【作者】 文亚峰；

【导师】 谢碧霞；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2006， 博士

【摘要】 人心果(Manilkara zapota)属稀有热带果树,有十分重要的开发利用价值。人心果上个世纪初引种至我国,但其经济价值并未得到充分认识。与其它热带果树(荔枝、龙眼、芒果等)相比,我国人心果产业发展和研究相对滞后,资源稀少、良种缺乏、.栽培技术落后,这与当前国内外人心果的市场需求很不相称。有必要在引进国外优良品种的基础上,积极开展品种资源调查、品种间亲缘关系、开花授粉生物学及优良品种选育等方面的研究工作。针对目前人心果产业发展中存在的品种混杂,分类不清,产量低等实际问题,本文采用形态和AFLP分子标记技术研究来源于美国、越南及国内共31个人心果品种资源的亲缘关系及其遗传多样性。在此基础上,选取部分品种,通过自交异交授粉试验,结合细胞显微观察,探寻人心果座果率低的原因,研究品种间亲缘关系与其异交亲和性的相关关系。为人心果授粉品种的选配、产量提高和今后育种工作奠定科学基础。表型性状研究结果表明,人心果品种资源遗传变异丰富。.20个形态特征平均变异系数达到了25.6%,品种(类型)间的欧氏遗传距离范围为1.11-10.94,平均遗传距离6.10。根据表型聚类结果,品种间亲缘关系与其地理来源有较明显的联系,不同地区的人心果品种在聚类图中有较为明显的界限。来源于美国的13个品种大致聚为三类,即AG-1亚群的A-1、A-2亚组和AG-2亚群中的B-2亚组。国内12个品种主要聚为两类,集中在AG-2亚群中的B-1和B-4亚组,特别是B-4亚组中海南的3个品种最先聚在一起,表现出极近的亲缘关系,有可能为同一品种。根据主成份分析(PCA)结果,20个表型性状中,叶片形状、果实形状与大小、花器特征和嫩梢(叶)颜色(有无褐毛)等反映了人心果64.8%的表型信息,利用这些形态特征可对25个人心果品种进行初步分类。13个美国人心果品种大致分为四大类型,一是大果类人心果,以OX为代表,第二类是叶片形状为长卵形的人心果,如Molix, Alana, Morena等,第三类果实为长卵形,如Hasya, Modella等,第四类特征不明显,但以圆果、叶片卵形为主。国内12个品种(类型)根据嫩梢(叶)颜色(有无褐毛)可分为两大类型,根据花器特征可再分出海南品种。采用梯度实验方法建立了人心果AFLP标准化实验体系,对31个人心果品种的DNA指纹图谱进行分析。从64个引物对(PstⅠ/MseⅠ)中筛选出9对引物,共扩增得到1131条带(75-500bp),其中多态性条带1096条,多态性比例96.80%,去除曼妹人心果属的4个品种,在种的水平,多态性比例也达到了89.04%。品种间的遗传变异有大有小,多态性比例在22.19%至45.89%之间。与其他热带水果(荔枝、龙眼、香蕉)相比,品种间遗传多样性并不丰富,这与人心果分布范围狭小,品种间基因交流较少有关。31个供试品种中,28个已找到特征性条带,特征带大小一般在350-500bp之间。这些特征带在人心果品种鉴别及性状基因克隆中具有重要作用。31个品种间的遗传相似性系数范围在0.40-0.87,平均相似性系数为0.65。UPGMA聚类结果表明：所有供试材料可聚为三大类群,分别为曼妹人心果类群(MAM)和另个两个人心果亚群(SAPl, SAP2)。在相似性系数0.52处,曼妹人心果4个品种首先被区分开,聚为一类。27个人心果品种全部聚为另一大群(SAP),其中13个美国品种,10个国内品种及1个越南品种构成SPA1亚群,表现出较近的亲缘关系。云南和湛江3个品种在相似性系数0.54处单独聚为SPA2亚群,与其他24个品种差异性较大,有可能为一新种。主坐标分析(PCOA)结果与系统聚类结果一致。分子聚类结果表明,品种间的亲缘关系与其地理来源相关性不高。群体研究结果表明：人心果种群多态性达到了94.80%0,居群间遗传变异占总遗传变异的比值(GST)为0.4883,即有51.17%的遗传变异存在于种群内,相当于自交植物的GST值,这与人心果种群结构有较大关系。我国广西人心果居群遗传多样性丰富,与美洲人心果亲缘关系最近,而云南人心果居群多样性水平较低,种群独特,应予以重点保护。通过人心果开花习性、花粉分析、细胞显微观察及授粉后座果情况调查研究表明：人心果花粉活力因品种和开放期不同而存在差异,从花蕾期到开放期,花粉活力逐渐减小,但总体高于60%。不同品种间花粉活力差异较大,海南品种高出广西品种约10个百分点；人心果花粉萌发率总体较低,开放期会达到10%左右；人心果座果率低的主要原因是由于自交不亲和,它属于一种较为特殊的配子体自交不亲和类型,花柱和子房都会对花粉管生长产生较强的抑制作用。不同品种间亲缘关系与其异交授粉亲和性之间存在相关性,亲缘关系越近,其异交亲和性也相对较差。根据亲缘关系研究结果可为今后人心果栽培中授粉株植的选配提供参考依据。更多还原

【Abstract】 Sapodilla (Manilkara zapota, Sapotaceae) is a rare tropical fruit tree native to Central and South America. It was introduced into South China in the early 1990s, .popularly in these areas for its delicious fruit and rich nutrition. However, the development and research about sapodilla drop behind the other tropical fruits in China, such as lichee, longan and mango. Germplasm resource, quality variety and cultivation technique were major obstacles for the development of sapodilla industry in China. It is necessary to do fundamental researches on the basis of cultivars introduction, such as the investigation of germplasm resources, the studying of genetic relationship, flower and pollination biology and breeding.In order to solve the problem of cultivar identification, classification and low fruit yield in sapodilla industry. The article used the morphological and AFLP markers to study the genetic diversity and relationship among 31 sapodilla cultivar resources from USA, China and. Vietnam. On the basis of genetic relationship result,5 cultivars were selected to examine their self and cross compatibility, and confirm the connection between genetic relationship and compatibility of sapodilla, which has much benefit to pollination, increasing fruit yield and breeding.Morphological research result showed that genetic diversity was much abundant in 25 sapodilla cultivars. The average variation coefficient(CV) was 25.6% among the 20 morphological characters. Genetic distance (Nei’s) ranged from 1.11 to 10.94, and the average genetic distance was 6.10.. According to UPGMA clustering result that cultivars genetic relationship connected with their geographical origins.13 cultivars from USA grouped in 3 clusters:A-1, A-2 and B-2, while 12 cultivars from China grouped in B-1 and B-4 clusters. Hainanl, Hainan2 and Hainan3 showed close genetic relationship each other, maybe they are the same cultivars.The results of principal components analysis showed that the shape of leaf and fruit, characters of flower, color of soft tip (leaf) were the decisive factors affecting classification of sapodilla cultivars. They revealed about 64.8% morphological information.13 cultivars from USA could be classified into 4 types:large fruit sapodilla, long-oval leaf sapodilla, long-oval fruit sapodilla and round fruit,oval leaf sapodilla.12 cultivars from China could be classified into 2 types on the basis of color of soft tip (leaf), and 3 Hainan cultivars could be selected according to the flower characters.To fully understand the genetic diversity and relationship among sapodilla cultivar resources.31 cultivars were analyzed using fluorescent-labeled AFLP markers. From 64 AFLP primer combinations,9 were selected for the analysis based on number and quality of polymorphic fragments. A total of 1131 AFLP markers (75-500bp) were identified, of which 1096 (96.80%) were polymorphic. On the species level, the polymorphic rate was 89.04%. The genetic diversity was different among cultivars. The polymorphic rate rang from 22.19% to 45.89%, with a little low than the other tropical fruit trees, such as lichee, longan and banana. The number of amplified bands also different among cultivars. Among 31 cultivars,28 had unique fragments ranging from 350 to 500 bp. These unique fragments had important use in cultivars identification and gene clone.The genetic similarity coefficients (Nei and Li’s) ranged from 0.40 to 0.87, and the average was 0.65. Using UPGMA clustering method,31 cultivars were placed into three different clusters. The 4 mamey sapote cultivars were separated in genetic similarity coefficients 0.52 and grouped in Cluster MAM. The 27 sapodilla cultivars were grouped into two major clusters:Cluster SAP1 and Cluster SAP2. Cluster SAP1 contains 13 American cultivars,10 Chinese cultivars and 1 Vietnamese cultivar, and showed closely genetic relationship among each other. Cluster SAP2 were grouped by 3 Chinese cultivars:Yunnanl, Yunna2 and Zhanjiang, which were distinctly different from all other sapodilla cultivars in Cluster SAP1. PCOA (Principal Coordinates Analysis) analysis also supported the UPGMA cluster grouping. The clustering results concluded that sapodilla cultivars genetic relationship was not correlated with their geographical origins.The polymorphism bands rate of sapodilla population was 94.80%. Coefficient of gene differentiation (GST) was only 0.4833, which means about 51.17% genetic diversity existed in population, much low than cross-compatibility plants. The genetic diversity of Guangxi population was abundant and had close genetic relationship with American population. Yunnan population was an especial sapodilla species and should be protected.5 cultivars were selected from sapodilla cultivar resources to examine flowering characteristics, pollen viability, self and cross- incompatibility. TTC method results showed that the pollen viability was different in different flower stages. The pollen viability decreased slowly from bud stage to full bloom stage. All of the pollen viability was higher than 60% in three stages. Hainan cultivars were higher 10% than that of Guangxi cultivars. Sapodilla pollen germination rate was about 10% in full bloom stage. Self-incompatibility was the major factor affecting fruit set, and it was a special GIS type. Pistils and ovary tissue can restrain the growth of pollen tubes strongly. Cultivars genetic relationship had close correlation with their pollination compatibility. The genetic relationship was more close, then their cross- compatibility more low. The result of genetic relationship could guild us to select the pollination cultivars to receive high fruit yield.更多还原