【作者】 杜晓云；

【导师】 罗正荣；

【作者基本信息】 华中农业大学 ， 果树学， 2008， 博士

【摘要】 我国是柿属植物的分布中心和原产中心之一,拥有丰富的种质资源。逆转座子是真核生物基因组中的重要组成成分,其在决定植物基因组大小、结构、功能以及及其进化中扮演重要角色。开发和应用逆转座子分子标记,可为我国柿属植物种质资源研究提供技术支撑。本研究首先分离柿Tyl-copia类逆转座子RNaseH-LTR序列并设计逆转座子引物,其次建立柿属植物IRAP、REMAP、SSAP和ISTR系列逆转座子分子标记技术体系并应用于柿属植物种质鉴定和亲缘关系分析等方面,同时应用非逆转座子分子标记AFLP和DAMD于柿属植物,并比较了二者以及ISTR与柿属特异性逆转座子分子标记在该属植物遗传分析中的性能,此外,对柿逆转座子引物在它类果树物种及禾本科、茄科和蔷薇科已发表的逆转座子引物在果树类作物上的通用性进行研究,并对一份特殊种质‘90-1-10’进行了鉴定。主要结果如下:1.利用抑制PCR方法从‘罗田甜柿’（Diospyros kaki Thunb.‘Luotiantianshi’）基因组中分离31条Tvl-copia类逆转座子的RNaseH-LTR序列,序列分析表明:至少有10个逆转座子家族得到扩增;其家族间序列普遍表现高度异质,发生不同程度的碱基替换、插入或缺失突变,以及翻译成氨基酸后的终止密码子突变、氨基酸取代和移框突变等;家族内部某些序列相似性极高,类反转录病毒中的准种居群;序列LTR区含有启动子的结构特征“CAAT box”、“TATA box”以及受不同胁迫条件作用下的调控元件。序列投递GenBank,登录号为EU068698-EU068728。2.基于其中15条RNaseH-LTR（Long terminal repeat）序列共设计26条逆转座子引物,包括8条正向LTR引物、10条反向LTR引物和8条PPT（polypurine tract）引物。为增加特异性,引物设计过程中充分考虑其较长长度和高Tm值:长度均在20bp～26bp之间,平均23bp;Tm值普遍高于55℃,平均为60.2℃。其中24条引物表现较好。3.优化PCR反应体系各组分浓度、退火温度（T_m）和扩增程序的基础上建立起适合柿属植物的逆转座子分子标记IRAP（Inter-retrotransposon amplifiedpolymorphism,逆转座子之间扩增多态性）和REMAP（Retrotransposon-miorosatelliteamplified polymorphism,逆转座子一微卫星扩增多态性）PCR扩增技术体系:20μL反应体系中,含1×Buffer、模板DNA 50 ng、Mg²⁺ 2.0mmol/L、dNTPs 0.25mmol/L、引物0.40μmol/L、Taq DNA聚合酶1.0U,矿物油20μL;PCR扩增程序:95℃5min;95℃1min;56℃1min ramp+0.6℃/s;72℃2min,循环44次;72℃延伸8min。4.建立柿属SSAP（Sequence-specific amplified polymorphism,特异序列扩增多态性）逆转座子分子标记技术体系,并对可能影响其扩增性能的几个重要因素和不同逆转座子在柿属种质遗传关系研究中的特点进行分析比较。结果表明,所有逆转座子都产生丰富的多态性,表明每个逆转座子都可单独用于SSAP分析;SSAP扩增性能与逆转座子自身的特点、选择性扩增酶接头引物的种类、组成及排序相关;不同逆转座子在28份试材中表现不同的转座特点。研究为进一步使用多逆转座子分子标记,以获得较为全面的柿属种质起源进化信息和进行遗传多样性评价及种质鉴定提供技术支持。5.建立适合柿属植物的通用引物逆转座子ISTR（Inverse sequenoe-tagged repeat,反向序列标签重复）分子标记体系,与IRAP同时用于32份柿属基因型的多态性和亲缘关系分析,并比较二者分析性能。结果表明,IRAP和ISTR均能很好的用于柿属植物的遗传分析;IRAP能区分供试所有基因型,包括芽变;ISTR不能区分‘富有’和其芽变‘松本早生’,但可以区分其它所有基因型;IRAP与ISTR的聚类结果基本一致,但在细节上存在差异;IRAP在种间及种下分析所得的所有参数值（平均每次实验扩增总位点数、平均每次实验扩增多态位点数、多态位点百分率Pi、多样性指数DI、有效多重系数EIVIR、标记指数Marker Index MI）均高于ISTR,总的来看,具有柿属特异性的IRAP标记系统的分析性能优于通用引物的ISTR。6.探讨IRAP用于柿属植物种间关系研究的可行性,并与小卫星分子标记DAMD（Directed amplification of minisatellite-region DNA,直接扩增小卫星DNA）进行多态性和聚类分析比较。结果表明IRAP可以很好的用于柿属种间亲缘关系研究;DAMD作为首次应用于柿属植物的标记类型,也表现出很好的扩增性能,并能区分芽变;IRAP和DAMD聚类基本均能将不同采集地的种按地理位置聚类,但对中国热带、亚热带采集的柿种与温带栽培柿之间的关系存在分歧;两种标记用于柿属植物种间亲缘关系研究,在多态性水平、平均扩增总位点等方面表现相当,然而综合扩增性能评价IRAP较优于DAMD。总的看来,IRAP是揭示柿属植物种间及种下丰富多态性的有效标记类型,而DAMD也是将来用作柿属植物遗传分析的极具潜力的标记类型。7.探讨IRAP用于品种内微小变异检测的性能,以我国最大的‘磨盘柿’产区—北京市房山区张坊镇上报的11株磨盘柿基因型作为试材,这些试材在成熟期、果实外观形态、抗性及耐贮性等方面性状与标准品种存在差异。结果表明,19个逆转座子单引物IRAP扩增中,4个引物（PPT3,PPT6,SAF5和SAR10）在磨盘柿变异间具多态性,采用二歧法,该4个引物可将11份‘磨盘柿’变异完全区分;UPGMA聚类图上,磨盘柿变异与标准磨盘柿紧密相聚,分支模式显著不同于种与种间及不同品种之间:相似系数计算表明,磨盘柿变异单株间的相似指数在0.90（‘MP2’与‘MP4’、‘MP8’、‘MP9’）～0.98（‘MP7’和‘MP8’）之间,平均0.94,与磨盘柿标准品种‘BZMP’的平均相似指数为0.91,大于种间和品种间相似水平,与芽变间的相似水平接近。总的看来,本研究鉴定了供试的11份北京房山区磨盘柿的变异为遗传物质的改变,并非饰变;11份基因型之间以及与标准磨盘柿品种在遗传上的高相似性表明,它们可能为磨盘柿的芽变类型;IRAP分子标记能够很好的区分遗传背景高度相似的微小变异,反映了该项技术是今后进行柿品种鉴定,尤其是遗传差异较为接近的基因型,如芽变的有效工具。8.IRAP、REMAP和SSAP逆转座子分子标记用于28份柿属植物种质鉴定,16个柿逆转座子引物在供试材料的单引物IRAP分析中检测到丰富的扩增位点,且高多态性比例（97.6%）;筛选出的7对REMAP引物和25对SSAP引物在相应的分子标记内同样检测到丰富的多态性（分别为95.2%和93.4%）;每一类型分子标记均可成功鉴定28份种质,其中2个IRAP引物（PPT3和SAR10）及4对SSAP引物（SAR10/EA06、SAR9/MC03、SAR9/MC11、SAR1/MC11）可以单独用于区分全部种质。进一步计算形态学相近和已知芽变关系的5组种质各组内基因型间的遗传距离,量化每种逆转座子分子标记的鉴别效率表明:IRAP为3种逆转座子分子标记中进行种质鉴别最为灵敏的标记类型。9.IRAP、REMAP、SSAP和AFLP（Amplified fragment length polymorphism,扩增限制性片段长度多态性）4种分子标记在柿属植物遗传关系研究中的应用效果比较表明:IRAP具有最高的多态性水平和多样性指数;AFLP的多态性低于IRAP和SSAP,但具有最高的标记指数;SSAP的标记指数值低于AFLP,但其多态性高于AFLP;4种分子标记的遗传相似系数矩阵间的相关性显著,相对而言REMAP可靠性略差;4种分子标记的聚类结果基本一致,均可将28份种质按种间及种下不同地理起源进行划分,然而聚类细节因标记种类不同有别。综合评述,IRAP适用于柿属植物遗传多样性分析和种质区分,尤其是遗传背景相近的种质鉴定,而SSAP和AFLP适用于起源、进化研究。10.从‘罗田甜柿’中开发的26个逆转座子引物在其它果树上的通用性试验表明:53.85%的引物可在供试果树类植物中获得扩增产物,但不同引物在不同物种中的通用性各异,其中在柿属、葡萄属、柑橘类和桃属植物中的通用性较好。利用可通用引物在柿属、柑橘类、葡萄属、桃属和梨属植物中的IRAP数据进行遗传关系分析的结果表明:这些引物在相关试材上还具有种质区分、亲子关系鉴定、分类和系统关系研究以及遗传多样性评价等研究的潜力;同时还研究了已发表的来自蔷薇科、禾本科和茄科的逆转座子引物在果树类作物上的通用性,进一步佐证了逆转座子引物可跨物种通用的特点,不同意前人的观点。11.我国原产柿种质中只有完全甜柿和完全涩柿两种类型,至今尚无不完全甜柿存在的报道。对引种自湖北省罗田县的‘90-1-10’进行形态学、细胞学鉴定及生化和分子水平分析,结果表明,‘90-1-10’具有与中国原产柿种质相近的果实特征,结合4种DNA分子标记IRAP、REMAP、SSAP和AFLP的聚类分析结果和引种地推断其属中国原产;可溶性单宁含量和单宁细胞大小的测定结果表明‘90-1-10’属非完全甜柿类型;果实脱涩和种子形成及其果肉褐斑情况分析结果表明‘90-1-10’应为非完全甜柿种质中的不完全甜柿:‘90-1-10’与‘罗田甜柿’及其它罗田甜柿变异单株在形态学、脱涩性状及DNA水平上表现明显的差异,表明其应为不同于‘罗田甜柿’的一份新种质。因此,‘90-1-10’可能系原产我国的不完全甜柿新种质。这是目前中国原产不完全甜柿类型的首例报道。更多还原

【Abstract】 China is one of the originating and distributing centers of Diospyros Linn.,where abundant valuable genetic resources exist.Retrotransposon are ubiquitous in eukaryotic organisms,constituting a major portion of the nuclear genome（often more than half of the total DNA） in plant,and play a major role in plant genome size,structure,gene function as well gene and genome evolution.Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. Therefore,the development and utilization of retrotransposon-based molecular markers in Diospyros Linn.will be of considerable interest for improving technical tools and extending obtainable genetic information concerning Diospyros Linn.germplasm resources.In the present study,we carried out a series of research in this aspect,and the main results were as follows:1.31 RNaseH-LTR（Long terminal repeat） sequences of Ty1-copia like retrotransposon were isolated from the genome of Diospyros kaki Thunb.’Luotiantianshi’ using suppression PCR method.Sequence multiple alignment and the phylogenetic analysis indicated that at least 10 subgroups were amplified.High sequence heterogeneity which were due to nucleotide replacement,insertion or deletion and amino acid replacement,stop codon and frameshift mutations in translated putative amino acid sequences were observed among the different subgroups.But some clones showed high similarity within the same subgroup,of which behavior like quasispecies-like population. Potential regulatory motifs from transcription,such as ’CAAT box’ and ’TATA box’ and some conserved cis-acting regulator elements were discovered within LTR regions in the great number of sequences using MatInspector and PLACE software.The sequences have been deposited in the NCBI GenBank database under accession numbers of EU068698-EU068728.2.26 retrotransposon primers were designed based on 15 out of 31 RNaseH-LTR sequences,which included 8 LTR forward primers,10 LTR reverse primers and 8 PPT （Polypurine tract） primers.To enhance the specificity and reliability of PCR amplification, long length and high annealing temperature（T_m） value of designed primers were thoroughly considered in primer disigh.In this study,the length of retrotransposon primer was 23bp on average;T_m value was set beyond 55℃universally with an average of 60.2℃.Of which 24 primers were successfully amplified by IRAP,giving rise to reproducible and reliable bands.3.The optimal IRAP（Inter-retrotransposon amplified polymorphism） and REMAP （Retrotransposon-microsatellite amplified polymorphism） retrotransposon-based molecular marker systems were successfully established in Diospyros Linn.on the basis of optimizing the concentration of several components in PCR reaction,the value of Tm and the PCR amplification program,which were:in 20μL reaction system,Buffer 1×, template DNA 50 ng,Mg²⁺ 2.0 mmol/mL,dNTPs 0.25 mmol/L,primer 0.40μmol/L Taq DNA polymerase 1.0U and mineral oil 20μL.The optimal amplification program was:1 cycle at 95℃,5min;1 cycle at 95℃,1min;56℃1min;ramp+0.6℃/s to 72℃;44 cycles of 72℃,2min.1 cycle at 72℃,72℃8min.4.SSAP（Sequence-specific amplified polymorphism） retrotransposon molecular marker was also developed successfully and several key elements influencing its performance were analyzed.Moreover,the features of different retrotransposon applied for the study of genetic relationship in 28 Diospyros kaki Thunb.were compared.The results showed that polymorphisms detected in the insertion pattern of all the retrotransposons revealed that each can be used for SSAP.All elements including different retrotransposons,the kind of adapter primers and the base composition and the order of the selective nucleotides in selective amplification had influence on the performance of SSAP analysis.Different retrotransposon had an unique behavior in accessions tested,which may likely reflect distinct evolutionary histories and activities of particular retrotransposon.Taken together,our results would offer a technique approach for obtaining more resolved and detailed genetic phylogenetic information,estimating genetic diversity and distinguishing germplasm in Diospyros kaki Thunb.by retrotransposon-based SSAP molecular marker.5.ISTR（Inverse sequence-tagged repeat） retrotransposon molecular marker system based on universal primers originally derived from coconut was established in Diospyros Linn.,and was further used for the detection of genetic variability and genetic relationship analysis in 32 Diospyros Spp..IRAP was also used for the same set of genotypes analysis, and the performance of these two methods was compared.The results showed that both IRAP and ISTR could be applied for the genetic analysis in Diospyros Linn..IRAP could distinguish all the genotypes examined including bud sports.ISTR also presented good discrimination capability,but it was not capable for differentiate ’Matsumoto-wase’ from its original variety ’Fuyuu’.The cluster pattern between IRAP and ISTR shared much similarities,however,some slight differences was showed.IRAP possessed the higher values of all the parameters for evaluating the efficiency of marker system including loci per assay unit,average number of polymorphic bands per assay unit,Effective Multiplex Ratio,Diversity Index and Marker Index than ISTR whether at the inter-specific level or at the intra-specific level.So,collection data suggested that Diospyros-specific IRAP retrotransposon marker techniques are superior to the universal primer-based ISTR retrotransposon marker in our study.6.Feasibility of IRAP for genetic relatedness analysis particularly for that of inter-species was investigated.Subsequently,comparative studies concerning the level of polymorphism and cluster results between IRAP and DAMD（Directed amplification of minisatellite-region DNA） molecular techniques were conducted.The results showed that IRAP could be capable for the genetic relatedness analysis between different species of Diospyros Linn.DAMD,although used for the ftrst time in the genus of Diospyros,also indicated good amplification,and it could distinguish bud sports examined.Species tested almost could be grouped well according to their different collection locality using two methods,however,there were incongruent concerning the genetic relatedness of species collected from tropic and subtropic regions with that from temperate regions in China. IRAP and ISTR produced almost comparable values of the polymorphic level,average amplified loci per assay unit and diversity index,whereas the former revealed the higher comprehensive performance than the later due to its higher multiplex effective ratio.In all, collective results suggested IRAP was an ideal marker system in the future for the detection of genetic polymorphism both at the inter-species level and at the intra-species level.Moreover,our results indicated that DAMD was also a potential powerful marker tool for the future genetic analysis in Diospyros Linn.7.Capability of IRAP for tiny differences detection within a cultivar was examined on the 11 ’Mopanshi’ genotypes which was selected and reported by Zhangfang town, Fangshan district of Beijing city,the most centralized regions of ’Mopanshi’.These genotypes had unique properties compared to the standard ’Mopanshi’ cultivar.The results showed that 4 out of total 19 amplifiable primers,PPT3,PPT6,SAF5 and SAR10, was polymorphic primers between different ’Mopanshi’ mutations.By using dichotomy classification,11 ’Mopanshi’ mutations could be completed distinguished by these 4 primers.In UPGMA dendrogram,’Mopanshi’ mutations grouped tightly each other and with standard cultivar ’BZMP’,and the branch pattern was significantly distinct from that between species,as well as those of different cultivars within the same species.The measurement of similarity coefficients revealed that the similarity coefficient between ’mopanshi’ mutations was from 0.90（’MP2’ and ’MP4’,’MP2’ and ’MP8’,’MP2’ and ’MP9’） to 0.98（’MP7’ and ’MP8’）,and the mean was 0.94.Moreover,Between ’Mopanshi’ mutations and the standard cultivar ’BZMP’,the mean similarity coefficient was 0.91,which was over that of between species and that of between different cultivars, but approximately similar to that of between bud sports.Therefore,in conclusion,the present study ascertained that the variation of 11 ’Mopanshi’ mutations from Fandshan district of Beijing was genetic variability,but not alternation due to environment, development stage and nutrition status etc.;11 ’Mopanshi’ might be the bud sports arose from standard ’Mopanshi’ cultivar in view of the great similarity between 11 genotypes, as well as between these mutations and the standard ’Mopanshi’ cultivar;IRAP molecular technique could distinguish the tiny mutations,which implied that this method was an powerful genetic tool for the future cultivar identification,particularly for genotypes of great genetic similarity,such as bud sports.8.IRAP,REMAP and SSAP molecular markers were exploited for the germplasm identification.The results showed that these primers can be used both within and between species;each of 16 primers out of 26 produced numerous amplified loci and abundant polymorphism bands by IRAP（Inter-Retrotransposon Amplified Polymorphism） in 28 genotypes of Diospyros spp..Moreover,successful amplifications were also obtained when these primers were further used for REMAP and SSAP analysis.Each types of molecular marker produced unique fingerprint in 28 genotypes tested,and among of all used primers or primer combinations 2 IRAP primers（PPT3 and SAR10）,4 SSAP primer combinations（SAR10/EA06,SAR9/MC03,SAR9/MC11 and SAR1/MC11） could be used individually for all germplasm differentiation.Furthermore,15 accessions that can been divided into 5 groups according to their similarity of morphologic characteristics and known relationships of bud sports were selected,and the genetic similarity within groups were computed to measure discrimination power of each molecular marker system, the result indicated that IRAP is the most sensitive and efficient one among 3 molecular techniques in germplasm identification.9.IRAP,REMAP,SSAP and AFLP were compared in terms of their informativeness and efficiency in a study of genetic relationships among 28 genotypes in the genus Diospyros.The results showed that IRAP represented a higher level of polymorphism and a greater information content,as assessed by the diversity index（DI）, than the rest of molecular markers.The lower level of polymorphism for AFLP were obtained than IRAP and SSAP,which,nevertheless was the most efficient marker system due to its capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes.The value of marker index （MI） is lower for SSAP than that of the AFLP,but the former had a higher level of polymorphism than the latter.The correlation coefficients of similarity were statistically significant for all four marker systems used,but the relatively lower correlation with other markers was observed for REMAP.For all markers a high similarity in dendrogram topologies was obtained,which generally divided 28 genotypes examined into 3 groups according to different species and their different geographic origin.However,some differences in the positioning of some genotypes at the main groups were revealed due to different marker systems.In summary,we recommend,in the genus Diospyros,IRAP for the assessment of genetic diversity and germplasm discrimination,particular for theidentification of genetically very similar germplasm;and SSAP and AFLP were preferred for the study of phylogenetic,as well as the creation of a genetic linkage map.10.The transferability of retrotransposon primers derived from ’Luotian-tianshi’ persimmon（Diospyros kaki Thunb.） in Diospyros spp.and other 13 kinds of fruit crops was investigated by way of IRAP retrotransposon molecular marker.Our results as follows:Retrotransposon primers from ’Luotian-tianshi’ could be used well not only in inter-species and intra-species in Diospyros,but also in the other 13 kinds of fruit crops analyzed,which may be attribute to the horizontal and vertical transfer characters of retrotransposon.14 primers representated 53.85%primers tested could be amplified in 14 kinds of fruit crops simultaneously.Primers showed different amplified performance in different fruit crops,and the outstanding performance of transferability was revealed in four fruit crops including Diospros spp.,grape,Citrus and peach.Furthermore,based on IRAP molecular data sets from the effective primers amplification corresponding to the different fruit crops,genetic similarity coefficients were calculated and UPGMA cluster dendrograms were constructed.The results indicated that these effective primers could further offer a good potential tool for germplasm differentiation,parentage identification, classification and phylogenetic study,as well as genetic diversity assessment.The conclusion that retrotransposon primers could be of transferability among different plant species was further confirmed by examining the transferability of retrotransposon primers derived from published primers in Rosaceae,Gramineae and Solanaceae in 13 kinds of fruit crops.Our results were incongruent with previous researchs that thought retrotransposon primers was of species-specific and species-specific primers was one of a perquisite for the development of molecular markers.This is the first successful report of transferability of retrotransposon primers derived from ’Luotian-tianshi’ of Diospyros to other fruit crops,and to our knowledge,it is also one of the limited number of related report concerning the transferability of retrotransposon primers available at present.It provided not only the proof of transferability of retrotransposon primers in different plant species,but also the precious resources of primers for other fruit crops,which is benefit for saving cost of retrotransposon primer development and for popularizing retrotransposon molecular markers.11.More than 963 cultivars named as Japanese persimmon exist in China,but hitherto most of them are PCA type.No PVNA and PVA types have been reported.In the present study,from the aspect of morphology,cytology,biochemistry and molecular biology,we investigated ’90-1-10’ which was introduced from Luotian county of Hubei province where the first Chinese native PCNA Japanese persimmon ’Luotian-tianshi’ was found.The results were as follows:’90-1-10’ had the similar tree type and fruit morphologic traits with other Chinese native persimmon germplasm,suggesting that it should be considered as a Chinese native persimmon,which was further confirmed by molecular marker analysis.The results of the tannin cell size test showed that ’90-1-10’ should be belong to non-astringent type persimmon.The results of the observation of brown in fruit flesh and soluble tannin content test indicated that it should be a pollination variant non-astringent（PVNA） type persimmon.In addition,’90-1-10’ exhibited remarkable differences with ’Luotian-tianshi’ and the other variations from ’Luotian-tianshi’ in fruit morphology,the pattern of astringency-loss and genetic composition,implying that it should be a new germplasm genetically distinct from ’Luotian-tianshi’.In summary,collective results demonstrated that ’90-1-10’ should be a Chinese native PVNA type persimmon.This is the first report on existence of a PVNA type persimmon in China hitherto.This germplasm would be an important source for the phylogenetic study on Chinese native PCNA type persimmon and would have a potential role for breeding in the future.更多还原