,具有抗逆性强、半矮化、丰产和提高果实品质等优良性状,是极重要的苹果砧木资源。变叶海棠的形态特征、种质特性(耐盐、耐旱、耐热、耐寒、耐涝、抗腐烂病和根腐病等)、分布的生态环境等均呈现出复杂的多样性;在不同的形态类型间其抗逆性呈现出明显的差异,也是苹果属植物中具有多种高抗性的种类,是极重要的苹果种质资源。本研究以变叶海棠马尔康、柯河和下阿坝3个居群的90个个体为材料,从表型、核基因ITS序列和叶绿体片段(TrnS-TrnG和TrnQ-rps16)序列三个方面检测了变叶海棠的遗传多样性。同时,利用对细胞核rDNA的ITS序列和叶绿体基因组的DNA序列变异,在DNA分子水平上揭示变叶海棠与陇东海棠和花叶海棠之间的亲缘关系,探讨变叶海棠的起源、ITS区进化和变叶海棠母系遗传系统。主要研究结果如下:1.以变叶海棠3个自然居群为研究对象,对其叶长、叶宽、叶形指数、刻距、刻基距、缺刻指数、每花序花朵数、每花朵花柱数、果实横径、果实纵径和果形指数等12个表型性状进行多样性分析。结果表明:变叶海棠表型性状的变异极其丰富,不同性状间表现出不同程度的多样性,叶形态变异最大。居群间和居群内均存在广泛的遗传变异,居群间的表型分化系数(Vst)为16.83%,居群内变异(83.17%)高于居群间变异。变叶海棠表型总多样性指数为2.034,居居群内多样性指数为1.699,居群间的多样性指数为0.335,表明居群内变异是变叶海棠表型性状的主要变异来源。2.以窄叶海棠(M.angustifolia Michx.)、草原海棠(M.ioensis Britt.)和台湾林檎(M.doumeri Chev.)作为外类群,对变叶海棠及其假定亲本的种间特异位点和系统进化关系进行了分析,并用软件RDP3beta27对变叶海棠的ITS序列进行了重组检测。结果表明,变叶海棠居群有三种ITS拷贝变异类型:(1)与陇东海棠相似的ITS拷贝;(2)与花叶海棠相似的ITS拷贝;(3)杂合的ITS拷贝。在ITS基因树上三类ITS拷贝分别与两个推测亲本单独聚在一支(自展值分别为100%和92%);杂合的ITS拷贝虽与花叶海棠聚在同一大支(B),但杂合的ITS拷贝在B支内形成具有94%自展值支持的次级分支。重组检测支持杂合ITS拷贝是假定亲本ITS重组进化的产物。上述结果为变叶海棠的杂种起源提供了进一步的分子证据。3.在变叶海棠的柯河(KH)和下阿坝(XB)两个居群中,找到了亲本之一(花叶海棠)的特异ITS拷贝,而陇东海棠特异的ITS拷贝未能被检测到。此外,在三个居群中发现的杂合的ITS拷贝类型,其变异式样介于两亲本之间,且呈现一定的连续性。这一结果表明变叶海棠的ITS序列存在致同进化现象,并且这种ITS致同进化属于定向致同进化。4.变叶海棠ITS核苷酸序列多样性(p=0.03359±0.00201,θ_w=0.03874±0.00943)均高于陇东海棠(p=0.01893±0.00128,θ_w=0.02174±0.00917)和花叶海棠(p=0.01518±0.00203,θ_w=0.01710±0.00773),表明变叶海棠遗传多样性丰富。陇东海棠大郎足居群与变叶海棠种间基因交流程度存在差异,与变叶海棠马尔康居群基因流(Nm=0.2662)大于与变叶海棠柯河居群基因流(Nm=0.1824)和与变叶海棠下阿坝基因流(Nm=0.1801)。花叶海棠与变叶海棠三个居群间存在较为频繁的基因交流,基因流Nm值都大于1。变叶海棠三个居群间遗传分化有限(Gst为0.00227-0.00988,Fst为0.01896-0.05329),基因交流强烈(Nm为3.7252-15.7688＞3)。AMOVA分析结果显示变叶海棠遗传变异主要来源于居群内部(97.41%)。5.综合比较叶绿体两个片段所有6个信息位点和7个Indel,变叶海棠的在陇东海棠和花叶海棠这两个叶绿体片段的变异位点上,几乎和花叶海棠完全一致。以Prunus hortulana作为外类群,分别基于TrnS-TrnG和TrnQ-rps16叶绿体数据构建的MP树,同时构建了TrnS-TrnG和TrnQ-rps16序列的合并分析后的MP树,结果一致支持变叶海棠和花叶海棠二者亲缘关系较它们任何之一与陇东海棠的关系更密切。陇东海棠与变叶海棠的遗传距离大于花叶海棠与变叶海棠的遗传距离;变叶海棠种内3个居群的叶绿体缺乏变异。AMOVA分析结果表明,变叶海棠三个居群内的变异高达89.8%,居群间的遗传变异仅为10.2%,变叶海棠的变异主要存在于居群内。更多还原

【Abstract】 Malus toringoides belongs to Series kansuenses Rehd.of the genus Malus Miller of the family Rosaceae.M.toringoides is a facultative apomictic species.It shows high diversities in morphology,germplasm characteristics,and ecological habitats.Used as rootstock,M.toringoides showed high resistance to various environmental stresses such as tolerances to salt,drought,heat,cold,water-logging,and resistance to valsa mail Miyabe et Yamada and Xylaria mali Fromme,and had important effects on the grafted trees including dwarfing,early-fruiting,fruit production and quality. Therefore,M.toringoides was considered as a very important apple germplasm resource in China.In the present study,the genetic diversity and population genetic structure of M.toringoides were analyzed using morphological characters,nuclear ribosomal DNA(nrDNA) internal transcribed spacer(ITS) and chloroplast DNA (cpDNA) sequence.A total of 90 accessions collected from three natural populations (Maerkang,Xiaaba and Kehe of Aba Zang Autonomous Region,Sichuan Province, China) of M.toringoides were used as plant materials.Based on the sequence variations of nuclear ribosomal DNA(nrDNA) internal transcribed spacer(ITS),and the intergenic spacer regions of chloroplast genome,trnG-trnS and rps16-trnQ,the phylogenetic relationship,between M.toringoides and its putative parents,M. transitoria and M.kansuensis were analyzed.In addition,the molecular evolutionary pattern of the ITS sequences of M.toringoides were analyzed,and the maternal evolutionary relationship of M.toringoides to its parents were discussed.The main results obtained are summarized as follows:1.An analysis of the phenotypic diversity including 12 morphological characters (leaf length,leaf width,leaf length/leaf width ratio,dent distance,dent base distance, leaf width/dent distance ratio,leaf length/dent base distance ratio,fruit longitudinal diameter,fruit transversal diameter,fruit longitudinal diameter/fruit transversal diameter ratio,number of flowers per inflorescence,numberof styles per flower) of M. toringoides were carried out using 90 accessions collected from three natural populations.The results showed that M.toringoides was rich in morphological variations and different characters showed a different degree of variations,among which the leaves had the highest coefficient of variation(CV=22.83%).Our data also revealed that rich phenotypic variation existed both within and between populations. The results indicated that extensive variation of phenotypic traits existed within populations and among populations of M.toringoides.The phenotypic differentiation coefficient Vst was 18.92%.The high variation of phenotypic traits in M.toringoides occurred within populations(71.08%).The Shannon’s diversity index was 0.335 among populations and 1.699 within population.The genetic variation of phenotypic traits within population was the main sources of genetic diversity in M.toringoides.2.Using M.angustifolia Michx.,M.ioensis Britt.,and M.doumeri Chev.as outgroups,the species-specific nucleotides of M.kansuensis and M.transitoria and the phylogenetic relationship between M.toringoides and its putative parents were analyzed using the sequences obtained in this study and those deposited in GenBank.The recombination detection of M.toringoides sequences was also carried out using RDP3beta27 computer program.Three different types of ITS sequences were found in M.toringoides:(1) type one sequences were identical to those of M.kansuensis.(2) type two sequences were identical to those of M.transitoria,and(3) type three sequences were the mosaic sequences between M.kansuensis and M.transitoria.On the gene tree,three different types of ITS sequences clustered separately with M.kansuensis (CladeA) and M.transitoria(CladeB) with strongly statistical supports.All type three ITS sequences from M.toringoides formed a subbranch within the CladeB with a bootstrap value of 94%.Recombination detection suggested that all mosaic ITS sequences were the evolutionary products of a recombination event between ITS sequences of M.kansuensis and M.transitoria.These results provided new evidence for the hybrid origin hypothesis of M.toringoides on the basis of our previous work.3.The M.transitoria specific ITS copy was detected within KH population and XB populations of M.toringoides.The M.kansuensi specific ITS copy was not detected within the two populations of M.toringoides.In addition,the mosaic sequences which showed a continuous variation pattern between M.kansuensis and M.transitoria were detected within three natural populations of M.toringoides.These results suggested that the concerted evolution within M.toringoides was unidirectional toward M.transitoria4.The average number of nucleotide differences(κ=17.597) and nucleotide diversity of M.toringoides were p=0.03359±0.00201,θ_w = 0.03874±0.00943 respectively,which were s higher than those of M.kansuensis(p=0.01893±0.00128,θ_w =0.02174±0.00917) and M.transitoria(p=0.01518±0.00203,θ_w=0.01710±0.00773). These results suggested an abundant genetic diversity in M.toringoides.The different levels of gene flow were also detected between M.kansuensis and each of M. toringoides population.The value of gene flow between M.kansuensis and MK population(Nm = 0.2662) was higher than those between M.kansuensis and KH population(Nm = 0.1824),and between M.kansuensis and XB population of M. toringoides(Nm = 0.1801).Higher gene flow between M.transitoria and each population of M.toringoides were detected(Nm＞1).Low level of inter-population genetic differentiation(Gst and Fst varied from 0.00227 to 0.00988 and 0.01896 to 0.05329,respectively ) and high gene flow(Nm ranged from 3.7252 to 15.7688＞3) were detected among the three natural populations of M.toringoides.Moreover,an analysis of molecular variance(AMOVA) of the populations of M.toringoides showed that the genetic variation mainly occurred within populations(97.41%).5.An analysis of the sequence variation between M.toringoides and its putative parents based on trnG-trnS and rps16-trnQ revealed that the cpDNA sequences of M. toringoides were similar to M.transitoria sequence.Using Prunus hortulana as outgroup,phylogenetic relationship between M.toringoides and its putative parents were analyzed based on TrnS-TrnG and TrnQ-rps16 sequences.A combined analysis of TrnS-TrnG and TrnQ-rps16 sequences was also carried out.The results obtained strongly supported that there were close maternal relationships between M.toringoides and M.transitoria.Higher genetic distance between M.kansuensis and M.toringoides than that between M.transitoria and M.toringoides were detected.There was limited variation between M.toringoides populations.An analysis of molecular variance (AMOVA) of the populations of M.toringoides based on cpDNA data showed that the genetic variation mainly occurred within populations(89.81%).更多还原