【作者】 王玉平；

【导师】 李仕贵；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 植物雄性不育是比较普遍出现的一种现象，由于利用核质互作雄性不育的三系杂交水稻的成功应用，植物雄性不育的研究受到重视。普通隐性核不育遗传简单，不受温光条件影响，有利于进行雄性不育的遗传规律、雄性不育的机理研究以及杂种优势利用等研究。本文利用四川隐性核不育水稻H₂S，对其进行遗传规律和遗传效应研究，然后进行了细胞形态学研究，利用分子标记进行基因定位，并利用该隐性核不育材料H₂S进行了水稻的轮回育种研究，主要结果如下：1．四川隐性核不育水稻H₂S是无花粉的雄性不育系，其花药细小，呈白色水浸状，成熟的花药无花粉粒；颖花的雌蕊等其它结构都正常，其不育性表现稳定，不受温光条件的影响。2．遗传分析表明：H₂S的不育性遗传表现为隐性的细胞核单基因控制，不受细胞质影响；恢复谱广，各种籼稻都是其恢复系。在不同胞质来源的珍汕97A（CMS-WA）、D702A（CMS-D）、G46A（CMS-G）、K18A（CMS-K）、协青早A（CMS-DA）、粤泰A（CMS-HL）的遗传背景下，均表现为隐性单基因遗传，与这些细胞质雄性不育的不育核基因是不等位的，没有发生两类不育核基因间的互作；表明该隐性核不育与核质互作不育是两个独立的、不同的系统。3．利用回交转育构建了该不育基因的突变型（H₂S）、杂合型（H₂Sh）、野生型（H₂Sw）近等基因系，遗传背景分析表明除不育基因外遗传背景高度相似；通过对近等基因系的性状考察，发现该不育基因对播始历期和有效穗具有正效应，对株高和穗平着粒具有负效应。4．利用回交转育及SSR和ISSR分析，构建了遗传背景相似含三系雄性不育细胞质CMS-WA、CMS-D、CMS-G、CMS-K、CMS-DA等的H₂S同核异质系。通过对同核异质系的主要农艺性状的比较，发现该不育基因在可育细胞质下，株高和穗平着粒数显著低于5个不育细胞质的同核系，包颈粒率和包颈度则极显著高于5个不育细胞质同核异质不育系，表明该不育基因在可育细胞质下对株高、穗平着粒、包颈粒率和包颈度存在显著的负效应，而当两套不育系统结合在一起，反而会部分解除隐性核不育基因的影响或三系雄性不育系统抑制了隐性核不育基因对上述性状的负效应。通过双列杂交试验结果分析，三系雄性不育细胞质的负效应主要表现在株高、结实率和千粒重，进一步影响单穗重和产量；隐性核不育H₂S在这些方面没有显著的负效应，因此在杂种优势利用中有着显著的潜力。5．通过扫描电镜观察，四川隐性核不育在早期的花器官形态发生时，颖花原基到雄蕊、雌蕊原基的先后分化都正常，雌雄蕊原基的数目与形态与H₂Sw无差异，表明该隐性核不育材料在早期的形态建成正常。石蜡切片细胞学观察，发现该隐性核不育能形成正常的四分体孢子，四分体释放出小孢子后，在单核花粉进入液泡化时小孢子的发育异常，不进行正常的液泡化，逐渐开始解体和消失；相应的绒毡层在小孢子形成后的降解延迟，绒毡层细胞的体积略有增大，在小孢子降解快结束时也很快解体。因此，四川隐性核不育是绒毡层降解延迟导致小孢子解体而发生的无花粉雄性不育。6．采取极端集团—隐性群法，利用SSR标记进行初步定位，在此基础上筛选新的SSR标记和开发INDEL分子标记，实现了基因的精细定位，把四川隐性核不育基因定位到分子标记W19、W11之间，遗传图距分别为0.5cM和0.2cM；在这两个标记间约73Kb的区域内找到11个已经预测的候选基因，经分析LOC Os06g42310有可能是目标基因。经检索，该隐性不育基因属首次利用分子标记定位的新基因，暂命名为ms-nop（t）。7利用四川隐性核不育材料进行了三系杂交稻的轮回选择育种研究，通过分别构建保持系和恢复系的轮回群体，进行了两轮轮回选择，选育出产量一般配合力高于原始群体骨干亲本一般配合力的新亲本；发现经2轮轮回选育出的亲本产量一般配合力优于1轮轮回选择，其它性状的选择效果因性状的遗传特点而异，一般非加性遗传效应强的数量性状通过轮回选择的改良比较有效。通过轮回选择育种，选育出D35A、蜀恢498、D83A等优良三系杂交稻亲本，已有配组的杂交稻新组合通过四川省的品种审定。更多还原

【Abstract】 Male sterility is a common phenomenon occasionally occurring in plant. The success of three-line hybrid rice application gave much drive to the pace of nucleolus-cytoplasm complementary sterility （CMS） research. The simplicity and the temperature-light impregnability of the recessive nuclear male sterility （RNMS） made its genetic mechanism a fine model for male sterility and heterosis application research. Using a mutated RNMS rice line （H₂S） from Sichuan Province, A RNMS gene was identified and fine mapped, and its genetic effect was studied. The results of the Mendelian genetics, genetic effect, morphological cytology, chromosome localization, and the breeding application study were as follows:1. The waterlogging like white anther of H₂S line was slender and small and produced no pollen, but the pistil in the glume was normal. The male sterility of the line was stable, not affected by temperature and light change.2. RNMS was a recessive trait controlled by only one gene in nucleolus, and its sterility was restorable in wide genetic spectrum （all indica rice lines can be its restorers）. Under the genetic background of Zhenshan97A（CMS-WA）, D702A（CMS-D）, G46A（CMS-G）, K18A（CMS-K）, XieqingzaoA（CMS-DA） and YuetaiA（CMS-HL）, RNMS showed no nucleolus-cytoplasm gene interaction, suggesting it was a independent gene system from CMS. It was deduced that the genotype of H₂S was N（rrmsms）.3. Homozygous, heterozygous and wild-type near-isogenetic lines of RNMS gene were constructed by backcrossing, and the agronomic trait investigation found that RNMS gene had positive effects on seeding-heading days and effective tillers, negative effects on plant height and grain number per panicle, no significant effect on panicle length.4. Five isonuclear alloplasmic lines with 5 different CMS cytoplasms sharing the same H₂S nucleolus were constructed. Compared with the five CMS lines, RNMS line was a little lower, its panicle was less enclosed and had smaller number of grains. The RNMS gene made no significant difference in seeding-heading days, effective tillers and panicle length. Combining ability analysis suggested that the General Combining Ability （GCA） of the RNMS line was significantly higher than other CMS lines in grain yield, plant height and seed setting rate, lower GCA in grain number per panicle. No significant GCA difference was found in other agronomic traits. Significant GCA difference was found among the 5 types of CMS lines in grain weight, grain number per panicle and grain yield.5. Through electron microscope scanning of the early floral organs, morphogenesis of the spikelet primordium, stamen primordium and pistilliod primordium was found normal. Paraffin slides observation found that the microspore of the RNMS line failed to vacuolate after its morphogenesis, disassembled and disappeared at last. After microspore morphogenesis, the breaking down of tapetum cells was delayed until microspore breading down. It was speculated that the sterility of the RNMS line was related to the delay of tapetum cells’ breaking down.6. Through bulked extremes and recessive class analysis with SSR markers and INDEL markers, the RNMS gene in H₂S line was fine mapped between W19 and W11, 0.5cM and 0.2cM away from the target respectively. 11 genes was found in the region of 73Kb between the two markers, bioinformatics analysis showed that LOC_Os06g42310 maybe the target gene. Literature search suggested that the gene was a new one, so we named it ms-nop（t） temporally.7. Recurrent selection and breeding research of the RNMS was conducted, the results showed that higher grain yield GCA lines can be produced by transferring the RNMS gene. Using RNMS gene as a outcrossing means, recurrent selection population breeding programs were launched, and a number of elite rice lines had produced, such as D35A and Shuhui498 and D83A. The hybrids combinations with these parental lines showed outstanding performance in the authority of the Province plot test, related hybrids varieties were examined and approved by the Province authority.更多还原