【作者】 沈紫微；

【导师】 陈本建； 张蕴薇；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2010， 硕士

【摘要】 本研究以航天搭载红豆草种子后代及地面对照为材料,结合生物学性状测定和ISSR（简单重复间序列）分子标记技术,分析供试红豆草的航天诱变效应。主要研究结果如下:1.航天诱变红豆草生物学性状测定:经田间观测,筛选出在株高、每复叶小叶数、叶片大小、厚度、叶色、分枝数、株型、特殊变异（叶表面被白毛）8个性状上发生变异的诱变单株。据统计,航天搭载的红豆草后代植株发生株型变异（匍匐型）的单株数量最多,占搭载总数的9.92%,特殊变异（叶表面被白毛）的单株数量最少,仅占搭载总数的0.94%。运用SPSS软件对株高、每复叶小叶数、叶片大小、厚度、叶色这5个性状进行差异显著性分析,结果表明,航天诱变对株高变异影响很大,与对照相比,差异极显著单株占测定总数的100%,而每复叶小叶数的变异单株中仅有个别单株表现出一定的差异,差异极显著单株占测定总数的16.7%。2.红豆草ISSR-PCR反应体系的建立:对引物、Mg²⁺、dNTPs、Taq DNA聚合酶四种ISSR-PCR反应体系的影响因素进行L9（3⁴）正交试验,获得红豆草ISSR-PCR最佳反应体系:2.5μL 10×PCR buffer、50 ng模板DNA、dNTP 0.2 mmol/L、Taq DNA聚合酶1.5 U、引物0.4μmol/L、Mg²⁺2.0 mmol/L,总体积25μL。反应扩增程序:94℃预变性3min,94℃变性30s,46℃-60℃（退火温度因引物不同而不同）退火30s,72℃延伸1min,进行35个循环,最后72℃延伸10min。3.ISSR引物的筛选与PCR扩增:在试验确定的最佳反应体系基础上,对53条ISSR引物经过两轮PCR扩增,筛选出12条扩增条带较多、信号强、背景清晰的引物。将供试红豆草材料经过12个引物扩增,结果显示,记录的条带大小范围在300-1000 bp之间,统计表明共获得228个扩增位点,其中多态性位点169个,多态性比率为74.1%,每个引物的扩增位点为13-29个,平均19个,其中多态性位点9-22个,平均14.1个。4.利用POPGENE32软件分析航天搭载群体和对照群体之间遗传变异的差别,结果表明搭载植株的多态位点比率、Nei基因多样度和Shannon多样性指数均高于地面对照,说明航天诱变增加了红豆草遗传变异,证明了空间环境对植物基因位点的影响。5.通过遗传相似系数（GS）的计算,供试材料间的GS值在0.243-0.838之间,表明供试材料的遗传基础较宽,说明航天诱变使材料间遗传差异变大。基于遗传相似系数的主成分分析显示,大部分材料与对照材料的相对位置较远,表明经过航天搭载的植株发生了较大的遗传变异,在亲缘关系上与对照产生了较大的分离。采用遗传相似距离将对照植株及随机抽取的不同变异类型中的变异单株作聚类分析,结果表明具有相同变异的单株大部分能聚为一类;另外,对各类性状变异单株及其对照进行UPGMA聚类分析,分别获得了与对照遗传关系较近和较远的变异单株,为红豆草品种选育、改良和种质资源评价提供了参考依据。更多还原

【Abstract】 The biological traits and the genetic diversity of Sainfoin dealt with space irradiation were analysed by the survey of the biological traits and ISSR markers. The results were as follows:1. The biological traits of Sainfoin mutants from seeds carried back by“Shijian No. 8”spaceship were studied in 8 biological traits, as height, number of leaflets per compound leaf, leaf size, leaf thickness, leaf color, branch number, plant type and the special variation. The results show that the number of plants of plant type variation was great. 5 biological traits of Sainfoin mutants from seeds carried back by”Shijian No.8”spaceship were studied using SPSS.The results show that significant difference existed in the height, blade thickness and leaf color of Sainfoin mutants treated by space irradiation. However, the variation of Sainfoin mutants in anoter biological traits was not clearly very much, such as the variation of the number of leaflets per compound leaf and blade width. In all of Sainfoin mutants, several variations were discovered on some ones.2. Establishment of ISSR-PCR reaction system for Onobrychis viciaefolia Scop.: An orthogonal design was used to optimize a ISSR-PCR system with 4 factors (dNTP, Taq polymerase, primer and Mg²⁺) at 3 levels plus the concentration of template DNA. The result shows that the optimized ISSR-PCR system for sainfoin was: 2.5μL 10×PCR buffer, 50 ng template DNA, dNTP 0.2 mmol/L, Taq DNA polymerase 1.5 U, primer 0.4μmol/L, Mg²⁺2.0 mmol/L in a total of 25μL reaction solution. The reaction process went as following: the temperature profile used for PCR was 94℃for 3 minutes, followed by 35 cycles of 94℃for 30 seconds, 46℃-60℃for 30 seconds, 72℃for 1minute, and was terminated with a 10 minutes DNA extension step at 72℃,then keep in 4℃.3. The selecting of ISSR primers and amplification of PCR: There were 12 ISSR primers by sieving which can produced DNA polymorphism in 53 ISSR primers. The result shows that the size of all the bands was in 300-1000 bp. In all experimental materials, 228 DNA bands were amplified by 12 ISSR primers, in which 169 bands were polymorphic, polymorphic ratio （PPB） was 74.1%, and the average of bands were 19 by every primer.4. The genetic variation of group was studied by POPGENE32. The percentage of polymorphic loci（P）,Nei’s gene diversity（h） and Shannon’s index（I） in Sainfoin of the control was lower than that of the plants carried by spaceflight. It shows variation of hereditary substance DNA of Sainfoin can be caused by space environment. These results indicated that the space radiation would be an effective method to develop new Sainfoin germplasm.5. The ISSR-based genetic similarity values among all experimental materials accessions ranged from 0.243-0.838. Abundant genetic diversity among Onobrychis viciaefolia Scop. accessions could be revealed by ISSR markers. Based on the UPGMA cluster analysis and PCA, the 29 materials with different biological traits variation may be divided into six groups, most of genetic variation was within the same group. There was a certain relationship between genetic distance and geographic distance among different Sainfoin variants in the experiment.更多还原