【作者】 赵曦阳；

【导师】 张志毅；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 杂交育种是杨树新品种培育最重要的手段。本研究以白杨派内不同种及杂种为亲本,通过人工控制授粉,利用组织培养的方法对幼胚进行挽救,获得以毛白杨为母本的杂种群体。同时对先前杂交获得的经苗期试验初选的29个白杨杂种无性系和1个选种无性系(LM50)进行4个地点2-4年树高、胸径测定分析,对这些无性系进行遗传稳定性评价。进一步利用生长性状、光合特性和抗氧化系统对30个白杨杂种无性系进行综合评价。同时采用SSR标记特异性扩增分析的方法构建这些白杨杂种无性系指纹图谱。主要研究结果如下：1.利用17个亲本材料,选配了19个杂交组合开展白杨派内不同种、杂种的杂交试验。对杂交亲本进行花序长度、宽度测定分析。结果表明不同杨树单株花序长度、宽度存在显著差异。雄株花序长度变化范围为44.40-126.43mm,宽度变化范围为11.29-15.73mm。雌株花序长度变化范围为13.84-25.48mm,宽度变化范围为5.01-7.30mm。从杂交亲和力来看,以银腺杨和毛新杨作母本的杂交组合亲和力较高,获得种子量较大。以毛白杨为母本的杂交组合在幼胚未成熟时果穗容易脱落。利用组织培养方法培育出以毛白杨为母本的6个杂交组合的杂交子代420株。对2年生子代苗高、地径调查分析结果表明不同杂交组合间子代苗高、地径差异显著。苗高平均值为49.67-84.42cm,地径平均值为5.61-10.86mm。相同杂交组合内苗高变异系数为13.66%-45.32%,地径变异系数为13.03%-39.51%,为无性系苗期选择提供了依据。2.以实验室先前杂交获得的杂种无性系进行苗期试验后,初选出29个苗期优良无性系,另加1个毛白杨选种无性系(LM50),共30个无性系为材试材,采用完全随机区组设计试验。对其4年生幼林的生长性状进行调查分析,结果发现30个白杨杂种无性系树高、胸径和材积在不同地点间、无性系间以及无性系与地点的交互作用间存在极显著差异(P<0.01)。4年生树高、胸径和材积的表型变异系数范围为19.84%-69.04%,遗传变异系数范围为14.65%-62.59%。遗传变异系数占表型变异系数比重逐年增大,表明随着植株增长,其变异增大,且这种变异主要由遗传因素引起。白杨杂种无性系树高、胸径和材积的重复力随着树龄的增加而增大,4年生各指标重复力为0.8887-0.9902。白杨杂种无性系树高、胸径和材积年-年相关性分析结果表明1年生胸径与4年生胸径表型相关系数高达0.8075,遗传相关系数达0.8841。3年生胸径和4年生胸径表型相关系数达0.9838,遗传相关系数达0.9979。表型相关和遗传相关可以为无性系选择提供理论依据。对不同地点白杨杂种无性系进行适应性分析结果发现无性系BL28、BL87、BL69、BL101和BL103具有广泛适应性,在4个地点栽培差异不大,其余无性系在不同地点显示出特殊地域适应性。3.对河北省邯郸市峰峰矿区试验点的30个白杨杂种无性系的17个生长和形态性状进行综合评价,结果表明白杨杂种无性系间17个性状差异达极显著(P<0.01)水平。无性系各性状的表型变异系数范围为15.63%-57.50%,遗传变异系数范围为8.99%-52.57%。各指标重复力变化范围为0.7734-0.9849,表明白杨杂种无性系间存在丰富的遗传变异,且变异受强的遗传控制,为无性系筛选提供基础。利用30个白杨杂种无性系的14个性状进行主成分分析,可以把14个性状分成3个主成分,第一主成分是树高、胸径等生物量性状,第二主成分主要包括通直度、节间距等干形性状,第三主成分包括叶片长度、宽度和叶面积等叶片性状。利用主成分评价无性系,BL106、BL107、BL23、BL46、BL78和BL83第一主成分值Y1是正值,且较大,表明这几个无性系树高、地径、胸径、材积、冠幅等生长量较大；无性系BL104、BL106、BL107、BL28、BL69和BL85第二主成分Y2值较大,说明这几个无性系通直度、节间距和分枝度较大；无性系BL106、LM50、BL104、BL107、BL98和BL49第三主成分Y3值较大,表明这些无性系叶片的长度、宽度和叶面积较大。4.测定30个白杨杂种无性系的光合指标,结果表明无性系Pn、Gs和Tr日变化呈双峰曲线,Ci日变化曲线呈先下降后上升的趋势。Pn-Par响应曲线和Pn-Ca响应曲线均呈S形,光饱和点lsp处于1396.55-1469.86μmol·m-2·s-1之间,光补偿点lcp处于33.08-81.17μmol.m-2.s-1之间。二氧化碳饱和点csp处于974.03-1080.50μmol.mol-1间,二氧化碳补偿点ccp处于74.03-93.35μmol.mol-1之间。30个白杨杂种无性系间瞬时光合指标呈显著差异(P<0.01)水平,平均Pn、Gs、Ci和Tr分别为19.82μmol.m-2.s-1,0.37mo1.m-2·s-1,263.68μmol·mol-1和4.38 mol·m-2.s-1。光合指标Pn、Gs、Tr与树高、胸径均达显著正相关水平,表明光合指标对无性系生长有很大影响。5.通过对白杨杂种无性系不同月份叶绿素含量、MDA含量、POD活性和SOD活性动态变化规律研究发现,白杨杂种无性系叶绿素含量平均值呈现“低-高-低”的变化趋势。丙二醛(MDA)含量平均值处于一直上升状态,而过氧化物酶(POD)和超氧化物歧化酶(SOD)活性呈现先上升后下降的趋势。相关性分析表明POD、SOD、叶绿素含量与生物量显著正相关,MDA含量与生物量显著负相关。利用聚类分析,对无性系预抗性进行评价,把30个白杨杂种无性系分为3类,第一类酶活力最高,预抗性最强,在受到胁迫时受伤害最小,第二类无性系预抗性较强,第三类无性系预抗性最差,在遭遇胁迫时最容易受到伤害。6.利用16对SSR引物对30个白杨杂种无性系进行特异性扩增,共获得40条DNA特异性条带,每对引物扩增的DNA谱带数目处于2-6条间,平均2.5条谱带,扩增的DNA带大小在100-600bp之间。30个白杨杂种无性系之间遗传距离变化范围在0.0864-0.2716,平均遗传距离为0.1436。利用DNA谱带进行无性系指纹图谱的构建,发现白杨杂种无性系间亲缘关系较近,可以用多对引物组合来实现无性系的区分。本研究利用生物量、干形、光合指标、抗氧化系统对白杨杂种无性系综合评价,同时利用SSR分子标记对无性系进行指纹图谱构建,为优良无性系选择提供理论依据,具有重要的理论意义和应用价值。更多还原

【Abstract】 Cross breeding is the most important method to breed new poplar varieties. This study used different poplar species and hybrid clones as parent materials, saving the immature embryos by artificial pollination and tissue culture in order to get hybrid population bred by Populus tomentosa. This study also analyzed the heights and diameters at breast height of trees aged from two to four years in four sites, and these trees included 29 poplar hybrid clones and one selected clone (LM50) by previous cross breeding during seedling test. Genetic stabilities of these clones were also evaluated. Comprehensive evaluation of 30 poplar hybrid clones was made by analyzing growth characters, photosynthesis and antioxidant system. Fingerprint mapping of hybrid clones was established by SSR specific amplification analysis. The results were as follows:1.19 cross combinations were selected from 17 parent materials to form cross experiment among species and hybrid clones in Leuce. By analyzing flower buds length and width of crossing parents, we found that there was significantly difference of flower anthotaxys length and width between individuals. The range of male plants flower anthotaxys length was between 44.40 and 126.43mm, and the range of width was between 11.29 and 15.73mm, while the ranges of female plant were from 13.84 to 25.48mm, and from 5.01 to 7.30mm respectively. The hybridization compatibility and the amount of seeds were high when using P. alba×P. glandulosa and P. tomentosa×P. bolleana as hybrid parents. The clusters of clones with P. tomentosa as hybrid parent were easily to shed when young embryos were immature.420 P. tomentosa hybrid progenies from 6 cross combinations were produced by tissue culture. The result of analyzing seedling height and ground diameter of 2-year offspring showed that there was significant difference of seedling height and ground diameter among different cross combinations. The average seedling height was from 49.67 to 84.42cm and the average ground diameter was between 5.61 and 10.86mm. The variation coefficient of height in the same cross combination was from 13.66% to 45.32%, and that of ground diameter was between 13.03% and 39.51%, and these results provided evidence for clone selection during seedling.2.29 superior clones and one poplar selected clone (LM50) were chosen as test materials, and field experiments were set in randomized blocks. By investigating growth characters of 4-year young plantation, we found that the heights, diameters at breast height and volumns of 30 poplar hybrid clones were significantly different among different sites, clones and interaction of clones and sites. The range of phenotypic variation coefficient was between 19.84% and 69.04%, and the range of genetic variation coefficient was from 14.65% to 62.59%. The proportion of genetic variation coefficient in phenotypic variation coefficient increased every year, indicating that the variation was caused mainly by genetic fators. The repeatabilities of heights, diameters at breast height and volumns of 30 poplar hybrid clones increased by the ages of trees, and the repeatabilities of each factor during 4 years were between 0.8887 and 0.9902. The correlation analysis of poplar heights, diameters at breast height and volumns showed that the phenotypic variation coefficient of diameters at breast height between one-year and four-year poplars was up to 0.8075, and that the genetic variation coefficient was 0.8841. While the phenotypic variation coefficient of diameters at breast height between three-year and four-year poplars was 0.9838 and with genetic variation coefficient 0.9979. Phenotypic variation coefficient and genetic variation coefficient could provide theoretical basis for clone selection. The adaptability analysis of poplar clones at different sites showed that clones of BL28, BL87, BL69, BL101 and BL103 had wide adaptation and made no difference planting in 4 sites. While the other clones showed particular regional adaptabilities at different sites.3. By comprehensively evaluating 17 indices in 30 poplar hybrid clones in Fengfeng mining area in Handan, Hebei Province, we found that these indices among different hybrid clones were extremely significantly different (P<0.01). The range of phenotypic variation coefficient of hybrid clones was between 15.63% and 57.50%, and the range of genetic variation coefficient was from 8.99%to 52.57%. The repeatabilities of each factor were between 0.7734 and 0.9849, showing that there was ample genetic diversity in poplar clones and the variation was highly controled by genes, and this result can provide basis for clone selection. We used principal component analysis to divide 14 indices of poplar clones into 3 principal components, and the first principal component was biomass characters such as height and diameter at breast height, the second was stem form characters such as stem straightness and distance between leaves, the third was leaves characters such as leaf length, width and leaf area. The result of principal component analysis showed that principal component values Y1 of clones BL106, BL107, BL23, BL46, BL78 and BL83 were positive and high, and indicated that the biomass such as height, ground diameter, diameter at breast height, volumn and crown width were high; while principal component values Y2 of clones BL104, BL106, BL107, BL28, BL69 and BL85 were high, revealing that the straightness, distance between branch, and branching degree of clones were high, and the principal component values Y3 of clones BL106, LM50, BL104, BL107, BL98 and BL49 were higher and showed the length, width and area of leaves of these clones were higher.4. The results of photosynthetic indices of 30 poplar hybrid clones showed that the diurnal variations of net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were classified as double-peak curves, while diurnal intercellular CO2 concentration (Ci) decreased at first and then increased. Pn-Par response curves and Pn-Ca response curves were classified as S-curve, the light saturation points (lsp) were between 1396.55 and 1469.86μmolm-2s-1, and light compensation points (lcp) were between 33.08 and 81.17μmolm-2s-1. CO2 saturation points (csp) were between 974.03 and 1080.50μmol CO2 mol-1, and CO2 compensation points (ccp) ranged from 74.03 to 93.35μmol CO2 mol-1. The difference of instantaneous photosynthetic indices among 30 poplar hybrid clones was extremely significant (P<0.01), and the average Pn, Gs, Ci and Tr were 19.82μmol·m-2·s-1, 0.37 mol·m-2·s-1,263.68μmol·mol-1 and 4.38 mol·m-2·s-1 respectively. Photosynthetic indices, Pn、Gs、Tr were significantly positively related to the height and diameter at breast heigh of trees, indicating that photosynthetic indices had great effect on growth of clones.5. Study on the monthly dynamic variation of contents of chlorophyll, MDA and activities of POD and SOD of poplar clones showed that trend of chlorophyll contents was low-high-low. The average malondialdehyde (MDA) kept increasing, while peroxidase (POD) and superoxide dismutase (SOD) increased at first and then decreased. Correlation analysis showed that POD,SOD and chlorophyll content were significantly positively related to biomass, while MDA content was significantly negtively correlated to biomass. We evaluated the pre-resistance of different clones by cluster analysis and classified the 30 hybrid clones into 3 groups, and the first group had high enzyme activity, revealing their most strong resistance and would be vulnerable by stress. The second group had stronger resistance, and the third group with low resistance would be susceptible to stress.6.30 poplar hybrid clones were specifically amplified by using 16 pairs of SSR primers, and 40 DNA specific bands were obtained, the numbers of DNA bands which were amplified by each pair of primers were from 2 to 6 and the average number of bands was 2.5, and the DNA bands were between 100-600bp. The range of genetic distance among 30 poplar clones was from 0.0864 to 0.2716, with an average distance of 0.1436. Fingerprinting map of hybrid clones was established with DNA bands, and the result showed that there was close genetic relationship among poplar hybrid clones.In present study, biomass, stem form, photosynthetic indices and antioxidant system were used to comprehensively evaluate poplar hybrid clones. Meanwhile, fingerprinting map was also established for the evaluation. All these provided the valuable theoretical basis for superior hybrid clones selection.更多还原