【作者】 韩国辉；

【导师】 梁国鲁； 向素琼；

【作者基本信息】 西南大学 ， 果树学， 2012， 博士

【摘要】 柑橘是世界上最具经济价值的果树之一。其遗传育种工作历来受到各国研究者的重视,人们通过芽变筛选、杂交和多倍体培育等方法已经培育了许多在世界各地广为栽培的优良柑橘品种。这些品种的推广对于促进当地经济的发展和人们生活水平的提高起到了重要作用。近年来,分子生物学的发展为柑橘的遗传育种提供了新的技术手段,对柑橘的选育种研究起到了有效的辅助作用,有力的推动了柑橘产业各个领域的快速发展。而随着基因和基因组学研究的兴起,对于重要性状基因的精确鉴别和定位也成为柑橘遗传育种学领域研究的热点。遗传连锁图谱就是承载这个热点研究领域的重要基础之一。以上各方面的进步都将为柑橘的研究和应用带来深远影响。为此,本研究构建柑橘的高密度遗传连锁图谱,并与已有图谱进行比较整合；同时以不同品种或倍性材料进行杂交培育,并利用细胞和分子生物学方法对获得的杂交后代群体其进行遗传鉴定和分析,以期为柑橘的分子遗传和育种研究提供技术平台和优良种质资源。具体结果如下：1.以柑橘幼苗微量叶片为材料,改进柑橘基因组DNA提取方法,并对三种PAGE凝胶浓度和三种银染色方法进行了筛选和优化,建立了一种经济、高效的柑橘苗期SSR分析体系。初步应用结果表明优化的技术体系稳定可靠,可以用于柑橘杂交后代的早期辅助选择和遗传图谱构建等研究。2.采用正交设计方法,对影响柑橘SCoT-PCR反应的Mg2+、dNTPs、引物、TaqDNA聚合酶及模板DNA用量等因素进行优化,首次建立了适于柑橘的SCoT标记分析体系,即20μL反应体系中含有：Mg2+1.5mmol-L-1, dNTPsO.35mmol·L-1,引物0.625μmol·L-1, TaqDNA聚合酶0.5U,模板DNA10ng。最适退火温度为49℃利用Wan2橘橙×Li2甜橙的杂交后代对该体系进行验证,结果表明建立的柑橘SCoT体系结果稳定,重复性好,可为柑橘遗传多样性分析和连锁图谱构建提供新的技术支持。3.分别利用168对SSR引物和20条SCoT引物对92株Wan2橘橙[Citrus unshiu (Mark.) Marc,×Citrus sinensis (L.) Osb.]×Li2甜橙[Citrus sinensis (L.) Osb.]杂交群体进行多样性分析。168对(112对EST-SSR和56对SSR) SSR引物共扩增出294条多态性条带,平均每对引物扩增出1.75条带。20条SCoT引物共扩增出32条多态性带,平均每对引物扩增出1.6条带。分别以两种标记的数据进行聚类分析,结果显示两种方法都可以将所有材料分为8类,并且每一类别所包含单株的比例也比较相近,二者均表明该群体具有丰富的遗传多样性,后代和亲本以及后代之间都存在较大遗传差异,可以推测该作图群体的分离方式具有多样性。4.以92株Wan2橘橙[Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.]×Li2甜橙[Citrus sinensis (L.) Osb.]杂交F1为群体,利用EST-SSR,SSR和SCoT三种标记技术构建柑橘遗传连锁图谱。对518对(条)引物进行了筛选,共获得219个多态性引物,占引物总数的42.28%,其中SSR引物67对,占全部同类引物的37.02%；EST-SSR引物127对,占同类引物的47.57%；SCoT引物25条占同类引物的35.71%。从这些多态性引物中选出206对(条)引物用于柑橘遗传图谱的构建,包括65对SSR引物,121对EST-SSR引物和20条SCoT引物,分别为各自引物总数的34.81%,45.69%和28.57%。利用筛选出的206对(条)引物构建柑橘遗传图谱。EST-SSR引物共扩增出213条多态性带,平均每对引物扩增出1.8条多态性带；SSR引物共扩增出多态性带114条,平均每对引物扩增出1.7条,SCoT引物扩增出32条多态性带,平均每个引物扩出1.6条。206个引物总共扩增出359条多态性带,平均每个引物产生1.7条多态性带。359条带中共产生了232个符合四种分离类型的标记,包括69个SSR标记,131个EST-SSR标记和32个SCoT标记,其中偏分离标记113个,占总数的48.7%。利用Joinmap4.0软件对获得的232个标记进行连锁分析,并构建柑橘遗传连锁图谱。最终226个标记被定位在了构建的9个连锁群上,包括67个SSR标记,129个EST-SSR标记和30个SCoT标记。各个连锁群长度在5到122.3cM之间,平均长度为77.1cM。总覆盖图距为693.7cM,平均遗传图距为3.1cM。9个连锁群上包含的标记数在4到104个之间,每个连锁群的平均标记数为25.1个,含标记数最多的是ESS-WL1,共含有104个标记,最少的为ESS-WL7,仅包含4个标记。分别比较分析了SCoT标记和偏分离标记的加入对图谱构建的影响。发现SCoT标记在连锁群上分布均匀,对原图谱有良好的整合效果,使连锁群的组建更加合理,更加符合与柑橘9条染色体相对应的理论连锁群数。而偏分离标记对图谱构建也并无太大影响。说明本文所构建的遗传图谱准确可靠,可以用于柑橘重要性状基因的定位等后续研究。5.将本研究构建的图谱和其他研究者构建的包含有共同标记位点的6张柑橘图谱进行比较分析。发现一些标记位点可以很好的对应起来,能够进行图谱的整合,使本研究构建的图谱具有更加重要的应用价值。但是也有一些标记由于各个图谱连锁群的组成差别较大而无法对应起来,说明了柑橘标准参考图谱构建的必要性。6.利用优化的SSR技术体系对沙田柚×红江橙杂交实生苗进行杂种性质鉴定和多样性分析。结果表明5对特异引物可以将55株杂种苗全部鉴定出来,并且发现了一个纯合显性标记AAT12,该标记在在亲本间有明显差异,可以用于沙田柚和红江橙杂交后代大量群体的遗传鉴定。在引物GA18和AGC9扩增结果中出现了亲本条带的缺失情况,可能与有性杂交过程中染色体的异常交换有关。SSR聚类分析显示55株杂种后代中,65.45%的植株和沙田柚聚在一起,34.55%的植株和红江橙聚在一起,说明杂交后代植株较多表现为偏母遗传,并且具有较高的遗传多样性。7.以柠檬四倍体为母本,分别以强德勒柚和枳为父本进行授粉杂交。共收获5个柠檬四倍体×强德勒柚杂交果,32粒种子；获得1个柠檬四倍体×枳杂交果,5粒种子。观察发现杂交果明显小于正常四倍体果,杂交所得种子也明显比正常种子小。经培养获得了27株柠檬四倍体×强德勒柚杂交苗和2株柠檬四倍体×枳杂交苗。利用细胞学方法对培养成活的植株进行染色体观察,发现29株杂交后代全部为三倍体(2n=3x=27)。根据父本枳的三叶特征和染色体数目鉴定出2株柠檬和枳杂交苗为真杂种。利用13对EST-SSR标记对25株柠檬和强德勒柚杂交后代进行遗传鉴定和分析。杂种鉴定结果显示有9对引物可以一次性鉴定出全部杂种后代。其余4对引物也可以一次鉴定出9-18株不等的杂种。EST-SSR标记的鉴定结果表明25株柠檬和强德勒柚杂交后代全部为杂种。遗传分析结果显示13对引物共扩增出87条带,多态性条带共82条,多态率达到94.03%。三倍体杂种后代的扩增图谱中出现了部分新条带或亲本条带的缺失,可能与亲本倍性不同及其在杂交过程中所提供的遗传信息量差异较大有关。聚类分析显示27份材料分为两大类：父本强德勒柚单独聚为一类；25株三倍体杂种后代全部和母本柠檬四倍体聚在一起。聚类结果充分说明了这些三倍体后代植株的偏母遗传,与母本和父本提高的遗传信息量的多少一致,据此可以推断出在杂交后代基因组中母本柠檬四倍体提供的遗传物质远多于父本,这也在一定程度上表明这些杂交后代染色体的组成及倍性水平,说明EST-SSR标记能够很好的反映出植株遗传物质所含信息量的多少,可以作为不同倍性植株杂交后代染色体倍性确认的一个参考。8.利用建立的柑橘SCoT标记分析体系对沙田柚四倍体进行分析。11个引物在4株沙田柚四倍体及其二倍体亲本中共扩增出84条带,其中多态性条带46条,多态性比率为54.8%,聚类分析显示5株沙田柚可分为三类,3株同源四倍体分别聚在不同的位置,表明4株四倍体基因组相对于二倍体母本均发生了不同程度的遗传变异,并且其相互之间也存在较大的遗传差异。这些含有丰富遗传变异的四倍体材料也可以作为柑橘育种的重要材料。更多还原

【Abstract】 Citrus is one of economically the most important fruit trees in the world. The genetic improvement programs of citrus has been subjected to the attention of global researchers, many new cultivars have been released in the world via bud sport selection, cross, polyploid and other breeding channels.The promotion of these cultivars has played an important role in promoting local economic development and the improvement of living standards of the people. The molecular biotechnology provide new technical means for improvement programs of citrus, have greatly promoted the citrus breeding. Identification and location of important qualitative genes and quantitative trait loci on a chromosome has also become a hot spot of citrus genetics and breeding programs with the rise of genetic and genomic studies,and genetic linkage mapping is one of the important foundation. The progress of the above aspects are a profound impact on research and applications for citrus. Therefore, we constructed the genetic linkage map of citrus with different molecular markers, and which were used for comparison with other citrus genetic linkage maps. Then, we obtained some hybrid progenies throgh different cross combinations, and identification and analysis by cell and molecular biology methods.The results are as follows:1. An optimized SSR analysis system on citrus were obtained, through building a new method to isolate DNA from microamount of citrus leaves rapidly and testing three PAGE concentrations and three silver staining methods. The results showed that the method had the characteristics of little material and shorter time, and its primary application showed that the optimization SSR system could provided a technical support for citrus molecular assisted breeding and constructing of molecular linkage map.2. Orthogonal design was applied to optimize SCoT-PCR amplification system of citrus in five factors such as Mg2+, dNTPs, primer, Taq DNA polymerase and template DNA. An optimal reaction system of Citrus was established, PCR reaction mixtures contained Mg2+1.5mmol· L-1, dNTPs0.35mmol· L-1, primers0.625μmol· L-1, Taq polymerase0.25U, template DNA10ng. The most suitable annealing temperature of primers was49℃. Amplifications and genetic analysis were carried out on progenies from Wan2[tangor, Citrus unshiu (Mark.) Marc. x Citrus sinensis (L.) Osb.] x Li2[sweet orange, Citrus sinensis (L.) Osb.]. The result showed that the SCoT system established in this report is useful for genetic diversity analysis and constructing linkage map of Citrus, which possesses considerable potential for citrus breeding.3. The genetic diversity of92F1individuals derived between Wan2(tangor, Citrus unshiu (Mark.) Marc, x Citrus sinensis (L.) Osb.) and Li2(sweet orange, Citrus sinensis (L.) Osb.) was analysized. A total of294polymorphism bands were amplified by112EST-SSR and56SSR merkers,with an average of1.7bands.and20SCoT primers produced32polymorphism bands,with an average of1.6bands. UPGMA cluster analysis showed that92F1individuals could be grouped into eight distinct families, and the resuits from two different marker were similar,which all showed that the F1population has high genetic diversity and greater genetic variation, can be speculated that the separation of population is diversity.4. Expressed sequence tag-simple sequence repeat (EST-SSR), simple sequence repeats (SSR),and start codon targeted (SCoT) polymorphism markers were used to establish genetic linkage map of citrus using a population of92F1individuals derived between Wan2(tangor, Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.) and Li2(sweet orange, Citrus sinensis (L.) Osb.). We initially tested518primers or primer pairs, and219(42.28%) gave polymorphic markers,which included67SSR(37.02%of187SSR),127EST-SSR (47.57%of267EST-SSR) and25SCoT primers(35.57%of267EST-SSR). Among all,206primers, including65SSR,121EST-SSR and20SCoT primers were used in linkage analysis.EST-SSR, SSR, and SCoT analyses produced213(1.8per primer),114(1.7per primer) and32(1.6per primer) polymorphic bands, respectively. Total number of polymorphic bands were359by206primers, with an average of1.7bands. Among all,232markers, including69SSR,131EST-SSR, and32SCoT markers were found segregating in the progeny, and113(48.7%) showed distorted segregation.A genetic map was constructed using JoinmaP4.0setting CP type data based on232markers, a total of226markers,including67SSR markers,129EST-SSR markers and30SCoT markers were assigned to9linkage groups. The groups ranged in size from5to122.3cM, with an average length of77.1cM, The total map length was693.7 cM, with an average distance of3.1cM between adjacent markers. the total number of markers per linkage group ranged from4to104, with an average number of25.1markers, ESS-WL1has the most number markers,and the least number of markers was detected in the ESS-WL7.The effects of SCoT markers and distorted markers on genetic linkage maps were analyzed. Distribution of SCoT markers was evenly on chromosome, which were beneficial in the construction of linkage maps, and were valuable for genetic mapping. The distorted markers also will not have a great effect in linkage maps. Which shows that the genetic map constructed in this paper is accurate and reliable, can be used for determining locations of genes on a chromosome or QTL mapping of citrus.5. Through comparison of shared markers in the map constructed by us and6maps constructed by other researchers, we found that some markers in this study could show better identity with other maps. However, some markers were placed on difference postion in different linkage groups.Which show that construction of a reference map of citrus is very necessary.6. The hybrid nature and the genetic relationship of offsprings from’Shatianyou’×’Hongjiangcheng’was analyzed with optimized SSR system.The results showed that all the offsprings from’Shatianyou’pummelo×’Hongjiangcheng’were proved to be hybrids with5pairs SSR primers, one homozygous maker AAT12was obtained, and some markers were absent in55F1seedlings in primer GA18and AGC9, which may be related to chromosome abnormality exchange. The cluster analysis of SSR data showed that65.45%progenies could be grouped with’Shatianyou’together, and34.55%grouped with’Hongjiangcheng’ together, which indicated that the most of offsprings were partial maternal inheritance and the genetic variation of hybrids from the cross combinations was obviously.7. A total of5fruits and32seeds were obtained from the cross between tetraploid lemon and’Chandler’pummelo,and1fruit and5seeds were obtained from tetraploid lemon xtrifoliate orange. The hybrid fruits and seeds are significantly smaller than normal tetraploid fruits and seeds. A total of29progenies (27from lemon×’Chandler’ pummelo,2from lemon×trifoliate orange) were obtained.Cytological analysis indicated that all the29hybrids were triploid (2n=3x=27). Two progenies from tetraploid lemon×trifoliate orange were considered to be hybrids by morphology identification and number of chromosomes.25triploid from tetraploid lemon× ’Chandler’pummelo were hybrids by EST-SSR verification. Genetic analysis were carried out on25triploid from tetraploid lemon×’Chandler’pummelo and the parents by13pairs EST-SSR primers. The13primers generated a total of87fragments,and82(94.03%) were polymorphic, some progenies had non-parent markers and some markers were absent in progenies, UPGMA cluster analysis showed that27plants could be grouped intotwo distinct families:25triploid hybrids were grouped with female parent together;’Chandler’pummelo was grouped alone.Which showed that the hybrids included different genetic information from female and male parent. EST-SSR markers can reflect plant genetic material contained in the amount of the hybrid, which can Provide a basis for ploidy confirmed.8. Amplifications and genetic analysis were carried out on four tetraploids of Shatianyou pomelo [Citrus grandis(L.)Osb.’Shatianyou’].84bands were amplified from Shatianyou pomelo with11primers, of which46(54.8%) were polymorphic. UPGMA cluster analysis showed that four tetraploids could be grouped into three distinct families based on similarity of0.825, and three homologous tetraploids were clustered in different locations, which showed that different degree of genome variation was detected in tetraploids compared with the diploid. And the tetraploids can provid important genetic resources for breeding programs of citrus.更多还原