【作者】 薛亚东；

【导师】 郑用琏； 严建兵；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 细胞质雄性不育(Cytoplasmic Male Sterility, CMS)是在大多数植物中都存在的一种自然现象。CMS/Rf系统不仅在玉米、水稻、油菜等主要农作物的杂交育种中发挥着重要的作用,而且也是研究核质互作的重要模式系统之一。玉米是最早将CMS/Rf系统应用于杂种优势利用的粮食作物。S-CMS是玉米细胞质雄性不育中最大的一种类别,精细定位并克隆其育性恢复基因对阐明核质互作控制其育性发生的分子机理,充分发挥S-CMS在玉米杂种优势利用中的作用具有重要的理论及实践意义。在前人关于玉米S-CMS育性恢复基因定位研究的基础上,本研究以含有Rf3/rf3基因的近等基因系(NIL) Mo17为材料,构建了S-Mol7rf3rf3×-Mo17Rf3Rf3)X N-Mo17rf3rf3和(S-Mo17Rf3Rf3xN-Mo17rf3rf3)×N-Mo17rf3rf3)两套BC1群体。利用Rf3/rf3区间的分子标记信息,并借助B73基因组序列开发分子标记,对Rf3进行精细定位构建S-Mo17Rf3Rf3的BAC文库。同时对定位区域内包括PPR基因在内的功能基因信息进行挖掘分析,确定可能的Rf3候选基因。研究取得以下进展：1.通过比较分析玉米雄穗不同小花内的花粉发育动态,确定了“鉴定小穗上位花的花粉育性”作为判定玉米配子体CMS材料花粉育性的形态学指标。确保了研究中育性鉴定的准确性。2.两套BC1群体的田间植株育性表型和室内花粉育性镜检结果一致,BC1群体中半不育和全不育株分离比符合1：1的分离比例。表明定位群体的雄花育性受-对Rf3/rf3基因控制。3.以B73基因组序列为参考,在前人初步定位Rf3/rf3基因的2.09bin区间内共开发标记638对,其中SSR标记有198对,STS标记440对。标记分析结果表明：两类标记在亲本材料间的多态性分别为1%和5%。4.STS引物扩增片段的序列分析发现,多数片段的序列完全相同,仅有少数SNP位点可以成功转化为CAPS和dCAPS标记。这一结果从另一侧面确定了本研究所利用的Rf3/rf3的NIL间,正向选择差异区段的图距相对较窄。5.采用图位克隆策略,利用不同年份构建的两套较大的BC1分离群体,构建了Rf3/rf3基因区间19.9cM的分子标记遗传连锁图,标记间平均图距为2.84cM。但这些被精细定位的标记均位于Rf3基因的一侧,最近的标记为2042S-3,遗传距离为1.6cM。在为恢复基因的图位克隆奠定了基础。6.以S-Mo17Rf3Rf3为材料,构建了含有Rf3基因的Mo17核背景的细菌人工染色体(BAC)文库。该文库包含175104个BAC克隆,平均插入片段长度为130kb,覆盖玉米基因组9.1倍,为图位克隆Rf3基因提供了有效的工具。7.对2.09bin区域内的序列分析发现了12个PPR基因,其中有6个与CMS恢复基因相关的P亚家族基因位于Rf3定位的区域内。以该6个PPR基因为探针,采用RFLP技术检测包含近等基因系在内的、分别含有Rf3或rf3基因的若干试材,为后续Rf3的克隆提供了重要的功能标记。8.以候选PPR基因的序列和最近的标记为探针,筛选BAC文库,在2.09bin区域内获得阳性克隆48个。contig构建及后续分析正在进行中。本研究另一个关注的领域是玉米干旱胁迫下耐旱QTL的定位。干旱胁迫是全球干旱、半干旱地区影响玉米生产的重要逆境之一,利用转基因技术和分子标记辅助选择等分子育种手段,选育抗旱玉米自交系是玉米育种的一个重要研究领域。本研究对350份热带玉米自交系与测验种CML312组配的F1群体,在中国、墨西哥、肯尼亚和泰国等4个环境下的干旱处理和对照(正常灌溉)获得的表型鉴定数据,利用Illumina MaizeSNP50芯片分析的基因型资料进行全基因组耐早性的关联分析,为进一步发掘和克隆抗旱QTL提供理论依据。研究获得了以下结果。1.9个耐旱相关表型的遗传力分析表明,株高、穗位高、相对穗位高、散粉-吐丝间隔期和百粒重等遗传力较高,籽粒产量的遗传力中等,穗粒数遗传力最低,表明籽粒产量和穗粒数受干旱影响较大。以上结果与CIMMYT多年育种实践一致。2.利用Illumina MaizeSNP50芯片对350份供试材料的基因分型结果表明,A/G类型的SNP占74.5%,其他类型的SNP占25.5%,这种SNP的“偏爱”现象暗示了基因组结构—基因功能—耐旱表型—热带地区自然选择和人工选择之间的可关联性。3.本研究在Bonferroni1/N下共检测到275个SNP,定位于221个基因与所测表型关联,其中50个基因与一个以上的性状相关联。GO功能分析显示这些基因涉及到生物合成、代谢、信号传导等多个途径。结果显示玉米耐旱是一个复杂的过程,需要多个代谢途径协同响应。4.38个基因共定位到的28个已知的抗旱Meta-QTL区间中,有9个为干旱胁迫环境下检测到的,22个在正常环境下检测到,7个在两个环境下都检测到。38个基因分布于玉米基因组的10条染色体,第二染色体最多,有7个,第三染色体最少,仅有一个。更多还原

【Abstract】 In higher plant, cytoplasmic male sterility (CMS) is a common phenomenon. The CMS/Rf system is not only widely used in hybrid breeding, but also a perfect model system for studying the molecular mechanism of the interaction between nucleus and cytoplasm. Maize is one of the earliest crops whose heterosis is improved through this system. S-CMS is the largest one of the three CMS groups in maize but utilization in breeding is limited by its unstable sterility. For using CMS in maize production by the greatest extent, it is necessary to clone the Rf3gene and theoretically elucidate the mechanisms of sterility and restoration of sterility.In the present study, we screened SSR and STS marker polymorphic between the near isogenic lines (NILs). Two sets of BC1F1populations were constructed from NILs with differences in Rf3locus for linkage analysis. The two populations were designated SSN for (S-Mol7rf3rf3xS-Mol7Rf3Rf3)xN-Mol7rf3rf3and SNN for (S-Mol7Rf3Rf3xN-Mol7rf3rf3)xN-Mol7rf3rf3. Additionally, we analyzed the sequences of maize2.09bin from B73genome (AGPv2). At the same time, a BAC library containing Rf3gene was also constructed. And some primary results are listed below:1. By examining the structure and development process of maize florets, we found it was more accurate to check pollens from the upper flowers inside the floret for determining whether the plant was sterile or not, which ensures the correction of fertility identification.2. The SSN population generated2781semi-fertile and2720sterile plants while the SNN generation yielded445semi-fertile and472sterile plants. The segregation retios of the two populations fit a monogenic Mendelian inheritance model of1fertile (Rf3rf3):1sterile (rf3rf3), it confirmed that the fertility restoration was conditioned by one dominant restorer gene in these maize materials.3. Total638pairs of primers were designed. Of those,198pairs were SSR markers,440were STS markers and only10SSR and4STS marker were polymorphic. The rates of the polymorphism for SSR and STS marker were10%and1%, respectively, relatively lower than usual, probably because markers were assayed between NILs in which most genomic region assayed were as the same as the recurrent, the other parent.4. Sequencing results of STS fragments confirmed the low polymorphism between the target region of the NILs. Only a small amount of SNP sites were discovered. Several could be converted to CAPS and dCAPS markers successfully.5. A linkage map was constructed around R/3locus, which was mapped on the distal region of chromosome2long arm. The total size of the map is19.9cM and all marker are mapped to one side of Rf3gene. The nearest marker to R/3gene is2043S-3, which is1.6cM away from the gene.6. A bacterial artificial chromosome (BAC) library was constructed from inbred line S-Mol7Rf3Rf3, and high-density filter set was prepared also. The library contains175,104clones with130kb average insertion fragment, which covers9.1times of the maize genome. The availability of this BAC library provides a useful tool for the cloning of Rf3gene.7.12PPR genes were discovered after rescanning the region of maize2.09bin. Of those PPR genes,10are belong to P subfamily and6PPRs of P subfamily located the mapping area of R/3locus. RFLP analysis revealed two PPR genes were polymorphic between tested lines containing R/3gene and lines containing rf3gene.8. the sequences of two PPR genes and the nearest marke were used as hybridization probes to screen the BAC library and48positive clones were obtained. Contig building and further analysis are under the way.Another area interested in our research is the discovery of maize genes related to response or tolerance to drought. Drought is one of the most important factors affecting the yield stability of maize (Zea mays L.) in China and many other areas in the world. In order to solve the drought and water shortage problem, breeding hybrids with drought tolerance is an important method besides water saving engineering. All350hybrids were phenotyped for9traits under water-stressed (WS) and well-watered (WW) conditions in multiple environments. Genes significantly related to drought were found through association analysis. These findings lay the foundation for further study on the mechanism of maize drought tolerance.1. Broad sense heritability of nine traits across the tested environments was calculated. Heritability of all but GY and KNO were high, which indicated the genotypic values of these traits were more consistent across environments. GY and KNO were more affected by the environments. These are in accordance with results from CIMMYT.2. Illumina MaizeSNP50array was used for genotyping the panel of350maize inbred lines. Statistical results showed that the SNP type of A/G was the most abundant in the population which was74.5%of total. All the other SNP types only held25.5%. This bias reflects the inherent connections among genomic structure, gene function, natural selection and artificial selection.3. In this study,275associated SNPs (P≤2.25x10-5|1/N) were identified, located i n221genes for126trait x environment x treatment combinations. under the Bonferroni correction of1/N.50genes were related to more than one trait. GO annotations showed that these genes were related to biosynthase, signaling, metabolism, etc. The results indicated that it is a very complex process of maize responsive to drought and drought tolerance of maize is a result by coordinating multiple pathways.4.38genes were co-localized to28Meta-QTL intervals which were known for drought tolerance of maize,9of them were found under WS,22under WW and7under both WW and WS.38genes were distributed over10chromosomes.7genes within5Meta-QTLs were on chromosome2while there was only one gene on chromosome3.更多还原