【作者】 庄飞云；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2003， 博士

【摘要】 全文以黄瓜属双二倍体Cucumis hytivus Chen and Kirkbride(2n=38)及其回交后代为主要研究材料，从细胞分子遗传学角度初步探讨了双二倍体染色体组间的交换重组，及渐渗杂交对黄瓜遗传基础的影响，对种间正反杂交后代的形态及育性差异进行了深入研究。采用RAPD和SSR两种分子标记对黄瓜属系统亲缘关系进行再评价。 1 双二倍体C．hytivus及其回交后代的细胞分子遗传研究 通过对双二倍体C．hytivus花粉母细胞(PMCs)的减数分裂终变期和中期Ⅰ的染色体行为进行了观察，46.3％的细胞出现单价体或多价体，其染色体构型为0.56Ⅰ+17.36Ⅱ+0.35Ⅲ+0.26Ⅳ+0.046Ⅴ+0.056Ⅵ。在其与栽培黄瓜回交获得的三倍体(2n=26)PMCs中，发现同型二价染色体(野生种C．hystrix染色体内部配对)和异型三价体(野生种C．hystrix单条染色体和栽培黄瓜二价染色体联会配对)，但频率较低，细胞染色体构型平均为11.53Ⅰ+7.16Ⅱ+0.05Ⅲ。 对双二倍体C．hytivus进行DNA指纹图谱分析，共获得6个特征性RAPD条带和4个SSR位点，分别是A-15／2000bp，480bp，G-02／1750bp，AP-03／700bp，AP-07／640bp，AP-20／700bp，CMCT160a+b／380bp，CSCTTT15a／280bp，CMCCA145／190bp，CMGA165／190bp。将其中3个特征性RAPD标记转化成SCAR标记，深入研究其特异性和准确性。SAP-03／700标记表现出特异性，而且在栽培黄瓜及野生黄瓜C．sativus var．hardwickii中分别扩增出一条不同带。SAP-07／640标记表现为非特异性标记，在野生种C．hystrix和双二倍体C．hytivus中扩增出同样的目的片段。SAP-20／700标记表现为假阳性标记。通过对SAP-03／700标记在双二倍体、栽培黄瓜及野生黄瓜中扩增出的3条不同带的序列深入研究，结果表明3条带的两端分别有200bp左右序列一致，中间部分不同。通过对SAP-03／700标记同源序列的查询，发现与黄瓜线粒体基因组有200 bp左右的同源序列，但序列方向相反。 将双二倍体、回交三倍体与不同生态型栽培黄瓜进行杂交，结果表明杂交的座果率很高，为83-100％。以双二倍体与栽培黄瓜杂交获得含有胚的果实比率较高(60-67％)，平均每个果实中含有10-20个，胚胎拯救成活率接近100％，染色体数统计为26条。以栽培黄瓜与双二倍体杂交，仅获得1个含有大约180个胚的果实，胚胎拯救成活率接近80％，染色体数26条，另有2个果实分别产生60粒(HH1群体)和15粒(HH2群体)，染色体数为14条。回交三倍体与栽培黄瓜间杂交产生的胚或种子的果实极少，只有6个果实，其中1个果实中含有23粒饱满种子(HH3群体)，另5个果实共产生5粒饱满种子，染色体数均为14条。回交三倍体的自交座果率比杂交座果率低，为48-75％，果实中没有任何成熟胚或饱满种子。 HH1群体形态呈现较大变异，4株表现原始亲本野生种的多分枝习性，6株的果刺摘要颜色表现野生种黑色性状，其果皮成熟颜色均为桔黄色，不同于两原始亲本，但两性状表现出紧密连锁关系.采用SSR和RAPD两种标记对HHI群体的遗传变异进行深入研究，19个SSR标记共产生63个等位基因，其中24个表现出遗传变异，占到38.1%，稍高于n种不同生态型栽培黄瓜的遗传多样性37.7%.其中有8个等位基因可能来自于野生种。在400条随机引物中，24条产生位点变异，在统计的186个位点中，共有31.7%的位点出现遗传分离，稍低于不同栽培黄瓜的遗传多样性33.0%.但两者的结果都显著高于由不同生态型栽培黄瓜产生的H圣121群体的遗传变异，SSR标记27.1%和RApD标记23.9%，这表明通过远缘杂交及渐渗杂交过程扩大黄瓜遗传基础是具有潜力的。2黄瓜属双二倍体C hytivus及其后代耐低温及光合性能的研究 对双二倍体C hytivus及其后代幼苗对低温逆境的适应能力进行了研究.结果表明:在低温条件下，可能由于亲本染色体组间的功能不协调，双二倍体幼苗出现代谢异常，丙二醛(MDA)、肺氨酸含量变化大，冷害指数达到3.5，表现为对低温的适应性相对较弱.但双二倍体与黄瓜的回交自交后代却表现出较强的抗性，超出了参试黄瓜品种.经4d变温处理后，其冷害指数仅为 1.。，MDA含量为9.2拼mol.岁，在所有供试材料中为最低。这表明通过回交和自交，杂种代谢系统已基本恢复正常，具有进一步研究和应用的价值. 通过对双二倍体C妙tiv。、原始亲本、回交三倍体、单体异附加系517及其它黄瓜栽培品种的光合能力测定.结果表明双二倍体和亲本北京截头对光照强弱变化应急能力最强.采用二项式曲线对测定的数据进行模拟，求得各试材在30℃下的补偿点.双二倍体为22.0林mol·m一2·s一’，低于其两原始亲本野生种(27.1林mol·m一s一’)和北京截头(26.0林mol.m一2了，)，表现出较强的耐弱光能力.但回交三倍体和单体异附加系517的光补偿点较高，分别是犯.3 omol·m‘，45”和41.7、mol·m一，·s一，.3黄瓜属植物种间正反杂交的差异研究 对栽培黄瓜与野生种c娜trix杂交而成的二倍体(2n=19)、双二倍体(2n==38)及其正反交植株的形态和育性差异进行了比较研究.结果表明:正反交二倍体植株的主蔓直径，叶柄长，第一朵雄花尊筒和花冠长度，叶形状和大小较相似更多还原

【Abstract】 1 Studies of cytological and molecular genetics on the amphidiploid species C. hytivus and its progeniesThe behavior of chromosome at diakinesis and Metaphase I in meiosis was observed in C. hytivus. About 46.3% of pollen mother cells (PMCs) had the univalent or multivalent and the average chromosome configuration was 0.56I + 17.36II + 0.35III + 0.26IV + 0.046V + 0.056VI. In PMCs of the progeny of the allotriploid backcrossed C. hytivus with cucumber, heterobivalent formed within the C. hystrix chromosomes and trivalent formed with two cucumber chromosomes and one C. hystrix chromosome, but the frequency was low. The average chromosome configuration of C. hystrix was 11.53I + 7.16II + 0.05III at meiotic.RAPD and SSR markers were used to investigate the DNA fingerprints of C. hytivus. Six specific RAPD bands (A-15/2000bp, 480bp, G-02/1750bp, AP-03/700bp, AP-07/640bp, and AP-07/700bp) and four specific SSR sites (CMCT160a+b/380bp, CSCTTT15a/280bp, CMCC- A145/190bp, and CMGA165/190bp) were found. The specificity of 3 SCAR markers converted from the specific RAPD markers of C. hytivus were analyzed. SAP-03/700 primer pair was specific to C. hytivus, while it produced one different band in cucumber cultigens and C. sativus var. hardwickii, respecitively. SAP-07/640 primer pairs yielded the same band in C. hystrix and C. hytivus and it was not specific. SAP-20/700 was false positive marker and yielded no band in the 16 accessions. Compared with the sequence of 3 different bands yielded by SAP-03/700 primer pair in C. hytivus, C. sativus var. sativus and C. sativus var. hardwickii, ~ 200 bp sequence at the ends of the bands were similar while the middle parts were different. In addition, about 200 bp of SAP-03/700 was homologous with cucumber mitochrodial genome, but the directions were contrary.The fruit set percentage and number of seeds produced in the crosses between amphidiploid C. hytivus, allotriploid, and different ecotype cucumber cultigens were high (83-100%). The ratio of fruit with embryo was 60-67% in the cross between amphidiploid C. hytivus and cucumber cultigens. Each fruit had about 10-20 embryos, all of which could survive and develop into whole plants through embryo rescue. The chromosome number of these plantlets was 26. When cucumber cultigens crossed with amphidiploid C. hytivus, only one fruit with about 180 embryos was obtained and 80% of the embryos could survive through embryo rescue, and the chromosome number of them was 26. The other two fruits produced 60 seeds (as HH1 population) and 15 (as HH2 population) with 14 chromosomes, respectively. Few fruits with embryos or seeds were obtained in reciprocal crosses between allotriploid and cucumber cultigens. One fruit had 23 seeds (as HH3 population) and theother five had one seed each. All of them had 14 chromosomes. The fruit set percentage of allotriploid selfed was lower (48-75%) and all fruit had no embryo and seed which could develop into plantlets. In HH1 population, some plants showed the origional parent C. hystrix traits such as black spines on fruit, multiple branching habits, while some had unique morphological characteristics such as orange skin of fruit. The black spines trait was tightly linkage with the orange skin trait. Nineteen SSR primer pairs produced 63 alleles in HH1 and 24 alleles (38.1%) were segregated, which was similar to the genetic diversity (37.7%) about 11 different ecotype cucumber cultgens. Eight of them might come from C. hystrix. Twenty-four arbitrary primers produced varied primed sites and among the 186 primed sites generated 31.7% were segregated. This was similar to the genetic diversity (33.0%) of the different ecotype cucumber cultgens. Both of them were significantly higher than the genetic variation (SSR: 27.1%, RAPD: 23.9%) of HHzl population produced from cross between Chinese cucumber CC3 and American pickling cucumber "A309". The results suggested that the genetic base of cucumber could be improved through interspecific hybridization and introgression.2 Studies on the response更多还原