Genetic Analysis Of The Male Sterility Of Tagetes Erecta And Its Application In Breeding

Tagetes erecta and T. patula, belonging to the Tagetes genus of the Asteraceae family, were multipurpose plants. Their flowers are one of the most important natural resources for xanthophylls. The marigold plant also contains bioactive compounds that are widely employed as insecticides, fungicides and nematicides. The species also has the advantage that it can be cultivated under a broad range of climatic conditions. But it was difficult to process the castration program because of the terminal capitulum composed of hundreds of florets. Male sterile lines are needed to yield F1 hybrid seeds and make the hybridization process relatively efficient and economic.In this study, morphological and cytological observation were conducted to explore the forming reasons of male sterility in T. erecta. Molecular marker technologies were used to identify the molecular markers linked to the male sterility and to evaluate the genetic relationship between T. erecta and T. patula. The compatibility, heterosis, combining ability were investigated in interspecific crosses. A high efficient technique system of chromosome doubling, ploidy identification and propagation was established for the purpose of transferring the male sterile genes of. T erecta to T. patula. The main results were presented as following:1. Morphological and cytological analysis and the exploration of the forming reasons of the male sterility of T. eracta. The morphologies of segregated male sterile and fertile plants of the male sterile two-type line were indistinguishable prior to flowering. The bud shape of fertile plants was column, while the sterile plants had truncated-cone-shape or conical-shape buds as a morphological marker linked to male sterility. Morphological and cytological analysis of flower structure indicated that the ’petal’organs of the male sterile florets appeared more similar to sepals in terms of colour, luster, shape, persistent characteristics and detailed cytological observations and it confirmed that the petals had converted to sepal-like structures. More critically, the stamen primordia deviated from the normal polarization and developed as style-like structures, thereby failing to produce any of the cell-types leading to pollen formation. This stamen-to-style transformation was morphologically incomplete and the organs were non-functional. Thus, although extraneously introduced pollen could germinate and attach to the papilla-like cells of the converted stamen/style organs, the germinated pollen tube could not fully penetrate the style-like structure. It indicated that spontaneous floral organ homeotic conversion was the cause of the male sterility trait in African marigold, while the male sterile flower maintained fully normal female functions as the true style was unaffected by homeotic conversion.2. Identification of molecular markers tightly linked to male sterility of T. erecta. An F2 segregation population was constructed from a cross of male sterile material M525A and inbred line f53f. ISSR, SRAP and AFLP techniques combined with bulked segregant analysis (BSA) were used to develop markers linked to the trait, and one SRAP marker S48 and four AFLP markers, AF1, AF2, AF3, and AF4, was identified. The S48 marker was converted into a dominant marker, SCS48, and the AF4 marker was converted into a co-dominant marker with sequence characterized amplified region (SCAR) marker conversion techniques and PCR walking techniques. These markers were mapped in a linkage group. AF1, AF3, AF4/SC4 were located on one side of the Tems gene, and AF2, S48/SCS48 were located on the other side of the gene. AF4/SC4 and AF2 were the most closely linked markers among these flanking markers, approximately 0.3 cM and 0.7 cM from the Tems gene, respectively. These results indicated that the molecular markers were close enough to the Tems locus to be applied in MAS and map-based clone.3. Studies on relationship and interspecific hybridization between T. erecta and T. patula. The relationship between six male sterile two-type lines of T. erecta and twenty-five inbred lines or cultivars of T. patula were analyzed by SRAP. T. erecta and T. patula could be distinguished based on genetic distance by UPGMA method. The cluster of T. patula was comparatively chaotic. The genetic distance of T. patula was relatively small, the similarity coefficient was high, and the genetic background was narrow. There was no significant difference in seed setting rate between interspecific hybridization of T. erecta and T. patula and sibling of T. erecta, and the seed germination rate was comparatively high. It indicated that the hybridization compatibility was high and there was no obstacle in the interspecific hybridization between T. erecta and T. patula. The first-flowering date of the interspecific F1 hybrids manifested high heterosis and other traits of the interspecific F1 hybrids showed mid-parent heterosis. Analysis of combining ability indicated that both male sterile two-type lines of T. erecta and inbreds of T. patula should be a particular focus of attention in interspecific hybridization. Analysis of genetic parameters showed that the additive effects were larger than non-additive effects, therefore, the inheritance of traits was mainly controlled by additive effects, and they should be effectively selected in the early generations.4. Polyploid breeding of male sterile two-type line in T. erecta. A high efficient technique system of chromosome doubling, ploidy identification and propagation was established. Twenty-nine tetraploid plants of T. erecta were obtained by three methods of colchicine treatment, among which germinating seeds immersed in 0.05% colchicine for 3-6 h had the highest doubling efficiency,88.89%. Cutting propagation and seed propagation techniques were used to preserve tetraploid plants of T. erecta that had very low seed setting rate, while the survival rate of cutting propagation was comparatively high. The tetraploid plants of T. erecta were dwarf, compact with diameter of inflorescence increased, which displayed excellent ornamental characters. The flower type of the interspecific F1 hybrids between tetraploid plants of T. erecta and T. patula was better than that of triploid hybrids, but the plant type was too compact, and had less branches. Therefore, it is necessary to improve the plant type of tetraploid plants in T. erecta for the transfer of male sterility from T. erecta to T. patula and further cross breeding of T. patula.