【作者】 王春梅；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 遗传学， 2007， 博士

【摘要】 分子标记技术是二十世纪80年代初建立起来的一种以遗传物质DNA为基础的新型的遗传标记体系，目前广泛应用于分子遗传图谱的构建、基因定位与克隆、重要性状的标记辅助选择以及比较基因组学等研究。本研究利用分子标记技术（如SSR，STS，RGA等）开展对小麦抗条锈病基因Yr26的分子标记筛选和定位研究，并开发了黑麦1R、鹅观草1Rk^#1、簇毛麦1V和6VS染色体特异的分子标记。1．小麦抗条锈病基因Yr26的SSR和STS标记小麦条锈病是由小麦条锈菌（Puccinia striiformis f．sp．stritic）引起的一种广泛流行的世界性小麦病害，培育抗病品种是防治小麦条锈病最经济有效且利于环境安全的措施。然而，由于小麦条锈菌具有高度的变异性，随着新致病小种的出现许多抗病品种相继“丧失”了抗性。普通小麦-簇毛麦6VS／6AL易位系携带有位于1B染色体上的Yr26基因，能抵抗目前流行的多个条锈菌生理小种，因此对Yr26基因进行分子标记研究对在育种中更好地利用该基因具有重要意义。本研究选用普通小麦1B染色体上的35对SSR引物和根据1B染色体不同物理区段的EST序列设计的81对STS引物，对抗、感亲本和抗、感池进行扩增分析，筛选到5对SSR标记（Xgwm11、Xgwm18、Xgwm413、Xbarc181和Xwmc419）和8对STS标记（WE173、WE171、WE177、WE201、WE202、WE210、WE11和WE17）在抗、感亲本和抗、感池间存在多态性。进一步利用筛选到的13对引物扩增扬麦5号×92R137 F₂群体的单株，大部分感病单株扩增出与感病亲本扬麦5号一致的带型，而大部分抗病单株扩增出与抗病亲本92R137相同的带型或双亲的带型。利用Mapmaker 3.0进行连锁分析，这13个标记位点与抗条锈病基因Yr26连锁，遗传距离在1.4～14.1cM之间。其中WE173、WE171、WE177、WE201、WE202、WE210、Xgwm11、Xgwm18和WE11等9个标记与Yr26的连锁距离在5.5 cM以内，特别是共显性的STS标记WE173与Yr26的遗传距离最小，为1.4cM。利用中国春非整倍体和缺失系对筛选到的多态性标记进行物理定位，位于Yr26基因两侧的标记WE173和Xwmc419定位在1B染色体长臂，紧靠Yr26基因的WE173、WE201等标记被定位在1BL-6区段，从而将Yr26基因定位于1B染色体长臂紧靠着丝粒的1BL-6区域。利用与Yr26基因连锁的分子标记对利用6VS／6AL易位系做抗病亲本育成的高代品系和新品种进行分子标记检测。内2938，内麦9号，内4221，95-108和南农9918等5份材料含有与Yr26基因紧密连锁的标记，石012056，A4-74-8和A5-82-3-5等3份材料不含有与Yr26基因紧密连锁的标记，利用分子标记检测的结果与苗期条锈病的抗性鉴定结果一致，表明本研究筛选到的分子标记可以用于Yr26基因的分子标记辅助选择。2．簇毛麦6V染色体短臂特异分子标记的开发和应用来自簇毛麦的抗白粉病基因Pm21被定位在6V染色体短臂上，为进一步定位和克隆Pm21基因，须创造更多染色体结构变异体和更多的分子标记。为开发簇毛麦6V染色体短臂特异的分子标记，本研究选用11个RGA和17对STS引物进行多态性分析，有1个RGA引物和1对STS引物分别检测到一条约1000bp和约800bp的多态性片段，根据这两条多态性片段的序列设计出引物CINAU17和CINAU18，利用一套普通小麦-簇毛麦异附加系和涉及6V不同片段的异染色体系，筛选出6V短臂特异的分子标记CINAU17-₁₀₈₆和CINAU18-₇₂₃，并进一步将CINAU17-₁₀₈₆标记定位在簇毛麦6VS FL0.58与FL0.70之间，将CINAU18-₇₂₃标记定位在簇毛麦6VS FL0.45和着丝粒之间。利用这两个标记对通过花粉辐射获得的部分簇毛麦6VS结构变异材料进行PCR鉴定，结果与细胞学鉴定结果相一致。因此CINAU17-₁₀₈₆和CINAU18-₇₂₃标记可用来快速检测和追踪导入普通小麦背景中的簇毛麦6VS染色体片段，并对缺失系的断点进行初步界定。3．普通小麦近缘物种黑麦1R、簇毛麦1V及鹅观草IRk^#1染色体特异分子标记的开发根据普通小麦第一部分同源群的EST序列设计104对STS引物，筛选出5对黑麦1R染色体特异标记：CINAU 19-₅₀₀、CINAU20-₉₅₀、CINAU21-₁₅₀₀、CINAU22-₃₁₀和CINAU23-₂₀₀₀；5对簇毛麦1V染色体特异标记：CINAU23-₁₇₀₀、CINAU24-₁₀₅₀、CINAU25-₁₆₅₀、CINAU26-₅₀₀和CINAU27-₆₂₀；5对鹅观草IRk^#1特异标记：CINAU27-₉₆₀、CINAU28-₁₃₆₀、CINAU29-₄₈₀、CINAU30-₅₆₆和CINAU31-₅₂₀。表明可以利用普通小麦的EST序列设计PCR引物，转化成STS标记，筛选普通小麦近缘物种黑麦、簇毛麦及鹅观草等染色体特异的分子标记，这些标记可以用来快速检测和追踪导入普通小麦背景中的黑麦1R、簇毛麦1V及鹅观草1Rk^#1染色体或染色体片段，从而为普通小麦远缘杂种材料进行深入研究提供新的工具。更多还原

【Abstract】 The molecular marker, which was established in the early of 1980s, is a DNA sequence-based genetic marker system. It was now widely used in many research fields, such as genetic map constructing, gene mapping and cloning, marker assistant selection and comparative genomics researches. In present study, different types of molecular markers （SSR, EST, RGA etc.） were used to map the stripe rust resistance gene Yr26 and develop the chromosome-specific markers for chromosome 1R of Secale cereale, 1Rk^#1 of Roegneria kamoji , 1V and 6VS of Haynaldia villosa.1. Development of markers for wheat stripe rust resistance gene Yr26Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating wheat diseases worldwide. Development of resistant cultivars is the most economic, effective and ecological approach to control the disease. However, most of the cultivars with genes for resistance to stripe rust have lost resistance because of the co-evolution of host resistance and pathogen virulence. T. aestivum-H, villosa 6VS/6AL translocation lines were highly resistant to stripe rust with resistance gene Yr26 located on chromosome 1B. In order to better utilize Yr26 in wheat improvement, we attempted to develop SSR and EST-based STS markers more closely linked with Yr26. 35 SSR markers and 81 STS markers derived from EST sequences located on chromosome1B were select to analyse resistant and susceptible bulks as well as their parents. The results showed that 5 pairs of SSR primers, GWM11、GWM18、GWM413、BARC181and WMC419, and 8 pairs of STS primers, WE173、WE171、WE177、WE201、WE202、WE210、WE11and WE17, could amplify polymorphic fragments between resistant and susceptible bulks and their parents. These thirteen markers were used to amplify the DNA of individuals in Yangmai 5/92R137 F₂ population. Most of the susceptible plants amplified the bands as same as that from susceptible parent and most resistance plants amplified the bands as same as that from resistant parents or both. Using Mapmaker 3.0b, the linkage between Yr26 and the 13 markers was detected with genetic distances ranging from 1.4 to 14.1 cM. Among them, nine markers, WE173、WE171、WE177、WE201、WE202、WE210、Xgwm11、Xgwm18 and WEll, showed the genetic distances less 5.5 cM with the Yr26, especially WE173 with genetic distance of 1.4 cM.Using Chinese Spring nulli-tetrasomic, telosomic and deletion lines, the polymorphic markers flanking Yr26, WE173 and Xwmc419, were physically mapped in the long arm of chromosome 1B, and the markers close to Yr26, WE173 and WE201, were mapped in deletion bin 1BL-6. Therefore, gene Yr26 was mapped in deletion bin 1BL-6 near the centromere of chromosome lB.Eight cultivars and advanced lines developed by different institutes used 6VS/6AL translocation lines as resistant parents, were identified using selected markers closely linked to Yr26. Five of them, Nei 2938, Neimai 9, Nei 4221, 95-108 and Nannong 9918, could amplify the specific linked markers, while three derivatives, Shi01Z056, A4-74-8 and A5-82-3-5, can not amplify the markers. The result of molecular assisted selection was in agreement with the result by race inoculation test in the greenhouses. The markers linked to Yr26 developed in this study should be efficient tools for identifying Yr26 in the breeding program.2. Development of markers specific to chromosome 6VS of Haynaldia villosaThe resistance gene of powdery mildew Pm21 was introduced from Haynaldia villosa into common wheat and located on 6VS. In order to precisely map and clone Pm21, more molecular markers and chromosome structural variants need to be developed. 11 RGA and 17 pairs of STS primers were selected to screen polymorphism between wheat-Haynaldia villosa 6VS/6AL translocation and Yangmai 5. One RGA primer and a pair of STS primer amplified polymorphic fragments, about 1000bp and 800bp, respectively. Two pairs of primers, CINAU17 and CINAU18, were designed according to the sequences of two polymorphic fragments. The results indicated that CINAU17-（1086） and CINAU18-（723） were located on chromosome 6VS because only the materials with 6VS could amplify the specific fragments. Furthermore, CINAU17-（1086） was located in the region of 6VS between FL0.58 and FL0.70, and CINAU18-（723） was located in the region between FL0.45 and centromere by using 6VS deletion addition lines and translocation lines. The results obtained by using these two markers were coincided with that by using cytogenetic identification in the structural variants of chromosome 6VS induced by radiating powder. Therefore, CINAU17-1086 and CINAU18-723 markers could be used to rapidly detect the correspondent chromosome segments of 6V and the breakage points of deletion lines.3. Development of chromosome-specific markers for chromosome 1R of Secale cereale, 1V of Haynaldia villosa and 1Rk^#1 of Roegneria kamojiIn order to develop chromosome-specific markers for chromosome 1R of Secale cereale, 1V of Haynaldia villosa and 1Rk^#1 of Roegneria kamoji, 104 STS primers were designed based on EST sequences distributed on chromosome 1A, 1B and 1D of Triticum aestivum. Five STS markers, CINAU 19-500、CINAU20-950、CINAU21-1500、CINAU22-310 and CINAU23-2000 amplified from S.cereal, T. aestivum-S, cereale amphiploid and 1R addition line, but not from CS and 2R-7R addition lines, were specific to 1R of S. cereale. Five STS markers, CINAU23-1700、CINAU24-1050、CINAU25-1650、CINAU26-500 and CINAU27-620 were specific to 1V of H. villosa because of the corresponding fragments amplified only from H. villosa, T. durum-H, villosa amphiploid and 1V addition line, not from CS and 2V-7V addition lines. And five STS markers, CINAU27-960、CINAU28-1360、CINAU29-480、CINAU30-560 and CINAU31-520 specific to 1Rk^#1 of R. kamoji were screened owing to specific fragments amplified from R. kamoji, Wheat-R. kamoji DA1Rk^#1 and DS1Rk^#1（1A）. The result indicated that STS markers derived from the EST of common wheat were useful tool for identify corresponding specific chromosomes of wheat relatives.更多还原