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.
JIN Ji-qiang
,
CHEN Liang
,
YAO Ming-zhe
,
WANG Xin-chao
,
MA Chun-lei
. Simplification of EcoTILLING Technique for Tea Plant[J]. Journal of Tea Science, 2010
, 30(1)
: 19
-26
.
DOI: 10.13305/j.cnki.jts.2010.01.004
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