【作者】 薛永；

【导师】 翟虎渠； 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2006， 博士

【摘要】 本研究利用Kinmaze／DV85重组自交系群体、Nipponbare／Kasalath回交重组自交系群体以及其置换系群体、Asominori／IR24重组自交系群体和以IR24为背景的Asominori染色体片段置换系群体进行了耐铝毒QTLs分析。并在第9染色体上定位了一个稳定表达的耐铝毒QTL。据此，利用目标QTL对应的置换系与背景亲本杂交构建次级F₂群体，将稳定表达的QTL分解为单基因，并精细定位，为水稻抗铝毒QTL图位克隆奠定基础。主要结果如下：1利用重组自交系群体检测水稻耐铝毒QTLs利用Kinmaze／DV85 81个重组自交家系（RIL）作图群体，采用苗期单营养液水培鉴定方法，以相对根伸长量（RRE）作为耐铝毒性状的表型值，分析亲本和重组自交系群体对铝毒的耐性表现。利用Windows QTL Cartographer软件共检测到5个耐铝毒QTLs，分别位于第1、5、8、9和11染色体上，各个QTL的贡献率在8.64％～18.60％之间，其中，位于第5、8和11染色体上的QTLs对铝毒的抗性基因来自亲本DV85，位于第1和9染色体上的QTLs对铝毒的抗性基因来自亲本Kinmaze。结合前人研究结果，讨论了稳定表达的耐铝毒基因座，为进一步水稻耐铝毒分子标记辅助选择育种和耐铝毒基因的图位克隆奠定了基础。2利用回交重组自交系群体检测水稻耐铝毒相关性状QTLs利用98个株系组成的Nipponbare（japonica）／Kasalath（indica）∥Nipponbare（japonica）回交重组自交系（backcross inbred lines，BIL）群体，采用苗期水培鉴定方法，测定了亲本及98个BILs在对照根伸长量、处理根伸长量以及由此计算得到的相对根伸长量。三个性状总共检测到7个QTLs，其中对照根伸长量分别在第6、8和9染色体上检测到3个QTLs；处理根伸长量在第4染色体上检测到1个QTL；相对根伸长量分别在第5、9和10染色体上检测到3个QTLs。位于9和10染色体上的qRRE-9和的贡献率为9.7％和11.8％，对铝毒耐性的增效等位基因来自Nipponbare；位于第5染色体的qRRE-5的贡献率为9.8％，增效等位基因来自Kasalath。相对根伸长量检测的3个QTL中qRRE-5显示与玉米第6染色体上的一个耐铝毒主基因正同源。另一个qRRE-9在染色体上的位置与先前利用不同群体报道的耐铝毒QTL位置相近。在第9染色体上检测到对照根伸长量和相对根伸长量共有的QTL也表明此QTL的候选基因的功能可能与根生长速率相关。而qRRE-10为新检测到的耐铝毒基因座。同时，利用对应的置换系我们验证了这些QTLs的功能。这些结果为通过分子标记辅助选择和聚合QTLs来增进水稻铝毒耐性提供了可能。3水稻耐铝毒QTL分析与精细定位（1）利用Asominori×IR24的重组自交系群体在2004年检测水稻耐铝毒QTLs，结果发现重组自交系群体在相对根伸长量的次数分布图呈现正态分布，并出现双向超亲遗传现象；分别在第1、9、11染色体上检测到一个耐铝毒加性效应QTL；其中位于第9染色体上的QTL qRRE-9在不同的群体中都重复出现，该QTL的稳定表达的QTL。利用以IR24为遗传背景的全基因组片段置换系（CSSL）群体的相关株系，验证了上述QTLs等位基因的功能和表达稳定性。在水稻中新检测到的qRRE-11也显示与玉米第10染色体上存在的一个主效的耐铝毒基因相一致。（2）利用QTL qRRE-9对应的1个置换系CSSL51与IR24回交和自交，构建次级F₂群体（192个单株，2004年南京）及其F₃群体（192个株系，2005年南京），将QTL qRRE-9分解为单基因（Alt-9）。应用6对SSR引物，结合CSSL51×IR24次级F₂群体192个单株的分子数据和F₂、F₃群体的耐铝毒表型数据，将Alt-9基因定位在SSR标记RM553和RM215之间。2005年在南京，将CSSL51×IR24次级F₂群体扩大到1043个单株。利用本研究中开发的SSR标记和InDel标记和Zhang QF等（1994）“隐性极端个体基因作图”的分析方法，调查224个耐铝毒极端个体的分子数据，将Alt-9基因精细定位在RM24702和ID47-2之间，距离RM24702和ID47-2分别为0.90 cM，并与该区间中的SSR标记RM5765共分离。4 T-DNA插入产生的水稻铝毒敏感突变体的遗传分析通过筛选水稻T-DNA插入突变体，获得一个铝毒敏感的突变体。在100μM Al³⁺浓度下，野生型亲本Nipponbare的根伸长被抑制了34％，而突变体根伸长量被抑制59％。对该铝毒敏感突变体进行遗传分析的结果表明，分离群体后代中出现铝毒敏感：耐铝毒分离比率为3∶1，表明控制耐铝毒的基因是一个隐性单基因。对突变体及其后代分离群体做Basta和潮霉素抗性检测及PCR分子检测，证实该突变体是由T-DNA插入所引起的，突变表型与T-DNA共分离。该材料可用于插入座位的基因克隆。更多还原

【Abstract】 Aluminum （Al） toxicity is considered as one of the primary causes of low rice productivity on acidupland and lowland acid sulfate soils. In the present study, different populations were used to detectquantitative trait loci （QTLs） for Aluminum tolerance. Meanwhile, one of stable QTLs were dissectedinto single genes using secondary F₂ populations, derived from the cross between target CSSLs andgenetic background parent, IR24. Then, fine mapping was further conducted for these single genesusing the data of SSR and InDel markers and phenotypic evaluation. The results thus obtained shouldbe useful for rice MAS breeding and map- based cloning of target QTLs. The main conclusions are asfollows:1 Mapping of quantitative trait loci associated with aluminum tolerance inrice （Oryza sativa L.）, using recombinant inbred linesIn this study, a mapping population of 81 F₁₁ lines （recombinant inbred lines: RILs）,derived from a cross between a japonica variety Kinmaze and an indica variety DV85 bythe single-seed descent methods, was used to detect quantitative trait loci （QTLs） forAluminum tolerance. Aluminum tolerance in roots of two cultivars （Asominori and IR24）of rice was studied using relative root length （RRL） and relative root elongation （RRE）.Results indicated that RRE is a parameter more directly related to Al tolerance andconvenient than RRL Furthermore, relative root elongation （RRE） of scedlings whichgrew on nets floating on 0.5mM CaCl₂ （pH 4.5） solution with Al stress or non-stressconditions, was evaluated for the Al toxicity tolerance of RILs and the parents at seedlingstage. QTL analysis was performed with Windows QTL Cartographer 1.13a program bycomposite interval mapping, as the result, five QTLs controlling Aluminum toxicitytolerance for RRE were detected on chromosomes 1, 5, 8, 9 and 11, respectively.Individual QTL accounted for 8.64％～18.60％of the phenotypic variation in the RILpopulation. In the five QTLs for RRE, Kinmaze contributed favorable alleles （lessimpaired by stress） for qRRE1 and qRRE9, while DV85 contributed favorable alleles forqRRE5, qRRE8 and qRRE11. Comparing with the other mapping results, QTLs for RRE,which mapped on chromosomes 1、8、9 and 11, appear to be consistent among different rice populations. These QTLs would be highly useful in breeding cultivars tolerant to Altoxicity in marker-assisted selection （MAS） program and map-based cloning forAl-tolerance genes.2 Identification of quantitative trait loci associated with aluminum tolerancein rice （Oryza sativa L.）, using backeross inbred linesQuantitative trait loci （QTL） analysis for Al tolerance was performed in rice using amapping population of 98 BC₁F₁₀ lines （backcross inbred lines: BILs）, derived from across of Al-tolerant cultivar of rice （Oryza sativa L. cv. Nipponbare） and Al-sensitivecultivar （cv. Kasalath）. Three characters related to Al tolerance, including root elongationunder non-stress conditions （CRE）, root elongation under Al stress （SRE） and the relativeroot elongation （RRE） under Al stress versus non-stress conditions, were evaluated for theBILs and the parents at seedling stage. A total of seven QTLs for the three traits wereidentified. Among them, three putative QTLs for CRE （qCRE-6, qCRE-8 and qCRE-9）were mapped on chromosomes 6, 8 and 9, respectively. One QTL for SRE （qSRE-4） wasidentified on chromosome 4. Three QTL_s （qRRE-5, qRRE-9 and qRRE-10） for RRE weredetected on chromosomes 5, 9, 10 and accounted for 9.7％-11.8％of total phenotypicvariation. Interestingly, the QTL qRRE-5 appears to be syntenic with the genomic regioncarrying a major Al tolerance gene on chromosome 6 of maize. Another QTL, qRRE-9,appears to be similar among different rice populations, while qRRE-10 is unique in theBIL population. The common QTLs for CRE and RRE indicate that candidate genesconferring Al tolerance in the rice chromosome 9 may be associated with root growth rates.The existence of QTLs for Al tolerance was confirmed in substitution lines forcorresponding chromosomal segments. These results also provide the possibilities ofenhancing Al tolerance in rice through using marker-assisted selection （MAS） andpyramiding QTLs.3 QTL analysis and fine-mapping for aluminum tolerance in rice（1） Aluminum （Al） toxicity is considered as one of the primary causes of low riceproductivity on acid upland and lowland acid sulfate soils. In the present study,quantitative trait loci （QTLs） controlling Al tolerance based on relative rootelongation （RRE） were dissected using a complete linkage map and arecombinant inbred lines （RILs） derived from a cross of Al-tolerant japonica cultivar Asominori and Al-sensitive indica cultivar IR24. A total of three QTLs（qRRE-1, qRRE-9 and qRRE-11） were detected on chromosomes 1, 9 and 11 withLOD score ranging from 2.64 to 3.60 and the total phenotypic variance explainedfrom 13.5％to 17.7％. The Asominori alleles were all associated with Altolerance at all the three QTLs. The existence of these QTLs was confirmed usingAsominori chromosome segment substitution lines （CSSLs） in IR24 geneticbackground （IAS）. By QTL comparative analysis, the two QTLs （qRRE-1 andqRRE-9） on chromosomes 1 and 9 appeared to be consistent among different ricepopulations while qRRE-11 was newly detected and syntenic with a major Altolerance gene on chromosome 10 of maize. This region may provide animportant case for isolating genes responsible for different mechanisms ofaluminum tolerance among different cereals. These results also provide thepossibilities of enhancing Al tolerance in rice breeding program bymarker-assisted selection （MAS） and pyramiding QTLs.（2） Phenotypic values were significantly different between the recurrent parent,cultivar IR24, and the six CSSLs harboring qRRE-9 allele, indicating the effectsof the qRRE-9 allele were significant and stable. Based on F₂ and F₃ populationsderived from the cross of CSSL51 and IR24, the QTL qRRE-9 was dissected intoa single gene, namely, the Alt-9 allele in Asominori was a recessive gene, Alt-9,controlling Aluminum tolerance. Then, the Alt-9 gene was further mappedbetween RM24702 and ID47-2 on chromosome 9, and co-separated withRM5765, using the 1043 CSSL51/IR24 F₂ plants and SSR/InDel markers.4 Selection and identification of aluminum-sensitive mutant by T-DNAinsertion in Rice （Oryza sativa L.）As the nearly completed rice genome sequence is currently public available, T-DNAinsertional mutagenesis has been successfully used as a powerful tool to identify a numberof genes in rice. To identify aluminum-responsive genes in rice, we screened T-DNAtagging lines that had been subjected to aluminum toxicity stress at 100μM Al³⁺ （pH 4.5）.The T-DNA-tagged lines were previously constructed through transformation of vectorpDsBar1300 into Nipponbare （Oryza sativa L. subsp, japonica cv. Nipponbare） usingAgrobacterium mediated methods. Vector pDsBar1300 was based on pCAMBIA1300,with an insertion of Ds elements harboring a PHOSPHINOTHRICIN （PPT）-resistance gene bar into the multicloning site of the T-DNA fight side. On the left side of the T-DNA,there is a hygromycin-resistant gene under the control of the CaMV35S promoter forselecting rice transformants. This T-DNA was of the nopalinetype. An aluminum-sensitivemutant caused by T-DNA insertion in rice was identified by measuring root elongationduring a 24-h period. Root elongation of Nipponbare was inhibited by 34％after exposureto100μM Al³⁺ for 24h, while that of the Aluminum-sensitive Mutant was inhibited by59％. Genetic analysis of the mutant showed that the phenotype of relative root elongationin the segregating populations derived from the T-DNA heterozygotes fit the ratio of 3:1.Test for Basta resistance and hygromycin resistance showed the aluminum toleranceplants were all susceptible whereas the aluminum sensitivity plants were resistant, and theratio of resistant and susceptible plants was nearly 3:1, which indicated that thealuminum-sensitive mutant was co-segregated with Basta resistance and hygromycinresistance. Furthermore, the aluminum-sensitive mutant caused by T-DNA insertion wasconfirmed by T-DNA detection using PCR method. This Aluminum-sensitive mutant willbe used for isolation of the tagged gene in rice.更多还原