發(fā)布時間：2018-05-25 03:31

  本文選題：水稻 + 誘變育種　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：輻射技術(shù)作為一種重要的突變誘發(fā)手段,在種質(zhì)創(chuàng)新和品種選育中得到了廣泛的應(yīng)用。但是,在分子水平、特別是基因組水平上對輻射誘發(fā)的遺傳變異尚缺乏足夠的認(rèn)識,制約了輻射育種的發(fā)展。為明確伽瑪射線和高能離子束對水稻的誘變效應(yīng),建立高效的誘變育種體系,本研究采用基因組重測序等基因組學(xué)和分子遺傳學(xué)技術(shù),對其誘導(dǎo)的遺傳變異的主要特征開展了研究,并在此基礎(chǔ)上探索了基于新一代測序技術(shù)(NGS)和高分辨熔解曲線(HRM)技術(shù)檢測缺失突變的效果。主要結(jié)果如下：1、在全基因組范圍、單核苷酸水平上揭示了γ射線誘發(fā)可遺傳變異的頻率與特征。γ射線誘發(fā)的變異種類既有單堿基替代(SBS)突變,也有插入缺失突變(Indels)和結(jié)構(gòu)變異；以堿基為單位,水稻M2代植株中的平均突變頻率約為7.5-9.8×10-6(比擬南芥報道的快中子輻射突變頻率高了一個數(shù)量級)；SBS變異頻率約為Indel突變的4倍；結(jié)構(gòu)變異頻率比Indel更低;SBS和Indel突變隨機(jī)的分布在12條染色體上,無明顯的突變熱點。2、比較分析了C、Ne和Ar三種離子束對水稻的輻射生物學(xué)效應(yīng)和誘變效應(yīng),確定了三種離子束的適宜劑量和突變類型。相同劑量的Ar離子束對水稻M1代成活率和結(jié)實率的影響都比C和Ne更大(后兩者相似)；三種離子適宜用于水稻誘變育種肩膀劑量不同,可能與離子束的LET值不同有關(guān)；同一M1單株的不同M2稻穗可攜帶不同的突變類型；離子束也可誘發(fā)SBS和Indel突變,隨機(jī)的分布在水稻的12條染色體上。3、探索了基于NGS (Seq-TILLING)和HRM (HRM-TILLING)技術(shù)高通量篩選Indel突變的效果和影響因素。采用Seq-TILLING時存在一定比例的假陽性,但可以生物信息學(xué)分析加以排除,樣本以192倍混池測序效果較好；采用以4倍混樣開展HRM-TILLING時,對較短片段缺失可確保極低的假陽性和假陰性率,但檢測片段較長的突變時效果較差。研究結(jié)果為在突變育種中設(shè)計科學(xué)合理的育種方案、更加有效地利用丫射線指明了方向,如不同的劑量產(chǎn)生的突變頻率相似表明相對低的輻射劑量更適用于輻射育種；M2代存在大量的突變位點表明選育突變體作為品種時應(yīng)進(jìn)一步進(jìn)行回交以清除有害突變；突變位點會在M3代產(chǎn)生分離表明在進(jìn)行突變體系選育時,單株選擇比群體選擇更適合。三種高能離子束適宜劑量和突變類型的確定、不同分蘗攜帶不同突變的發(fā)現(xiàn)為水稻離子束誘變育種體系的建立奠定了基礎(chǔ)；基于NGS和HRM技術(shù)的靶向突變高通量篩選體系的建立可望推進(jìn)核輻射水稻育種技術(shù)的進(jìn)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 緯 -ray. The variation types induced by 緯 -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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S511

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 蘆海平;張華麗;富昊偉;李友發(fā);崔海瑞;舒慶堯;;水稻類病斑突變系的培育與研究[J];核農(nóng)學(xué)報;2016年06期

2 張得芳;馬秋月;尹佟明;夏濤;;第三代測序技術(shù)及其應(yīng)用[J];中國生物工程雜志;2013年05期

3 Ping LIU;Xing-Xing;CAI Bao-Rong LU;;Single-seeded InDel fingerprints in rice:An effective tool for indica-japonica rice classification and evolutionary studies[J];Journal of Systematics and Evolution;2012年01期

4 杜艷;余麗霞;劉青芳;周利斌;李文建;;TILLING技術(shù)的最新進(jìn)展及其在輻射誘變育種中的應(yīng)用前景[J];原子核物理評論;2011年03期

5 ;A Cost-effective High-resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants[J];Journal of Integrative Plant Biology;2010年12期

6 劉錄祥;郭會君;趙林姝;古佳玉;趙世榮;;我國作物航天育種20年的基本成就與展望[J];核農(nóng)學(xué)報;2007年06期

7 安學(xué)麗,蔡一林,王久光,王國強(qiáng),孫海燕;化學(xué)誘變及其在農(nóng)作物育種上應(yīng)用[J];核農(nóng)學(xué)報;2003年03期

8 彭鎖堂,莊杰云,顏啟傳,鄭康樂;我國主要雜交水稻組合及其親本SSR標(biāo)記和純度鑒定[J];中國水稻科學(xué);2003年01期

9 楊美英,武志海,崔秋華,楊福,趙斌;粳稻氬離子(Ar~+)束誘變育種的細(xì)胞學(xué)效應(yīng)研究[J];吉林農(nóng)業(yè)大學(xué)學(xué)報;2002年05期

10 余增亮，，霍裕平;離子注入生物學(xué)研究述評[J];安徽農(nóng)業(yè)大學(xué)學(xué)報;1994年03期

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