白蜡核心种质及绒毛白蜡无性系SSR评价体系建立

发布时间：2018-04-29 15:37

本文选题：白蜡 + 核心种质　；参考：《甘肃农业大学》2014年硕士论文

【摘要】：白蜡是良好的盐碱地造林树种，用材树种以及园林绿化树种，构建白蜡指纹图谱对我国白蜡资源的充分合理利用、品种鉴定和知识产权保护均具有重要意义。本实验利用SSR分子标记技术对白蜡种质以及绒毛白蜡无性系进行了分析，筛选出了一批多态性丰富的SSR引物，构建了SSR指纹图谱并对白蜡种质及绒毛白蜡无性系进行了遗传多样性评价。主要结果如下： 1．本研究建立了适合白蜡SSR-PCR反应的最佳体系，用于白蜡SSR标记的进一步研究。本研究采用L16(45)正交设计和单因素试验对影响白蜡SSR-PCR的Taq聚合酶用量、Mg2+浓度、DNA模板浓度、dNTP浓度和引物浓度等5个因素在4水平上进行筛选。优化后的白蜡SSR反应体系为：Mg2+(25mmol/L)0.8μL、引物(10μmol/L)0.2μL、Dntp (10mmol/L)0.3μL、Taq酶(5U/μL)0.05μL、DNA模板(10ng/μL)2.00μL、10×PCR缓冲液1.0μL，ddH2O5.45μL，总体积10.0μL。该结果为今后利用SSR-PCR标记技术研究分析白蜡奠定了基础。 2．选用的12份白蜡核心种质材料，，从156对SSR引物中筛选出10对反应稳定，扩增条带清晰，多态性强的引物。10对引物共扩增出35个多态位点，且均为多态位点，多态性比例为100%。平均每对引物检测到3.5个多态位点。多态性信息含量(PIC)变幅为0.3648～0.6725，平均为0.5480，引物20的PIC值最高，引物30的PIC值最低。利用引物42、18、26、30构建引物组合，可将12个白蜡树种完全区分开，每一份白蜡树种都有自己独特的指纹图谱。UPGMA聚类及遗传多样性分析基本能将不同属白蜡树种分开，并能体现白蜡不同种间的亲缘关系，结合白蜡SSR指纹图谱，在白蜡种间鉴别、种质资源管理、杂交育种和知识产权保护等方面具有重要的参考意义。 3．以46份绒毛白蜡无性系优树为实验材料，在156对SSR引物中筛选出17对反应稳定，扩增条带清晰，多态性强的引物。这17对引物共扩增出68个多态位点，且均为多态位点，多态性比例为100%。平均每对引物检测到4.0个多态位点。PIC变幅为0.7059～0.1970，平均0.5184，引物42的PIC值最高，引物16的PIC值最低。利用引物12、16、E3、17、24、5、7、F3、4、42、C3、37、30构建引物组合，可将46个绒毛白蜡无性系树种完全区分开，每一份绒毛白蜡无性系树种都有自己独特的指纹图谱。通过UPGMA聚类分析，不同性状和株型的绒毛白蜡无性系大致能被区分开来，遗传多样性分析数据表明绒毛白蜡无性系遗传多样性比较丰富，说明本研究建立的评价体系具有参考意义。
[Abstract]:White wax is a good afforestation tree species in saline-alkali land. It is of great significance to construct the white wax fingerprint for the rational utilization of Chinese white wax resources, variety identification and intellectual property protection. In this study, SSR markers were used to analyze the white wax germplasm and fluffy white wax clones, and a number of polymorphic SSR primers were screened out. SSR fingerprint was constructed and genetic diversity of white wax germplasm and fluffy white wax clone was evaluated. The main results are as follows: 1. In this study, the optimum system for SSR-PCR reaction of white wax was established, which was used for further study of SSR labeling of white wax. In this study, the orthogonal design and single factor test were used to screen five factors, such as the concentration of Taq polymerase and the concentration of Taq template and primer, which affect the dosage of Taq polymerase and the concentration of primer. The optimized SSR reaction system was as follows: 10 渭 mol/L)0.2 / L Dntp 10 渭 mol / L 10 渭 mol / L Taq 5 U / 渭 L 0. 05 渭 L SSR template, 10 渭 L 10 渭 L 10 脳 PCR buffer 1. 0 渭 L LddH 2O 5.45 渭 L, and 10 渭 L 路L ~ (10) 渭 L ~ (10) 渭 L ~ (10) 渭 L ~ (10) 渭 L 路L ~ (-1) for 10 渭 L 路L ~ (-1), 10 渭 L ~ (10) 渭 L 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). The results laid a foundation for the study and analysis of white wax by SSR-PCR labeling technique in the future. 2. Ten pairs of primers with stable reaction, clear amplified bands and strong polymorphic bands were selected from the 12 white wax core germplasm materials, and 35 polymorphic loci were amplified by 10 pairs of primers, all of which were polymorphic, and the proportion of polymorphism was 100%. An average of 3.5 polymorphic loci per pair of primers were detected. The variation of polymorphic information content (Pi) was 0.3648 ~ 0.6725 with an average of 0.5480. The PIC value of primer 20 was the highest, and the PIC value of primer 30 was the lowest. The primer combination was constructed by using primer 42O18 / 2630, and 12 species of white wax tree can be completely distinguished. Each species has its own unique fingerprint map. UPGMA clustering and genetic diversity analysis can basically separate the species of different genus of white wax tree. It can reflect the relationship between different species of white wax, combined with the SSR fingerprint of white wax, it has important reference significance in identification of white wax species, management of germplasm resources, cross breeding and intellectual property protection, etc. 3. Using 46 fluffy white wax clones as experimental materials, 17 pairs of primers with stable reaction, clear amplified bands and strong polymorphism were screened out of 156 pairs of SSR primers. A total of 68 polymorphic loci were amplified from 17 pairs of primers, all of which were polymorphic, with a polymorphic ratio of 100. The average number of polymorphic loci per primer was 0.70590.197, with an average of 0.5184.The PIC value of primer 42 was the highest, and the PIC value of primer 16 was the lowest. The primer combination was constructed by using the primer 12A16 E3C3C3C3730. The primer combination can completely distinguish 46 species of fluffy white wax asexual species, and each species has its own unique fingerprint map. The primer combination can be used to construct a primer combination of primer 12C16E3C3C3C3H730, and each of them has its own unique fingerprint map. The primer can be used to construct a primer combination. By UPGMA cluster analysis, different traits and plant types of fluff white wax clones can be roughly distinguished, and genetic diversity analysis data show that the genetic diversity of fluffy white wax clones is relatively rich. It shows that the evaluation system established in this study has reference significance.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S792.41

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