常用菌草的细胞核型和ISSR分析

发布时间：2018-01-03 17:41

本文关键词：常用菌草的细胞核型和ISSR分析　出处：《福建农林大学》2015年硕士论文　论文类型：学位论文

【摘要】：为了构建菌草种质资源圃,完善菌草种质的信息,为进一步的研究提供理论基础,因此探讨常用菌草的亲缘关系及构建其DNA指纹图谱成为亟需解决的问题,然而异地引种导致了命名混乱。本文主要内容有常用菌草的狼尾草属中24种植物ISSR遗传多样性研究及采用传统压片法对象草(Pennisetum purpureum)和巨菌草(Pennisetum spp.)的细胞染色体核型分析。目的是从分子水平上探讨狼尾草属植物间的亲缘关系,为其遗传育种及大面积推广应用奠定理论基础；从细胞和分子水平上为鉴定巨菌草品种提供理论依据。具体结果如下：1)优化象草的核型分析的反应条件本研究采用传统压片法进行染色体分析,分别比较取材部位、预处理、解离,选择最优的条件。结果表明：取材部位为根尖,预处理的方法是0.05%秋水仙素处理2h,1 M盐酸解离8 min,为最优结果。2)象草和巨菌草染色体核型分析象草和巨菌草的染色体形态较为一致,大部分由中部(m)和近中部(sm)着丝粒的染色体组成,其中象草和巨菌草的核型公式分别为2n=4x=28=20m+8sm、 2n=4x=28=16m+8sm+4st;象草和巨菌草染色体相对长度组成分别为2n=4x=28=6L+6M2+10M1+6S、2n=4x=28=4L+8M2+10M1+6S。研究认为：象草和巨菌草的不对称系数都接近50%,均属于较原始的类型,核型分类均为2A型。3)采用单因素实验建立了象草ISSR-PCR引物UBC847的反应体系及程序和优化其他引物的退火温度引物UBC847的反应体系：在20μL的反应体系中,引物浓度0DNA量40 ng。反应程序：95℃预变性2min,95℃变性45s,50.4℃退火45s,72℃延伸60s,35个循环,72℃延伸5 min,4℃保存。保持反应体系及程序,只设定退火温度变化范围,优化其他77条引物的退火温度。4)ISSR标记的聚类分析采用ISSR分子分析狼尾草属24个品种间的遗传关系,结果表明：从78条引物中共筛选出13个引物,共扩增出303条带,其中多态性条带为297条,多态位点达98.0%,13条引物扩增谱带范围为15～37条,分布范围较宽,每条引物扩增26.9个多态性条带。实验表明在这24个品种间具有较高的遗传多样性。利用UPGMA聚类分析结果,当相似性系数为0.826时可分为4类。5)阐述了巨菌草和象草的亲缘关系巨菌草和象草染色体数目是一致的,形态有些差别即在染色体核型公式和组成上有一些差别,无明显的差异,巨菌草的进化程度高于象草。结合ISSR分析中两者的相似性系数为0.814,被分为同一大类。核型分析和ISSR分析结果较为一致,这两者有一定的亲缘关系。
[Abstract]:In order to improve the construction of JUNCAO germplasm resources, Germplasm JUNCAO information, provide a theoretical basis for further study, so the study on genetic relationships and build the common JUNCAO DNA fingerprint has become a urgent problem, however, led to the introduction to the main contents in this paper. Naming confusion is commonly used in the grass Pennisetum 24 plant genetic ISSR research on diversity and the traditional compression method using object (Pennisetum purpureum) and grass Jujun grass (Pennisetum spp.) analysis of chromosome karyotype. Aims to explore the relationship between Pennisetum plants at the molecular level, which lays the theoretical foundation for the popularization and application of the genetic breeding and large area; from the cellular and molecular level and provide a theoretical basis for the for the identification of Jujun grass varieties. The main results are as follows: 1) the reaction condition optimization of Elephantgrass karyotype analysis in this study using the traditional compression method of dyeing Analysis, compare the different parts, preprocessing, dissociation, select the optimum conditions. The results show that the derived part is the root, the pretreatment methods of 0.05% colchicine 2h, 1 M hydrochloric acid dissociation of 8 min,.2) for optimal results of karyotype analysis of chromosome Elephantgrass and Jujun grass like morphology of grass and Jujun grass is the most consistent, central (m) and (SM) composition near middle centromere chromosome, the karyotype formula of Elephantgrass and Jujun grass were 2n=4x=28=20m+8sm and 2n=4x=28=16m+8sm+4st; the relative length of Elephantgrass and Jujun grass chromosomes were 2n=4x=28=6L+6M2+10M1+6S, 2n=4x=28=4L+8M2+10M1+6S. study shows that the asymmetry coefficient of Elephantgrass and Jujun grass are close to 50%, belong to the type is original, karyotype classification were 2A type.3) by single factor experiment established the reaction system and program of elephant grass and optimize their ISSR-PCR primers UBC847 The reaction system UBC847 primer primer annealing temperature he: in the reaction system of 20 L, 40 0DNA primer concentration ng. reaction procedure: 95 C predegeneration 2min 95 C 45s 45s degeneration, annealed at 50.4 C, 72 C extension 60s, 35 cycles, 72 degrees extension 5 min, 4 degrees to save. Keep the reaction system and procedures, only set the range of annealing temperature, annealing temperature of.4 to optimize other 77 primers) clustering analysis with ISSR markers by ISSR molecular analysis of Pennisetum genetic relationship among 24 varieties. The results showed that: from 78 primers were screened out of 13 primers, 303 bands were amplified, among them 297 polymorphic bands, 98% polymorphic loci, amplified bands ranged from 15~37 to 13 primers, wide distribution, amplified 26.9 polymorphic bands per primer. The experimental results show that the genetic diversity was high in the 24 varieties. The use of UPGMA cluster analysis results, when The similarity coefficient of 0.826 can be divided into 4 categories:.5) describes the number of Elephantgrass Jujun grass and related Jujun grass and Pennisetum chromosome is consistent with some differences in morphology, karyotype formula and composition have some differences, no significant differences, evolution degree is higher than that of Elephantgrass Jujun grass. Combined with ISSR analysis the similarity coefficient of 0.814, were divided into the same categories. Karyotype analysis and ISSR analysis results are consistent, there are some relationships both.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q943

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