茶树TIDH核苷酸多样性及与咖啡碱含量的关联分析

Nucleotide Acid Diversity of Inosine-5’-monophosphate Dehydrogenase Gene and Association Analysis of the Gene with Caffeine Content in Tea Plant

咖啡碱是嘌呤碱中最重要的一种,对茶叶的滋味等品质起重要的作用,对于咖啡碱代谢途径的研究有重要意义。根据NCBI登录的TIDH基因(编码咖啡碱合成途径的一个关键酶——次黄嘌呤核苷酸脱氢酶)cDNA设计引物,克隆获得了一条基因组DNA的中间片段,长4172bp(TIDH3)。从中国茶树资源核心种质中选取咖啡碱含量有代表性的95份资源进行了两个年度、春秋两季的咖啡碱含量HPLC测定,大部分材料的咖啡碱含量在2.50%～4.50%,4次重复的平均值为3.50%,变异系数在15.52%～19.25%,表明茶叶咖啡碱含量在不同年份与季节都是相对稳定的。经PCR扩增、测序和序列多态性分析,TIDH3中的一个759bp片段的总核苷酸多样性指数πT和θW分别为0.006和0.012,同义突变多样性πsyn=0.009大于非同义突变多样性πnonsyn=0.006。Ka/Ks=0.600<1,认为TIDH3基因受到负向选择作用。TIDH3基因连锁不平衡分析表明,基因内的连锁不平衡衰减速度较快,在约300bp范围内r2值降低到了0.2。通过关联分析,获得两个与咖啡碱含量显著相关的SNP位点,遗传贡献率分别为10.43%和5.68%。

Caffeine is one of the most important purine alkaloids, as it plays an important role in flavor of tea. It＇s of great importance to study the caffeine biosynthesis pathway of tea plant. The primers were designed based on TIDH （which codes for inosine-5＇-monophosphate dehydrogenase, a key gene in the caffeine biosynthesis pathway of tea plant） cDNA from the NCBI. One internal fragment of TIDH genomic DNA was cloned （4 172 bp, TIDH3）. A set of 95 accessions which are typical in caffeine content was selected from the core collection of Chinese tea germplasm previously established. The caffeine content of two seasons of spring and autumn in two years was tested by HPLC. The result showed the caffeine content of most germplasms was 2.50% - 4.50%, the average content was 3.50%. The coefficientof variation is 15.52% - 19.25%, which means that the caffeine content of tea is stable between different years or seasons. A 759 bp genomic DNA fragment of TIDH3 was cloned, sequenced and analyzed. The average nucleotide diversity for the sequenced region was calculated to be ira- = 0.06 and 0w = 0.012. The diversity at synonymous sites, Ztsyn = 0.009, was higher than diversity at non-synonymous sites, 0.006. The ratio of Ka/Ks was 0.600 （lower than 1.0）, indicating the action of purifying selection. The linkage disequilibrium （LD） of SNPs in TIDH3 was detected and the result showed that LD declined rapidly within the gene region（the value oft2 reduced to 0.2 in 300 bp）. By means of association analysis, two SNPs were found to be significantly correlated with caffeine content, their genetic effect values were 10.43% and 5.68%, respectively.