Exploration Of The Molecular Mechanisms For Apple(Malus×Domestica Borkh.) Columnar Tree Architecture

Apple is one of the most important fruit in the world and both of its cultivation area and yield are the front in Chinese fruit production, In21st century, the development trend of apple cultivation pattern is intensive, mechanized and modernized, which can be reflected in the way of dwarfism and density planting. It has the advantage of early fruiting, high yield, easy management, and mechanized operations, and so on. Columnar apple is a special dwarf mutant and is very suitable to high density planting.So far, the molecular mechanism of apple columnar phenotype is unclear. Studies on molecular markers of columnar gene are not only valuable to the molecular assisted selection for columnar trait, but also have great meaning to map based cloning of this gene.Gibberellin (GA) is an important plant hormone. Bioactive GAs influence the entire life of higher plants. The regulation to plant height is one of the most remarkable physiological roles of GAs. It was reported that the height of plant were closely related to the type of GAs and its metabolic pathways. To date, The genes encoding the key enzymes in several steps of gibberellin biosynthesis are still unknown. Therefore, cloning these genes is of great importance to reveal their molecular characters and to understand their functions in the formation of important traits.The main researches and results were as follows:1. Using the F1population of ’Fuji’×’Telamon’as plant materials, we screened a SSR marker Hi01b01which linked to the columnar traits of apples by High resolution melting (HRM) method. The result of evaluation of different reaction systems for HRM analysis indicated that5μL reaction volume was as efficient as10μL or20μL for the polymorphism detection. In5μL reaction volume, even DNA concentration as small as0.25ng/μL was good enough for PCR amplification and the following HRM detection. It demonstrated that a touchdown PCR program could also performed very well in HRM analysis for polymorphism detection.2. In this research, the open reading frame sequences of three new genes, MdCPS, MdKS, and MdKAO were isolated from stem apical of ’Fuji’(GeneBank Accession Number is KC433942, JQ281521and KC433941, respectively). The coding sequences were2400,2214and1512bp in length and encoding799,737and503amino acids, respectively. In addition, by searching in the apple genome,7genes encoding the key enzymes of GA biosynthesis, MdCPS, MdKS, MdKO (FJ571521), MdKAO, MdGA20oxl(AB037114), MdGA3oxl(FJ571520) and MdGA2ox (FJ571521) were located on chromosome11,10,8,2,1,9and10of apple genome, respectively.3. Sequences of the amino acids encoding by MdCPS, MdKS, MdKAO and MdGA2ox were analysed by the bioinformatics. The results showed that protein MdCPS and MdKS belonged to TPS superfamily and both contained the two structural domains of TPS. MdKAO belonged to cytochrome P450superfamily. MdGA2ox belonged to the class2-oxoglutarate-dependent dioxygenases. Moreover, subcelluar location analysis showed that MdCPS was in mitochondrion; MdKS and MdGA2ox was in cytoplasm; MdKAO was in endoplasmic reticulum membrane. Homology analysis indicated that they were more identical with the homolog genes in other plants.4. Quantitative real-time PCR was used to analyze the expression of the five genes including MdCPS, MdKS, MdKO, MdKAO and MdGA2ox, in shoot apical tissue of ’Telamon’,’Fuji’and their F1progenies. The results indicated that the transcription level of almost all these genes in’Telamon’was lower than that in ’Fuji’. Moreover, as for the F1progenies, the expression difference of MdCPS and MdKS was not significant between the columnar and standard population. However, MdKO, MdKAO and MdGA2ox were distinctively lower expressed in columnar progenies than standard progenies. In addition, qRT-PCR was also employed to analyze the expression of the five genes in shoot apex of two columnar progenies and their standard mutants, The result showed that almost all genes lower expressed in columnar progeny ’Z95-121’ compared to it standard mutant ’P95-121’, which was the same as that in the columnar and standard parents. Nevertheless, the expression level of MdCPS, MdKS and MdKO in columnar progeny ’Z95-177’were slightly higher than its standard mutant ’P95-177’, but MdKAO and MdGA2ox were obviously lower expressed in the former one. Thus, we can speculated that the formation of apple columnar trait maybe related to the regulation of some genes encoding the key enzymes in the metabolic pathway of gibberellin biosynthesis.