【作者】 李光友；

【导师】 白嘉雨；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2010， 博士

【摘要】 本文以尾叶桉、巨桉及其杂种等为研究对象,开展N⁺注入诱变育种新技术对其苗期及大田生长影响的分析研究,阐明注入浓度及离子注入方式对性状的影响方式和程度,估算注入与未注入家系性状的家系遗传力、单株遗传力以及遗传增益,评价苗期及大田试验家系和单株综合性状的差异,快速选择优良家系,评选高产、抗逆性强的正向突变株和优良单株用于高世代育种和无性系推广。根据苗期、田间多性状指标测定,并结合SSR分子标记辅助选择,对突变株及优株进行分析和大田定位,最终获得以下结论:（1）不同N⁺注入浓度对桉树萌发产生不同程度影响,种子发芽率和发芽势随注入浓度升高,而出现升高-降低-再升高-再降低的现象。7种注入浓度以浓度1×10¹⁴ N⁺.cm^-2和1×10¹⁶ N⁺.cm^-2处理下的发芽率和发芽势较高,注入能促进种子萌发;浓度对苗期多个性状（仅地径等3个性状除外）的影响达显著水平。综合分析表明中等浓度的注入对地径和根长等具有抑制和损伤效应,低和高注入浓度则能促进生长;以地径的临界值标准筛选,7种注入浓度共有28株突变株入选,突变率为7.910%,不同浓度的正向突变率为0²1.67%;7种注入浓度以1×10⁽15 N⁺.cm^-2和1×10⁽17 N⁺.cm^-2处理获得突变株率较高;注入促进了苗期生长及生物量增加,提高了不同家系植株生理水平和抗性能力的变异幅度和分化程度,有利于选择。低浓度注入引起植株EC值降低,表明其抗性能力得到提高。（2）桉树注入与非注入家系苗期在净光合速率、蒸腾速率、气孔导度、胞间CO2浓度、光能和水分利用效率上差异显著,且性状在季节间的差异也达显著水平。注入与非注入家系在光合生理生态指标上出现分化,反映出家系间的差异,注入有助于提高光合能力的作用,高浓度注入家系的光能和水分利用效率高于低浓度注入或不注入家系;利用光能和水分利用效率对家系进行选择,二者均高的有5个家系,即4、14、15、16和17号家系,其中3个注入和2个非注入家系。（3）大田3个地点31个家系和单点15个家系/无性系的各年度方差分析表明大多数生长性状、形质性状在家系、地点、区组间及其交互效应上均存在显著或极显著差异。各年度间的生长性状间具有一定相关性,在生长性状上可实现早期选择和生长预测。3.5年生多点试验的单株材积大于总体平均值的家系有20个,占参试家系的66.67%;而大于总体平均的离子注入家系有9个,占参试家系的30%;相同家系注入浓度高时具促进生长作用;单点试验的单株材积大于总体平均值的家系有5个,占参试家系的33.33%;多点和单点试验结果均表明注入家系的平均单株材积高于非注入家系。（4）通过多点适应性评价,不同家系适生不同地点;稳定性评价将30个家系分为四类,即高产稳定型12个、高产不稳定型6个、低产稳定型8个及低产不稳定型4个家系。合并分析4个点8个性状指标的相关系数,几乎所有性状（仅干形与其它性状间相关不显著外）间均存在表型和遗传的强相关性;各性状间强相关为联合选择和简化指标提供了可行性;大多数生长性状受较强遗传控制的影响;多点试验的家系遗传力在0.0977⁰.7739之间,家系遗传增益在1.31%¹8.05%之间,单点试验的家系遗传力在0.2732⁰.9028之间,家系遗传增益在23.16%⁵7.40%之间;同地点家系遗传力均高于单株遗传力,说明通过家系选择好于单株选择,通过优良家系选择目的单株具有更大的遗传增益和利用价值。（5）SSR分子标记检测尾叶桉2个群体的10个家系,在17个位点上共检测到245个等位基因,每对引物能扩增出10-20个等位基因,平均每对引物扩增等位基因为14.41个,有效等位基因数为7.68个,245个等位基因中有94个等位基因（占38.37%）仅在其中1³个家系中出现。Shannon信息指数的变动范围在1.3533¹.7887之间,说明参试家系内的变异程度都达到较高水平;经注入与非注入家系的SSR标记后比对,注入与非注入群体共扩增出54个等位基因的差异片段,其中16个存在于注入群体中,另外38个存在于非注入群体中,相同家系不同处理的等位基因数量也存在不同,可能与离子注入引起的等位基因变异有关;标记与性状关联分析表明,17个位点上存在多个与性状QTL相关联的标记,与光能和水分利用效率关联的标记位点各有1个和3个;与Ht、DBH、SV性状QTL同时关联的标记有4个;与树皮率关联的标记有1个。（6）利用坐标综合评定法评定苗期和大田生长株,评价指标分别为6个和8个,按入选标准苗期共获得43株突变株或优株,2005年试验突变株14株,入选率为4.375%,优株6株,入选率2%;2006年试验突变株21株,入选率17.50%,优株2株,入选率6.67%; 3.5年生3个点、2.5年生1个点按突变株入选标准共获得89株突变株或优株,入选率达到2.285%。（7）对10个参与SSR分子标记的家系内单株进行苗期综合评定,分别有0株或2株入选2005年或2006年苗期全部试验单株优选率的10%范围内。对参与SSR分子标记的10个家系大田单株综合评定,分别有5株和2株入选2005年多点试验和2006年单点大田试验所有单株优选率10%的范围内。入选单株除具有优良表型性状外,在染色体上可能存在控制优良性状的基因,因此今后对入选株的SSR分子标记与生长性状具显著关联的位点进行克隆、测序、验证和应用。通过分子标记辅助选择技术提高优良单株的中选率,对目的性状进行基因定位,从而缩短育种周期和实现基因改造。更多还原

【Abstract】 E. urophylla,E. grandis and E. urophylla hybrids were researched by N-ion-beam implanting during seedling in nursery and standing trees in field, and the aim was to illuminate the method and effect of implanted-mutation breeding that it was introduced into tree-breeding. In the trials growing and form-quality traits of families were measured and scored to estimate single tree or family heritability, genetic gains, seedling quality and growing level, even to select excellent families, MET and ET trees that could be utilized to high-generation breeding programs and new clone exploitation. According to the selected effect of nursery and field by multi-traits and microsatellite （SSR） markers the METs and ETs could be get a fix on the site for future. The main conclusions were as follows:（1） The implanting effects of different N⁺ dosage indicated that germination capacity and germination rate had a remarkable difference among different dosage and had a up-down-up-down of trend along with the dosage rising. 1×10¹⁴ N⁺.cm^-2 and 1×10¹⁶ N⁺.cm^-2 in 7 kinds of dosage had a higher germination capacity and germination rate, and it indicated that implanting could promote the bourgeon of Eucalyptus seeds; There was a remarkable difference of traits by variance analysis among different dosage except 3 traits including GD. Synthesis analysis of growing effect indicated that medium dosage could restrain and injure GD and RL growing, but higher or lower dosage could improve growing and biomass level. The anlalysis effects of physiological traits indicated that 3 traits had more remarkable difference between implanting and no-implanting, and it revealed a difference of physiological and resistance level among Eucalyptus families. EC could be lower along with lower dosage and it indicated that resistance of tree could be improved after implanting; With a criterion by MCV of GD of seedlings, there were 28 selected-trees of MET or ET in 7 kinds of dosage, and mean MR or the range of positive MR was 7.910% or 0²1.67%, respectively. 1×10⁽15 N⁺.cm^-2 and 1×10⁽17 N⁺.cm^-2 in 7 kinds of dosage were the most suitable dosage to create MET. After implanting the growing and physiological effect of trees or seedlings had an obvious difference and it would help to MET selection. （2） Pn, E, gs, Ci,ε,WUE had a remarkable difference between implanting and no-implanting families by variance analysis and it indicated that implanting could improve the photosynthetic level to make for MET selection.εor WUE of the higher-dosage-implanted families was higher than no or lower-dosage families, and there were 5 most excellent families including No. 4, 14, 15, 16 and 17 that belong to 3 implanting and 2 no-implanted families could be selected in all familes according toεand WUE value.（3） The 3-site or single-site trial including 31 families or 15 families was afforested at 2005 or 2006. The effect indicated that growing or form-quality traits had a remarkable difference among family, site and interaction by variance analysis. There were a remarkable correlation of traits among different age, and the effects could make for early-age selection or growing prediction. 20 of 30 families in 3-site trial or 5 of 15 in single trial exceed mean SV and the proportion was 66.67% or 33.33%, respectively; 9 of 16 implanting families in 3-site or 4 of 7 implanting families exceed mean SV and the proportion was 56.25% or 57.14%, respectively. The growing taxis had an obvious difference between 2 treats of families in 4 field trails and it indicated that implanting could improve growth to make for MET or ET selection.（4）According to the effect of adaptability and stability analysis of families in 3-site trial, 30 families could be classified into 4 groups that had been named high-yeild and stable-growing group including of 12 families, high-yeild and unstable-growing group including of 6 families, low-yeild and stable-growing group including of 8 families, low-yeild and unstable-growing group including of 12 families. High-yeild and stable-growing group was fit for excellent-family selection and generalization. The effect of 8 traits-selection in 4 sites indicated that there was a remarkable correlation among traits except St, and the remarkable correlation effect of traits had supplied a method for multi-traits and less-quota selection to find target families; Growing-trait impact was stronger than that of form-quality traits by genetic factors, and the former was fitter for MET selection than the latter. Family heritability or genetic gain in 3-site trial was 0.0977⁰.7739 or 1.31%¹8.05%; family heritability or genetic gain in single site trial was 0.2732⁰.9028 or 23.16%⁵7.40%; family heritability was higher than single-tree heritability in the same site, and the effect indicated that family selection were better than single-tree selection, and had a more higher genetic gain and utility worthiness.（5）There were 10 families of 2 populations had been analyzed by microsatellite （SSR） markers. The effect indicated that there were 245 alleles （Na） to be detected in 17 loci and 10²0 Na in one locus by primer amplification reaction, and there were mean 14.41 Na and 7.68 Ne, too. 94 of 245 alleles （38.37%） appeared in 1³ families with lower frequencies. Some rare alleles in 2 populations were revealed by SSR. 54 Na were difference between implanting and no-implanting population including 18 in implantings’and 36 in no-implantings’by SSR markers. The different treat in same family which had different alleles could affect the increase, decrease or mutation of alleles, so we think that implanting is the cause of alteration or mutation; The correlation analysis between loci and traits indicated that there were a few QTLs in 17 loci including 1 to controlε, 3 to control WUE and 4 to control growing traits. Only one locus in all loci was peculiar to control Ba% which can be found by the correlation analysis after added the implanting data into the no-implanting data, so it indicated that the QTLs were the effect of mutation by implanting.（6） By trait value of taxis of coordinate assessment at 4 trial sites, there were 43 MET or ET to conform to MCV standard. The selected MET or ET of seedling-age trial in 2005 were 14 or 6 trees, and the selected rate were 4.375% or 2.0%, respectively; The MET or ET of seedling-age trial in 2006 were 21 or 2 trees, and the selected rate were 17.50% or 6.67%, respectively; MET or ET of field trial in 2005 and 2006 had 89 trees, and the selected rate was 2.285%; MET of all 4 site trial including Shiwan, Gonghe, Fengan, Luokeng were 11, 9, 7, 12 trees, and selected rate were 1.83%, 1.41%, 1.17% and 4.90%, respectively.（7）2 MET seedlings or 7 MET trees had been selected during seedlings or field trial by allied method of SSR markers and the taxis of coordinate assessment, and it indicated that MET owned excellent genes to control growing traits. The selected MET could be used to gene mapping, gene clone, the sequencing of the aim gene by SSR markers, and be used to multi-generation breeding and clone afforestation in production and popularization, too.更多还原