【作者】 刘旭；

【导师】 陈化榜；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 玉米(Zea mays L.)是雌雄同株异花授粉作物，通常情况下自交和异交都可以正常结实。玉米配子体遗传因子（gametophyte factor）所介导单向异交不亲和基因广泛存在于爆裂玉米中，当普通的马齿和硬粒玉米作为父本为其授粉时不能结实，而反交却可以正常结实。本研究以携带单向异交不亲和性最彻底的基因Ga1-s的爆裂玉米种质SDGa25为材料，进行理论及应用研究，得到如下结果：1.本实验利用148份国内外玉米自交系材料作为父本对单向异交不亲和种质SDGa25（Ga1-s/Ga1-s）人工授粉两年的实验结果证明，其中有3份自交系材料对SDGa25异交亲和，其中ZD12F为国外玉米种质，JKN2000F为糯玉米种质，仅HN178为普通玉米种质，其他的145份自交系对SDGa25异交结实率为0，表现为完全异交不亲和。2.携带Ga1-s的自交系SDGa25与携带Ga1-m的自交系HN178分别与普通玉米自交系组建BC1F1个体表现型经卡方检验符合1:1分离比例，携带Ga1-m的自交系JKN2000F与普通玉米自交系组建F2个体表现型经卡方检验符合3:1分离比例，证明Ga1-s与Ga1-m均为显性单基因。携带Ga1-m的自交系HN178与JKN2000F组配的F2个体表现型经卡方检验符合15:1分离比例，证明两组自交系HN178与JKN2000F所携带的Ga1-m为非等位基因。3.本研究分别通过分子标记辅助筛选交换单株、构建单一表型群体以及Ga1-m辅助Ga1-s定位等方法，将单向异交不亲和基因Ga1定位在玉米的第四号染色体短臂上端粒侧标记dCAPS1和着丝粒侧标记ID7之间。标记dCAPS1位于BAC208523，标记ID7位于BAC204382，参照数据库maizesequence中B73RefGen_v2sequence物理距离为100Kb，比原有定位区段减少了2.1Mb，新开发与Ga1紧密连锁的分子标记13个，可以用于后续BAC文库筛选和分子标记辅助选择育种。4.本实验构建了SDGa25基因组BAC文库，挑取约300000个单克隆，重组克隆平均插入片段长度为70Kb，空载率小于1%，重组文库稳定性良好，覆盖率约为玉米基因组的10倍。通过对构建的超级池、亚池以及单克隆的逐级筛选，得到覆盖100Kb全部定位区段的2个重组单克隆并送样测序。5.根据重组单克隆部分测序序列信息以及数据库maizesequence中B73RefGen_v2sequence的序列信息进行比较基因组学分析预测到3个基因，其中有已知功能的基因2个，分别为GRMZM2G027021和GRMZM2G039983，各含有14个和6个外显子。GRMZM2G027021具有GTP酶结合结构域，序列分析表明：Ga1-s、Ga1-m与ga1相比，第3个外显子完全缺失，可能最终导致Ga1-s、Ga1-m花粉具有使Ga1-s/Ga1-s母本正常结实的功能。GRMZM2G039983为Xklp2的靶蛋白，与拟南芥中的WDL1基因同源，可能与ga1花粉管在Ga1-s花丝内的伸长过程中受阻相关；序列分析表明，该段区域内Ga1-s、Ga1-m、ga1三个基因均有明显的区别：与ga1相比，Ga1-s、Ga1-m在3’非编码区段相同位点有序列插入；与Ga1-m、ga1相比，Ga1-s在第1外显子与第2外显子之间有序列插入，包含一个CACTA转座子元件。6.本研究通过回交转育的方法将Ga1-s基因导入糯玉米品种JKN2000的父母本，将Ga1-s基因介导的生殖隔离引入制种生产防止不同类型玉米之间的花粉污染。以JKN2000的父母本作为轮回母本，在回交过程中利用筛选到的紧密连锁分子标记辅助选择获得Ga1-s单株。对6代回交的目的单株回交，利用分子标记筛选Ga1-s/Ga1-s父母本单株组配杂交种。Ga1-s/Ga1-s父母本自交系异交结实率均为0，Ga1-s/Ga1-s杂交种异交结实率为0，而普通杂交种的异交结实率为46%。更多还原

【Abstract】 Maize (Zea mays L.) is both a self and cross compatible species. However, many popcornstrains carry unilateral cross incompatibility genes and cannot be fertilized by pollen of dentand flint maize strains although the reciprocal crosses are successful. These genes are namedgametophyte factors (Ga) because the cross incompatibility are governed by the genotypes ofindividual pollen grains rather than the parental sporophytes. A Chinese popcorn strainSDGa25carrying the strongest allele of Ga1(Ga1-s) was chosen as material for genetic studyand fine mapping. The main results were as follows：1. One hundred and forty-eight dent and flint maize inbred lines were crossed as male withSDGa25by hand pollination in two years. Only3lines, ZD12F, a foreign line, JKN2000F, awaxy line, and HN178, a dent strain, showed cross compatible to SDGa25. The rest of themshowed complete cross incompatible to SDGa25.2. Ga1-s and Ga1-m were dominant genes. Ga1-m alleles from different strains were notallelic.3. The Ga1locus was fine mapped into a100kbp interval based on the B73RefGen_v2sequence between markers dCAPS1on BAC AC208523on the telomere side and ID7onBAC AC204382on the centromere side. The mapping interval was2.1Mbp shorter thanpreviously reported and was achieved by integrating results from three different mappingpopulations. Thirteen tightly linked markers to Ga1-s were developed in this study.4. SDGa25BAC library consisted of300000clones, and more than99%of the clonescontain maize nuclear DNA inserts with an average size of70kb. The library containedgenomic DNA that was equivalent of10×haploid genome. Two positive BAC clonescovering the whole mapping interval were sequenced.5. Based on the sequences of the positive BAC clones and B73RefGen_v2sequence,3genes was predicted. GRMZM2G027021and GRMZM2G039983had14and6exons, respectively. GRMZM2G027021was GTP-binding protein hflX. Compared to that of ga1, thethird exons of Ga1-s and Ga1-m were deleted. The deletion may be involved in pollen-pistilinteractions. GRMZM2G039983had homology to WDL1of Arabidopsis, which regulatedanisotropic growth of cells, and perhaps could inhibit ga1pollen tube growth on Ga1-s pistil.Compared to that of ga1, Ga1-s and Ga1-m alleles had insertions at the same site of3’untranslated region. Only Ga1-s allele had a CACTA transposon element in the insertionbetween the first and the second exon.6. The present paper reports introgression of Ga1-s into elite waxy corn hybrids JKN2000by marker assisted selection and the possibility of using Ga1-s as biological reproductivebarrier for the containment of gene flows between different types of corns. Ga1-s/Ga1-shybrids showed complete cross-incompatible to foreign pollen, whereas46%cross-compatible rate occurred in the ga1/ga1hybrids.更多还原