核辐射诱发水稻基因组变异的特征及定向选择技术研究

发布时间：2018-05-25 03:31

本文选题：水稻 + 诱变育种　；参考：《浙江大学》2016年博士论文

【摘要】：辐射技术作为一种重要的突变诱发手段,在种质创新和品种选育中得到了广泛的应用。但是,在分子水平、特别是基因组水平上对辐射诱发的遗传变异尚缺乏足够的认识,制约了辐射育种的发展。为明确伽玛射线和高能离子束对水稻的诱变效应,建立高效的诱变育种体系,本研究采用基因组重测序等基因组学和分子遗传学技术,对其诱导的遗传变异的主要特征开展了研究,并在此基础上探索了基于新一代测序技术(NGS)和高分辨熔解曲线(HRM)技术检测缺失突变的效果。主要结果如下：1、在全基因组范围、单核苷酸水平上揭示了γ射线诱发可遗传变异的频率与特征。γ射线诱发的变异种类既有单碱基替代(SBS)突变,也有插入缺失突变(Indels)和结构变异；以碱基为单位,水稻M2代植株中的平均突变频率约为7.5-9.8×10-6(比拟南芥报道的快中子辐射突变频率高了一个数量级)；SBS变异频率约为Indel突变的4倍；结构变异频率比Indel更低;SBS和Indel突变随机的分布在12条染色体上,无明显的突变热点。2、比较分析了C、Ne和Ar三种离子束对水稻的辐射生物学效应和诱变效应,确定了三种离子束的适宜剂量和突变类型。相同剂量的Ar离子束对水稻M1代成活率和结实率的影响都比C和Ne更大(后两者相似)；三种离子适宜用于水稻诱变育种肩膀剂量不同,可能与离子束的LET值不同有关；同一M1单株的不同M2稻穗可携带不同的突变类型；离子束也可诱发SBS和Indel突变,随机的分布在水稻的12条染色体上。3、探索了基于NGS (Seq-TILLING)和HRM (HRM-TILLING)技术高通量筛选Indel突变的效果和影响因素。采用Seq-TILLING时存在一定比例的假阳性,但可以生物信息学分析加以排除,样本以192倍混池测序效果较好；采用以4倍混样开展HRM-TILLING时,对较短片段缺失可确保极低的假阳性和假阴性率,但检测片段较长的突变时效果较差。研究结果为在突变育种中设计科学合理的育种方案、更加有效地利用丫射线指明了方向,如不同的剂量产生的突变频率相似表明相对低的辐射剂量更适用于辐射育种；M2代存在大量的突变位点表明选育突变体作为品种时应进一步进行回交以清除有害突变；突变位点会在M3代产生分离表明在进行突变体系选育时,单株选择比群体选择更适合。三种高能离子束适宜剂量和突变类型的确定、不同分蘖携带不同突变的发现为水稻离子束诱变育种体系的建立奠定了基础；基于NGS和HRM技术的靶向突变高通量筛选体系的建立可望推进核辐射水稻育种技术的进步。
[Abstract]:As an important mutation inducing method, radiation technology has been widely used in germplasm innovation and variety selection. However, at the molecular level, especially at the genomic level, there is a lack of sufficient understanding of radiation-induced genetic variation, which restricts the development of radiation breeding. In order to clarify the mutagenic effects of gamma rays and high energy ion beams on rice and to establish an efficient mutagenic breeding system, genomics and molecular genetics techniques, such as genome resequencing, were used in this study. The main characteristics of the induced genetic variation were studied, and the effect of detecting deletion mutation based on the new generation sequencing technique (NGS) and the high resolution melting curve (HRM) technique was explored. The main results were as follows: 1. In the whole genome, single nucleotide level revealed the frequency and characteristics of the genetic variation induced by 纬 -ray. The variation types induced by 纬 -ray included single base substitution for SBS), insertion deletion mutation (Indels) and structural variation. The average mutation frequency of M2 plants was about 7.5-9.8 脳 10 ~ (-6) (an order of magnitude higher than that of Arabidopsis thaliana reported by fast neutron radiation). The mutation frequency of Indel was about 4 times of that of Arabidopsis. The frequency of structural variation was lower than that of Indel. Indel and Indel mutations were randomly distributed on 12 chromosomes and had no obvious mutation hotspots. The radiobiological and mutagenic effects of Con ne and ar ion beams on rice were compared and analyzed. The suitable dose and mutation type of three ion beams were determined. The effects of the same dose of ar ion beam on the survival rate and seed setting rate of M1 generation of rice were greater than those of C and ne (the latter two were similar to each other), and the three ions were suitable for rice mutagenesis breeding with different shoulder doses, which might be related to the different LET value of ion beam. Different M2 panicles of the same M1 plant can carry different mutation types, and ion beam can also induce SBS and Indel mutations. Random distribution was observed on 12 chromosomes of rice. The effects and influencing factors of high-throughput screening of Indel mutations based on NGS Seq-TILLINGand HRM HRM-TILLINGwere explored. There was a certain proportion of false positive in using Seq-TILLING, but it could be excluded by bioinformatics analysis. A very low false positive and false negative rate can be ensured for the deletion of short fragments, but the detection of long mutations is less effective. The results indicate the direction of designing scientific and reasonable breeding schemes in mutation breeding and making more effective use of Ya-ray. If the frequency of mutation produced by different doses is similar, the relatively low radiation dose is more suitable for the M _ 2 generation of radiation breeding with a large number of mutation sites. It is suggested that further backcross should be carried out to eliminate harmful mutations when breeding mutants as varieties. The mutation sites were isolated in M3 generation, which indicated that the selection of individual plant was more suitable than that of population selection in the selection of mutant system. The determination of suitable dose and mutation type of three kinds of high energy ion beams and the discovery of different tillers carrying different mutations laid a foundation for the establishment of rice breeding system of ion beam mutagenesis. The establishment of high throughput screening system for targeted mutation based on NGS and HRM is expected to promote the development of nuclear radiation breeding technology in rice.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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