【作者】 熊万山；

【导师】 钱伟； 李加纳；

【作者基本信息】 西南大学 ， 作物遗传育种， 2009， 硕士

【摘要】 杂种优势利用是作物增产的一个重要途径。自20世纪30年代杂种优势在玉米中应用获得高产以来,其在农作物生产中得到广泛的应用并取得了显著的增产效果。然而一个世纪以来关于作物杂种优势产生机制仍不清楚。至20世纪80年,人们开始从分子水平来探讨作物杂种优势的分子机理,证实了基因表达与杂种优势存在关系。而作物基因组DNA甲基化修饰是调节基因表达的一种重要途径,因而本研究从检测油菜基因组DNA甲基化状态来探索油菜杂种优势产生的分子基础。主要研究结果如下:1、全基因组DNA甲基化状态检测本实验共选用了10对MSAP选择扩增引物组合,对45个杂交组合(包括22个甘蓝型油菜与白菜型油菜种间杂种和23个甘甘、白白种内杂种)及其8个亲本材料的苗期的叶片和蕾进行了全基因组甲基化分析,共检测出252个可清晰记录条带,其中177条带具有多态性,平均每对引物检测到17.7条多态性位点。2、甲基化组织特异性分析对亲本及其杂种在苗期和蕾期5’-CCGG位点甲基化状态的对比分析表明,苗期的甲基化水平均高于其在蕾期的甲基化水平。亲本的甲基化含量在苗期均显著高于其杂交后代,但在蕾期差异不显著(p=0.49)。苗期和蕾期DNA甲基化的改变表现为去甲基化和超甲基化,其中去甲基化为主要变化形式,除甘蓝型油菜种内杂种外,其它材料间的去甲基化比例均大于超甲基化。白菜亲本甲基化位点变化比例最高,平均为48%。对45份杂交后代和8个亲本材料从苗期到蕾期甲基化变化的主成分分析表明,三类杂交后代及其两类亲本主要聚成三类,第一类为甘蓝型油菜亲本及其种内杂种后代,第二类为白菜型亲本及其种内杂交后代,第三类为甘蓝型油菜与白菜型油菜的种间正反交杂交后代。基因组结构和材料的异质性对苗期到蕾期甲基化的变化都有影响,但基因组结构的影响大于材料的异质性对其的影响。3、亲本与杂种甲基化状态比较杂种与其亲本比较主要存在4种条带类型,A类为杂种与双亲的甲基化带型相同(P1=PI=F1);B类为杂种的甲基化类型与母本相同(P1=F1≠P2);C类为杂种的甲基化类型与父本相同(P2=F1≠P1),A,B,C三种带型符合孟德尔遗传规律;D类为杂种与双亲皆不相同,即杂交后代出现甲基化新带型(F1≠P1,P2)。杂种具有与两亲本或亲本之一相同的甲基化带型(A,B,C类带型)占总的甲基化的比例约占67.9%。在D类带型中,杂种在苗期去甲基化的比例总是大于超甲基化,而蕾期种间杂种出现去甲基化的比例大于超甲基化,种内杂种出现超甲基化的比例大于去甲基化。4、农艺性状分析本研究对包括株高、第一次分枝高度、主花序长度、主花序有效荚果数、全株有效荚果数、每荚果粒数、第一次分枝数目等7个杂种优势相关农艺性状指标进行了测定,结果显示,杂种后代在株高、主花序长度、主花序有效荚果数以及产量上都高于对照和亲本的均值,7类农艺性状的都有较强的中亲优势,其中,株高、第一次分枝高度、主花序长度、主花序有效荚果数、全株有效荚果数等5个农艺性状都为正向中亲优势,但每荚果粒数、第一次分枝数具有负向中亲优势。亲本与杂种、年度间、不同基因组结构(甘蓝型油菜与白菜型油菜种间杂种、甘蓝型油菜种内杂种、白菜型油菜种内杂种)方差分析的结果表明,株高、第一次分枝高度、主花序荚果数、全株荚果数和每荚果粒数等5个农艺性状在0.01水平上存在显著差异。主花序荚果在年度间差异不显著,分枝数在年度间达到极显著异,但亲本与杂种间以及不同基因组结构间差异不显著。5、与杂种优势相关的甲基化位点分析对株高、主花序长度、主花序有效荚果数、生物重等4个性状的中亲优势与甲基化带型(六种带型:P1=P1=F1、P1=F1≠P2、P2=F1≠P1、去甲基化,超甲基化,其它类型)作单因素方差分析,筛选与杂种优势相关甲基化位点。对筛选位点进行甲基化带型效应值的多重比较发现,六种甲基化变化带型都与杂种优势有关。更多还原

【Abstract】 Utilization of heterosis has been to an important approach to increase crop yields since it was successfully used to improve seed yield in corn in the 1930s.However,the mechanisms of heterosis have remained poorly understood so far.Since 1980s,the evidences from molecular studies proved the possibility to reveal its mechanism.For example,the differential gene expression was detected between hybrid and parent.DNA methylation is considered to regulate the gene expression.In this study,the relationship of DNA methylation with heterosis was investigated.Main results were as follows.1.DNA methylation status on the whole genomeTen MSAP amplification primer combinations were used for the methylation analysis among crosses of B.napus with B.rapa,23 intraspecific crosses,together with 8 parental accessions in Brassica.Totally,177 polymorphic loci were detected from 252 clear recorded bands.2.Tissue Specificity of DNA methylationIt was found that the methylation level in seedling was higher than bud among 53 materials,and that parents exhibited high ratio of alternations of DNA methylation than hybrids at the seedling stage,but not significantly differed each other at the bud stage(p=0.49)Alternations of DNA methylation,hypomethylation and hypermethlation,were found from seedling to bud.The proporation of hypomethylation was higher than hypermethylation,except for the intraspecific hybrids of B.napus.The principle component analysis for the methylation alternation from seedling to bud revealed three groups.One group contained the B.napus parents and their intraspecific hybrids,while another one contained the B.rapa parents and their intraspecific hybrids.The third group was composed of the interspecific hybrids or B.napus and B.rapa.3.Differential methylation patterns among hybrids and parentsFour types of methylation patterns were detected among the tested accessions,the common band patterns in the hybrid and its both parents refered as A type(P1=P2=F1),the common band patterns in the hybrids and its female parents refered as type B(P1=F1≠P2),the common band patterns in the hybrids and its male parents refered as type C(P2=F1≠P1),the new band refered as type D,in which it differ between hybrid and parents(F1≠P1,P2).About 67.9%of band patterns belonged to the classification of type A,B and C.Among band patterns of D type,the degree of hypomethylation among all the accessions tested was higher than that of hypermethlation in seedling,while the degree of hypomethylation among the interspecific hybrids was higher than hypermethlation and the degree of hypomethylation among the interapecific hybrids was lower than hypermethlation.4.Analysis on agronomic traitsSeven agronomic traits related to heterosis,containing plant height,height of primary branch,length of main inflorescence,number of effective pods of the main inflorescence,number of effective pods per plant, number of seeds per pod and the number of primary branches,were tested in this study.Hybrids were better than relative parental accessions in the plant height,the length of main inflorescence,the number of effective pods of the main inflorescence and the yields.ANOVA analysis showed significant differents between parents and hybrids for plant height,height of primary branch,number of effective pods of the main inflorescence,number of effective pods per plant and number of seed per pod(p=0.01).5.Relationships of DNA methylated with heterosisThe loci significally relative to heterosis were selected by ANOVA analysis for plant height,length of main inflorescence,number of effective pods of the main inflorescence and biomass.All of methylation patterns in F1(P1=P1=F1,P1=F1≠P2,P2=F1≠P1,hypomethylation,hypermethlation and other patterns) were related to heterosis by calculating the effects of band patterns.更多还原