【作者】 刁卫平；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2009， 博士

【摘要】 黄瓜(Cucumis sativus L.,2n=14)隶属于葫芦科,黄瓜属,是一种重要的世界性蔬菜作物。通过单倍体诱导技术可以快速获得纯系(单倍体和双单倍体),进而加快黄瓜的育种进程。同时,通过选育不同倍性(整倍体和非整倍体)的新种质资源材料,不仅可以增加黄瓜的商业价值,而且对促进它的分子细胞遗传学研究也具有重要价值。为此,本论文利用不同基因型的黄瓜品种为试材进行未授粉子房培养,以期获得单倍体和双单倍体材料,建立其诱导单倍体的技术体系；利用未授粉子房培养获得的同源四倍体材料进行杂交、回交,筛选出了一系列同源三倍体、初级三体及早世代稳定遗传的二倍体黄瓜材料；此外,采用细胞遗传学和分子标记技术对这些获得的新种质材料进行了鉴定与评价,以期为黄瓜的倍性育种打下基础。具体结果如下：1.黄瓜未授粉子房培养诱导单倍体／双单倍体技术体系的建立研究了热激处理时间、TDZ和AgNO3浓度对黄瓜未授粉子房培养过程中胚状体发生率的影响。结果表明,子房培养开始阶段进行35℃热激处理的胚发生率显著高于对照(0 d),其中处理2 d的效果较好；TDZ对提高胚状体发生率具有显著效果,添加0.04 mg·L-1TDZ的诱导培养基培养的最大胚状体发生率可达72.7%；在诱导培养基中添加AgN03可以提高胚状体发生率,同时能缩短胚状体出现的时间并提高胚状体产量。最后,在3种不同基因型黄瓜材料中共获得了40个再生植株,经染色体计数后发现2株为单倍体,5株为同源四倍体,剩下33株为二倍体。利用SSR手段对获得的二倍体植株进行同质性分析后发现,17株为双单倍体。2.同源四倍体黄瓜的培育及分子细胞遗传学研究利用SSR手段对未授粉子房培养获得的同源四倍体植株进行同质性分析后,发现它们均为纯合子。利用形态学观察和染色体计数法对同源四倍体单株自交后代的遗传稳定性进行了研究,结果表明自交后代倍性没有发生变异。对同源四倍体植株花粉可染率、花粉萌发率及自交结籽数分别进行观察后发现,所获得的同源四倍体具有较高的育性。对同源四倍体黄瓜(Tetra-Jinlv)花粉母细胞减数分裂与雄配子体发育观察结果表明,同源四倍体黄瓜花粉母细胞减数分裂过程与二倍体基本相同但有其特殊性。主要表现为：中期Ⅰ染色体的构型复杂,有多价体、四价体、三价体、二价体和单价体；中期Ⅰ和中期Ⅱ有赤道板外染色体；后期Ⅰ和后期Ⅱ出现落后染色体、染色体桥；后’期Ⅱ和末期Ⅱ还出现染色体分离不同步及不均等分裂的现象；四分体时期出现二分体、三分体、含微核的异常四分体及多分体。花粉母细胞减数分裂平均异常频率为37.2%。正常四分体中的小孢子,大多数可以发育成具有三孔、两细胞的可育雄配子,小孢子的发育主要经过以下几个阶段：单核早期、单核晚期、两核期。花粉萌发试验发现同源四倍体的花粉萌发率仅为46.9%。此外,利用分子细胞学研究手段对两种不同来源途径四倍体(Tetra-5来自未授粉子房培养；Tetra-Jinlv来自秋水仙素处理)以及原始二倍体进行了遗传分析。细胞学研究结果发现,同源四倍体Tetra-5在减数分裂各个阶段染色体异常行为频率明显较Tetra-Jinlv要低,尤其在后期落后染色体数目上。对比两种同源四倍体在中期Ⅰ的染色体构型发现,同源四倍体Tetra-5较Tetra-Jinlv含有较多的四价体、较少的单价体和三价体。同时,采用AFLP技术对这两类同源四倍体及原始二倍体进行基因组DNA多态性分析后发现,从38对引物扩增获得的2214条60-500bp条带中,多态性位点仅有50个,占2.26%。在多态性位点表现中,二、四倍体特异性条带分别为0.50%和0.18%。比较两种不同来源途径同源四倍体扩增条带后发现,同源四倍体Tetra-5较Tetra-Jinlv表现出更多的遗传变异,共扩增出新增条带26条。研究结果表明,不同倍性及不同来源途径黄瓜在分子细胞遗传学水平上存在广泛的遗传变异。体细胞无性系变异引起了同源四倍体在DNA水平上更广泛的变异。3.同源三倍体黄瓜的培育及其细胞遗传学研究采用常规杂交法探讨了黄瓜二、四倍体杂交过程中亲本育性、授粉组合及亲本基因型对杂交结实率的影响。结果表明：同源四倍体自交结实率比较低(13.0%-14.5%),可能与其花药内所包含的正常花粉粒比例小及花粉管萌发长度较短有关；二、四倍体杂交组合的结实率很低(0.26%-1.02%),但在两种配组方式之间存在着明显差异,即以同源四倍体黄瓜为父本、二倍体黄瓜为母本的杂交结实率比较高,反之则杂交结实率比较低；在二、四倍体杂交过程中,二、四倍体的基因型对杂交结实率的影响较大,以杂交双亲同属一个基因型的杂交效果较好。对同源三倍体黄瓜花粉母细胞减数分裂行为及雄配子体发育进行观察后发现,同源三倍体黄瓜花粉母细胞减数分裂过程与二倍体基本相同但有特殊性。主要表现在：中期Ⅰ染色体的构型复杂,有多价体、四价体、三价体、二价体和单价体；中期Ⅰ和中期Ⅱ有赤道板外染色体；后期Ⅰ和后期Ⅱ出现落后染色体和染色体桥；后期Ⅱ和末期Ⅱ还出现染色体分离不同步及不均等分裂的现象；四分体时期出现二分体、三分体、含微核的异常四分体及多份体。正常四分体中的小孢子,有约91.2%的能发育为具有三孔两细胞的可育配子,小孢子的发育主要经过以下几个阶段：单核早期、单核晚期、两核期。花粉育性检测发现,同源三倍体的可染率和萌发率分别为18.8%和13.5%，表明其育性较低。4.黄瓜初级三体系的培育及其细胞遗传学研究为促进黄瓜的分子细胞遗传学研究,本研究利用黄瓜同源三倍体与二倍体正反交来培育初级三体。同时,对同源三倍体黄瓜减数分裂后期Ⅰ染色体分离情况进行了观察,以期为二、三倍体杂交培育初级三体提供细胞学证据。研究结果表明：二、三倍体正反交后获得了大量的种子,存活下来的56个子代的染色体数从14到28不等,其中染色体数为15的植株占了较大的比例(51.8%)。本研究首次获得了4种不同类型的黄瓜初级三体材料,并能各自相互区分出来。对同源三倍体花粉母细胞减数分裂染色体行为观察后发现,后期Ⅰ染色体分离符合正态分布规律,其中8／13型染色体分离的比例为6.25%。同源三倍体减数分裂,特别是8配子的形成为二、三倍体正反交后产生2x+1配子及初级三体的获得提供了细胞学证据。5.不同倍性黄瓜材料遗传差异的AFLP分析采用AFLP技术对黄瓜品种‘津绿四号’的单倍体、二倍体、三体、三倍体和四倍体进行基因组DNA多态性比较。从51对引物扩增获得2922条60-500bp的条带,检测出多态性位点152个,占5.2%,其中13对引物组合在不同倍性材料扩增条带没有差异。在多态性位点表现中,以三体和四倍体同时扩增出条带为主,占2.293%。与相应二倍体相比,三倍体、初级三体和四倍体都有特异片段的增加。结果表明,不同倍性黄瓜材料之间在DNA水平上存在广泛的遗传变异。6.二、三倍体杂交获得早世代稳定遗传的F2群体早世代稳定遗传群体的获得可以加速育种进程。利用由未授粉子房培养获得的同源四倍体(Tetra-1)与双单倍体(DH-Jinlv)杂交得到的同源三倍体(Tri-Jinlv),与双单倍体黄瓜翠玉(DH-Cuiyu)进行正反交,在杂种Fl代中发现有非整倍体和二倍体个体产生,其中一个二倍体的自交后代(F2)在田间农艺性状表现稳定。对这个稳定F2群体进行微卫星验证,结果表明：F1和F2群体30个单株的扩增带型整齐一致,在各个SSR多态位点只出现一条同父或同母带,丢失了来自另一亲本的等位位点,说明该F2群体确系由二、三倍体杂交得到的一个早世代稳定群体。更多还原

【Abstract】 Cucumber, which is one species of cucurbitaceous, is an important vegetable crops worldwide. It can accelerate process of breeding through using haploid or doubled haploid. Meanwhile, its commercial value could be enhanced, and further molecular-cytology studies could be promoted by selecting different ploidy germplasm in cucumber. In this study, unpollinated ovary from different genotypic materials were cultured to produce haploid or doubled haploid, further to esbtalish an efficient protocol for haploid induction. Autotriploid were obtained from reciprocal crosses between diploid and autotetraploid, which obtained from ovary culture. Further, primary trisomic and a non-segregation F2 population derived from reciprocal crosses between autotriploid and diploid. In additional, cytogenetic and molecular analyses have investigated and confirmed precisely these unique germplasms, in order to support basic for ploidy breeding in cucumber. The details results as following:1. Production haploid and doubled haploid from unpollinated ovary culture in cucumberSeveral factors, i.e. the duration of thermal shock pretreatment at 35℃, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in cucumber ovary culture. The results showed that embryo formation after thermal shock treatment at 35℃was significantly higher than control (0 d), and highest embryo formation was found in treating two days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO3 to induction medium had significant effect on frequency of embryo formation, and shortened embryo sprouting period and improved number of embryos formed in each ovary slice. In total, forty plantlets were regenerated from three genotype cucumber. Two were observed as haploid (2n=x=7), five were autotetraploid (2n=4x=28) and other thirty-three were diploid after chromosome counts. Homozygous of these diploid plants were identified using simple sequence repeat (SSR) analysis, and seventeen were considered as doubled haploid.2. Production and molecular cytogenetical analyses on autotetraploid in cucumberAutotetraploid plants originated from unpollinated ovary culture were identified as homozygote after simple sequence repeat (SSR) analysis. Genetic stability of the progenies from self-crossing of these autotetraploid was studied, no ploidy variation occurred among these progenies after morphology and chromosome observation. These autotetraploid plants have relatively higher pollen fertility after observation stainability of pollen grains, viability of pollen grains and number of seeds per fruit.Meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetaploid cucumber was studied, and results showed that the PMCs meiosis of the autotetraploid was similar to the diploid. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalent were observed in most PMCs of the autotetraploid at metaphase I. Chromosomes’lagging was frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyad, triad, tetrad with micronucleus and polyad could be observed at tetrad stage. The frequency of abnormal behavior in each stage of meiosis was counted, and the average percentage was 37.2%. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages:single-nucleus early stage, single-nucleus late stage,2-celled stage after pollen mitosis. The germination rate of pollen grains was noted, and it was 46.9% in autotetraploid.In additional, genetic variation among two kinds of autotetraploid with different origin (Tetra-5 and Tetra-Jinlv originated from unpollinated ovary culture and cochicine treatment) and their original diploid were studied using molecular cytogenetical analyses. From the cytogenetics analysis, we found the frequency of abnormalities at the different stage from metaphase to tetrad of meiosis was lower in autotetraploid Tetra-5 than that of in Tetra-Jinlv, especially the number of lagging’ chromosome at anaphase. Comparing the chromosome configuration at metaphaseⅠin these two kinds of autotetraploid, there are relatively more quadrivalents, and relatively less univalents and trivalents in Tetra-Jinlv. On the other hand, AFLP analysis detected the genetic differences among two kinds of autotetraploid with different origin and these original diploid.2214 bands ranging with 60-500bp were amplified with 38 pairs of primers. Only 50 polymorphic loci were identified which accounted for 2.26%. The expression of polymorphic loci showed that the differential bands remained present in the diploid and autotetraploid, which account for 0.50% and 0.18%, respectively. We also detected 26 novel bands present in Tetra-5, which has much more genetic variation than Tetra-Jinlv. These findings indicated that extensive genetic variation occurred during ploidy change or origin in cucumber. Meanwhile, autotetraploid Tetra-5 showed extensive genetic variation at level of DNA which caused by somaclone variation.3. Production and cytogenetical analyses on autotriploid in cucumberThe effects of fertility of parents, pollination combination and genotype of parents on seed-setting in hybridization between autotetraploid and diploid were analyzed with conventional cross method in this study. The results showed:(1) The low seed-sets (13.0%-14.5%) of the autotetraploidy combinations may be attributed to the presence of more abnormal pollen in the anthers or length of pollen tube. (2) The seed-sets of cross combinations between autotetraploid and diploid were very low (0.26%-1.02%), but there were obvious different seed-settings between the two mating methods. If the autotetraploid cucumber was used as the male parents and the diploid as the female parents, higher seed-sets were found in the hybridized combination. However, the seed-sets in the combinations were lower if the diploid cucumber was used as male parents. (3) Besides, the effect of genotype of parents on production of autotriploid was biggish; the average percentage of autotriploid production was higher when autotetraploid and diploid were belonging to same genotype.Meiosis of pollen mother cells (PMCs) and male gametophyte development in autotriploid cucumber (Cucumis sativus L.) was studied, and results showed that the PMCs meiosis of the autotriaploid was similar to the diploid with particularities. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalent were observed in the most PMCs of the autotriploid at metaphaseⅠ. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization were frequently observed as well, which led to formation of micronucleus and inviable gametes. The frequency of normal PMCs in autotriploid at stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. The stainability and germination rate of pollen grains were noted, and they were only 18.8% and 13.5%, respectively.4. Production and cytogenetical analyses on primary trisomics in cucumberThe reciprocal crosses were made between autotriploid and diploid for selecting the primary trisomics. Many viable F1 seeds were obtained from reciprocal crosses between autotriploid and diploid. The number of chromosomes of 56 surviving progenies varied from 14 to 28, with plants having 2n=15 occurring at the highest frequency (51.8%). Primary trisomics were firstly obtained in this study. Four types of primary trisomics were isolated and they could be distinguished from each other, as well as diploid. Variable chromosome configurations, e.g. univalent, bivalent and trivalent were observed in many pollen mother cells (PMCs) of the autotriploid at metaphaseⅠ. Binomial chromosome distribution was observed at anaphaseⅠand frequency of 8/13 was 6.25%. The meiosis of autotriploid, especially the class of gametes with eight chromosomes, gave the cytological evidence of producing 2x+1 type gamete and could be induced into primary trisomic plants from progeny of autotriploid-diploid crosses. These studies have established a ground work for selecting a series of primary trisomics, and further using them for associating linkage groups with specific chromosomes in cucumber.5. AFLP analysis of genetic differences among cucumber materials with different ploidiesGenetic variation for the response to ploidy change, haploid, diploid, triploid, tetraploid and trisomic polymorphisms of cucumber variety’Jinlv No.4’were compared by AFLP. Fifty-one pairs of primer combination were employed on cucumber materials with different ploidy and a total of 2922 bands between 60-500 bp were observed. Results showed only 152 polymorphic loci were found which accounted 5.2% and AFLP fingerprints amplified from the materials with different ploidies with 13 of the primer pairs did not remarkably differ. The expression of the polymorphic loci showed that the bands remained present in trisomic and tetraploid and absent in other materials in most case, which accounted for 2.293%. In contrast with diploid material, the trisomic, triploid and tetraploid materials got their specific bands. These results indicated that extensive genetic variation occurred among these cucumber materials with different ploidy.6. Identification and analysis of F2 stable population derived from the cross of autotriploid diploid in cucumberAutotriploid (Tri-Jinlv) obtained from cross between autotetraploid (Tetra-1) and DH-Jinlv, which regenerated from unpollinated ovary culture. It was then used as parent reciprocal crossing with a doubled haploid DH-Cuiyu. From its F2 generation we obtained a genetic-stable population. To prove the uniformity of such a population, SSR markers were used to survey the F2 individual plants. The results showed that F2 individuals carried only one parental molecular marker at each polymorphic locus, and their genotypes were identical with F1 progeny. Based on the above experiments, we consider that this F2 population is definitely an early-generation stable population.更多还原