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小麦抗叶锈基因Lr38的RAPD分子标记

标题: 小麦抗叶锈基因Lr38的RAPD分子标记
英文标题: Development of a RAPD Molecular Marker for the Leaf Rust Resistance Gene Lr38 in Wheat
作者: 张翠茹
出版时间: 2001-01-01
所在大学: 河北农业大学
关键词: 小麦,抗叶锈病,基因,RAPD,分子标记
英文关键词: wheat,leaf rust,resistance gene,RAPD,molecular marker
论文级别: 硕士
学位: 学位论文
导师: 刘大群
专业: 植物病理学
提交时间: 2001
摘要: 随机扩增多态性DNA(RAPD)是近年来发展起来的一种分子标记技术,本研究利用这种技术对Thatcher及已知的21个以Thatcher为遗传背景的小麦抗叶锈病近等基因系(NIL)及7个不同的小麦品种进行了分析,结果如下: 1.建立了适合本实验室应用的小麦RAPD分析的优化反应体系及反应程序.研究以29份小麦基因组DNA为模板,对RAPD反应程序中的一些重要参数进行摸索和优化试验,通过与以前的研究结果进行比较,提出小麦RAPD分析最佳反应体系的建立应根据实际情况,对影响扩增的各种因素进行调整,建立一套适合本实验室使用的应用体系及反应程序.本研究所用反应体系及反应程序为:25ul反应体系中含有10mM KCl、8mM(NH_4)_2SO_4、10mMTris-HCl(pH9.0)、NP-40、2.0mM Mg~(2+)、200μM dNTP、45ng引物、20ng模板DNA、1.5U Taq DNA聚合酶.反应程序为第一个循环基因组DNA经94℃预变性5min,后40个循环改为94℃变性30sec,37℃退火60sec,72℃延伸90sec,最后一个循环完成后,在72℃下延伸8min,最后在4℃下保温. 2.筛选出一个与Lr38抗病基因连锁的RAPD标记S33-800.本研究采用160个随机引物对小麦抗叶锈近等基因系进行了分析,其中绝大多数引物均能扩增出1~9条明显可辨的条带.第一次初选时,有4个引物S13(TTCCCCCGCT)、S30(GTGATCGCAG)、S33(CAGCACCCAC)和S196(AGGGGGTTCC)在小麦抗叶锈基因Lr38的抗、感近等基因系间表现多态性,但在随后对此4个引物的重复实验中,只有引物S30和S33经5次以上重复,均获相同的结果,表现了较好的扩增稳定性,其多态性标记分别命名为S30-200、S33-800.当用这两个引物对已知含Lr38基因的抗病材料远中4号及其它不含Lr38基因的感病材料进行检测时,标记S30-200不能将不同遗传背景中的Lr38基因检测出来,表明引物S30-200的特异性差,同时说明S30-200的结合位点与Lr38基因不连锁.而标记S33-800可以从抗病材料远中4号中检测到这一条800bp的多态性片段,而在其它感病材料中,这条800bp的条 小麦抗叶锈基因Lr38的MPD分于标记带表现缺失,表明标记533-800与Lr38基因连锁,该RAPD标记与Lr38基因连锁的紧密程度尚有什于通过F.分离群体分析进一步确定. 3.回收了一条800hP 的特异性片段.采川热溶化-苯酚-氯仿抽提法对引物533在Lr38/6*Thatcher中扩增出的800hp的特异性片段进行回收,回收片段经相应引物进行扩增后,用琼脂糖电泳检测,结果显示,条带清楚,特异性好,反应物中的引物和非特异性产物已去除干净.
英文摘要: The random amplified polymorphic DNA (RA PD) analysis was carried out in Thatcher, near-isogenic lines carrying twenty-one different genes conferring resistance against leaf rust and seven different varieties. The main results were as follows: 1. The optimized RAPD reaction system was established. In a total volume of 25 LI I, containing 10mM KCI, 8mM(NH4)2S04, 10mM Tris-HCI(pH- 9.0), NP- 40, 2.0mM Mg2~, 200 Li M dNTP, 45ng primer, 20ng template DNA, I.5U Taq DNA polymerase. Amplification performed in a AmpGene DNA Thermal Cycler 4800 was programmed for 5 mm at 94C, 40 cycles of 30s at 94t, 60s at 37t, 90s at 72℃, and a final extention at 72C for 8 mm after the last cycle. 2. One polymorphic RAPD marker (S33-800) linked to leaf rust resistance gene Lr38 was screened. All of the 160 random primers screened for RAPD analysis gave clear amplification products, 4 of them amplified the polymorphic DNA in the NILs Thatcher and Lr38/6°hatcher when screened first time. Further investigation demonstrated that primers S30 and S33 showed the same discriminating results in more than five replications. The polymorphic bands were named as S30-200, S33-800, respectively. Using these two primers to detect a resistant material with Lr38 and other susceptible material without Lr38, it was shown that only S33-800 could be amplified in the resistant material and absent in all of the susceptible materials. This demonstrated that the marker S33-800 was linked to resistant gene Lr38. Further research will be focused on RAPD analysis of F2 population using primer S33 in order to evaluate the genetic distance between S33-800and Lr38 resistant gene. 45 3. The 800bp specific fragment was recoveried. The 800bp specific fragment, which was amplified in Lr38/&Thatcher with primer S33, was recoveried using phenol-chloroform extraction method, and then amplified with the above-mentioned primer S33. The amplification product was separated in 1.4%agarose gels and visualized by ethidium bromide staining. A clear band was gained and it implied that the primer and the non-specific products have been clean up.