【作者】 曾斌；

【导师】 李疆；

【作者基本信息】 新疆农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 新疆野扁桃(Amygdalus ledebouriana Schlecht.)为蔷薇科李亚科桃属扁桃亚属植物,是一种十分珍贵而古老的树种,属新生代第三纪孑遗的物种,在当今世界上已十分稀少,被誉为“活化石”。本文在对新疆野生扁桃资源普查、生长发育状况调查的基础上,开展了该野生种植物的繁殖生物学特性及其种质资源群体遗传多样性的研究。同时,还对包括新疆野扁桃在内的扁桃属中的不同种与新疆栽培扁桃品种间的亲缘关系进行了初步研究。希望通过本文的研究,以便掌握新疆野扁桃繁殖特性及不同分布群体的遗传变异情况,为扁桃杂交育种和选育优良品种提供理论依据和遗传学背景资料,也为下一步新疆野扁桃及其近缘栽培种遗传图谱的构建和功能基因定位奠定基础。主要研究结果如下:1.新疆野扁桃花芽形态分化的研究结果采用石蜡切片法对裕民野扁桃自然保护区群体的新疆野扁桃花芽分化过程进行观察研究,结果表明:新疆野扁桃花芽形态分化可分为未分化期、花蕾原基分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期6个时期。6月中旬开始进入花芽形态分化期,从花芽形态分化开始到雌蕊原基分化形成各分化时期有重叠现象,整个分化时期集中在6月下旬至9月上旬。完成花芽形态分化所需时间为90～100d。2.新疆野扁桃授粉特性的研究结果新疆野扁桃完成授粉试验15d后,异花授粉平均坐果率较高,达到65.3%、而自花授粉率为19.7%;自然授粉率很低,仅为5.3%。整体的坐果率相对较低,为30%。3.新疆野扁桃实生繁殖的研究结果新疆野扁桃种子选择合适的秋播深度尤为重要。播种时覆土以4～5 cm较为适宜,出苗率极高,播后注意镇压保证种子与土壤密接,恢复土壤毛细管作用,有利于水分的吸收。4.新疆野扁桃组织培养繁殖的研究结果以生长在新疆北部塔城地区裕民县巴尔鲁克山的新疆野扁桃树上当年结的种子为试材,采用赤霉素24h处理打破休眠,之后使用常规法消毒后接种于诱导培养基上。生长约一个月后,将由试管中种子培养长成的小植株剪切成长约1cm的单芽茎段,接入增殖培养基进行培养。新疆野扁桃自然条件下进行实生及根蘖繁殖,而在组织培养离体繁殖条件下非常难生根,本试验采用100mg.L-1IBA进行浸渍处理60min后,再接入1/2MS培养基中培养,初期暗处理2周后置于白炽光下培养生根率可达100%。而后生根的试管苗在珍珠岩基质中培养,进行有效的移栽驯化管理,获得了较理想的移栽成活率。5.新疆野扁桃表型多样性的研究结果为了从数量上分析新疆野扁桃天然群体表型性状的变异,我们调查了新疆野扁桃在新疆全部5个不同天然分布群体。每个群体选取30个个体为试材,针对新疆野扁桃15个表型性状指标进行了系统比较分析研究,讨论了群体间和群体内的表型多样性,结果表明:新疆野扁桃种内表型性状在群体间和群体内均存在着丰富的遗传差异,其天然分布群体内的变异大于群体间的变异,群体间的分化相对较小;利用群体间欧氏距离进行UPGMA聚类分析表明,新疆野扁桃天然群体可以划分为3类,表型性状的欧式距离与地理距离相关不显著。主成分分析结果显示叶片长度/叶片宽度比值、果核横径、果核长度/果核宽度比值、叶片长度、叶片宽度、果核千粒重等6个表型性状指标是反映新疆野扁桃形态差异的主要因素。6.新疆野扁桃遗传多样性SSR分子标记研究结果采用10对SSR引物对新疆野扁桃5个不同天然分布群体的150个叶片样本进行群体遗传多样性标记的研究。结果表明:10对SSR引物在5个居群中可平均扩增出9.2条带,其中裕民县群体多态性带数百分比最高为92.77%,各位点平均Nei’s基因多样度为0.262;5个群体共扩增出92个位点,总的种级水平及布尔津县、哈巴河县、塔城市郊、托里县和裕民县5个群体水平多态性位点百分比分别为100%、88.04%、85.87%、85.87%、90.21%、92.39%,种级水平Nei基因多样度(H = 0.2971)和Shannon信息指数(I=0.4421)大于种下群体,5个种下群体Nei基因多样度和Shannon信息指数比较:裕民县>托里县>哈巴河县>塔城市郊>布尔津县。SSR谱带频率方差分析(AMOVA)表明,遗传多样性主要分布在群体内,群体内方差分量的贡献率占63.56%,而群体间方差分量的贡献率占36.44%,表明具有丰富的群体内变异。UPGMA聚类分析结果表明:在一定的遗传距离基础上,5个分布群体分为3个类群,表型性状的欧式距离与地理距离相关不显著。塔城市郊群体和哈巴河群体遗传关系最近,而裕民县群体与托里县群体的遗传关系也很近,布尔津县群体远离其他4个群体,根据基因分化系数值(GST =0.0598),测得的基因流Nm为0.9197,证明种群间遗传交换较小,这与环境适应性和高山阻隔有一定的关系。所有参数分析表明,裕民县群体遗传多样性最丰富,故在制定原位种质保护计划时应优先考虑裕民县群体。7.新疆野扁桃遗传多样性不同研究分析方法结果的比较与评价表型与DNA二种水平的遗传标记方法揭示的遗传多样性水平有差异,表型性状比DNA分子标记具有更大的变异性;其受到环境的影响较大,也即对环境更敏感。2种水平的遗传标记聚类结果将野扁桃5个不同分布群体均可分为3个大类群,但是出现有个别不同的群体排序位置,反映了2种标记在分析种群间关系上的异同性,从本研究结果来看,说明表型与DNA标记有一定的耦合性和相关性,但也存在差异,在研究上可以互为补充。8.扁桃属植物种质资源鉴定的SSR分子标记研究利用SSR分子标记技术对扁桃属植物中的国内外55份扁桃材料的亲缘关系进行了鉴定,使用12对选择性引物组合共扩增出75条扩增条带,并对其进行聚类分析,结果表明:不同种以及不同品种间的遗传距离不同,在相似系数小于0.68时,大多数栽培品种聚为一类,栽培品种中,同一种源区的大多数栽培品种能聚类在一起。从聚类图上看,普通栽培扁桃与新疆野扁桃间的亲缘关系比长柄扁桃、蒙古扁桃、西康扁桃以及榆叶梅间的亲缘关系更近。这个结果也表明了扁桃属植物种质资源的演化关系和遗传多样性水平。更多还原

【Abstract】 Amygdalus ledebouriana Schlecht. (wild Almond) belong to Rosaceae, Prunoideae, Amygdalus, A. ledebouriana Schlecht. It is a very primitive tree species, precious, ancient and very scarce in the world today. Botanists call it "living fossil plants".On the basis of preliminary investigation of Germplasm Resources in A. ledebouriana Schlecht., growth and development of the situation, we carried out A study on propagate biological characteristics and genetic diversity of germplasm resources in A. ledebouriana Schlecht. and identification of genetic relationship of Amygdalus plants by SSR, including the different species and cultivation of different varieties, in order to grasp propagate biological characteristics, different groups genetic variation of A. ledebouriana Schlecht.. and genetic relationship of Amygdalus plants. This would provide a theoretical basis and genetics background information for hybrid breeding and breeding improved varieties, and lay the foundation for the genetic gene mapping of the cultivated species and their relative construction in Amygdalus. The main research results were as follows.1. Research results of flower bud differentiation in A. ledebouriana Schlecht.In this experiment, the paraffin section method is adapted to observe the developing process of flower bud differentiation of A. ledebouriana Schlecht. in TaCheng, Xingjiang. The results indicated that the floral morphodifferentiaion can be divided into five development stages: flower initial stage, sepal differentiation stage, petal differentiation stage, stamen differentiation stage and pistil differentiation stage. The floral morphological differentiation occurred at the mid ten-day period of June. There was intersectional phenomenon in each differentiation stage. From the starting of the flower bud differentiation to the pistil primordial differentiation, the whole process appeared intently between the last ten-day period of June and the first ten-day period of September. It took 90~100 days to finish morphodifferentiation.2. Research results of fertilization pollination biology in A. ledebouriana Schlecht.15 days after pollination on A. ledebouriana Schlecht., cross-pollination has higher average fruit setting rate than self-pollination and natural-pollination. 65.3% for the cross-pollination, 19.7% for self-pollination, 5.3% for natural-pollination. Overall fruit setting rate was the relatively low, It was 30%.3. Research results of seed multiplication in A. ledebouriana Schlecht.It is especially important for autumn sowing seed multiplication of depth of A. ledebouriana Schlecht.This is more appropriate for planting a depth of 4 ~ 5 cm, and has very high germination rate, Attention to the suppression of soil after sowing seeds, restore capillary effect of the soil, this would conducive to the absorption of water.4. Research results of tissue culture and plantlet regeneration in A. ledebouriana Schlecht.The seed of precious tertiary carry-over species in low region of Baerluk mountainous of Tacheng district in Xinjiang were used to establish the system in vitro plant and develop a feasible and practicable micropropagation system of A. ledebouriana Schlecht. Seed was plunged into gibberellin to breaking dormancy, after sanitizing Planting in primary medium. One month later, single bug was cultured in elongation medium.twenty day later, 100% in rooting percentage was obtained with 100mg.L-1IBA dip in 60 min, initial dark incubating for two weeks and continuous naturally lighting treatment in room. Improvement in rooting percentage was obtained . In vitro plant was transplanted in the same time to reduce humidity gradually, in this way the survival rate was higher.5. Research results of morphological diversity in A. ledebouriana Schlecht.In order to analyses morphological characteristics of the pattern of variation of natural populations of A. ledebouriana Schlecht., we determined the morphological variation of natural populations of A. ledebouriana Schlecht. in Xinjiang to found out the relationship between morphological variation and distribution.We compared fiften phenotypic traits based on 30 trees from five populations. The results showed that there was rich genetic variation among populations and within individuals. The variation within the populations is greater than that among populations. Based on the UPGMA cluster analysis, the five populations could be divided into three groups. There was no significant relationship between geographic distances and Euclidean distance. Principal component analysis showed that Length of leaf /Maximal width of leaf index, Width of seed, Length of seed/width of seed index, Length of leaf, Maximal width of leaf, The weight of per 1000 seeds were the most important phenotypic traits which lead to the morphological variation of A. ledebouriana Schlecht.6. Research results of SSR genetic diversity in A. ledebouriana Schlecht.The genetic diversity and genetic structure as well as genetic differentiation of natural populations of A. ledebouriana Schlecht. were investigated with ten pair SSR primers for a total of 150 individuals from five populations sampled. The purpose of the study was to determine the genetic structure and diversity in these eco-geographical populations. The results indicated that: an average of 9.2 bands was detected in the five populations. The percentage of polymorphic bands in Yumin population (92.77%) was the highest in the five populations. The average Nei’s gene diversity index was 0.262 for all the loci. Totally, 92 polymorphic loci were detected and the percentage of polymorphic loci (P) was 100%、88.04%、85.87%、85.87%、90.21%、92.39%, respectively, at the species level and Burjin, Habahe, Tacheng, Tuoli and Yumin population levels. The Nei’s gene diversity index (H = 0.2971) and Shannon’s information index (I=0.4421) in the species level were higher than in the population level. The Nei’s gene diversity index and Shannon’s information index in the five populations were Yumin > Tuoli >Habahe > Tacheng>Burjin. The results of AMOVA had been indicated that the variance components of within populations and among populations contributing to total genetic variance were 63.56% and 36.44%, The results showed that there was rich genetic variation within individuals. The UPGMA cluster analysis indicated the five populations could be divided into three groups. There was no significant relationship between geographic distances and Euclidean distance.Tacheng population and Habahe population were the highest in genetic identity and the closest in genetic distance. Yumin population and Tuoli population were the close relations. Burjin population were relatively independent populations. Concurrently, Gene flow between the populations was 0.9197 based on genetic differentiation coefficient (GST= 0.0598). indicating that genetic recombination between populations is quite small , which probably is related to environmental adaptations of A. Ledebouriana Schlecht. and population isolation in their high mountain habitat. on the basis of the study of population genetic structure and the highest genetic diversity, Yumin population should be given a high priority consideration in A. ledebouriana Schlecht. population’s in situ germplasm conservation.7. A preliminary study on comparison and coupling of morphological and DNA markers in A. ledebouriana Schlecht.The diversity levels are different among morphological and DNA markers. Phenotypic characteristics had greater variability than the DNA molecular markers. The former was more sensitive to environment. Genetic cluster analysis is similar based on the results of morphological and DNA markers, the five populations could be divided into three groups. But there are different groups sort of individual locations, reflecting the similarities and difference about the analysis of the relationship between population with the two kinds of markers, It could be concluded that there was coupling and relevance to some extent in evaluating genetic diversity with the same experiment sampling by morphological and SSR marker variation. However, there was also differences. In research, the two can complement each other.8. Identification of Genetic Relationship of Amygdalus plants by SSRThe genetic relationship among 55 Amygdalus plants collections, belonging to 6 species and collected from both homeland and abroad were identified via SSR molecular marker technique. 12 pairs of primers were selected out to amplify 75 polymorphism bands. The genetic distance between these collections were analyzed with cluster analysis based on these bands. The result showed that the genetic distance between them were different. And most of the cultivars were classified in the same group when the genetic similarity coefficient was less than 0.68. The cultivars from the same area were clustered in the same group. Compared the relationship among A. communis L., A.ledebouriana Schlecht. and A.pedunculata Pall., A.mongolica Moxim., A.tangutica Batal. and A.triloba (Lindl) Ricker., the relationship between A. communis L. and A.ledebouriana Schlecht. is closer than the relationship between A. communis L. and A.pedunculata Pall., A.mongolica Moxim., A.tangutica Batal. and A.triloba (Lindl) Ricker. This results gave a clearly outlook of the genetic relationship and genetic diversity level among Amygdalus plants germplasm.更多还原