【作者】 牛娜；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2012， 博士

【摘要】 小麦像玉米、水稻等作物一样具有明显的杂种优势，有效利用小麦杂种优势是提高其产量的一条重要途径。目前，主要是利用小麦雄性不育来实现杂种优势，而小麦雄性不育又分为遗传型雄性不育（核不育，核质互作雄性不育，光温敏不育）和生理型雄性不育（化杀剂诱导不育等）。大量研究表明每种途径都有其明显的优势和需要克服的缺点，相对来讲，核质互作型小麦雄性不育和化杀途径是两条较有生产潜力的途径。黏类（黏型、偏型和二角型等一些易保持、易恢复小麦雄性不育系的总称）小麦细胞质雄性不育系其最大特性是细胞质雄性不育系育性基因单一，不育性彻底，易恢复性高；但与CHA（化杀诱导雄性不育）途径相比，黏类小麦雄性不育系又因受限于不育系和恢复系制约配制强优势组合的几率较低。本研究以黏类小麦雄性不育系为材料，在对其育性进行分子细胞遗传研究的基础上，旨在探索原有黏类小麦雄性不育系的局限性，拓宽其新的不育-恢复基因源，然后将CHA途径的优点与黏类小麦雄性不育三系途径相结合，充分发挥这两种途径的优点，以建拓出一套杂交小麦杂种优势利用的新途径，为三系杂交小麦能早日进入生产应用提供理论依据和技术支撑。研究获得下述重要结果：1.黏类1BL/1RS小麦雄性不育系杂种F1中期Ⅰ部分染色体配对异常和后期Ⅰ染色体变异是由于核质互作的结果；中期Ⅰ单价体细胞频率和后期Ⅰ染色体变异率呈显著正相关；K、Ven、B型异源细胞质对杂种F1育性恢复性无显著相关性；中、后期染色体变异率与杂种F1育性恢复性呈负相关，中、后期染色体变异有降低杂种F1育性的作用。2.采用MINQUE（1）统计方法、加性-显性（AD）及其与环境互作的遗传模型，对黏类小麦雄性不育系和恢复系杂交F1结实性状进行遗传分析，结果表明（1）黏类小麦雄性不育系育性恢复性主要以加性效应为主，其次是显性效应；（2）不育系（V）-90-110、（Ven）-90-110、（K）-224、（V）-224在四个结实性状上，具有正向显著或极显著加性效应；亲本5253、02-7-215、00-6-247、M8003具有使不育系恢复度提高的加性效应；（3）双亲加性效应高的组合多表现出其显性效应比较差；（4）国际法恢复度高的组合（K-224×5253），究其原因是双亲国际法恢复度的加性效应高；（5）黏类小麦雄性不育系杂交结实率的高低与环境互作相对较小，育性恢复性在年份间表现比较稳定。3.对几类不同来源的黏类非1BL/1RS小麦雄性不育基因载体进行了筛选与鉴定，并对其育性特异性进行了研究。结果表明：（1）通过根尖体细胞随体鉴定和APAGE电泳筛选出SP4，莫迦小麦两种黏类小麦雄性不育载体，其为非1BL/1RS类型，其它不育载体均属于1BL/1RS类型；（2）选择不同来源的不育基因载体培育成的新型黏型、偏型不育类型进行育性恢复性测定发现，非1BL/1RS类型SP4、莫迦两种不育载体的小麦雄性不育系育性恢复性有一定差异，且育性恢复度相对较低，莫迦不育类型育性恢复性要高于SP4类型，在实际应用上应加以重视；（3）供试黏类非1BL/1RS小麦雄性不育系其不育性是在整个配子发育过程中连续产生的。4.采用SDS-PAGE和A-PAGE对黏类小麦雄性不育新种质材料及回交转育后代的特定染色体进行了鉴定，结果表明：（１）SDS-PAGE分析鉴定出小偃22、SP4、西农2611、莫迦不具有43和40kD谱带，为非1BL/1RS核型；（２）A-PAGE技术对部分亲本材料鉴定小偃22、SP4、西农2611、莫迦则不含GldlB3位点，为非1BL/1RS核型，其余为1BL/1RS核型；（3）A-PAGE技术对部分采用1BL/1RS易位系转育不育系后的保持系后代材料进行鉴定，显示出大部分转育后代含有GldlB3醇溶蛋白标记位点，为1BL/1RS易位系；5.对杂交小麦化杀强优势组合西杂一号、西杂五号亲本遗传背景进行鉴定，发现西杂一号两个亲本均为1BL/1RS类型，西杂五号两个亲本均为非1BL/1RS类型，针对不同核型，专门设计了1BL/1RS类型和非1BL/1RS类型化杀强优势组合定向转化三系强优势组合各自保持系、不育系及恢复系转育体系与程序。6.按照设计好的转育程序图，将西杂一号的母本西农Fp1定向转育为具有SP4、莫迦小麦1Brfv1的新保持系，并同时进行不育系的转育；将西杂一号的父本Mp1转育为具有R5253、R5383的1BRfv1的恢复系。在此过程中，建立了利用多重PCR技术，同时结合传统的根尖随体鉴定技术对1BL/1RS易位纯合体和易位杂合体鉴定的技术体系。7.按照设计好的转育程序图，将西杂五号的母本西农Fp2定向转育为具有SP4、莫迦小麦1Brfv1的新保持系，在转育过程中，建立了SDS-PAGE和A-PAGE鉴定目标基因所在染色体的方法技术体系。SDS-PAGE鉴定结果发现SP4高分子量谷蛋白亚基6+8可作为小麦雄性不育基因rfv1基因的示踪特征亚基，为小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系提供了可靠的转育与检测依据。A-PAGE分析结果发现，在ω-醇溶蛋白区也发现莫迦和SP4不同于西农Fp2的特异蛋白条带，可作为小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系的示踪蛋白条带。8.经过多年回交转育，现已获得杂交小麦西杂一号、西杂五号母本同型黏类小麦非1BL/1RS雄性不育系的保持系，其不育系正在转育中，获得杂交小麦西杂一号、西杂五号父本同型黏类小麦非1BL/1RS雄性不育系的恢复系。更多还原

【Abstract】 Wheat (Triticum aestivumL.) has obvious heterosis like other crops, such as maize andrice. Heterosis utilization can improve quality and yield of wheat by a large amount. NowWheat heterosis utilization is realized by means of male sterility, which includes hereditablemale sterility (such as CMS, NMS and PMS）and physiological male sterility (such as CHAS).Each of male sterility has their own merits and demerits.CHAS and CMS had become the main ways to produce the hybrid wheat in recent years.Nian-type is the generic terms of cytomplas male sterile line with the ctyoplasms ofAegilops.kotschyi, Ae. variabilis, Ae. ventricoca, and Ae. Bicornis. CMS of NIAN-type hasthe permanent merits of easy restoring, single male sterile and restoring gene, and thedemerits of obtaining hybrid componention hardly compared to CHAS. The parents-lines ofCHAS was selected easily and freely. Either two cultivars can be combined to produce thehybrid wheat.The CMS of NIAN-type were utilized to carrying out the fundmental research,applied research in this research. The aims were to explore the faults of old CMS, screen thenew sterile-restoring genes, and create a new way of hybrid whear utilization by means ofcombining the CMS and CHAS. The results were as follows:1. MⅠand AⅠin meiosis of hybrids F1of3alloplasmic wheat male sterile lines weresystematically observed and investigated, the relationships between FCU, FCLB and thefertility restoration of hybrids F1were analyzed. The results were as follows:(1)Thechromosomes synapsis at MⅠand variation at AⅠof hybrids F1were caused bynucleo-cytoplasmic interaction.(2) FCU at MⅠwas highly positively correlated to the FCLBat AⅠ.(3) The fertility restoration of hybrids F1had no correlation with Ae.kotschyi,Ae.ventricosa or Ae.bicornis cytoplasms.(4) Fertility restoration of the hybrids F1wasnegatively correlated to the FCU and the FCLB, the chromosomes variation at MⅠandAⅠdecreased the fertility restoration of F1hybrids.2. Some seed set characters of genetic variances, such as seed set in both sides、seedset in the middle of F1spikelets from sterile lines and restoring lines of Nian group(withAe.kotschyi, Ae.variabilis, Ae.ventricosa and Ae.bicornis cytoplasms) in wheat for twoyears are analyzed by the genetic model of additive-dominance (AD) and their interactions with environments by MINQUE(1) approaches. Additive effects was themain behavior to the fertility restoration performance of male sterile lines of Nian groupin wheat, and the next was the dominance effects. The seed set characters of somecombinations from sterile lines (V)-90-110,(Ven)-90-110,(K)-224,(V)-224hadpositively significant or the most significant additive effects, the parents5253,02-7-215,00-6-247, M8003had the additive effects of making the restoring degree increased. Ingeneral, the combinations with higher additive effects of two parents were lower indominance effects. The restoring degree of international method was high, because theadditive effects of the restoring degree of international method of two parents were high.There was small interaction between the environment and the rate of seed set of malesterile lines of Nian type in wheat, fertility restoration performance was more stablebetween years.3. specific fertility characteristic and screen the sterile gene carrier of non-1BL/1RSwheat male sterile lines of nian type from different sources were studied in this paper, in orderto breeding the new excellent non-1BL/1RS wheat male sterile lines of Nian type andpromote the hybrid wheat. Results as follows:(1) Two kinds of wheats SP4(T.spelta varduhamelianum) and T.macha var subletschchumicum carry the sterile gene of non-1BL/1RSwheat male sterile lines of Nian type by the body cell identification and A-PAGE. The othermaterials carry the one of1BL/1RS wheat male sterile lines of Nian type.(2) The fertilityrestoration performance of the two male sterile lines with Aegilops kotschyi, Ae.ventricosacytoplasms and SP4, T.macha nucleus were investigated. There is difference between the twomale steriles, and there is a smaller fertility degree. Averagely, the fertility degree of the twomale sterile lines with T.macha nucleus is higer than SP4’s. So T.macha should be thoughtmore in the application.（3）The sterility develop is in the whole developping process of thegamete. The cytology morphology of SP4, T.macha in the cytoplasms with Ae.bicornis isdifferent from the cytoplasms with Aegilops kotschyi, Ae.ventricosa, and the rates ofbourgeon is higer in the cytoplasms with Ae.bicornis.4. In order to study the specific function of PAGE in breeding male sterility germplasmof NIAN type in wheat directionally, SDS-PAGE and A-PAGE were adopted to indentify thespecific chromosome from sterile germplasm and their backcross offspring, the result were asfollows:(1) There were43kD and40kD band in906, Xiaoyan22, XC-2and Xinong186bymeans of SDS-PAGE. They are1BL/1RS translocations.(2) The prolamine protein markerloci GldlB3was founded in906,05－S-9-10，XC-2, Xinong1376, Xinong186, which showsthis materials are1BL/1RS translocations. It was not founded in were P1, P2and Xinong2611,Xiaoyan22, which shows they are non-1BL/1RS translocations.(3) Based on the results above, offsprings of maintainer transducted were identificated by A-PAGE. It shows that most ofoffsprings are1BL/1RS translocations, because of having the prolamine protein marker lociGldlB3.(4) Both SDS-PAGE and A-PAGE can be used in indentifing1BL/1RS translocationsand non-1BL/1RS translocations in Male sterility of Nian Type on wheat, But the resolutionof A-PAGE is more higer than SDS-PAGE.5. The parents of XZ-1belonged to1BL/1RS translocations, and that of XZ-5belongedto non-1BL/1RS translocations based on identification. In order to transduct non-1BL/1RSmale sterile and restoring genes of Nian type directional and quickly, we designed someprocedures for trsducting the maintainer, sterility and restorer against these two types ofparents.6. According to the procedures designed, the female parent of hybrid wheat XZ-1wasused as receptor and recurrent parent, and crossed and backcross with SP4, Macharespectively to breed maintainer and sterile lines of male sterility. Similarly, the male parentof hybrid wheat XZ-1was used as receptor and recurrent parent, and crossed and backcrosswith restoring lines R5253、R5383respectively to breed new restorer lines of male sterility. Inthis process, we established a technology system of indentifing1BL/1RS homozygote andheterozygote of translocation.7. According to the procedures designed, the female parent Xinong Fp2of hybrid wheatXZ-5was used as receptor and recurrent parent, and crossed and backcross with SP4, Macharespectively to breed maintainer and sterile lines of male sterility. In this process, weestablished a technology system of indentifing target chromosome by means of SDS-PAGEand A-PAGE. The results of SDS-PAGE showed that protein subunit6+8in HMW could beused as the marker to trace the male sterile gene rfv1, and there is the character band of SP4and Macha in D region of LMW. The results of A-PAGE showed that there was a characterprotein band in ω zone between SP4Macha and Xinong Fp2, which can be used as the tracerof the male sterile gene rfv1.8. After cross and backcross of many generations, we have got the new maintainers ofnon-1BL/1RS male sterile lines with the background of female parents of hybrid wheat XZ-1,XZ-5, and the male sterile lines was breeding now. We have got the new restorer ofnon-1BL/1RS male sterile lines with the background of male parents of hybrid wheat XZ-1,XZ-5.更多还原