【作者】 马艳青；

【导师】 刘志敏；

【作者基本信息】 湖南农业大学 ， 蔬菜学， 2001， 硕士

【摘要】 通过对辣椒RAPD反应的各个环节，包括DNA提取、PCR反应进行筛选和优化，确定了一个简单、高效、相对稳定的RAPD系统；在此基础上，对来自国内外的46个辣椒种质资源进行了RAPD分析，结合统计分析方法，对46份材料进行了聚类分析和亲缘关系演化分析。结果表明：①辣椒DNA的提取以幼嫩叶较好，DNA产量高，质量优，0.5克幼嫩叶片提取的DNA足够用于RAPD分析。②辣椒DNA的提取，试剂盒法提取的DNA产量最高，纯度最高，质量最好，操作最简单，但费用贵。在样少、需DNA量多质高时，可采用此法。③辣椒RAPD优化体系为：dNTP 0.5ul，引物1ul、Buffer 2.5ul、Taq酶0.5U，模板DNA 2ul，纯净灭菌水18.5ul，反应总体积为25ul；扩增反应在PTC—200型PCR仪上进行，PCR扩增程序95℃预变性1min，36℃退火40s，72℃延长1min 20s，94℃变性20s，41次循环，36℃退火1min，72℃延长3min，4℃保存。④引物T11、Y14、D13在辣椒遗传多样性研究、品种鉴定、指纹图谱构建以及它们的杂种一代鉴定方面有较大的应用价值。⑤根据基因组指纹图谱聚类分析，46份辣椒品种资源可分为六大类，来自贵州的罗甸椒、亚洲蔬菜研究与发展示中心的ICPN9-20、辽宁的紫色观赏椒、云南的金沙江辣椒和小米辣各自独立成一类，其余品种聚为一大类。⑥RAPD基础上的辣椒亲缘关系演化是：小果多籽味辛辣类型圆锥椒→小果味辛辣果多类型簇生椒→长果味辣易干制类型长线椒→长果、肉厚味辣鲜食干制兼用类型长线椒→肉厚果大有辣味类型牛角椒→肉厚果大近似灯笼形或灯笼形，少辣或无辣味类型灯笼椒。更多还原

【Abstract】 In this report , a simple and efficient protocol for RAPD assay in pepper was established through optimization in two main procedures-DNA extraction and PCR .Based on the protocol, 46 pepper varieties from homeland and abroad were analyzed by RAPD and clustering and their evolution relationship were also analyzed . The main results are as follows : The immature leave of pepper is suitable for the DNA extraction, with high yield and good quality . The DNA extraction from 0.5gram immature leave is sufficient for RAPD analysis .or the methods of pepper DNA extraction, the reagent-box method is the best and has highest yield, best quality, highest purity and simplest procedure, but higher cost. This method can be used when a lot of high quality DNA with few variety are needed . (D The optimization program of RAPD in pepper is : DNA 2 ul ,dNTP 0.5 ul,primer 1 ul,Buffer 2.5 ul ,Taq ployrase 0.5 U ,pure water 18.5 ul ,total volume 25 ul; PCR program : 95 XH predenaturation for 1 min ,36 annealing for 40s ,72 prolong for Imin20s ,94X denaturation 20s ,41times circle ,36 annealing for 1 min ,72 prolong 3 min ,4X1 for ever.Primer T11,Y14,D13 are very important in study of pepper genetic diversification ,variety identification ,finger map building and identification of their hybrid Fl .Cluster are analyzed based on RAPD .46 varieties can be divided into six groups .Luodianjiao from Guizhou province,ICPN9-20 from AVRDC ,Ziseguanshaongjiao from Liaonin province ,Jinsajianglaojia and Xiaomila from Yunnan province were divided into single group independly , others were clustered into a large group . The evolution of pepper based on RAPD is : The taper shaped pepper with small fruit ,many seeds ,hot chilli taste-fruit cluster type with small fruit ,hot chilli taste ,many fruit setting long shaped pepper with long fruit , chilli taste , easy to dehydration long shaped pepper with long fruit ,thick wall ,chilli taste , suitable for fresh market and dehydration -ox-horn shaped pepper with thick wall ,large fruit -lantern shaped pepper with thick wall ,large fruit, without hot taste.更多还原