【作者】 陈天生；

【导师】 许莉萍；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2008， 硕士

【摘要】 分子标记辅助育种是提高育种效率的重要措施,但其在育种实践上的应用,依赖于甘蔗分子遗传多样性尤其是基于靶标基因标记的遗传多样性的研究以及与目标基因连锁标记的开发。糖分是甘蔗育种最重要的性状,蔗糖代谢基因与该性状的表现型密切相关。同时,甘蔗栽培上不时也遭受寒害胁迫。为此,本试验率先在国內应用TRAP标记,以5个蔗糖代谢关键酶基因及1个耐寒性相关基因为靶标基因,通过设计正向锚定引物和9个随机引物,采用其中34个多态性好的引物组合,对33份包括我国主栽品种、新选育品种(系)和新引进的重要种质,进行TRAP标记分析。共产生579个条带,其中多态性条带288条,占49.74%,显示正向锚定引物和上游随机引物组合也能扩增出丰富的多态性条带,暗示了甘蔗的开放读码框外显子存在着高度的变异性与多样性。基于靶标基因的遗传距离幅度为0.26～0.63,UPGMA聚类分析显示,在GD为0.52水平上,可将33个样本分为4类,其中ROC系列品种间靶标基因的相异系数小。基于靶标基因TRAP标记的聚类结果,不能完全反映血缘关系的远近,但能揭示不同基因型蔗糖代谢关键酶基因家族的差异程度,同时,也发现了一些较特异的种质,为应用分子标记数据指导甘蔗高糖及抗寒杂交育种的亲本选择与组合配置积累了基础资料。另一方面,本文还利用17个TRAP引物组合,对28份糖分性状表型差异比较明显的基因型及部分杂交后代个体进行多样性分析,共扩增出170个条带,其中109个为多态性条带,多态性比率为64.12%;基于靶标基因的遗传距离变化幅度为0.12～0.80,UPGMA聚类结果能反映甘蔗蔗糖代谢基因家族的遗传多样性,对高糖育种杂交亲本的选择和组合配置具有指导意义,同时,聚类呈一定的糖分高、低同类型率先聚类的规律,为建立糖分性状标记辅助评价技术奠定基础。此外,本研究结果同样显示了TRAP标记具有稳定性、高效性和易操作性的特点,暗示其在甘蔗分类、重要性状标记等方面,具有很大的应用潜力。研究结果具有重要的理论意义和实践价值。更多还原

【Abstract】 Marker-assisted selection(MAS)is one of main techniques,which can greatly accelerate the process and improve the efficiency of plant breeding,but it depends on the data of molecular genetic diversity,especially on the research based on target genes and the development of linked marker to target genes.Sucrose content is one of the most important characters,which closely related to the sucrose metabolism genes.In cultivation,cold stress is an important factor that affects the growth of sugarcane.For above,TRAP marker was occupied to study on the genetic diversity of thirtythree important sugarcane clones,including some main cultivars,recent released varieties and some germplasms introduced abroad recently.The total of six genes related to sucrose metabolism were selected as target genes and were used to design the anchored primers for pairing with nine arbitrary primers.The six genes contain five key genes involved in sucrose metabolism in sugarcane,and one gene reported to play an important role in the character of cold tolerance of sugarcane.In total,there were 90 primer combinations and thirty-four combinations of primers were screened to produce clear banding patterns and polymorphisms.The result showed that a total of 579 bands were scored with the 34 pairs of primed screened,of which 288 taking 49.74%in total were polymorphisms.Thus,the polymorphism results from combinations of forward fixed and arbitrary primers reflected that the exonic regions outside the open reading frames(ORFs)of sugarcane genes have a very high mutation rate and diversity.The cluster analyses using UPGMA revealed a narrow genetic diversity among the entries with genetic distance(GD)ranging from 0.26 to 0.63.At the GD level of 0.52,33 sugarcane clones were divided into four groups and the target-genes in ’ROC’ series cultivars had a small dissimilarity coefficient.Therefore,the cluster analysis based on TRAP analysis with six target-genes can reflect the variation degree of key genes in sucrose metabolism,in 33 sugarcane clones used in this study,while can’t contribute completely to parentage grouping of the 33 clones.At the same time,some special germplasms were found.Therefore,the data from the TRAP analysis can enhance the accumulation of basic data of parent selection and combination configuration and help in high-sucrose and cold-tolerance breeding practice.In this study,genetic diversity analysis was also conducted in another 28 sugarcane clones by 17 primer combinations.The 28 samples set included parents and their hybrid progenies,of which the differences of sucrose content character is relatively obvious.In other words,they are high-sucrose content and low-sucrose content individual.A total of 170 bands were scored and among them 64.12%ie.109 were polymorphisms. Cluster analysis showed that the genetic distance(GD),based on the diversity of the target genes,ranges from 0.12 to 0.80.The results of TRAP analysis on the 28 sugarcane genotypes can’t completely grouping these genotypes according to their pedigree records,as in some cases full and half siblings failed to cluster together. However,in this cluster analysis,it was observed to have an order distribution of high sucrose content clones and low sucrose content clones to some extent.And thus,it can provide the practical and theoretical foundation for the establishment of the sucrose trait evaluation in marker-assisted selection.Furthermore,it can also be concluded from this study that TRAP marker technique have many other merits,such as good repeatability, high efficiency,and easy operation,which suggests its great potential applicability in sugarcane classification,important trait marker etc.The results of this study have an important theoretical and practical value.更多还原