【作者】 王欣欣；

【导师】 卢萍；

【作者基本信息】 内蒙古师范大学 ， 植物学， 2016， 硕士

【摘要】 老芒麦(Elymus sibiricus L.)是一种广泛分布于我国北部和西北部等高寒地区的优质牧草,为牲畜所喜食,具有重要的营养价值和生态价值。老芒麦种质主要以种子形式保存在低温种质库中,然而低温保存只能延长贮存寿命,长时间保存会发生老化现象,老化降低种质发芽率,导致生理生化特性改变,蛋白质和DNA分子水平也可发生变异,种质贮藏一定时间后须繁殖更新,以维持遗传完整性。本研究以老芒麦“农牧”、“川草1号”2份育成品种和“01090”、“03658”2份野生材料为研究对象,利用高温(40℃)高湿(70% RH)老化法,获得不同发芽率(80%、50%和30%)的种子,以未经老化处理发芽率为90%以上种子作为对照,分别利用醇溶蛋白标记和ISSR分子标记研究种子老化对老芒麦种质资源遗传完整性变化的影响,为老芒麦种质资源的保存和繁殖更新提供基础数据。主要研究结果如下：1.4份老芒麦材料随老化时间的延长发芽率下降,不同材料老化至同一发芽率水平时所需时间不同。2份育成品种材料比野生材料所需的时间长：发芽率降至80%、50%和30%左右时,老化时间分别为4d、6d和7d；而野生材料老芒麦“01090”所需时间为4d、5d和6d, “03658”为3d、 4d和5d。2.用醇溶蛋白标记对4份材料的不同老化处理群体进行遗传完整性变化分析结果表明：种子老化使种质醇溶蛋白条带数发生变化,主要表现在γ和ω区；种质群体的遗传参数如等位基因数、有效等位基因数、Nei’s基因遗传多样性和Shannon多样性指数均低于对照群体的各参数,且随发芽率降低呈下降趋势；当发芽率降至30%左右时,4份材料每个位点等位基因数与对照相比差异显著,老芒麦“农牧”和“川草1号”的遗传多样性与对照相比差异显著,老芒麦“01090”香农指数与对照相比差异显著。3.用ISSR标记对4份材料的不同老化处理群体进行遗传完整性变化分析结果表明：13个ISSR引物扩增出108条带,多态性位点总数为93,平均多态位点百分率为86.09%；老化后种质基因组DNA在250bp-500bp之间和750bp-2000bp之间的条带发生缺失或增加；对照群体的遗传参数如等位基因数、有效等位基因数、Nei’s基因遗传多样性、Shannon多样性指数和多态位点百分数高；随老化处理时间延长,各群体的遗传参数逐渐降低。当发芽率降至30%左右时,4份种质材料每个位点等位基因数和Nei’s基因遗传多样性指数与对照相比差异显著。认为当发芽率降低到30%左右时,可对老芒麦种质进行繁殖更新。4.醇溶蛋白标记和ISSR标记都可用来检测种子老化影响老芒麦种质遗传完整性的变化,两种标记的遗传一致度相关性分析结果呈极显著正相关。ISSR标记的多态位点数多于醇溶蛋白标记,因此ISSR标记更合适。更多还原

【Abstract】 Elymus sibiricus L. is a kind of high-quality forage widely distributed in north and northwest alpine region of China. With important nutritional and ecological values, it is stored mainly as seed form at low-temperature germplasm library. However, it only prolongs the shelf-life of cryopreservation, aging could be caused by long-term storage. Seed aging will not only reduce the germination rate of germplasm, but also lead to physiological and biochemical characteristics changes, and genetic variations of protein and DNA might be aroused. The germplasm storage shall be updated after a certain time in order to maintain the genetic integrity.In this research Elymus sibiricus L. bred varieties (Elymus sibiricus L. cv Nongmu, Elymus sibiricus L. cv Chuancao No.1) and two wild materials (Elymus sibiricus L.01090, Elymus sibiricus L.03658) were selected. The seeds of them were treated in the artificial aging conditions at high-temperature (40℃) and high humidity (70% RH) of aging method to obtain different germination rate (80%,50% and 30%) by taking the seeds with more than 90% germination rate with no aging treatment as a control. Elymus sibiricus L. were used to study effects of seed aging in the alteration of genetic integrity by gliadin and ISSR molecular markers. The purpose of this study was to provide foundation for making a criteria of preserving and regenerating germplasm resources to Elymus sibiricus L.. The main results were showed as follows:1.Four Elymus sibiricus L. materials with different germination rate presented the declined trend with the increase of aging time. The time was different when the aging of different materials got to the same germination rate. The time of two bred varieties required was longer than that of the wild materials. The aging time for the bred varieties was 4d,6d and 7d when germination rate dropped to about 80%,50% and 30%. However, the aging time for the wild materials 01090 was 4d,5d and 6d, while the aging time for the wild materials 03658 was 3d,4d and 5d.2.The results of effects on genetic integrity of different seed aging treatments to four Elymus sibiricus L. materials by gliadin markers showed that numbers of gliadin bands changed after aging mainly in areas of γ and ω. The genetic parameters of the germplasm population such as the number of alleles, the number of effective alleles, index of genetic diversity and Shannon’s information index decreased compare to that of the control with the decreasing of germination rate. When germination rate dropped to about 30%, the number of alleles per loci of four materials showed significant differences compare to that of control. The index of genetic diversity of Elymus sibiricus L. cv Nongmu and Elymus sibiricus L. cv Chuancao No.1 showed significant differences compare to that of control, and the Shannon’s information index of Elymus sibiricus L.01090 showed significant difference compare to that of the control.3.The results of effects on genetic integrity of different seed aging treatments to four Elymus sibiricus L. materials by ISSR markers showed that 108 bands were amplified by using 13 primers, of which 93 loci were polymorphic, and the average percentage of polymorphic loci was 86.09% for the test materials. Bands of 250bp-500bp and 750bp-2000bp appeared or disappeared after aging. The genetic parameters of the control such as the number of alleles, the number of effective alleles, index of genetic diversity, Shannon’s information index and the percentage of polymorphic loci were higher than that of the aging populations. The genetic parameters of population reduced gradually with the increase of aging time. When the germination rate dropped to about 30%, the number of alleles per loci and the index of the genetic diversity of four materials showed significant differences compare to that of control. The germplasm of Elymus sibiricus L. should be suggested to update when the germination rate dropped to about 30%.4.Both gliadin markers and ISSR markers could be used for detecting effects on genetic integrity of seed aging for Elymus sibiricus L. germplasm resources. The results of the correlation analysis of genetic identity of the two markers showed highly significant positive correlation. The number of polymorphic loci detected by ISSR markers was more than that of polymorphic loci detected by gliadin markers. Therefore, ISSR markers are more applicable.更多还原