【作者】 代程；

【导师】 高爱农；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2012， 硕士

【摘要】 冰草6P上含有很多可用于小麦改良的优异基因。因此小麦-冰草6P易位系的鉴定、遗传效应分析以及冰草6P特异分子标记的开发对于冰草6P的遗传研究具有重要意义。SM2-244是一株表现多花多粒的材料，由Luan鉴定为大小片段相互易位，是由藁城8901与普通小麦-冰草(Agropyron cristatum L. Gaertn，2n=4x=28, PPPP)6P附加系4844-12杂交获得的种子进行电离辐射获得的，并对其后代进行了GISH、FISH和P基因组特异标记分析确定出现的各种易位类型。利用本实验室开发的EST标记筛选新的冰草6P特异分子标记，将这些标记在获得的易位片段上进行定位，另一方面通过这些特异分子标记确定获得的易位片段之间的关系。同时对获得的不同的小麦-冰草6P易位系的农艺性状进行考察。分析易位片段在小麦背景下所表现出的遗传效应。研究结果如下：1、利用本实验室开发的EST-STS引物，获得了130个冰草6P特异分子标记。2、通过原位杂交（ish）和分子标记技术对一个表现多花多粒的小麦-冰草6P易位系SM2-244的自交后代进行鉴定，获得了4类不同的小麦-冰草6P易位系，分别为大片段易位系T6PS.6PL-5AL、小片段易位系T6PL-5AL.5AS、小片段纯合易位材料T6PL-3BS.3BL以及一个小片段纯合+臂端纯合易位系T6PS-5AL.5AS+T6PS-5AL。其中大片段易位系和小片段易位系中的易位片段是由一条完整的6P断裂形成的；一个小片段纯合+臂端纯合易位系在染色体组成上相当于6PL整臂附加；小片段易位系T6PL-5AL.5AS中易位片段为小片段易位系T6PL-5AL.5AS中易位片段的一部分。这些易位系中易位片段大小不同，可将6P分为4个部分。130个6P特异分子标记分布在6P短臂上为56个；分布在6P长臂1.00-0.69区段上24个，0.69-0.32区段上30个，0.32-0.00区段上20个。3、对不同的易位系的生物学性状进行考察，结果表明小片段易位系T6PL-5AL.5AS的穗长和小麦亲本相比表现极显著增加；在小穗粒数及穗粒数上表现显著增加；大片段易位系表现穗粒数减少，但是穗部形态和冰草相似。两者的小穗粒数及穗粒数和小麦-冰草附加系4844-12相比还有很大差距。说明在冰草6P的长臂和短臂上都含有多花多粒基因，在长臂1.00-0.32区段上有可以显著增加穗长的基因。4、对这些特异标记与NCBI nr蛋白质数据库及小麦EST序列进行了比对，发现4条冰草EST序列的功能注释与抗病、抗逆相关；36条冰草EST与已经定位的小麦EST具有较高的相似性，其中33条（91.67%）位于小麦第６部分同源群染色体。进一步对这些标记进行特异性验证，结果证明其确实可用于检测6P染色体。本研究获得了130个可以分布在冰草6P上的特异EST标记和4个不同的小麦-冰草异源易位系，对于小麦-冰草6P异源易位系的检测以及6P染色体的遗传分析具有重要意义；通过对不同易位系的生物学性状进行调查，认为在冰草6P长臂上携带有控制小麦穗长的基因，而在6P的长臂和短臂上都含有控制小麦的小穗粒数以及穗粒数的基因。更多还原

【Abstract】 Agropyron cristatum (L.) Gaertn chromosome6P contains a lot of excellent genes for wheatimprovement. So it is very important for the genetic research of6P chromosome to identify wheat-Agropyron6P translocation lines and develop molecular markers specific to6P. In this study, wedeveloped EST markers specific to the A. cristatum6P chromosome using the wheat-A. cristatum6Pdisomic addition line4844-12(2n=44) and its common wheat parent ‘Fukuho’ as well as A. cristatumaccession Z559(2n=4X=28, PPPP), then detected the offspring of wheat-Agropyron6P addition linesand common wheat‘Gaocheng8901’with these markers. Positive plants which showed positive by6P-spicific markers were detected by GISH and FISH to identify their type of translocation. Locating the6P-specific molecular markers with these wheat-A. cristatum6P translocation lines， which is used todetermine the relationship among these6P translocation fragments. At the same time，traits of obtainedwheat-A. cristatum6P translocation lines were conducted to analyze the genetic effects brought bytranslocation fragments in common wheat background. The results are as following:1. A total of1306P-specific molecular markers were developed from1453PCR primers designedaccording to the sequences of ESTs gained from the transcriptome sequencing of A. cristatum.2. four wheat-Agropyron translocation lines were found in the self-progeny offspring of SM2-244by in situ hybridization (ISH) and EST markers, which are one large-fragment translocation line27-9(T6PS.6PL-5AL), one small-fragment translocation line37-1(T6PL-5AL.5AS)，small-fragment line65-5(T6PL-3BS.3BL) and one arm-side additional translocation line9-1(T6PL-5AL.5AS+T6PS.6PL).The translocation fragments from27-9and37-1constitute a completely6P chromosome. Thetranslocation fragment from65-5were confirmed to be one part of37-1′s by EST markers. The threetranslocation lines divided6P chromosome into four parts: there are56markers located on the short armof6P chromosome,20markers located on the0.00-0.32section of long arm of6P chromosome,30markers located on the0.32-0.69section of long arm of6P chromosome,24markers located on the0.69-1.00section of long arm of6P chromosome.3. The biological characteristics of different translocation lines shows that small-fragmentstranslocation line37-1has longer spikes, more grains per spikelet and spike compared to wheat parent.While large-fragment translocation line27-9has shorter spikes, less grains per spikelet and spikecompared with wheat parent, but its spike type is similar with wheat parent. Which suggests that the twoalien segments carries spike-related genes. The number of kernels per spike of the two translocation linesare much less than wheat-A. Cristatum6P disomic addition line4844-12(2n=44), and the reason ofcausing superior numbers of florets and kernels per spike is complicated. There are some genes relatedto spike length on the0.32-1.00section of long arm of6P chromosome.4. These specific markers were matched with protein database from NCBI and wheat EST sequenceto understand the potential function genes on chromosome6P and the gene synteny between A. cristatum and common wheat. The results showed that the function annotation of four A. cristatum EST sequencesare related to disease and stress resistance; Thirty six A. cristatum EST sequences could be matched withthe located wheat EST bin map, in which33(91.67%) sequences were located in the sixth homoeologousgroup.Conclusion:1306P-specific molecular markers and4wheat-A. Cristatum6P translocation lineswere gained in this study, which is helpful to the6P chromosome genetic research. The genes causingsuperior numbers of florets and kernels per spike may exist on both of the long and short arm. Here aresome genes related to spike length on the0.32-1.00section of long arm of6P chromosome.更多还原