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Journal of Tea Science ›› 2010, Vol. 30 ›› Issue (1): 19-26.doi: 10.13305/j.cnki.jts.2010.01.004

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Simplification of EcoTILLING Technique for Tea Plant

JIN Ji-qiang, CHEN Liang*, YAO Ming-zhe, WANG Xin-chao, MA Chun-lei   

  1. Research Center for Tea Germplasm and Improvement, Tea Research Institute of the Chinese Academy of Agricultural Sciences/ National Center for Tea Improvement, Hangzhou 310008, China
  • Received:2009-07-31 Revised:2009-09-30 Published:2019-09-10

Abstract: EcoTILLING(Ecotype Targeting Induced Local Lesions In Genomes) had been used as a SNP(Single Nucleotide Polymorphism) discovery tool to examine DNA variation in natural populations. A simple EcoTILLING technique for tea plant was established. Firstly, specific PCR primers for the amplification of the target gene were screened out. Then through comparison, a fine CELⅠ enzyme digestion could be resulted using 20 min with 10× buffer containing 250 mmol/L Tris-HCl (pH7.4), 150 mmol/L KCl, 15 mmol/L MgSO4, 4 μg/mL BSA and 0.04%Triton x-100, and 1.8 μL CJE(Celery Juice Extract)/10 μL reacting solution at 47.5℃. Moreover, a good picture could be obtained when using 5% polyacrylamide gels (PAGE), adding 4~5 μL digesting products in PAGE electrophoresis. The 870 bp fragment within the PPO (Polyphenol Oxidase) gene of 6 tea germplasms was distinguished from each other using the simplified EcoTILLING. The CELⅠdigested bands were ascertained that according to the position of SNP in or between the 6 accessions through sequencing verification.

Key words: tea plant, CEL I, EcoTILLING, SNP

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