【作者】 吴鹏昊；

【导师】 赖锦盛； 陈绍江；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 玉米单倍体育种的大规模应用需要对单倍体育种流程中每一个环节的效率进行系统研究。随着诱导率和鉴别效率的不断提高,较低的单倍体加倍效率已经成为高通量单倍体育种的主要限制因素。单倍体雄穗育性自然恢复性能是影响单倍体自然加倍效率的关键因素,本研究针对该性状的遗传规律及其相关控制基因进行了探讨,主要结果如下：(1)本研究对单倍体雄穗育性恢复性状进行分级评价,用露药率、露粉率、露药得分和露粉得分四个指标来评价单倍体雄穗育性恢复能力。通过对不同杂种优势群的16个自交系及其组配的23个杂交种F1诱导单倍体的雄穗育性自然恢复进行分析,发现单倍体雄穗育性恢复主要由基因型决定,除露粉得分因显著受环境影响外,其它三个性状遗传力都在60%以上。(2)通过对自交系来源单倍体雄穗育性恢复能力进行评价,发现高频雄穗育性恢复材料豫8701和4F1,散粉比例都在80%以上,散粉单株中90%为高等级散粉单株；高露药能力材料许178、齐319和高露粉能力材料K22、B73。通过对杂交种来源单倍体雄穗育性恢复能力进行评价,发现豫8701组配的杂交种后代单倍体均表现出高频雄穗育性恢复,而4F1组配的杂交种后代单倍体雄穗育性恢复能力有高有低,说明豫8701中可能含有更多有利于单倍体雄穗育性恢复的基因位点。本研究发现从两个低频雄穗育性恢复系郑58和昌7-2组配郑单958后代单倍体中观察到超亲育性恢复现象,这说明单倍体育性恢复能力可以通过选择得到提高。(3)对单倍体育性恢复其他遗传因素进行研究发现该性状主要受核基因的控制,不受细胞质基因的调控；诱导率不同的诱导系对单倍体雄穗育性恢复能力没有显著影响。(4)利用流式细胞仪对不同散粉等级的单倍体在其散粉吐丝期对叶片的倍性进行测定,发现只有10%左右的较高等级的可育单倍体中才会含有部分二倍体细胞,大部分发生育性恢复的单倍体其体细胞仍然停留在未加倍状态,说明单倍体在发生自然加倍时其生殖细胞的加倍应当早于或者优于体细胞的加倍,并且不以体细胞的加倍为前提。(5)利用郑单958F2：3群体对单倍体被诱导率进行定位,共检测到两个相关位点qmhirl和qmhir2,分别位于第1和第3号染色体上,解释表型变异的14.7%和8.42%。用该群体对单倍体雄穗育性恢复进行定位,共检测到8个相关位点,分别位于第2、3、8和9号染色体上。其中位于第8号染色体上的位点qHmf7在两年的重复中都被检测到,位于第3.07区的位点qHmf5也在豫8701×郑58以及4F1×郑58的后代单倍体群体中得到验证。这两个位点的贡献率都在10%左右。通过对候选基因预测认为单倍体育性恢复可能受到多个功能基因的调控。(6)对单倍体群体中出现的早期加倍单倍体EH进行了系统研究。表型特征统计分析发现EHo当代在二倍化程度上要显著优于一般加倍单倍体,完全可育,植株农艺性状与常规自交系一致。同时分子标记SSR检测以及SNP检测发现EH0代在基因型上与母本被诱导材料或者DH0代基本一致。EH是单倍体自然加倍中一种特殊现象,其可能发生于胚形成过程中。EH在单倍体群体中出现的频率较低,目前发现的最高仅为3%左右。更多还原

【Abstract】 Large-scale applications of DH breeding require breeders to do systematic research on every step during the process. Haploid induction and identification are not a big problem right now, but haploid doubling techniques are still not efficient enough to meet the high throughput production for DH lines, spontaneous doubling is one of the most convenient ways in DH breeding, however, the rate is so high. Therefore it is necessary to study the genetic characteristics and related genes of haploid male fertility (HMF) in order to explore new ways to increase spontaneous doubling rate. The main results are as follows:(1) This study classified the characteristics of HMF into different levels according to different haploid diploidization with different index:anther ratio, pollen ratio, anther score and pollen score were used to describe HMF. Haploids derived from16elite inbred and composed23F1hybrids covering the five heterotic groups were investigated of HMF in different environment in two years. It was found that haploid male fertility was mainly controlled by genetic background; the heritability of all of the traits was more than60%except that pollen score was a little lower and affected by the environment.(2) Cluster analysis found that the haploids originated from inbred lines Yu8701and4F1had better HMF capabilities than other materials, the fertile ratio was more than80%and90%of the fertile haploids were with high level. Xu178-derived haploids and Qi319-derived haploids performed well in anther traits; K22-derived haploids and B73-derived haploids performed well in pollen traits. The haploids from the hybrids of Yu8701always performed well in HMF but no same results were found in the haploids from the hybrids of4F1, which meant that there were more positive HMF related genes in Yu8701. Transgressive inheritance was found in the progeny of two low-frequency HMF inbred lines Chang7-2and Zheng58, which indicated that the trait could be improved through selection.(3) Other genetic factors of HMF were also investigated and found that HMF was mainly controlled by gene in nuclear, not in the cytoplasm. Inducer lines did not affect HMF.(4) Ploidy investigation of the leaf in pollination using flow cytometry found only10%high level fertile haploids contained half of the proportion of diploid cells, vegetative organs of most fertile haploids remained in the state of haploid, indicating that the reproductive cells should be doubled earlier than the vegetative cells.(5) A mapping population consisting of186F23family lines derived from the cross between Zheng58and Chang7-2was used to map the maternal QTL of in vivo haploid induction. Two quantitative trait loci (QTLs), qmhirl and qmhir2, were detected on chromosomes1and3and could be explained14.70%and8.42%of the phenotypic variation, respectively. This Zhengdan958F2:3population was also used to locate the HMF related genes. A total of8HMF related genes were detected. The loci on chromosome8qHmf7were detected in two replications; the loci qHmf5on3.07were verified in other genetic background Yu8701×Zheng58and4F1×Zheng58with extreme value chi-square test methods. Each of the contribution of these two loci was about10%. The predicted candidate gene was found to have different functions.(6) Early doubled haploid (EH) in haploid populations was studied systematically. Phenotypic characteristics analysis found that EH0performed better than normal doubled haploid in diploidizaion with totally fertile. Agronomic traits of the EH plants consistent with the inbred lines. SSR marker and SNP detections found that genotype of EH0was the same with the donor parents, EH is a kind of special DH from haploid spontaneous doubling in embryo development. The ratio of EH is about3%in haploid population.更多还原