【作者】 官华忠；

【导师】 吴为人；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2011， 博士

【摘要】 杂交水稻的推广应用为解决我国粮食问题作出了很大贡献,但其生产中所用的不育系均存在包穗现象,严重影响了杂交水稻繁殖制种产量。水稻中包穗现象主要是由最上节间缩短造成的。阐明包穗形成的分子遗传机制,对解决水稻不育系的包穗问题,创造水稻新种质具有重要意义。本研究从数量性状和质量性状两方面对包穗的遗传基础进行研究。主要研究内容和结果如下:1.包穗相关性状的QTL定位:在两个环境下种植一个由热带粳稻品种DZ60与籼稻品种H359杂交而建立的重组自交系（RIL）群体,调查最上节间长（UIL）、剑叶鞘长（FLSL）和最上节间长与剑叶鞘长比率（UFR）。利用已构建的分子标记连锁图,对这3个与包穗有关的性状进行QTL定位。共检测到10个QTL,其中3个控制UIL,位于1、4、12号染色体,对表型变异贡献率为3.20%-14.59%;4个控制FLSL,位于1、6、8、12号染色体,对表型变异贡献率为2.59%-20.70%;3个控制UFR,位于4、6、8号染色体,对表型变异贡献率为2.89%-6.00%。qUIL-1与qFLSL-1的位置完全重叠,且效应最大,在不同环境中表达稳定,是一个同时控制UIL和FLSL的主效QTL。对水稻基因组序列的查询结果显示,该QTL所在区间正好包含半矮秆基因sd1,因而推测其效应来自sd1基因。2. esp1突变体的遗传分析:利用物理诱变方法从籼稻恢复系早R974中获得了一个包穗突变体,表现为植株变矮、抽穗延迟、最上节间短缩、半包穗、穗型短小、穗粒数减半、二次枝梗数目减少以及部分颖花退化等特征。我们将该突变体命名为esp1。解剖观察发现,esp1最上节间的薄壁细胞纵向平均长度（41.29μm）仅为野生型（59.11μm）的69.85%,说明esp1最上节间中部细胞延伸受阻,使其最上节间伸长不足,是导致包穗的原因。遗传分析表明,esp1受一隐性基因控制,能稳定遗传,不受遗传背景的影响。喷施GA3试验表明,esp1对赤霉素表现钝感。将esp1与粳稻品种日本晴杂交,构建了F2和BC₁F₁群体,将ESP1基因定位在水稻第11号染色体上SSR标记RM26281和GRM40之间,与这两个标记的遗传距离分别为0.29 cM和0.048 cM。根据ESP1所在区域的物理图谱,初步确定突变体esp1在SSR标记GRM88和GRM40之间发生了大约260 kb的缺失。基因预测表明该区域包括52个基因。这些研究结果为克隆ESP1基因奠定了基础。3. esp2突变体的遗传分析:从籼稻品种明恢86的组织培养后代中获得了一个包穗突变体,其穗部被剑叶叶鞘完全包裹,最上节间几乎完全退化,而其余各节间长度则没有明显改变。我们将该突变体命名为esp2。解剖观察发现,esp2的最上节间细胞数目大幅减少,细胞生长分化停顿,茎秆空心髓和维管束发育受阻。遗传分析表明,esp2受一隐性基因控制,能稳定遗传,不受遗传背景的影响。显然,ESP2是控制水稻最上节间发育的一个关键基因。基因互作分析表明,就最上节间长度这个性状而言,ESP2对ESP1以及两个最上节间伸长基因（EUI1和EUI2）都表现为隐性上位,双突变体esp1esp2、eui1esp2和eui2esp2皆表现为几乎没有最上节间。这说明ESP2功能的丢失使水稻最上节间无法发育,因而也就无从表现出由ESP1、EUI1和EUI2基因突变所造成的最上节间长度的数量变异。喷施GA3试验表明,esp2与esp1相似,对赤霉素也表现钝感。利用esp2与粳稻品种秀水13杂交的F2群体,将ESP2精细定位在1号染色体短臂末端一个14 kb的区域内。根据水稻基因组序列的注释,该区域内只存在1个完整的基因,亦即一个假定的磷脂酰丝氨酸合成酶（putative phosphatidylserine synthase）基因。DNA测序分析表明,该基因内部插入了一个5287 bp的反转座子序列。因此把该基因作为ESP2的候选基因。实时荧光定量PCR分析结果显示,ESP2候选基因在野生型明恢86的各个时期和各个组织均有表达,且表达量受外源赤霉素的诱导上调。本研究结果为ESP2基因的功能分析奠定了基础。更多还原

【Abstract】 The successful application of hybrid rice has made great contribution to solving the food problem in China. However, the phenomenon of panicle enclosure exists in all the male sterile (MS) lines used in hybrid rice production. This seriously affects the yields of MS seed reproduction and hybrid seed production. Panicle enclosure in rice is mainly caused by the shortening of uppermost internode. Elucidating the molecular genetic mechanism of panicle enclosure will be helpful for solving the problem of panicle enclosure in MS lines and creating new germplasms in rice. In this study, we investigated the genetic basis of panicle enclosure in rice from the aspects of natural variation and mutants, respectively. The main research contents and results are as follows:1. Mapping of QTLs underlying panicle enclosure-related traits: A recombinant inbred line (RIL) population derived from a cross between a tropical japonica rice cultivar DZ60 and an indica rice cultivar H359 was grown under two environments and phenotyped for uppermost internode length (UIL), flag leaf sheath length (FLSL) and UIL-to-FLSL ratio (UFR). Using a molecular marker linkage map constructed previously based on this population, QTL mapping was performed for these three panicle enclosure-related traits. A total of 10 QTLs were detected, of which 3 were for UIL, located on chromosomes 1, 4 and 12, explaining 3.20%-14.59% of the phenotypic variance; 4 were for FLSL, located on chromosomes 1, 6, 8 and 12, explaining 2.59%-20.70% of the phenotypic variance; and 3 were for UFR, located on chromosomes 4, 6 and 8, explaining 2.89%-6.00% of the phenotypic variance. qUIL-1 and qFLSL-1 were mapped at the same position and displayed the largest effects expressed steadily across the environments, suggesting that they are a major QTL underlying UIL and FLSL simultaneously. Searching of the rice genome sequence indicated that the interval of this QTL covers the semidwarf gene sd1. Hence, we guess that the effect of this QTL might come from the sd1 gene. 2. Genetic analysis of mutant esp1: A panicle enclosure mutant was obtained from the progeny of an indica rice restorer line Zao-R974 after irradiation mutagenesis. The mutant displayed the characteristics of reduced plant height, delayed heading, shortened uppermost internode, partial enclosure of panicle, shorter panicle, 50% fewer grains per panicle, decreased number of secondary branches and degeneration of partial spikelets. We named the mutant as esp1 (enclosed shorter panicle 1). Anatomical observation indicated that the average longitudinal length of parenchyma cells in esp1 uppermost internode (41.29μm) was only 69.85% of that of the wild-type (59.11μm), suggesting that the extension of cells in esp1 uppermost internode was cumbered, leading to less elongated uppermost internode and thus causing panicle enclosure. Genetic analysis showed that the mutant phenotype was controlled by a recessive gene, which was regularly inherited and unaffected by genetic background. GA3 spraying experiment indicated that esp1 is insensitive to gibberellin. Using the F2 and BC1 populations of a cross between esp1 and the japonica cultivar Nipponbare, we fine mapped ESP1 to a region between SSR markers RM26281 and GRM40 on chromosome 11, with genetic distances of 0.29 cM and 0.048 cM to the two markers, respectively. According to the physical map of the region covering ESP1, it was suggested that there was a 260-kb deletion between markers RM26281 and GRM40 in the mutant genome. According to the rice genome sequence annotation, there are 52 genes in this region. These results will facilitate the cloning of ESP1 gene.3. Genetic analysis of mutant esp2: A mutant of panicle enclosure was acquired from the tissue culture progeny of indica rice cultivar Minghui-86. In the mutant, panicles were entirely enclosed by flag leaf sheaths and the uppermost internode was almost completely degenerated, but the other internodes did not have obvious changes in length. We named the mutant as esp2 (enclosed shorter panicle 2). Anatomical observation revealed that in the uppermost internode of esp2 the number of cells was dramatically reduced, the growth and differentiation of the cells was stopped, and the development of stem pith cavity and vascular bundles was arrested. Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene, which could be steadily inherited and was not affected by genetic background. Apparently, ESP2 is a key gene for the development of uppermost internode in rice. Gene interaction analysis showed that in regard to the trait of uppermost internode length, ESP2 has recessive epistasis over ESP1 and two uppermost internode elongation genes (EUI1 and EUI2). The double mutants of esp1esp2, eui1esp2 and eui2esp2 all hardly have visible uppermost internode. GA3 spraying experiment indicated that esp2 is also insensitive to gibberellin like esp1. Using an F2 population of a cross between esp2 and a japonica rice cultivar Xiushui-13, we fine mapped ESP2 to a 14-kb region on the end of the short arm of chromosome 1. According to the rice genome sequence annotation, only one intact gene exists in this region, namely, a putative phosphatidylserine synthase gene. Sequencing analysis on the mutant and the wild type indicated that this gene was inserted by a 5287-bp retrotransposon sequence. Hence, we took this gene as the candidate of ESP2. Quantitative RT-PCR analysis indicated that the ESP2 candidate gene is expressed in various growth stages and tissues in the wild-type Minhui-86, and the expression is enhanced by exogenous GA3. The results of this study will facilitate functional analysis of ESP2 gene.更多还原