利用二代测序技术高通量分析水稻品种间抗病性差异的分子基础

稻瘟病和白叶枯病是由稻温病菌(Magnaporthe oryzae)和白叶枯病菌(Xanthomonas oryzae pv.oryzae)引起的两种主要水稻病害,也是制约中国水稻生产的主要病害。为了从DNA水平探索造成水稻感病品种‘丽江新团黑谷’(LTH)和高抗品种‘特特普’(Tetep,TTP)间抗病性差异的分子基础,该研究对其已知的3个抗稻瘟病基因和3个抗白叶枯基因所在DNA区段分别进行PCR扩增,将等量混合的PCR产物再与基因组重测序样品按Ct值差值(ΔCt)~10的比例混合,采用二代测序技术进行一次性测序和比较分析,并对有差异的基因区域进行常规传统测序验证,以确定这2个品种中抗性基因(R基因)的数目和结构与品种抗病或感病表型的关联性。实验结果表明,二代测序能够快速并准确地寻找到2个不同水稻品种中多个特定基因的序列差异,且差异位点与常规测序结果相符。从LTH和TTP这2种抗性不同水稻品种在多个抗性基因的DNA水平差异来看,有差异的抗性基因位点在高抗品种TTP中大都与原始抗性基因序列相同,而对应的普感品种LTH的抗性基因往往多表现为氨基酸突变,这些序列差异很可能就是导致TTP与LTH抗性差异的分子基础。

Exploration on the Molecular Basis of Different Resistance in Rice Using Next-generation Sequencing

Rice blast and bacterial blight,caused by Magnaporthe oryzae and Xanthomonas oryzae pv.oryzae,respectively,are two principal diseases worldwide.Meanwhile,these diseases are also the major limitations in rice production in China.Lijiangxintuanheigu(LTH) is a highly susceptible cultivar to rice blast,while Tetep(TTP) has broad-spectrum resistance against the pathogens.However,the molecular mechanism underlying the difference on disease resistance in LTH and TTP is largely unknown.To identify the nucleotide difference in multiple resistance genes between these two cultivars efficiently,6 known R genes were amplified from LTH and TTP,respectively.The PCR products were mixed with a 10-cycle difference in Ct value and sequenced together by the next-generation sequencing(NGS) technology.The results were validated by traditional sequencing strategy.Our results revealed that the NGS can rapidly identified nucleotide polymorphisms from multiple PCR-amplified products of different cultivars in single sequencing run,which is more economical and more efficient to find sequence difference of target genes among different cultivars.Additionally,we found that the sequence of R genes in TTP is more similar to that of original functional alleles,while the sequence of R genes in LTH has more mutations that altering amino acid.