| Lilium spp., as an important ornamental flowers in the world, has an important economic value. There are abundant wild resources in China. However, the resources were destroyed severely, and the endangered species was growing. In addition, the utilization of Lilium resources was not optimistic. Investigation, evaluation and utilization of germplasm resources is the basis of innovation, yet the investigation and evaluation of the Lilium mostly concentrated on some region, lacking of a comprehensive and systematic understanding of Lilium resources. Resistance breeding has always been a hot spot for lily breeding. The harm of gray mold is severe and extensive in cutting flower production of lily, however, the evaluation and screening of Lilium resources resistance to Botrytis elliptica, breeding for gray mold resistance, as well as the mapping and cloning of resistance gene is very weak. Therefore, evaluation and excavation of excellent resistance resources is very important! Due to the above problems, on the basis of investigation and collection of Lilium resources, this research evaluated resources at three levels:the phenotypic traits and the AHP, the genetic relationships and molecular systematics and the resistance to gray mold. Moreover, cloning and expression analysis of resistance gene homologous from Lilium was carried out, laying a foundation for cloning gray mold resistant genes. Through these studies, the main results obtained are as follows:1. A systematic investigation was carried out in the Northeast, Central and Southwest regions of concentrated distribution of Lilium, a total of34species (varieties) representing86samples of Lilium were found. The survey found that the level of Lilium resources are widely distributed, especially the southwest China. Besides, Lilium resources occupy a wide variety of habitats consists of forests, mountain, shrub and meadows. L. brownii, L. lancifolium and L. sargentiae have very wide ranges on the vertical distribution. Rich variation within populations was found. Features of Bulbs added and characteristics of underground stemsbulbs were various. Although reproductive capacity of Lilium is strong, the update process is slow, and therefore it is vulnerable to the environment. An analysis of soil characteristics of the original habitat found that different kinds of wild lily in situ soil nitrogen, organic matter content, phosphorus, potassium, full of salt and pH value were different, and soil characteristics of the same species from different provenances were also different. L. davidii can be used as an excellent parent for salt resistance breeding. Soils in situ of L. regale, L.lancifolium, L. henryi, L. rosthornii, L. sargentiae and L. leucanthum was relatively infertile, suggesting that they have good adaptability.2. An analysis of phenotypic trait found that wild lily resources showed significant morphological diversity. Principal component analysis showed that floral organ factors were the relative importance of indicators in morphological differentiation and the classification of resources. An analytical hierarchy process (AHP) utilizing16indicator characteristics was used to evaluate the collected Lilium resources based on their ornamental value, utilization potential, and ecological adaptability. L. dauricum, L. henryi, L. rosthornii, L. tsingtauense, L. concolor var. pulchellum, L. lancifolium, and species with trumpet-shaped flowers were selected which showed better potential for exploitation than other species. This study also provides references regarding the effective exploitation and use of wild Lilium resources.3. The largest currently ITS phylogenetic tree of Lilium for214samples representing98species and five varieties, including44species and five varieties native to China was constructed. The results suggest that the subsection (subsect.)5c Comber should be classified into the true subsect.5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum â… , subsect. Lophophorum â…¡, and subsect. Lophophorum â…¢). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion6a. The results support Comber’s subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.4. Though the scanning electron microscopy (SEM), pollen tetrads was first found in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, the following hypothesis was proposed:evolution of an exine sculpture shows pollen type trends from Martagonâ†'Calloseâ†'Concolorâ†'Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions.5. Based on the analysis of genetic parameters of SSR markers, the results show that the SSR loci were polymorphism. Cluster analysis indicate that the resources of wild lily has a rich genetic diversity, Principal coordinate analysis showed a significant clustering of wild lily with geographical features; L. henryi and L. rosthornii are closely related, and they showed close relationship with subsect. Leucolirion6a, moreover, L. brownii should be classisfied into subsect. Leucolirion6b. Besides, SSR markers in genetic analysis and phylogenetic analysis can be effectively used for lily evaluating, genetic diversity analysis as well as germplasm identification.6. Eight high resistance resources (L. regale, L. leucanthum, L. sargentiae L. dauricum, L. henryi. L. rosthornii, L. tsingtauense, L. taliense) were selected based on in vitro inoculation by mycelium block. These resources could provide for materials for resistance breeding and excavation of disease resistance genes. RGA-PCR amplification reaction system of lily was established. Resistance evaluation and cluster analysis by RGA fingerprinting showed similar resistance tending to gather together.7. Based on transcriptome sequencing, the NBS-LRR resistance gene analogs from Lilium was firstly isolated and cloned. Phylogenetic studies revealed close relationships of some Lilium RGAs to R genes and classified these sequences into five main subclasses. Pairwise comparisions indicated that the deduced amino acid sequences of RGAs contain the conserved domains such as P-loop, Kinase-2, Kinase-3a and GLPL, and the RGAs all belonged to nonTIR-NBS type resistance gene segments. Southern blot analyses showed that the RGAs were organized as multicopy loci in the Lilium genome. Quantitative real-time PCR analysis revealed dramatic variations in the transcript level of RGAs with the increase in the level of resistance of the hosts against gray mold, indicating its close relationship with the resistance against this pathogen. These RGAs are of great value for positional cloning of disease resistance genes. Our results provide a primary RGA pool for the further functional validation of resistance genes in Lilium. |
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