【作者】 赵志刚；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 遗传学， 2007， 博士

【摘要】 上世纪80年代广亲和基因S5ⁿ的发现，为籼粳亚种间杂种优势利用提供了新的机遇。随着研究不断深入，育种家发现仅S5ⁿ广亲和基因并不能解决所有籼粳亚种间杂种不育的问题，与S5ⁿ非等位的育性基因也不断被鉴定出来。因此，有必要研究更广泛的亚种间杂种不育，挖掘更多新的亚种间广亲和基因为充分利用杂种优势提供理论基础。本研究发现广东地方籼稻品种广解9号与前苏联地方粳稻品种USSR₅杂交杂种F₁表现半不育。为探明引起杂种半不育的位点数目及其在染色体上的位置，对USSR₅/广解9号//USSR₅回交群体进行了全基因组分析，结果表明其杂种育性主要受两个独立遗传的主基因所控制，其中第4染色体上的不育位点与已报道的S9等位；而第5染色体上不育位点与现已报道的都不等位，为本研究新发现的位点，暂命名为S31（t）。杂种育性的下降是由于这两个位点内等位基因间互作所导致的，并且，这两个基因有累加效应。通过以粳稻Asominori为遗传背景、籼稻IR24为染色体片段供体的一套覆盖全基因组的染色体片段置换系（66个株系）群体为材料，分别与亲本Asominori和广亲和品种02428杂交构建了两套杂种群体，用于杂种不育位点的检测。分析两年的实验结果，找到一置换系CSSL34与Asominori杂交杂种半不育，与02428杂交杂种育性正常，该置换系置换的片段来自IR24第5染色体的短臂，通过染色体位置的比对，发现置换系置换的片段与我们前面定位的不育基因S31（t）位置相同。为进一步证实杂种CSSL34/Asominori的败育原因，一方面通过遗传分析，以杂种CSSL34/Asominori作父本，以Asominori作母本，构建了回交群体Asominori//CSSL34/Asominori，并调查该群体的花粉和小穗育性，结果表明群体的花粉和小穗育性都正常，这说明了杂种F₁的花粉是正常的；另一方面是通过细胞学的观察获得直接证据，以亲本为对照，系统调查了F₁的花粉育性、胚囊育性、受精率以及结实率，结果表明杂种F₁的花粉萌发、花粉管在柱头上的生长均正常，激光共聚焦显微观察结果直接证实了胚囊部分败育是F₁半不育的主要原因。进一步利用石蜡切片方法对杂种F₁胚囊发育过程进行连续切片，实验结果表明F₁中功能大孢子解体出现几率最高，因此认为F₁雌配子败育的主要原因是功能大孢子解体，从而无法进行下一步发育。首先构建一个三交群体CSSL34/02428//Asominori（400个单株），用SSR标记将S31（t）初步定位在1.5cM的区域内，然后通过扩大三交群体单株数目（总共1630个单株），采用极端个体定位的方法，最终将S31限定在标记Indel193和Indel212之间54kb的物理距离内。基因注释表明该区域内有8个预测的ORF，其中4个ORF编码有功能的蛋白，另4个编码未知蛋白。初步研究了其中一个候选基因的表达情况，获得了全长cDNA，构建了表达载体，目前，正在进行转基因实验。以高温处理下的小穗育性以及小穗育性热敏感指数为指标，对水稻籼粳回交群体USSR₅/广解9号//USSR₅孕稳期高温耐热性及其相对耐热性进行数量性状位点分析。结果表明在第4、8染色体上分别检测到与孕穗期相对耐热性相关的QTL，qhts-4和qhts-8，LOD值分别为3.81和2.86，对表型变异的解释率分别为16.8％和9.9％。对其进一步的上位性分析表明，有8条染色体的4对位点存在基因间互作，小穗育性耐热性除受主效QTL控制外，还受基因间互作的影响。更多还原

【Abstract】 This S5ⁿ has been incorporated into indica or japonica variety to overcome sterility problem in wide crosses and hybrid rice breeding. So far, the S5ⁿ allele has been effective to produce fertile hybrids in a large number crosses. In the analyses of many indica-japonica hybrids, it has also been observed that there is considerable variation in the fertility level in hybrids from the same WCV crossed to different indica or japonica varieties. Thus, hybrid sterility in different varietal combinations may be explained by allelic interaction at different loci.A japonica cultivar, USSR₅ from former Soviet Union is identified to show strong genninability under cold stress, and Guangjie 9 is an indica cultivar from Guangdong province, China, with weak genninability under cold stress. The hybrid of USSR₅/Guangjie 9 reveals hybrid sterility. Therefore, we performed a genome-wide analysis by assaying a backcross population of USSRs/Guangjie 9//USSR₅ to determine the number and genomic location of genes controlling the hybrid sterility. As the result, two loci conferring the hybrid fertility were found, which acted independently of each other. Interestingly, the locus on chromosome 4 coincided with the previously identified S9, while the locus on chromosome 5 was distinct from all the previously reported ones. The new locus was thus designated as S31（t）. Based on allelic interaction causing female gamete abortion, two alleles were found at S31（t）, i.e. S31（t）-un in USSR₅ and S31（t）-gi in Guangjie 9. An Aus variety, Dular, was assumed to have a neutral allele, S31（t）-n. The SSR markers of RM5586 and RM13 were considered to be useful for marker-aided transfer of the wide compatibility genes in the hybrid rice breeding.To further understand the genetic mechanism of the hybrid sterility, in this study, two sets of F₁S were constructed using Asominori, a typical japonica variety, and 02428, a wide-compatibility variety, as the pollen parents, and the 66 CSSLs as the maternal parents, respectively. Typical spikelet semi-sterility was observed in F₁ hybrids between Asominori and CSSL34 which was previously reported to have a sterility gene of S31（t）. The embryo sac fertility, pollen fertility and spikelet fertility of three parents CSSL34, 02428, and Asominori were normal, while spikelet sterility was observed in F₁ hybrid between CSSL34 and Asominori, and the reciprocal F₁ hybrid did not significantly differ in their spikelet fertility, which indicating no cytoplasmic effect on the spikelet fertility in the F₁ hybrids. To elucidate the mechanism of the sterility in F₁ hybrids, pollen fertility, in vitro pollen germination, embryo sac fertility, fertilized ovaries, and spikelet fertility were examined and compared to control parent strains. The results revealed that both pollen fertility and in vitro pollen germination rate of the F₁ hybrids were similar to those of the parents. Besides, pollen and spikelet fertilities in the BC1F1 of Asominori//CSSL34/Asominori were normal. In addition, the spikelet fertility of the F₁ hybrids of CSSL34/Asominori was not restored to normal levels after hand pollination with pollen from each parent. Furthermore, the number of pollen grains adhered to stigmas, pollen germination, and pollen tube elongation has no distinction between parents and F₁ hybrids by inspection with confocal laser scanning microscopy. On the other hand, the frequency of abnormal embryo sacs in the CSSL34/Asominori hybrid was significantly higher than in parents. The embryo sac plays a pivotal role in sexual reproduction of angiosperms. In order to study the stage of abnormal embryo sac, differences in embryo sac development between the CSSL34/Asominori hybrid and parent were analyzed using series sections. However, in some of the ovaries from the CSSL34/Asominori hybrid plant, megamametogenesis appeared to be blocked at the first round of mitosis. The degenerating functional megaspore is characterized by no vacuole differentiation, less or withered cytoplasm. Taken together, these results suggested that the partial abortive embryo sac was the main reason for the hybrid sterility in the F1 hybrids between CSSL34 and Asominori.A population of 1630 F₁ plants derived from the three-way cross CSSL34/02428//Asominori was developed to further fine-map S31（t）. Based on the physical location of molecular markers, S31（t） was finally delimited to a region of 54-kb between InDel193 and InDel223. Sequence analysis of this fragment revealed eight predicted open reading frames, six of which encoded known proteins and two encoded putative proteins. These results are useful in map-based cloning of S31（t） and marker-assisted transferring of the neutral allele in rice breeding programs.The spikelet fertility under high temperature and heat susceptibility index （HSI） of spikelet fertility were used to evaluate the tolerance of rice to heat stress at booting stage, respectively. Two QTLs related to relative heat tolerance at the booting stage were detected, with LOD scoring 3.81 and 2.86 on chromosome 4 and 8, and explained 16.8% and 9.9% of the phenotypic variance, respectively. Further analysis of epistatic effected QTLs for relative heat tolerance showed that four pairs of loci exhibited interaction on 8 chromosomes, which indicated that the heat tolerance of spikelet fertility was not only controlled by main effect QTL but also influenced by gene interaction.更多还原