【作者】 姜华；

【导师】 梁国华； 钱前；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2008， 博士

【摘要】 人们依赖稻米作为主要口粮使水稻成为最重要的粮食作物之一,较小的基因组、已知的基因组序列以及易于农杆菌介导的遗传转化和与其它禾谷类植物的共线性使水稻占据了单子叶模式植物的位置。正是水稻在社会生活中如此重要的地位,在生物研究中如此优秀的品质,人们对水稻的优良种质的筛选和各类生物学过程机理的研究从未停止过,尤其是关系水稻产量形成和稳定的各种性状和生物学过程。这其中,分蘖和光合无疑非常重要,分蘖和光合过程的稳定及相关各类性状的正常发展对水稻最终产量起着无可替代的作用。同时,分子生物学的快速发展为研究某类性状或生物学现象提供了成熟便捷的解决方案。突变体库构建、QTL分析和图位克隆等平台的成熟与广泛流行,促使水稻的生物学研究步入了一个崭新的时期。正是基于这样的背景,本研究利用目前已经非常成熟的QTL分析,对关系水稻最终产量形成的分蘖和光合相关性状—叶片耐光氧化力进行了QTL定位研究,以期明晰这些性状的遗传特性,为分子育种提供可资利用的分子标记;本研究还借助本试验室庞大的水稻突变体库,鉴定出了多个少分蘖和叶色突变体,并采用图位克隆策略对引发这些突变体表型变异的基因进行了分子定位,为将来深入研究分蘖和光合相关性状的分子机理奠定了基础。以下为本研究所进行的相关研究、目前所取得的研究进展及研究意义的简要概述:1本研究利用两份在粳稻日本晴EMS化学诱变库中发现的两个少蘖突变体rcn8和rcn9为实验材料。两者最高分蘖数都在4个以下,株高较野生型未发生显著改变,此外,rcn9还伴有少量斑点锈。遗传分析表明,这两份rcn突变体分别受一对隐性基因控制;与7个已知的少蘖突变体进行等位性分析表明,两者皆属于新发现的少蘖突变体,并命名为rcn8和rcn9。将两者再分别与93-11杂交,建立了两个F₂分离群体。根据已公布的水稻SSR标记及水稻基因组序列设计SSR引物,利用分离群体进行了连锁分析,将这两个隐性少分蘖基因分别定位于第1染色体的长臂（119.6 cM）和第6染色体的短臂（63.6 cM）上,为进一步对RCN8和RCN9基因的克隆和功能研究奠定了基础;2茎蘖消长动态是水稻发育过程中重要的生理现象,同时茎蘖数也是重要的农艺指标。本研究利用非条件及发育QTL分析方法研究水稻茎蘖消长的遗传控制。利用由127和120个株系组成,分别来源于窄叶青8号和京系17及春江06和台中本地1号的两个DH群体为材料（分别称为DH-1和DH-2群体）,以多个时期的茎蘖数为指标进行相关非条件及发育QTL定位,并对两个群体的定位结果进行全基因组比较QTL定位。DH-1群体定位到条件与非条件QTL共44个,分布于第3和12条染色体以外的其他10条染色体上;DH-2群体定位条件和非条件QTL共计70个,除第7染色体外其他11条染色体均有分布。通过全基因组比较QTL定位,在两个群体间共计确定了10对（共26个）处于相似的物理位置可能为相同QTL的位点,其中有6对（14个）位点均在相同时期内被检测到,很可能包含有控制茎蘖数的主效数量基因。发育QTL的分析结果表现出了丰富的时空性,并能够在不同程度上解释影响茎蘖数的非条件QTL的变化。本实验虽然在不同群体的相似物理区域定位到了同类型的QTL,但显著性、效应值和贡献率差异较大,因此这些QTL的精确位置及基因本质还需深入研究;3为进一步研究分蘖的分子机理,本研究选取了一个来源于窄叶青8号和京系17花药培养获得的DH群体中的株系DH78,该株系植株表现明显的少分蘖表型,但株高也显著降低,能够正常结实。利用DH78与亲本京系17的F₂群体中87个突变型单株进行了突变基因初步定位,将突变基因DFT1定位于第2染色体上。利用进一步扩大的群体,最终将DFT1基因所在染色体区间缩小为91kb,序列注释表明该区域包含有有14个开放阅读框。更为有趣的是DFT1基因位点与本实验室之前进行的双氮条件分蘖数和株高QTL定位研究中定位到的两个QTL座位ph2-3和tp2-2处于相同区域。而DFT1和ph2-3及tp2-2之间的关系则还需要进一步的研究;4高位分蘖是水稻生产中常见的现象,同时高位分蘖与水稻的驯化也存在密切的联系。本实验利用来源于窄叶青8号和京系17及春江06和台中本地1号的两个DH群体为材料（即DH-1和DH-2群体）,对水稻高位分蘖的遗传特征进行了研究。采用复合区间作图法,在DH-1群体中共定位到3个QTL座位qHOT3、qHOT6-1和qHOT8,分别位于第3、6和8染色体;对DH-2群体的QTL定位共检测到相关位点2个,分别位于第6和12染色体。同时,在两个群体中分别检测到3对和7对上位性互作位点。虽然比较QTL定位表明在两个群体中并未定位到区间一致的QTL座位,但两个群体均在第6染色体定位到QTL座位说明水稻第6染色体对高位分蘖具有重要的影响;5通过对一个水稻淡黄叶突变体材料ygl3进行遗传分析,表明它是受到一个单隐性基因控制。为了定位该突变基因,本实验利用ygl3和中花11的F₂群体进行基因初步定位,将其定位于水稻第二号染色体短臂的SSR标记RM6874和RM5764之间。进一步扩大群体后最终将YGL3定位在位于BAC克隆OSJNBb0088N06上的STS标记STS2和STS6之间约60kb的区间内。对这一区域进行基因预测分析发现,其中包含一个与叶绿素合成相关的基因—谷氨酸tRNA合成酶基因。对该基因测序发现突变体较野生型相比有三个碱基（GAG）的缺失,导致了一个氨基酸（Glu）的缺失,因此很可能是该基因的突变引起了黄绿叶的表型变异;6依托本实验室庞大的突变体资源,本研究还对另一个黄绿叶突变体ygl4进行了基因定位。借助成熟的图位克隆策略和ygl4与台中本地1号配组获得的1116株F₂突变单株,最终将控制ygl4突变表型的隐性基因YGL4定位于第1染色体SSR标记SSR1与RM297间250kb的区间内,基因注释表明该区间还有一个叶绿体发育相关的类囊体膜磷蛋白,因此将其确定为候选基因,对该基因的测序研究正在进行之中;7利用由127个株系组成,来源于籼稻品种窄叶青8号和粳稻品种京系17的加倍单倍体群体,以耐性指数和敏感性指数为指标,采用QTL Mapper 1.6统计软件进行水稻耐光氧化反应特性的QTL定位和上位性分析,共检测到控制耐性指数的1个加性效应QTL,位于第3染色体上;控制敏感性指数的加性效应QTL 5个,分别位于第1、1、6、8和9染色体上。还检测到3对影响耐性指数的加性×加性上位性互作效应QTL,5对敏感性指数的上位性QTL。同时还对41个常见亲本进行了光氧化实验筛选。本研究利用目前较为流行的QTL分析和图位克隆策略对水稻分蘖及光合相关性状进行了研究,并取得一定的成果,对目前取得的研究结果还需要进一步的深入研究才能服务于水稻分子育种,并深化对分蘖控制、叶绿素合成和叶绿体膜发育分子机理的理解。更多还原

【Abstract】 People depend on rice as a major food resourse makes rice one of the most important crops,and what’s more,a small genome size,a known genome sequence,easy for Agrobacterium-mediated genetic transformation and genome synteny between rice and other grasses make rice a model monocotyledon plant.Because of this importance of rice in social life and biological research,the screening for elite germplasm and research on various biological processes have never stopped;especially rice yield-related traits and biological processes.Among these biological processes,tillering and photosynthesis are undoubtedly very important;tiller number and photosynthetic-related characters show great association with rice yield.At the same time,the rapid development of molecular biology provides a mature and convenient solution for studying certain traits or biological phenomenons.Construction of mutant library,QTL analysis and map-based cloning,and other platforms are mature and widely popular,so that the biology of rice entered a new period.Based on such a background,QTL analysis for tiller number and photooxidiation tolerance was carried out to clarify,the genetic characteristics of these traits and to provide available molecular markers for the practice of molecular breeding. Relying on huge number rice mutants in our lab,we identified a number of few-tillering and leaf color-related mutants,and map-based cloning strategy was used to mapping the caused genes.All these researches shed light on the study on molecular mechanism of tillering and photosynthesis.The following is a brief introduction of the relevant research, the progress and the significance of these studies:1 In the present study,in order to systematically dissect the genetic mechanism of rice tilling for the super rice standard mould and the model system of branching development, two ethyl methane sulfonate-induced rice（Oryza sativa L.） reduced -culm-number（rcn） mutants from the progeny of Nippobare（O.sativa ssp. japonica）,namely rcn8 and rcn9,were used.Their maximum tillers were both less than 4.In addition,rcn9 had another major feature of rust-spotted leaves.Allelic tests between these two mutants and seven other recessive few-tiller mutants revealed that they were previously unknown loci.Genetic analysis showed that the rcn traits were all controlled by a pair of different recessive genes,designated as RCN8 and RCN9,respectively.Two F₂ populations derived from crosses between the rcn8 or rcn9 mutants and 93-11 was constructed.Linkage analysis using two rcn F₂ mapping populations with published simple sequence repeat markers demonstrated that the RCN8 and RCN9 genes were mapped on the long arm of chromosome 1（119.6 cM） and the short arm of chromosome 6（63.6 cM）,respectively. The results of the present study were beneficial to map-based Cloning and functional analysis of the RCN8 and RCN9 genes.2 Culm-tillering dynamic is used as a vital agronomic index and is a very important physiology phenomenon in rice development.The objective of this study was to investigate the genetic basis of the culm-tillering dynamic of rice.Two different DH populations,one was derived from a cross between japonica rice JX17 and indica rice ZYQ8,including 127 lines and the other with 120 lines was derived from a cross between japonica rice CJ06 and indica rice TN1（herein designated as DH-1 and DH-2,respectively） were used for unconditional,and developmental quantitative trait locus（QTL） mapping of culm-tiller-number at different growing stages. The whole genome comparative QTL mapping was also conducted.44 conditional and unconditional QTL were detected in 10 chromosomes except chromosomes 3 and 12 from DH-1 population,while When 70 conditional and unconditional QTL were detected on 11 chromosomes except chromosome 7 from DH-2 population. And through whole genome comparative mapping,10 pairs of 26 QTL which were located in the similar physical regions were also identified.The results also showed that 6 pairs of 14 QTL loci were detected in the same development stage,and there may be major quantitative trait loci within these QTL.Although we identified resemblant QTL in the similar physical region in these two DH populations,because of the inconsistent magnitude,further investigation is still needed for the exact position and the essential of the gene. 3 In this study,a line DH78 form a double haploid（DH） population derived from a ZYQ8（indica）/JX17（japonica） crossed by anther culture was used.DH78 exhib ited dwarfism and a less-tiller（dft） character.The gene（DFT1） was mapped to chromosome 2 using a F₂ population of DH78/JX17,and more interestingly,the genomic region of DFT1 was coincided with the mapping region of the small-LOD peak,QTL ph2 and tp2,that was identified at low-nitrogen in a research carried out in our lab before.Further backcrossing and fine-mapping successfully delimited the DFT1 locus to a 91 kb region with 14 open reading frames（ORFs）.4 High order tillering（HOT） is a common phenomenon in rice production,while high order tillering is an important sydrome of rice domestication.Two different DH populations,one was derived from a cross between JX17 and ZYQ8 and the other derived from a cross between CJ06 and TN1（herein designated as DH-1 and DH-2, respectively） were used to study genetic characteristics of high order tillering.Using composite interval mapping,3 QTL qHOT3,qHOT6-1 and qHOT8,located on chromosomes 3,6 and 8 respectively were identified in DH-1 population;and in DH-2 population,2 QTL were detected,which were located on chromosomes 6 and 12.Meanwhile,3 and 7 pairs of epistatie loci affecting HOT were mapped in DH-1 and DH-2,respectively.Although,no identical QTL was identified by comparative mapping,gene/genes affecting HOT must exist in chromosome 6 because QTL was detected in this chromosome by QTL mapping in both two populations.5 A rice mutant ygl3 with yellow leaf was isolated and found to be controlled by a recessive gene genetic analysis.Used F₂ population derived from cross between ygl3 and Zhonghual 1 as primary mapping population,YGL3 was mapped between SSR marker RM6874 and RM5764 on the short arm of the chromosome 2.Further more,enlarged mapping population makes the gene successfully narrowed down to a interval between STS marker STS2 and STS6 on the BAC clone OSJNBb0088N06,the physics distance was about 60 kb.Annotation reveald a chlorophyll synthesis-related gene Glutamyl-tRNA synthetase gene was in this region, Sequence analysis suggested a 3 bp deletion（GAG） which led to a deletion of an amino（Glu） might associate with the mutation phonetype.6 In present study,another yellow-green mutant ygl4 was characterized.Through map-based cloning strategy,YGL4 was located on a 250 kb region between simple sequence repeat（SSR） maker SSR1 and RM297 in chromosome 1 using 1116 mutantion individuals in a F₂ population derived from the cross ygl4/TN1.Sequence annotation suggested a candidate gene - thylakoid membrane phosphoprotein gene which was chloroplast development related,and the sequence analysis was undertaken.7 A double haploid（DH） population including 127 lines,derived from a cross between indica rice（Oryza sativa L.） Zhaiyeqing 8 and japonica rice Jingxi 17 was used to detect quantitative trait loci of tolerance to photooxidation.One QTL controlling tolerance index（TI） was detected on chromosome 1 and five QTL controlling sensitive index（SI） for photooxidation tolerance were mapped on chromosomes 1,1,6,8 and 9,respectively.Meanwhile eight pairs of epistatie loci affecthag TI and SI were also detected（three pairs for TI and five pairs for SI）.Besides, 41 rice varieties were identified for photooxidation tolerance and sensitive index.In this study,QTL analysis and map-based cloning strategy were used to study rice tillering and photosynthesis-related traits,and certain results were achieved.The current findings need further study to serve rice molecular breeding,and deepen the understanding of the molecular mechanism of the tillering control,biosynthesis of chlorophyll and the development of chloroplast membrane.更多还原