【作者】 荣立苹；

【导师】 雷家军；

【作者基本信息】 沈阳农业大学 ， 观赏园艺学， 2010， 博士

【摘要】 百合是百合科(Liliaceae)百合属(Lilium)多年生球根草本花卉,世界五大鲜切花之一,其花朵硕大、花色艳丽、花姿百态、芳香怡人,世界各地广为栽培,在世界鲜切花市场上占有十分重要的地位。我国野生百合资源十分丰富,约有55个种,其中36个种、15个变种为特有种,但是大部分野生资源没有得到充分利用,且破坏流失严重、多样性逐渐下降。本论文对东北地区野生百合资源进行调查和收集,并采用形态学、孢粉学、细胞学和RAPD标记对收集的百合资源进行亲缘关系和遗传多样性研究,为东北地区野生百合资源的收集、保存、保护、分类、评价、利用等奠定基础。主要结果如下：对东北地区野生百合种质资源进行考察与收集,共收集野生百合119份,分属6种3变种,包括卷丹25份、细叶百合15份、毛百合12份、垂花百合2份、朝鲜百合7份、东北百合7份、有斑百合40份、大花百合3份、大花卷丹8份,考察与收集过程中未发现条叶百合。其中,有斑百合和卷丹在东北地区分布范围最广、分布数量最多,尤其在铁岭、本溪等地分布较多；其次是细叶百合,分布较广泛；而垂花百合和大花百合分布范围比较狭窄,收集到资源份数较少,垂花百合仅在凤城和抚顺收集到2份资源,大花百合仅在铁岭和蛟河收集到3份；而朝鲜百合分布范围则比较集中,仅分布于靠近朝鲜的丹东地区。采用AHP法对野生百合进行综合评价,并依据最终得分情况,将所有种类分为3个等级。卷丹和垂花百合分值最高,为第Ⅰ级,应在保护的前提下加强开发力度；细叶百合有斑百合、大花卷丹和朝鲜百合处于第Ⅱ级；东北百合、毛百合和大花百合处于第Ⅲ级。虽然东北百合、毛百合和大花百合分值较低,处于第Ⅲ级,但并不代表它们没有利用价值,相反,它们均有各自的优良性状,因此应注重有目的的开发和利用。对50份野生百合资源的16个主要形态性状进行统计分析,结果表明,东北地区野生百合种质资源的主要性状有明显差异,表现出显著的形态多样性。各形态性状的变异系数除了叶长的变异系数相对较小,为23.35%,其他性状的变异系数都大于25%,其中花柱长和叶宽的变异系数较大,分别为64.57%和59.37%；野生百合形态多样性指数均较高,平均多样性指数达2.66,这表明东北地区野生百合种间各性状分化程度较大,其中叶长、内花瓣宽的多样性指数较高,分别为2.87和2.88。东北地区野生百合种质资源形态性状的主成分明显,前四个主成分方差累计贡献率达到了87.51%,包含了24个性状指标的87.51%的信息,基本能代表这24个性状的变异。在24个形态性状中,外轮花瓣宽、内轮花瓣长、内轮花瓣宽、花被片颜色、花型、花梗长、叶长是引起形态变异的主要性状,这一结论与形态多样性指数得出的结论相一致。利用DPS数据统计软件,将50份野生百合种质资源的形态数据进行聚类,50份材料被聚为三大类,第一类30份材料,包括花朵相对较小的种类：细叶百合、有斑百合、大花百合、朝鲜百合和垂花百合；第二类19份材料,包括茎叶粗壮、花朵较大的种类：卷丹、大花卷丹和毛百合；第三类包括东北百合,为轮叶组百合。利用光学显微镜和电子显微镜观察野生百合花粉形态及表面纹饰。百合的花粉形态均以单粒形式存在,对称性较强,赤道面观呈舟形,具单萌发沟且沟延伸至两极；极面观为椭圆形,边缘具萌发沟。对极轴、赤道轴、极轴/赤道轴3个指标进行方差分析,结果表明3个指标在种间均达到极显著差异。以极轴/赤道轴进行多重比较,结果表明,部分百合种存在显著或极显著差异,但也有部分百合种间差异不显著。种内差异显著性分析表明,不同来源间表现一致的只有朝鲜百合,极轴、赤道轴和极轴/赤道轴三个性状在种内未达到显著差异；而不同来源的卷丹和细叶百合的花粉各指标都达到极显著,存在差异。东北地区百合花粉表面纹饰基本类型为网状,网脊盘珠状或瘤状颗粒,形成多边形或不规则网眼。网眼的大小及分布密度和网脊上颗粒大小及密度在种间有一定的差异。在扫描电镜下观察,一般在近极面中部网眼最大,向两端或沟边缘渐变小。对染色体制片技术的取样时间、预处理液、解离时间、染色剂等方面进行试验研究,结果表明,根尖取样时间以上午8：30-9：30为佳,用0.1%秋水仙碱溶液4℃下预处理24 h,经卡诺固定液固定6 h后,在1 m01·L-1盐酸60℃下恒温水浴中解离5 min(卷丹为8 min),改良卡宝品红溶液染色压片镜检,能取得良好的制片效果。东北地区9种野生百合染色体为大型染色体,除卷丹为三倍体外,其他种均为二倍体,核型特征均由两对具中部(m)或具近中部(sm)着丝点的大型染色体以及10对具近端部(st)和端部(t)着丝点染色体所组成。种间的差异主要表现在各不同类型染色体的数目以及它们在核型中排序位置的差异,其次是随体的数目和位置的不同。核型分类除毛百合属于3A型,其它都属于3B型。在观察的百合根尖细胞中,除正常的染色体数目外,还存在少数体细胞染色体数目不同程度的变异,但是出现变异的频率较低。东北百合在观察的30个细胞中,没有发现染色体数目变异,其他种均发现染色体数目的变异,卷丹染色体数目变异范围较大,约占所观察细胞(100个)的11%,而且还观察有单倍体细胞(2n=x=12)出现,约占2%。在卷丹、细叶百合、毛百合、有斑百合、大花百合5种百合的根尖细胞中观察到B染色体,卷丹、有斑百合和大花百合的根尖细胞中观察到1条B染色体存在,毛百合根尖细胞中发现2条B染色体,而在细叶百合的根尖细胞中观察到有1条或2条B染色体存在,且出现频率较高,占观察细胞总数(100)的10%。用12个RAPD随机引物对50份野生百合试材进行扩增,共扩增出105条清晰谱带,其中86条为多态性谱带,多态率为81.8%,平均每个引物扩增出7.2条多态性谱带,表明东北地区野生百合之间具有丰富的遗传多样性。而且,大部分引物在不同试材上都具有主特征带,这反映了百合属内基因组的同一性。基于50份野生百合资源之间欧氏距离的矩阵,利用UPGMA进行了聚类分析。从聚类结果可以看出,供试的50份百合资源可分为两大类：第一类包括供试的38份试材,包括大花卷丹、卷丹、毛百合、朝鲜百合、东北百合、有斑百合和大花百合；第二类包括细叶百合和垂花百合,共12份试材。第一类又可以分为2个亚类,第一亚类包括大花卷丹、毛百合、朝鲜百合、东北百合4个种(变种)；第二亚类包括卷丹、有斑百合、大花百合3个种。聚类结果表明,卷丹与有斑百合的亲缘关系较近,而与同为卷瓣组的朝鲜百合、细叶百合、大花卷丹等亲缘关系较远；垂花百合与细叶百合亲缘关系较近。更多还原

【Abstract】 Lilium, a perennial bulbous flower belonging to the Liliaceae, is one of the five most important cut flowers.With its big and colorful flower, beautiful stature and fragrance, it is cultured extensively worldwide and paly an important role in the floral market. In China, there are abundant wild resources of Lilium with a total of 55 species, in which 36 species and 15 varieties are native species. But many of them have yet not been used, and a great number of precious resources are endangering because of natural disasters. Therefore, in this paper, 119 wild Lilium resources native to Northeast China were investigated and collected, and the genetic relationship and diversity were studied at morphological, palynological, cytological and molecular level, which lay a foundation for the collection, preservation, conservation, classification, evaluation and utilization of wild Lilium species. The results were as follows.119 accessions resources of wild Lilium germplasm were collected native to Northeast China. The germplasm accessions collected.were classified into 6 species and 3 varieties, including 25 Lilium lancifolium Thunb.,15 L. pumilum DC.,12 L. dauricum Ker-Gawl,2 L. cernuum Komar.,7 L. amabile Palib.,7 L. distichum Nakai,40 L. concolor Salisb. var. buschianum (Lodd.) Baker,3 L. concolor Salisb. var. megalanthum Wang et Tang,8 L. leichtlinii Hook.f. var. maximowiczii (Regel) Baker. But L. callosum Sieb. has not been collected until now. Based on the geographical distribution, L. concolor var. buschianum was believed to be the most widely distributed species, and it was collected from 16 cities in the northeastern region. L. lancifolium was one of the most widespread species with high adaptability in China, and was collected from diverse habitats such as bushes, road side, hillside, or grassland. Only 2 accessions of L. cernuum were collected from Fengcheng city and Fushun city, and only 3 accessions of L. concolor var. megalanthum were collected from Tieling and Jiaohe city. Therefore, the distribution of L. cernuum and L. concolor var. megalanthum was rather narrow as compared to other species. L. amabile also had narrow geographic distribution, which seemed to be concentrated in Dandong city near North Korea. The analytic hierarchy process was employed to evaluate ornamental characteristic and utilization potential of 9 collected species. It seemed that L. lancifolium and L. cernuum got the highest score Therefore, L. lancifolium and L. cernuum had an excellent exploitation potential. Among the 9 Lilium species, only L. cernuum had obvious purple flowers. Also, its population was very small. Besides, L. pumilum, L. concolor var. buschianum and L. leichtlini var. maximowiczii. got higher scores. Therefore, great importance had been continuously attached to L. lancifolium, L. cernuum, L. pumilum, L. concolor var. buschianum and L. leichtlini var. Maximowiczii. L. dauricum, L. distichum and L. concolor var. megalanthum got the lower score. However, that does not mean that they are worthless. They have a relatively high value in some certain aspects that implying that we should also pay attention to these species.16 morphological characteristics from 50 populations of 6 species and 3 varieties germplasm resources were researched. The results showed that the morphological characteristics were significantly different with high diversity among the wild lilies native to northeast China. The coefficient of variation were high and more than 25% except for leaf long (23.35%), of which the cofficient of variation of style length and leaf width were most highest, up to 64.57% and 59.37% respectively. The morphological diversity was high, and the average diversity index was 1.85, of which the diversity index of leaf length and inner petal width, were highest, up to 2.88 and 2.87 respectively. In PCA analysis, the first four principal components represented 87.51% of the morphological diversity. The significant morphological divergence consisted of outer petal width, inner petal length, inner petal width, flower color, flower type, peduncle length and leaf length.Based on the morphological data, 50 wild lilies were cluster into three groups. The first group included 30 accessions with the smaller flower, L. pumilum, L. concolor var. buschianum, L. concolor var. megalanthum, L. amabile and L. cernuum, the second group included 19 accessions with large flower, L. lancifolium, L. leichtlinii var. maximowiczii and L. dauricum, and the third group included L. distichum.The pollen was observed by light microscope and scaning electronic microscope. The result showed that pollen exists for single grain, and the symmetry is strong. The pollen shapes of wild Lilium are boat shape in equator view and there is a ditch which extends several to the two-pole. The pollen shapes of Lilium are oval in polar view and the edge has hollow, for sprouts the ditch. There was very significant difference on polar axis, equarorial axis, P/E among Lilium species. The result from multiple comparions showed that there was very significant difference among some Lilium species, and no significant difference among other Lilium species. Also, there was no significant difference in L. amabile from different areas. and There was very significant difference in L. lancifolium and L. pumilum from different areas.The ultrastructure of pollen surface basic type observes is the network, the net keel belt-shaped, granulated, plate bead shape or is irregular, forming the polygon or the irregular mesh. The mesh size, the density of distribution and the shape characteristic have the certain difference in different species, mesh size of some species is bigger, and others is smaller. The mesh in near polar middle is obviously bigger than the ditch edge by scaning electronic microscopeThe roots were used to study the effects of the sampling time, the pretreatment chemicals, hydrolysing time and staining on the chromosome squashing. The results showed that the optimal sampling time of roots was about 8:30～9:30 am. Of the three pretreatment chemicals used,0.1% colchicine solution for 24 hours was proved to be best. Then the root-tips were fixed with Carnoy solution for 6 hours and hydrolysed with 1 mol·L-1 HCl for 5 min (8min for L. lancifolium) at 60℃and stained with modified Carbol fuchsin solution. A very good effect of chromosome squashing could be obtained with this method. The chromosome of 9 Lilium species collected was bigger, and showed the same basic chromosome number x=12 and similar karyotype features, two large metacentric chromosome pairs and ten subtelocentric or telocentric pair. L. lancifolium was triploid (2n=3x=36) and the other species were diploid (2n=2x=24). Interspecific differences was mainly the number of chromosomes of different types and their ranking position in the karyotype, followed by the difference of the number and position of the satellite. The karyotype of all the species was 3A except for L. dauricum Ker-Gaw. with 3B. The variations of chromosome number were observed in the root-tip cells of 8 species except for L. distichum, but the variation frequency was relatively low. The aneuploidy cells of 2n=2x=22,25,26 were observed in seven diploid species with the rate of 2～10%. The aneuploidy cells of 2n=3x=34,37 were observed with the rate of 9%, and the cells with haploid chromosome number(2n=x=12) had also been observed in the triploid species L. lancifolium. B-chromosome was also observed in L. dauricum; L. concolor var. buschianum, L. concolor var. megalanthum, L. pumilum and L. lancifolium. There was 1 B-chromosome in L. lancifolium, L. concolor var. buschianum and L. concolor var. megalanthum,2 B-chromosomes in L. dauricum, and 1 B and 2 B-chromosomes in L. pumilum. Especially, the occurrence frequency of B-chromosome in L. pumilum was highest, up to 10%The DNA polymorphism from 50 accessions of 6 species and 3 varieties in Lilium native to Northeast China was studied using randomly amplified polymorphic DNA (RAPD) markers in the experiment. A total of 105 clear and reproducible bands were generated from twelve RAPD primers, of which 86 were polymorphic, with an average of 7.2 polymorphic bands, which indicated that the 50 wild lily accessions had high level of genetic diversity. The dendrogram based on RAPD data were constructed by using UPGMA analysis. The 50 wild lilies clustered into two groups. The first group included 38 accessions involved in L. leichtlinii var. maximowiczii, L. lancifolium, L. dauricum, L. amabile, L. distichum, L. concolor var. buschianum and L. concolor var. megalanthum. The second group included 12 accessions involved in L. pumilum and L. cernuum. The first group can be divided into two subgroups. The first subgroup involved in L. leichtlinii var. maximowiczii, L. dauricum, L. amabile, and L. distichum, and the second subgroup involved in L. lancifolium, L. concolor var. buschianum and L. concolor var. megalanthum. The results showed that L. lancifolium had close genetic relationship with L. concolor var. buschianum, but distant genetic relationship with L. amabile, L. pumilum and L. cernuum of Sect Sinomartagon. The genetic relationship was close between L. pumilum and L. cernuum.更多还原