【作者】 王小利；

【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， 2004， 博士

【摘要】 小麦细胞质雄性不育(CMS)是小麦杂种优势利用的重要途径之一，也是小麦遗传育种研究的一个主要领域，而新的核质互作类型的发掘及研究是其重要的基础工作。具有偏凸山羊草(Ae.ventricosa)、粘果山羊草(Ae.kotschyi)、易变山羊草(Ae.variabilis)和二角山羊草(Ae.bicornis)细胞质的粘类 1B/1R 不育系具有易保持、易恢复，农艺性状优良，种子饱满等特点，为其组配强优势组合和优化制种技创造了极有利条件，是很具有应用价值的不育系。本研究对粘类小麦雄性不育系、保持系进行分子细胞遗传学鉴定和 RAPD 分析，为小麦细胞质雄性不育系的选育提供一套有效的鉴定方法。在此基础上利用一批远缘杂交后代的稳定材料对其杂交，并对二角山羊草细胞质不育系育性特异性进行了 APAGE、过氧化物同工酶和花药细胞学分析，同时，对新转育的二角型非 1BL/1RS 小麦雄性不育系进行了育性和恢复性研究，建立了二角型非1BL/1RS 易位型小麦雄性不育新体系。得出以下结论： 1．利用 ＡＰＡＧＥ、Ｃ－分带、ＲＡＰＤ 和原位杂交技术对不同核型的具有粘果山羊草、易变山羊草、偏凸山羊草和二角山羊草细胞质的粘类小麦雄性不育系进行分子细胞遗传学标记检测。ＡＰＡＧＥ 分析发现，不育系 ５－１ 和 ７－２１ 与 １ＢＬ／１ＲＳ 易位型不育系 ７７（２）、８２２２、偃师 ９ 号、８３（３７）６５、８０（６）　一样均含有 １ＲＳ 醇溶蛋白标记位点 Ｇｌｄ１Ｂ３。根尖染色体 Ｃ－分带显示 ５－１ 和 ７－２１ 含有 １ＲＳ 端带。ＲＡＰＤ 表明不育系 ５－１ 和 ７－２１ 在３５０ｂｐ 具有黑麦的特异带。原位杂交显示 ５－１ 和 ７－２１ 均为　１ＢＬ／１ＲＳ 易位，且易位片段发生在 １ＢＳ 端部。从蛋白质、细胞学、分子水平以及连接细胞与分子的原位杂交技术为小麦细胞质雄性 １Ｂ／１Ｒ 和非 １Ｂ／１Ｒ 不育系的选育提供了一套有效的鉴定方法。同时，证实了不同 １ＢＬ／１ＲＳ 易位不育系中 １ＲＳ 片段大小的差异，直接影响着育性稳定性和易恢复性的表现。　 ２．通过总 ＤＮＡ 浓度测定和 ＰＣＲ 反应条件优化，利用 ２５０ 条 １０－聚寡核苷酸随机引物对具粘果山羊草、易变山羊草、偏凸山羊草和二角山羊草细胞质不育系及其保持系 ５－１ 的总 ＤＮＡ 进行了 ＲＡＰＤ 多态性分析，结果表明：在 ２５０ 条随机引物中，３１ 条引物对 ４ 种不育系及其保持系总 ＤＮＡ 均无扩增，２１７ 条引物扩增条带完全相同，有 ２ 条随机引物在 ２ 种不育系之间有特异的扩增片段，其中引物 Ｓ２２ 对偏凸山羊草细胞质雄<WP=6>2 粘类小麦 ＣＭＳ 分子细胞遗传学研究及其育性基因载体替换后新不育体系的建拓性不育系有特异扩增带，分子量大小约为 １６００　ｂｐ，引物 Ｓ２０２ 对粘果山羊草细胞质雄性不育系在 １３００ｂｐ 有特异扩增带。利用 Ｓ２２ 和 Ｓ２０２ 对其 ｍｔＤＮＡ 进行扩增，Ｓ２２ 也能扩增出分子量大小约为 １６００　ｂｐ 的偏凸山羊草 ｍｔＤＮＡ 特异片段，而 Ｓ２０２ 对 ４ 种不育系及其保持系 ｍｔＤＮＡ 则没有扩增出表现 １３００ｂｐ 的特异带。ＲＡＰＤ 分析证明了 ４ 种不育系及其保持系 ｍｔＤＮＡ 存在明显的差异。　 3．利用大量小麦品种（系）对具有粘果、易变、偏凸和二角山羊草细胞质的粘类小麦雄性不育系杂交，发现大部分粘类的共同保持系表现出1BL/1RS易位系的1RS醇溶蛋白标记位点Gld1B3，为非1BL/1RS不育系的选育提供了必要的手段；利用一批远缘杂交后代的稳定材料对粘类小麦雄性不育系杂交，发现二角山羊草细胞质与小麦核内的遗传物质组成两个不同的不育系统，粘、易、偏型不育系育性基本表现一致，而二角型不育系除了与前三种不育系具有相同的保持系以外，对某些非1BL/1RS小麦品种（系）还表现出育性特异性；粘、易、偏和二角型同核异质不育系５－１及其与Ｖ９１２５杂交Ｆ１过氧化物同工酶分析表明，粘、易、偏和二角型不育系５－１过氧化物同工酶带型基本表现一致，而二角型不育系５－１杂交Ｆ１过氧化物同工酶则表现出酶带减少变弱。　 4. 采用小簇麦、小滨麦和小新麦杂交后的稳定优良后代材料 Ｖ９１２５、Ｍ８５３ 和Ｈ９０２１，对 １ＢＬ／１ＲＳ　二角型雄性不育系回交置换，经育性染色体替换，获得新不育再现，由此建立了新型二角山羊草细胞质小麦雄性不育系 ｍｓ（Ａｅ．　ｂｉｃｏｒ）－Ｖ９１２５、　ｍｓ（Ａｅ．　ｂｉｃｏｒ）－Ｍ８５３　和 ｍｓ（Ａｅ．　ｂｉｃｏｒ）－Ｈ９０２１。利用染色体制片、原位杂交、分子标记对 Ｖ９１２５、　Ｍ８５３　和 Ｈ９０２１ 进行分子细胞遗传学检测，并对新型二角山羊草细胞质小麦雄性不育系育性恢复性机理进行了初步研究。结果表明：Ｖ９１２５ 为小麦－簇毛麦易位系，Ｍ８５３ 为小麦－滨麦端部易位系，Ｈ９０２１ 为小麦－华山新麦草端部易位系。利用一些非 １ＢＬ／１ＲＳ 小麦品种（系）与这三个新型不育系及 １ＢＬ／１ＲＳ　型不育系 ｍｓ（Ａｅ．　ｂｉｃｏｒ）－５－１ 杂交，新型二角山羊草细胞质小麦雄性不?更多还原

【Abstract】 The cytoplasm male sterile wheat is an important way in using studies and utilizationof heterosis in wheat, and it is an important field in crop genetic and breeding study.Building new male sterile system is an major foundation work. Recently, the male sterilewheat varieties with Aeglops kotschyi, Ae. variabilis, Ae. ventricosa ,Ae. bicorniscytoplasm were appointed valuable CMS, which were easily maintained and easilyrestored, and because the CMS were full seed and good agronomical character, andproduced advantageous condition in composing heterosis combination and improvingmaking seed. But most CMS with 1BL/1RS translocation chromosome produced somehaploid, their fertility restore were easily changed, act. These question hindered that1BL/1RS CMS lines were used in production in great area. So the good maintainer of fourmale sterile wheat with Aeglops kotschyi, Ae. variabilis, Ae. ventricosa and Ae. bicorniscytoplasm were detected by C-banding and GISH and were crossed by a great of progenymaterials to analysis why the CMS 5-1 and 7-21 were easily restored. The fertilityspecificity of CMS wheat with Ae.bicornis cytoplasms were crossed by some wheatvarieties and studied by APAGE, peroxidase isozyme zymograms and microsporogenesiscytological observation so that new sterile system with Ae. bicornis cytoplasm was buildedin common wheat. The results were as follows: Two male sterile wheat varieties with Aeglops kotschyi, Ae. variabilis, Ae. ventricosa,<WP=8>4 粘类小麦 ＣＭＳ 分子细胞遗传学研究及其育性基因载体替换后新不育体系的建拓Ae. bicornis cytoplasm were detected by APAGE, C-banding and GISH. APAGE analysisindicated that new male sterile wheat 5-1and 7-21 possessed the same the gliadin markerGld1B3 of 1RS translocation chromosome as other male sterile wheat, such as 77(2)、8222、yanshi 9、83（37）65、80(6). The C-banding and GISH detected that 7-21 and 5-1was 1BL/1RS translocation because of their terminal banding of 1RS chromosome, twosatellite chromosomes and translocated in 1BS chromosome terminal. These resultssuggested that Gld1B3, satellite chromosomes and terminal C-banding of 1RSchromosome could be molecular cytogenetics marker of 1BL/1RS male sterile wheat. Asuit of effective way was built in CMS wheat breeding from gliadin, cytogenetics,molecular to GISH. It was conformed that the abnormal meiosis of 1BL/1RS translocationchromosome in their crossing F1 was one of the important reason that the fertilityrestoration of 1B/1R CMS lines was low and easy to change. 250 random 10-mer primers were screened on DNA samples of male sterile linewheat with Aeglops kotschyi, Ae. variabilis, Ae. ventricosa, Ae. bicornis cytoplasm andtheir maintainer 5-1, among which 217 primers produced some bands, primer S22produced repeatable polymorphic band to ms (Ae. ventricosa)-5-1 in １６００　ｂｐ, primer　Ｓ２０２produced repeatable polymorphic band to (Ae. kotschyi)-5-1 in １３００ｂｐ． S22 as well asproduced repeatable polymorphic band to mt DNA of ms (Ae. ventricosa)-5-1 in １６００　ｂｐ，　ｂｕｔ　primer　Ｓ２０２　ｄｉｄ　ｎｏｔ　produced repeatable polymorphic band to mt DNA of ms (Ae.kotschyi)-5-1 in １３００　ｂｐ　ａｎｄ　ｓｈｏｗｅｄ　polymorphic band to mt DNA of CMS.　 Four male sterile wheat with Aeglops kotschyi, Ae. variabilis, Ae. ventricosa and Ae.bicornis cytoplasm, were crossed with some wheat materials, and from the crosses aseries of maintenance lines were selected by investigating F1 fertility. （1）TheAPAGE(acid polyacrylamide gel electrophoresis) analysis indicated that most maintenancelines possessed the gliadin marker Gld1B3 of 1BL/1RS translocation chromosome. （2）There were two male sterile systems when Ae. bicornis cytoplasm and different nucleusin common wheat. Three male sterile wheats with Aeglops kotschyi, Ae. variabilis, Ae.ventricosa cytoplasm had same fertility , but the male sterile wheat with Ae. bicorniscytoplasm could produce male sterile with some other wheat materials with non-更多还原