【作者】 汪勇；

【导师】 翟虎渠；

【作者基本信息】 南京农业大学 ， 遗传学， 2011， 博士

【摘要】 水稻不仅是世界上重要的粮食作物之一,而且是单子叶植物基因组研究的模式植物,全球一半以上的人口以稻米为主食。近年来,由于改良品种的广泛应用和育种家对亲本选择的偏好,使得水稻基因资源变得越来越单一,遗传基础变得越来越狭窄,新的有利基因出现的概率也越来越低,导致水稻产量出现徘徊不前的尴尬局面。为了丰富水稻的遗传基础,进一步提高水稻产量,突破育种的瓶颈效应,从目前水稻育种实践来看,最有效途径就是从水稻远缘物种中引进优良的基因资源,对水稻材料进行改良,从而创造出具有重要意义的水稻新种质。杂草稻和非洲栽培稻稻种资源中广泛存在着各类抗病虫、耐盐碱、抗旱、耐高温等相关性状的优良基因,如能引入亚洲栽培稻中,必将使水稻育种产生新的飞跃。但亚洲栽培稻与杂草稻、非洲栽培稻存在严重的生殖隔离,致使其杂种F1表现高度不育,极大限制了杂草稻和非洲栽培稻的有利基因向亚洲栽培稻的转移及栽培稻种间远缘杂种优势的利用。因此深入探讨亚洲栽培稻与杂草稻以及非洲栽培稻间杂种不育的细胞学机理,挖掘更多的杂种不育基因,并发现相应的广亲和基因,这对于克服亚洲栽培稻与杂草稻、非洲栽培稻间的杂种不育,进而有效利用栽培稻种间的远缘杂种优势,并最终创造出水稻新种质具有重要的理论价值和实践意义。本研究利用云南杂草稻和广亲和品种02428杂交,从细胞学角度深入探讨杂种F1花粉败育的机理,利用02428//云南杂草稻/02428 BC1F1群体在全基因组范围内构建了一张分子连锁图谱,检测到一个控制杂种花粉不育的主效QTL:qPS-1,并对其进行了精细定位；同时从系统发育和进化角度进一步分析云南杂草稻与栽培稻、野生稻间的亲缘关系,探讨云南杂草稻可能的起源。此外,以滇粳优1号为受体亲本,非洲栽培稻IRGC 102295为供体亲本,构建了一个近等基因系NIL,证明滇粳优1号与NIL杂种F1花粉半不育受一对杂合基因座位控制,并把这个基因命名为S37,阐明其杂种F1花粉败育的细胞学机理,并对S37进行精细定位和候选基因的分析,这为进一步图位克隆S37,并最终阐明栽培稻种间杂种花粉不育的分子机理奠定坚实的基础。本论文的主要研究结果如下：1.云南杂草稻与广亲和品种02428杂交,杂种F1的花粉育性表现为典型不育,败育类型包括：典败、圆败和染败,且正反交两组合的花粉育性差异不显著。细胞学研究表明：杂种F1花粉的败育发生在二胞花粉早期,败育的原因是：小孢子第一次有丝分裂出现异常,不能正常形成生殖核；同时,花药横切面的石蜡切片也证明：杂种F1花粉的败育与绒毡层无关,其绒毡层能正常形成和降解。苯胺蓝染色发现：两亲本的柱头都有大量花粉粒附着,并有花粉管伸入到花柱中；而杂种Fl柱头上,很少看到花粉粒的附着,不能观察到花粉管伸入到花柱中。综合辅助授粉的结果表明：杂种F1小穗育性的降低是由较低的花粉育性和花粉在柱头上存在着萌发障碍两因素共同造成,与胚囊无关。2.利用02428//云南杂草稻/02428 BC1F1群体在全基因组范围内构建了一张分子连锁图谱,分别在第1和8染色体上各检测到一个控制杂种花粉不育性的QTL,命名为：qPS-1和qPS-8。qPS-1位于SSR标记RM5和RM493之间,LOD值为11.3,贡献率为22.7%；qPS-8位于SSR标记RM210和LD13之间,LOD值为2.7,贡献率为12.4%。这两个位点间不存在互作效应,彼此独立控制花粉育性,且qPS-1是一个主效而稳定的控制杂种花粉不育的位点。3.利用02428//云南杂草稻/02428和Ketan Nangka/N22//Ketan Nangka两套BC1F1回交群体,选择花粉育性在85%以上的极端高育单株及花粉育性在65%以下的极端低育单株,最终将qPS-1限定在两个Indel标记LI1和LI14-1之间,其物理距离为110kb,含有27个完整的开放阅读框,其中包含一个已经克隆的杂种花粉不育基因Sa。为了验证qPS-1是否与Sa等位,对02428和云南杂草稻两个亲本进行测序,结果表明：qPS-1实际上与已经报道的Sa等位。4.调查云南杂草稻和栽培稻杂种F1的花粉育性发现Dular和IR36在Sa位点携带有中性等位基因San。随后分析9份杂草稻,64份栽培稻和23份野生稻在两个SNPs位点碱基的差异,结果表明：在SaF和SaM两邻近基因中,野生稻都存在有碱基的分化,籼稻中存在少数碱基的分化,而粳稻中没有发现碱基的分化,杂草稻中存在类似野生稻的碱基分化。同时,聚类分析的结果也表明：云南杂草稻被聚在籼稻中,具有籼型遗传背景,并且跟野生稻具有紧密的遗传关系。据此推断：云南杂草稻可能起源于野生稻和被驯化的远古栽培稻品种的自然杂交。5.以滇粳优1号为受体亲本,非洲栽培稻IRGC102295为供体亲本,经过连续6代的回交构建了一个近等基因系NIL,滇粳优1号与NIL杂种F1花粉表现典型的半不育,败育类型为染败,受一对杂合基因S37控制。透射电镜显示：染败花粉粒内部发生凹陷,细胞体积偏小,并且花粉粒内部只有少量的淀粉粒积累。杂种F1花粉的败育发生在成熟花粉期,败育原因是染败花粉粒的生殖核不能进行第二次有丝分裂,因此不能形成三核花粉粒。6.选择花粉育性作为育性指标,首先利用743株滇粳优1号与NIL杂交F2群体进行连锁分析,初步将S37限定在第12染色体短臂末端的两标记NJ5和G4之间,随后利用扩大的18014株F2群体,最终将S37精细定位到标记HP14和G21之间,物理距离为73kb,该区域包含13个预测基因。花药的定量分析结果表明：LOC_Os12g02800是最有可能的候选基因,它编码一个富含半胱氨酸的蛋白前体,进一步的互补实验和功能研究正在进行中。更多还原

【Abstract】 Rice (Oryza sativa L.) is not only one of the most important food crops in the world, but also a model plant for studying the developmental biology of monocots, and more than half of the world’s population depends on it as main source of nutrition. Recently, due to the wide application of improved varieties and breeder’s preference for parents, the gene resources of rice were becoming more and more single and genetic basis was becoming more and more narrow, which seriously affected increase of rice yield. In order to enrich the genetic basis of rice and break the bottleneck in rice breeding, at present, the most effective way is to improve rice materials by introgression of valuable genes from distant species. Because weedy rice and Oryza glaberrima Steud. possed so many valuable genes, such as disease and insect resistance, saline tolerance, drought resistance and high temperature resistance. If we can introduce these valuable genes into Oryza sativa L., it will certainly bring a new leap for rice breeding. However, the reproductive isolation caused extreme sterility of F1 hybrids between Oryza sativa L. and weedy rice, or Oryza sativa L. and Oryza glaberrima Steud., which greatly limited transfer of favorable genes from weedy rice and Oryza glaberrima Steud. to the Oryza sativa L and the use of distant heterosis. In order to overcome interspecific hybrid sterility and take full use of the strong distant heterosis, it is necessary to more widely evaluate cytological mechanisms of hybrid sterility and find more hybrid sterility gene between Oryza sativa L. and weedy rice, or Oryza sativa L. and Oryza glaberrima Steud..In this study, we further investigate cytological mechanism of pollen abortion in F1 hybrid between the japonica wide compatibility rice cultivar 02428 and a weedy rice accession from Yunnan province. Genetic mapping in a BC1F1 population (02428//Yunnan weedy rice (YWR)/02428) showed that a major QTL for hybrid pollen sterility iqPS-1) was present on chromosome 1, which was also fine-mapped. Simultaneously, in order to explore the possible origins of YWR, a phylogenetic analysis of YWR, cultivated rice and wild rice based on microsatellite genotyping was carried out. Moreover, we developed an NIL at the locus S37 via repeated backcrossing and molecular marker-assisted selection (MAS), where the japonica variety Dianjingyou 1 was used as the receptor parent and O. glaberrima Steud. variety IRGC102295 as the donor parent. An F1 pollen semi-sterility locus, S37, was identified on rice chromosome 12 between NIL and Dianjingyou 1. We further elucidated cytological mechanism of pollen abortion in F1 hybrid between NIL and Dianjingyou 1, and also described the fine mapping and candidate-gene screening of S37. This study established a solid foundation for better understanding hybrid sterility and finally utilizing strong heterosis between indica and japonica subspecies.The main results were as follows:1. The pollen stain ability and in vitro germination tests revealed that both YWR and 02428 pollen fertility was normal, whereas their F1 hybrid showed clear pollen sterility. The type of pollen abortion contained typical abortion, spherical abortion, and stained abortion, wherein stained abortion was observed in most of aborted pollens. The two reciprocal F1, hybrids showed a similar level of pollen fertility to one another (P<0.05). Cytological studies have shown that pollen abortion in the F1 hybrid occured at the early bicellular pollen stage and probably occurred because the failure in the first mitosis prevented the formation of a functional reproductive nucleus. No abnormality in the development of tapetum or other anther walls was apparent. Aniline blue staining revealed that many pollen grains adhered to the stigmas and were able to germinate in the parental plants, but in the F1 hybrid, adherence and germination were restricted, and no pollen tubes were able to penetrate the style. Thus, the reduced spikelet fertility of F1 hybrids was the cumulative result of pollen abortion and poor stigma adherence of any remaining viable pollen, but the embryo sac of F1 hybrids appeared to be uncompromised.2. Of 805 SSR primers tested,313 were informative in the YWR×02428 population, and an outline linkage map based on 02428//YWR/02428 BC1F1 population was constructed from 133 of these. A QTL analysis performed using this map indicated the likely existence of two hybrid pollen sterility QTL, one (qPS-1) on chromosome 1 and the other (qPS-8) on chromosome 8. The location of the former was close to RM5, and this QTL accounted for～23% of the phenotypic variation for hybrid pollen sterility. qPS-8 accounted for～12% of the phenotypic variation and was linked to RM210. No interaction was detected between the two loci, which affected pollen sterility independently with additive effect, and qPS-1 was a major and stable hybrid pollen sterility loci.3. A total of 795 extreme individuals containing 403 plants from 02428//YWR/02428 population and 392 plants from KN/N22//KN population were genotyped using the flanking markers RM493 and RM5, respectively. As the results, qPS-1 was fine mapped between LI1 and LI14-1, about 110-kb in length on a single PAC clone (P0013G02). Gene prediction analysis of the 110-kb region showed that there were 27 putative open reading frames (ORFs) in this region, of which ORF9 and ORF10 encoded putative SaF+ and putative SaM, respectively. The sequencing results revealed that the qPS-1 locus is actually allelic with Sa.4. Dular and IR36 were assumed to carry the sterility-neutral allele, Sa", at Sa locus. SNPs in the two subgenes were examined in 23 wild species,9 weedy strains, and in 82 cultivars. The results indicated that a single substitution of "T" or "C" and "T"or "G" at gene of SaM and SaF, respectively, arose in wild rice, weedy rice, and indica cultivars, while only "C" and "T" was found at SaF and SaM, respectively, in japonica cultivars. The obtained dendrogram showed that the weedy rice was classified into indica or japonica type in cultivar (O. sativa L.), and the YWR was distributed among the wild rice and indica type, being separated from the japonica type. Accordingly, we extrapolated that YWR most probably originated from hybridization between Oryza sativa indica cultivars and Oryza rufipogon.5. We developed an NIL via repeated backcrossing and molecular marker-assisted selection (MAS), where the japonica variety Dianjingyou 1 was used as the receptor parent and O. glaberrima Steud. variety IRGC102295 as the donor parent. F1 pollen fertility of NIL/DJY1 exhibited typical semi-sterility, and the type of pollen abortion exhibited stained abortive. At the same time, we examined the DJY1 and F1 pollens using scanning electron microscopy and transmission electron microscopy and found the stained abortive pollens in F1 hybrid have small volume and most of them are shrunken, which have no accumulations of starch granules. Cytological studies have shown that pollen abortion in the F1 hybrid occured at the mature pollen stage and probably occurred because the failure in the second mitosis prevented the formation of a functional trinuclear pollen.6. According to pollen fertility, we constructed a linkage map covering the S37 region by 743 plants randomly selected from F2 progenies. The results indicated that the S37 locus was located to 2.2cM in the interval between NJ5 and G4. Finally, S37 was mapped to the region between HP 14 and G21 using a large F2 population contained 18014 plants, with physical size of about 73 kb. Thirteen open reading frames can be predicted by a sequence analysis of this fragment. Quantitative analysis of anther showed that LOC_Os12g02800 was the most likely candidate genes, which codes a cysteine-rich family protein precursor. Further complementation experiments and functional studies are ongoing.更多还原