利用ISSR及SRAP分子标记研究菜用大黄与药用大黄的亲缘关系

发布时间：2018-02-24 09:48

本文关键词： 大黄 ISSR SRAP 体系优化亲缘关系分析　出处：《河南科技学院》2017年硕士论文　论文类型：学位论文

【摘要】：菜用大黄(Rheum rhaponticum L.)属于多年生草本植物,其叶柄粗壮多汁,富含纤维素A、维生素C、钙、磷以及琥珀酸等营养物质,是理想的保健蔬菜,在欧洲、北美和大洋洲地区大面积用于商业栽培。十七世纪,中国药用大黄被引进至欧洲后经过长期的自然进化演变为菜用大黄,然而关于菜用大黄与药用大黄亲缘关系未见报道。本研究对22份大黄品种(6份菜用大黄和16份药用大黄)进行了种子形态学观测和ISSR及SRAP分子标记分析。通过菜用大黄ISSR-PCR反应体系的优化及验证、ISSR及SRAP标记引物的筛选、亲缘关系分析等研究,对菜用大黄和药用大黄的亲缘关系进行了研究,并初步揭示菜用大黄的起源,为课题组今后的育种工作提供重要的理论依据。主要研究结果如下:(1)建立了菜用大黄ISSR-PCR最佳反应体系。即10×PCR Buffer2.5μL,模板DNA125ng,Mg2+2.5mmol/L,dNTPs 0.35mmol/L,引物0.5μmol/L,Taq DNA聚合酶0.04U/μL,反应体系的总体积为25μL。(2)通过对22个大黄品种的种子长度、宽度、翅宽和千粒重四个形态指标进行测量,利用SAS v9.2软件进行聚类分析。结果显示当最大距离为3.5时,可以将22份材料划分为4类,其中RT品种的种子在四个指标上明显与其他品种存在较大的差异。(3)利用菜用大黄品种R34和药用大黄品种SC分别对100条ISSR引物和182对SRAP引物组合进行筛选,共获得多态性好,谱带清晰,结果重复性高的ISSR引物13条、SRAP引物组合32对。ISSR-PCR扩增共获得74个等位基因位点,其中59个是多态性位点;SRAP-PCR扩增共计获得227个条带,其中206条具有多态性。结果表明这两种分子标记均能提供丰富的遗传信息,均可用于大黄遗传多样性分析。(4)利用POPGENE1.32软件对22份大黄品种进行遗传相似性和遗传距离分析,ISSR-PCR分子标记得出大黄各品种间的遗传相似系数在0.3220-0.8983之间,遗传距离在0.1072-0.9865之间;其中,SC和YN遗传相似性系数最大,说明这两者之间的亲缘关系最近;土大黄和RG的相似性系数最小,表明这二者之间的亲缘关系最远。SRAP-PCR分子标记得出大黄品种间的遗传相似性系数在0.3301-0.8495之间;其中,RG和RO遗传相似性系数最大,说明这两者之间的亲缘关系最近;南大黄和RO的相似性系数最小,表明这二者之间的亲缘关系最远。(5)利用NTSYS pc2.10c软件对22份大黄品种的亲缘关系进行聚类分析,基于ISSR及SRAP两种标记分析数据综合聚类结果,在相似性系数0.60处可将22份材料分成4类。第一类为R54、R34和RO,均为菜用大黄;第二类包括川大黄、苦大黄、毛大黄、R19、SC、ZJ、HN、GZ和HB,除R19外均为药用大黄;第三类为南大黄、西宁大黄、山大黄、马蹄大黄、RG、RT、HU、SX和YN,其中,RG和RT为菜用大黄;土大黄单独聚在了第四类。
[Abstract]:Rheum rhaponticum L.) is a perennial herb with a thick, juicy petiole, rich in nutrients such as cellulose A, vitamin C, calcium, phosphorus and succinic acid. It is an ideal health vegetable in Europe. In 17th century, Chinese medicinal rhubarb was introduced to Europe and evolved into vegetable rhubarb after a long period of natural evolution. However, the relationship between vegetable rhubarb and medicinal rhubarb was not reported. In this study, the seed morphology and ISSR and SRAP molecular markers of 22 rhubarb varieties (6 vegetable rhubarb and 16 medicinal rhubarb) were observed. The ISSR-PCR reaction system of rhubarb and the screening of SRAP marker primers were optimized and verified. The relationship between vegetable rhubarb and medicinal rhubarb was studied, and the origin of vegetable rhubarb was preliminarily revealed. The main results are as follows: 1) the optimal reaction system of Rhubarb ISSR-PCR for vegetable is established, that is, 10 脳 PCR Buffer2.5 渭 L, mg 22.5 mmol / L mg 22.5 mmol / L, primer 0.5 渭 mol / L Taq DNA polymerase 0.04 U / 渭 L, primer 0.5 渭 mol / L Taq DNA polymerase 0.04 U / 渭 L. The seed length of 22 rhubarb varieties was determined by 25 渭 L. The width, wing width and 1000-grain weight were measured and cluster analysis was carried out with SAS v9.2 software. The results showed that 22 samples could be classified into 4 groups when the maximum distance was 3.5. The seeds of RT varieties were obviously different from other varieties in four indexes.) 100 ISSR primers and 182 pairs of SRAP primer combinations were screened by using R34 and SC, respectively, and a good polymorphism was obtained. The results showed that there were 74 alleles in 32 pairs of ISSR primer combinations with high reproducibility, 59 of which were polymorphic loci and 227 were amplified by SRAP-PCR. Of these, 206 were polymorphic. The results showed that both of these markers could provide abundant genetic information. It can be used to analyze the genetic diversity of Rhubarb. 4) the genetic similarity and genetic distance of 22 Rhubarb cultivars were analyzed by POPGENE1.32 software. The genetic similarity coefficient was 0.3220-0.8983 and the genetic distance was 0.1072-0.9865. The genetic similarity coefficient of SC and YN is the largest, which indicates that the genetic relationship between them is the closest, and the similarity coefficient between Rhubarb and RG is the smallest. The genetic similarity coefficient was 0.3301-0.8495, and the genetic similarity coefficient between RG and RO was the largest, which indicated that the genetic relationship between RG and RO was the most recent, and the genetic similarity coefficient of RG and RO between the two cultivars was the most distant, which indicated that the genetic similarity coefficient was between 0.3301-0.8495 and 0.3301-0.8495, respectively. The similarity coefficient between South rhubarb and RO was the smallest, indicating that the genetic relationship between the two cultivars was furthest. The genetic relationship of 22 Rhubarb cultivars was analyzed by NTSYS pc2.10c software. The clustering results were synthesized based on the data of ISSR and SRAP markers. At the similarity coefficient of 0.60, 22 samples can be classified into four categories. The first type is R54, R34 and RO. they are both vegetable rhubarb, the second are Radix et Rhizoma et Rhizoma Chuan, Radix et Rhizoma Kuihuang, Radix et Rhizoma Rhei, R19SCC, JJ, HNN, GZ and HBB, all of which are medicinal rhubarb except R19, the third category is Nanda Huang and Xining Rhubarb. Radix rhubarb, Rhizoma et Rhizoma horseshoe, RGG, RGG, HUU, SX and YN, among them, RG and RT are used as vegetable rhubarb, and Rhubarb is clustered in 4th classes alone.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S644.9;S567.239

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