【作者】 王静；

【导师】 叶兴国；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2019， 硕士

【摘要】 中间偃麦草和大麦作为小麦的2个近缘物种,含有很多优良基因,对改良小麦抗病性、抗逆性和品质等性状具有重要意义。利用远缘杂交技术将这些基因转入小麦是丰富小麦遗传多样性、扩大遗传变异范围的重要手段。本研究拟利用小麦-大麦第二群染色体异代换系和小麦-中间偃麦草第二群染色体异代换系相互杂交,辅佐分子标记、分子细胞学、农艺性状调查等方法进行筛选和鉴定,培育小麦-大麦-中间偃麦草异源三属间第二群染色体代换聚合体。在此基础上,进一步利用原位杂交技术对聚合体材料减数分裂起始前、后同源染色体及部分同源染色体的动力学进行分析。同时,通过体细胞无性系变异技术诱导小麦、大麦、中间偃麦草第二群染色体间的易位。主要研究结果如下:1.通过小麦-大麦第二群染色体异代换系和小麦-中间偃麦草第二群染色体异代换系相互杂交和连续自交,利用第二群染色体特异分子标记筛选不同世代的材料,初步获得了2类小麦-大麦-中间偃麦草三属间第二群染色体聚合体材料2D2Ai-2（2B）2H（2A）和2A2Ai-2（2B）2H（2D）。农艺性状调查结果表明,聚合体材料在株高、开花期、穗长、穗粒数等性状方面与双亲存在明显差异。2.以标有不同荧光素的大麦基因组DNA和中间偃麦草基因组DNA为探针进行原位杂交,双色GISH显示聚合体材料中染色体组成为2n=42,包括38条小麦染色体、2条红色大麦染色体和2条绿色中间偃麦草染色体;分别利用特异于大麦2H染色体和小麦染色体着丝粒的探针,以及特异于中间偃麦草2Ai-2染色体臂的探针进行FISH鉴定,发现聚合体材料中含有19对显示小麦着丝粒信号的染色体,1对显示大麦着丝粒信号的染色体,1对显示中间偃麦草染色体臂信号的染色体。3.利用特异分子标记对3种类型的异源三属代换系F₁代杂种与中国春正反交后代检测,发现大麦2H染色体通过雌、雄配子的传递率分别为32.7%和24.7%,中间偃麦草2Ai-2染色体通过雌、雄配子的传递率分别为21.8%和19.3%。表明在不同遗传背景下大麦2H染色体的传递率高于中间偃麦草2Ai-2染色体,但均优先通过雌配子进行传递。4.利用小麦-大麦异代换系和小麦-中间偃麦草异代换系进行杂交,对授粉14-15 d的F₁杂种幼胚进行组织培养,通过2次继代培养和分化培养,获得102株再生植株。然后利用2HL、2HS、2Ai-2L、2Ai-2S特异分子标记进行筛选,结合基因组原位杂交（GISH）,在SC₂群体中获得了1株大麦-中间偃麦草2HL·2Ai-2S杂合易位系。5.采用滴片法对异源三属代换聚合体花粉母细胞进行细胞学观察,FISH鉴定发现减数分裂前端粒和着丝粒分布细胞两端,减数分裂前期着丝粒优先于端粒进行识别,两者共同促进后期染色体的正确配对;以大麦和中间偃麦草DNA为探针进行GISH分析,发现减数分裂前2对外源染色体发生配对,减数分裂前期2对外源染色体的遗传行为与其它小麦染色体相同,减数分裂中期发生部分染色体结构变异,如染色体断裂、染色体数目变化等。更多还原

【Abstract】 Thinopyrum intermedium and barley are two close relatives of wheat and carry many useful genes,including tolerance to abiotic stresses,high disease resistance and so on,which are potentially valuable for the improvement of various wheat traits.Distant hybridization,in which desirable chromosomes or fragments are transferred from genetically similar plant species to cultivars,is an effective technique for developing new germplasm or varieties.This study was designed to cultivate wheat pyramiding substitution lines by hybridizing different wheat-Th.intermedium and wheat-barley disomic alien substitution lines and induce the whole-arm translocation line between chromosome 2H of barley and chromosome 2Ai-2 of Th.intermedium in wheat background through the technique of somatic variation population,using molecular markers,in situ hybridization,cytology,agronomic examination,tissue culture and other methods.Based on above information,genetic behavior of homologous chromosomes and homeologous chromosome before and during meiosis were further analysed by in situ hybridization technique.The main findings of this investigation are as follows:Hybrids between wheat-Th.intermedium and wheat-barley disomic alien substitution lines were made and the progeny were repeatedly selfed.Plants containing target chromosomes in generations F₁ to F₅ were identified using chromosome-specific molecular markers,and two types of homozygous substitution lines,2D2Ai-2（2B）2H（2A）and 2A2Ai-2（2B）2H（2D）,were preliminarily obtained.In addition,agronomic traits indicated,compared to parent materials,the wheat pyramiding substitution lines behave differently in flowering date,plant type,and other agronomic characteristics.Genomic in situ hybridization（GISH）using total genomic DNA of Th.intermedium and barley as probes was performed,and the result showed that there were 42 chromosomes in wheat pyramiding substitution lines,with two red hybridization signals and two green hybridization signals.The fluorescence in situ hybridization（FISH）analysis using different probes indicated that there were red hybridization signals in the centromeric regions of 38 wheat chromosomes,green hybridization signals in the centromeric regions of 2 barley chromosomes,and green hybridization signals on both arms of 2Th.intermedium chromosomes.Using molecular markers specific to the two alien chromosomes to genotype plants in the F₁generation derived from reciprocal crosses between wheat cultivar CS and the three substitution lines2Ai-2（2D）/2H（2A）,2Ai-2（2B）/2H（2D）,and 2Ai-2（2B）/2H（2A）,the result showed that the average transmission rates for alien chromosome 2H via the male and female gametes were 24.7%and 32.7%respectively,whereas those for the alien chromosome 2Ai-2 were 19.3%and 21.8%.The alien chromosome 2H thus appeared to be transmitted to the next generation at a higher rate than the alien chromosome 2Ai-2 and the two alien chromosomes were preferentially transmitted via the female gametes.Crossing wheat-Th.intermedium with wheat-barley disomic alien substitution lines,the F₁ hybrid immature embryo for 14-15 days after pollination were cultured on callus induction medium,then two times of subculture was conducted to induce whole-arm translocation between chromosome 2H of barley and chromosome 2Ai-2 of Th.intermedium in wheat background.Ultimately,a total of 102 regenerated plants were obtained.Using molecular markers specific to 2HL,2HS,2Ai-2L,and 2Ai-2S,accompanied by in situ hybridization,a heterozygous translocation line between barley 2H and Th.intermedium 2Ai-2 was successfully obtained.Using drop method to observe PMCs of wheat pyramiding substitution lines,GISH and FISH analysis using different probes was also carried out to identify genetic behavior of the alien chromosomes before and during meiosis.The FISH result revealed that centromeres grouped at the opposite pole with respect to telomeres,which conformed to the basic Rabl configuration,then centromere pairing or clustering appeared to occur prior to telomere bouquet formation,both of them facilitated chromosome pairing.The GISH analysis using labeled total genomic DNA of Th.intermedium and barley with different fluorescent labels indicated that the two types of alien homologous chromosome initially separated,and then associated during the pre-meiotic stages of meiocyte development,while they behaved similarly to wheat chromosomes during the prophase of meiosis.The observation of PMCs at meiotic metaphase I showed that part chromosomes happened structural changes,such as chromosome breakage,number variation and so on.更多还原