【作者】 徐小彪； 廖娇； 黄春辉； 辜青青； 高洁；

【Author】 Xu Xiao-biao,Liao Jiao,Huang Chun-hui,Gu Qing-qing,Gao Jie (College of Agronomy,Jiangxi Agricultural University,Nanchang,330045)

【机构】 江西农业大学农学院；

【摘要】 从NCBI公共数据库中下载获得56400条中华猕猴桃EST序列,应用SSRHunter软件查找SSR重复序列,共获得7939条SSR,其中包括二核苷酸重复5131条,三核苷酸重复1237条,四核苷酸重复284条,五核苷酸重复397条,六核苷酸重复890条,分别占总SSR的64.63%、15.58%、3.58%、5.00%和11.21%。在所分析的猕猴桃EST-SSR中,二核苷酸重复是主要的重复类型,出现最多的重复基元是AG/CT。其次是三核苷酸重复和六核苷酸重复。应用Primer5.O设计了97对EST-SSR引物,在对模板、引物、dNTP、MgCl₂的浓度及退火温度等参数进行测试后,建立了适合的PCR反应体系,即Mg²⁺浓度为2.0 mM,dNTP浓度为0.25 mM,引物浓度为0.3μM,DNA用量为60ng/20μl,Taq DNA酶1U/20μ1。以‘漓江猕猴桃’DNA为模板,对所设计的引物进行筛选。在97对引物中,有77对引物能够扩增出清晰条带,有效扩增率为79.38%。在能扩增出条带的引物中随机选择20对引物对10个猕猴桃种类进行检测,结果发现有17对引物对所有材料都有扩增产物,扩增效率为85%,且有扩增产物的17对引物在所检测的10个猕猴桃种类间均能扩增出多态性。更多还原

【Abstract】 Fifty-six thousand four hundred ESTs of kiwifruit in the database of NCBI were downloaded and analyzed,resulting in 7939 SSRs by SSRHunter.The SSRs were consist of 5131 dinucleotide repeats,1237 trinucleotide repeats,284 tetranucleotide repeats,397 five nucleotide repeats and 890 six nucleotide repeats respectively accounting for 64.63%,15.58%,3.58%,5.00% and 11.21%.The dinucleotide repeat was the dominant type with repeat motif AG/CT being the most common.The next were the trinucleotide and the six nucleotide repeats. Ninty-seven pairs of EST-SSR primers were designed by Primer 5.0,and a suitable PCR system that contained 2.0 mmol/L Mg²⁺,0.25 mmol/L dNTP,0.3 mmol/L primer,60ng DNA and 1U Taq DNA Polymerase was eatablished after testing on the annealing temperature and the content of DNA,primers,dNTP and MgCl₂.The primers designed were screened by genomic DNA of Actinidia.lijiangensis under the condition of develped reaction system,and 77 pairs of primer showed amplification,accounting for 79.38%of total primers.20 primer pairs of the 77 were selected to PCR for DNAs from 10 Actinidia varieties.17 primer pairs of the 20 showed amplification from all of the DNAs,accounting for 85%of all the selected primer pairs,and all of the 17 primer pairs showed polymorphisms.更多还原