【作者】 肖金平；

【导师】 陈力耕；

【作者基本信息】 浙江大学 ， 果树学， 2007， 博士

【摘要】 无核作为一个重要的园艺性状,在长期的生产实践中被选择保留下来;无核果实以其食用方便、品质优良而倍受人们的青睐,其无论在鲜食和加工上都具有重要意义。上世纪60年代以来,国外已逐步将鲜食品种无核化,当今国际柑桔市场上的名牌品种都是无核或少核的;我国柑桔品种资源十分丰富,地方良种很多,但栽培的品种多是有核甚至多核的,因而影响了商品价值和在国际市场上的竞争力。因此,培育无核或少核品种是柑桔育种的重要目标,研究柑桔无核的内在成因,有着极其重要的意义。目前对柑桔无核机理的研究多集中在形态学和解剖生理等方面,而利用分子生物学的手段进行基因水平上的研究开展的工作还较少,对柑桔无核的分子机制缺乏相应的了解。本研究以无核椪柑新品种‘丽椪2号’为材料,以丽水当地普通有核椪柑为对照,从雄配子体育性、雌配子体育性和自交亲和性等方面方面系统查明‘丽椪2号’椪柑的无核成因;为了寻找与柑桔无核基因相连锁的分子标记,分别以8个椪柑品种和4个本地早桔株系为材料,采用AFLP分析的方法,来分离椪柑无核品种和本地早无核变异株系的特异AFLP标记片段,并比较了这些片段序列与已知功能基因的同源性,以期对以后柑桔无核基因的精细定位和全基因克隆做一些初步的探索。本试验主要研究结果如下:1.‘丽椪2号’的无核成因1.1‘丽椪2号’花粉低育。花药花粉粒数量、花粉萌发能力和花粉染色活力都显著低于有核品种。‘丽椪2号’椪柑和有核对照每粒花药的平均花粉粒数量分别为25300和41500个,二者的差异达到了显著水平;在筛选出的最佳蔗糖浓度培养基（1%琼脂+20%蔗糖+0.01%硼酸）上,‘丽椪2号’和有核对照花粉发芽率两年的测定结果分别为1.18%和15.05%,0.82%和14.35%;花粉染色活力两年的测定结果分别为9.27%和50.23%,9.18%和45.07%。1.2扫描电镜观察发现两品种花粉粒在微观结构方面存在很大的差异。有核花粉为长球形或近长球形,饱满,外壁网眼大,有4～5条萌发沟明显可见,萌发沟浅,沟界极区大;‘丽椪2号’花粉表现为畸形的凹瘪状或枣核状,多处条状凹陷,外壁网眼小,呈陷点状,萌发沟有4～5条,但为窄带状,沟腹较深,沟界极区小,萌发孔内陷。1.3‘丽椪2号’花粉母细胞减数分裂存在异常。通过花粉母细胞减数分裂期染色体行为观察发现,‘丽椪2号’的花粉母细胞有分裂异常的情况出现,主要表现为分裂前期Ⅰ出现染色体链和染色体环,后期Ⅰ在有些母细胞中还出现不均等分裂和落后染色体现象。其中染色体链比例高达37%,组成链的染色体个数从3～9个不等,染色体环的比例约为8%,最典型的是细胞中出现了染色体链上套有染色体环。这些染色体异常都会导致‘丽椪2号’的花粉畸形,进而造成花粉低育。1.4‘丽椪2号’胚囊败育石蜡切片观察发现,有核对照到受精后第7天可见胚囊内受精卵已经进行有丝分裂发育成球形胚,而‘丽椪2号’在胚囊发育过程中,在花后第2天观察到胚囊二核期,花后4～5天的胚珠中观察到了胚囊四核期,但是在其后的连续切片中未发现有八核期的胚囊,‘丽椪2号’胚囊败育的时期从四核期开始,随着发育进程,‘丽椪2号’胚囊中空,其形状由长形逐渐变为圆形,圆形空腔逐渐增大,到花后第11～12天在珠心中出现明显的球形空腔。1.5杂交试验‘丽椪2号’无论是自花授粉还是授以高育性的雪柑花粉,在授粉后17天和96天的座果率差异都不显著,说明其无核性状不是由自交不亲和造成的;杂交‘丽椪2号’最终得到65个果实中只得到1粒种子,几乎全为无核果,证明‘丽椪2号’的无核性状是由雌性器官败育引起的。但是通过授粉能够促进子房的发育,使其座果。2.柑桔无核性状的分子标记研究2.1采用AFLP和DNA分池技术相结合的方法,分析了8个有代表性的椪柑品种,72对引物组合中有13个引物组合初步筛选到19个多态性片段,将这些差异片段进一步在8个个体中验证,共得到5个无核基因连锁标记AFLP-1、AFLP-2、AFLP-3、AFLP-4和AFLP-5;片段大小从128bp到267bp不等,所有序列提交Genebank核酸序列数据库,Blast结果显示,标记AFLP-1与拟南芥种子发育缺陷突变体（titan）TTN基因家族成员ttn8基因和染色体结构维持蛋白（SMC1）编码基因片段都有73%的一致性;AFLP-2与拟南芥一种GTP酶激活蛋白（GTPase-Activating Protein,GAP）编码基因部分序列的一致性为80%;AFLP-3与拟南芥ATP依赖性膜转运蛋白家族成员PGP18编码基因和ATP结合运输蛋白编码基因片段分别有71%和69%的一致性;AFLP-4与梨属植物ATP合成酶基因和铁氧还蛋白谷氨酸合成酶基因比对区域的一致性同为83%;AFLP-5片段序列与拟南芥糖基水解酶家族蛋白17编码基因和beta-1,3—葡聚糖酶相关蛋白编码基因序列片段也都有73%的一致性。这些结果表明,分离得到的5个特异标记片段与上述功能基因都有一定的同源性。2.2采用相同的引物组合和方法对来自浙江黄岩的4个本地早桔株系（3个无核变异株系,1个有核株系）进行分析,72对引物组合中筛选出12对多态性较好的引物进行最终扩增;得到了9条清晰稳定的特异片断SL₁～SL₉;克隆测序,BLAST结果表明:特异片段SL₁与一种植物生长素输出蛋白编码基因PIN9片段有78%的同一性;特异片段SL₂与一种植物反转录转座子基因片段有86%的同一性;特异片段SL₄与植物叶绿体基因片段有94%的同一性;与这些已知功能基因的同源性分析有助于了解本地早桔无核变异的发生机理和为以后定位无核基因打下基础。更多还原

【Abstract】 Seedlessness is an important horticultural trait and so it is selected and reserved in producing practice for a long time.The peoples prefer choosing the seedless fruit for its easy-eating and better quality,and the seedless trait has important significance for fresh eating and processing of citrus.Since 1960s fresh-eating citrus cultivars have become seedless step by step overseas and all famous-brand species are seedless or low-seedy in international market of citrus.In china citrus resource are very rich along with many regional excellent cultivars but most of them are seedless,even with more seeds,so which reduces commercial values and competitive in international market.Therefore,breeding seedless or low-seedy cultivars is an important goal.To date researchs on citrus seedless mechanism are put focus on morphology and anatomy et al.,however few works are made in the aspect of gene level using the molecular biology means as well as get little knowledge about molecular basis of citrus seedlessness.In present research ’Lipeng No.2’,a novel seedless Ponkan cultivar,served as material and common seedy cultivar was used as control,investigating fertility of male,female gametophyte and compatibility systemly.Simultaneously in order to search the linkage markers related to seedless trait we performed the AFLP analysis using the 8 Ponkan cultivars and 4 Bendizao mandarin accessions as materials respectively.And sequences of the obtained AFLPs were compared homology with known functional genes.Primary results were as follows:1.Seedless reasons of ’Lipeng No.2’ Ponkan（1）.Pollen ferlity descending.Pollen grains per anther,pollen germinability,pollen stainability of ’Lipeng No.2’ were low obviously compared with seedy control.Pollen grains per anther of ’Lipeng No.2’ and CK was 25300,41500 respectively,which giving distinct difference;pollen germinability of ’Lipeng No.2’ and CK were 1.18%,15.05%and 0.82%, 14.35%at screened optimal concentration medium in two years;and pollen stainability were 9.27%,50.23%and 9.18%,45.07%.（2）.Microstructure of two cultivars had remarkable difference by using EMC.Pollen of seedy Ponkan was long-global or near long-global shape,plumb,and net-hollow on outer wall are big with 4-5 germinable grooves.Nevertheless pollen of ’Lipeng No.2’ showed concave or jujube-nuclus shape,strip sunken in many areas,small net hollow;and number of germinable groove was 4-5,narrowband shape,morever germianable hollow sinked insidely.（3）.Pollen mother cells of ’Lipeng No.2’ were abnomal at meiosis phase.PMCs divided abnormally including chromosome chain,chromosome loop,lagging chromosome and chromosome unequal distribution.Choromosome chain rate was as high as up to 37%and chromosome chain was composed of 3-9 chromatids;chromosome loop rate was approximate 8%.These abnomal chromosomes resulted in monstrous pollen and then bring on pollen fertility descending.（4）.’Lipeng No.2’ embryo sac sterility.Paraffin section result incidated that zygote of embryo sac had developed to become globular stage embryo at the 7th day after fertilization. However in the process of embryo sac development of ’Lipeng No.2’,two-nucleus stage was observed at the 2th day after blooming as well as four-nucleus stage in ovule at 4-5th day after blooming,but 8-nucleus stage embryo sac was not observed.Embryo sac sterility begined from 4-nucleus stage and subsequent with development,embryo sac of ’Lipeng No.2’ appeared middle-hollow and the shape became round from long.（5）.Cross test.Not only self-pollination but also pollinated by xuegan orange had high fruit-setting rate,which showed self-incompatibility was not the reason for seedless trait.In cross test ’Lipeng No.2’ produced only one seed and the fruits were almost seedless,which proved that seedless trait was resulted from the female sterility.2.Studies on molecular markers linked to seedless trait of citrus（1）.Combining the AFLP with DNA pool 8 respresent Ponkan cultivars were analysised,19 polymorphic bands were obtained by 13 primer combinations screened from 72 primer pairs and then these specific bands were identified in 8 individuals.Five markers,named as AFLP-1,AFLP-2,AFLP-3,AFLP-4,AFLP-5,were linked to seedless gene probably.Band size ranged from 128bp to 267bp and all sequences were submitted to Genebank database, BLAST result showed that marker AFLP-1 had 73%identify with Arabidopsis seed development defect mutant coding gene ttn8 and chromosome structural maintance protein cds;AFLP-2 had 80%identify with GTPase-activating protein cds;AFLP-3 had 71%and 69%identify with ABC transporter protein family PGP18 cds and ABC transporter protein; the identify of AFLP-4 with Pyrus communis putative ATP synthase partial cds and partial sequence of ferredoxin-dependent glutamate synthase-like gene;AFLP-5 had the same identify（73%）with Arabidopsis thaliana glycosyl hydrolase family 17 protein cds and beta-1,3 glucanase-like protein cds.These results showed that 5 isolated fragments had partial homology with functional genes aboved.（2）.Four bendizao mandarin accessions were conducted AFLP analysis using the same primer combinations with Ponkan.Twelve good polymorphic primer pairs screened from 72 primer combinations were performed final PCR amplification,obtaining 9 clear and stable bands named as SL₁-SL₉.These fragments were cloned and sequenced and then Blast result showed that SL₁ had 71%homology with putative auxin efflux carrier protein 9（PIN9）gene, SL₂ had 86%homology with retrotransposon Rtsp-1 DNA sequence and SL₄ had 94% homology with chloroplast genomic DNA sequence.These valuable information helped to realize the mechanism of seedless variation and locate the seedless gene of citrus.更多还原