Study On Genetic Diversity And Cultivar Identification Of Osmanthus Fragrans Germplasm

This thesis mainly dealt with the genetic diversity, genetic relationships and cultivar identification of Osmanthus fragrans Lour. germplasm by morphology, DNA markers (SRAP and ISSR) as well as DNA sequences (nrDNA ITS and cpDNA trnL-F), with 88 cultivars, wild species of O. fragrans from Wuxi, Nanjing, Hangzhou and Liuyang (Hunan Province) and 2 contrast species in Osmanthus (O. heterophyllus and O. cooperi) as materials. SRAP amplification system suitable for O. fragrans genome was set up and optimized. Genetic diversity and genetic relationships of O. fragrans germplasm resources were studied and discussed with SRAP and ISSR markers. ITS sequence and trnL-F sequence of several cultivars, wild species of O. fragrans were measured, and relationships among the 4 groups of O. fragrans were discussed primarily based on the combination of two sequences, with O. heterophyllus and O. cooperi as outgroups. The keys to 88 cultivars were made based on their major morphological characteristics, and two fingerprinting maps in DNA of O. fragrans were constructed based on SRAP and ISSR markers, and they can be used as morphological and molecular traits respectively for identification, cultivation, development and utilization of O. fragrans germplasm. The main results were as follows:1. Establishment of SRAP-PCR Amplification System and Analysis of Genetic Diversity and Genetic Relationships for O. fragransWith O. fragrans'Zao Yingui'as material, concentration gradients of template DNA, Mg2+, dNTPs, primer and Taq DNA polymerase enzyme in SRAP-PCR system were compared for genomic DNA of O. fragrans by monofactorial experiment, and the optimal SRAP-PCR amplification system was established. The optimal concentrations of each factor in the total 10μL system are 30 ng DNA,2.5 mmol·L"1 Mg2+,0.2 mmol·L-1 dNTPs, 0.4μmol·L-1 primer and 0.75U Taq polymerase enzyme.Genetic diversity and cultivar classification of 88 cultivars and wild species of O. fragrans were evaluated by using sequence-related amplified polymorphism (SRAP) marker, with O. heterophyllus and O. cooperi as contrast species.18 primer pairs produced 296 loci, out of which 248 were polymorphic, the percentage of polymorphic loci was 83.78%.16.4 loci and 13.8 polymorphic loci were amplified by each pair of primers. The Shannon information index (0.3412), genetic diversity (0.2191) and polymorphic loci rate (84.46%) of Albus Group were the highest among the 4 groups of O. fragrans. The measurement of genetic variation showed that the coefficient of genetic differentiation of O. fragrans was 52.95%, most of the genetic variation existed among groups. The cluster analysis showed that six groups could be clustered when genetic similarity coefficient was given as 0.762. The genetic distance between Asiaticus Group and other 3 groups was far. Cultivars with deep color in Luteus Group often clustered together with several cultivars in Aurantiacus Group. Cluster result based on SRAP marker was approximately consistent with taxonomic result based on morphology.2. Analysis of the Genetic Diversity and Genetic Relationships of O. fragrans Based on ISSR MarkersCultivar classification of 88 cultivars and wild species of O. fragrans were evaluated by using ISSR markers, with O. heterophyllus and O. cooperi as contrast species.28 primers produced 286 loci, out of which 269 were polymorphic, the percentage of polymorphic loci was 94.06%. The Shannon information index (0.3884), genetic diversity (0.2532) and polymorphic loci rate (87.41%) of Albus Group were the highest among the 4 groups of O. fragrans. The measurement of genetic variation showed that the coefficient of genetic differentiation of O. fragrans was 56.84%, most of the genetic variation existed among groups. The cluster analysis showed that seven groups could be clustered when genetic similarity coefficient was given as 0.720. Cultivars within the same group often clustered together first. The genetic distance between Asiaticus Group and other 3 groups was far. Cultivars with deep color in Luteus Group often clustered together with several cultivars in Aurantiacus Group. Cluster result based on ISSR marker was approximately consistent with taxonomic result based on morphology.3. Comparison of Analysis on Genetic Diversity and Genetic Relationships Based on SRAP and ISSR MarkersGenetic diversity and genetic relationships based on SRAP and ISSR markers were both similar and different. The result showed that more (16.4) loci were amplified by each pair of SRAP primers than that by each ISSR primer, whereas the percentage of polymorphic loci produced by ISSR marker was higher (94.06%) than that by SRAP marker. Results based on two markers both showed that observed number of alleles, effective number of alleles Nei's gene diversity, Shannon information index, genetic diversity and polymorphic loci rate of Albus Group were the highest among the 4 groups of O. fragrans, and those indexes of Luteus Group were the second. The sequence of polymorphic loci rate was: Albus Group> Luteus Group> Aurantiacus Group>Asiaticus Group. The genetic distance between Asiaticus Group and other 3 groups was far; the genetic distance between Aurantiacus Group and Asiaticus Group was the farthest while the genetic distance between Aurantiacus Group and Luteus Group was the nearest. Two markers also showed that most of the genetic variation existed among groups. Cluster results based on SRAP and ISSR markers were approximately consistent with taxonomic result based on morphology, and cluster result based on SRAP marker was more similar to morphological classification result. O. fragrans cultivars'Taoye Chi Yin','Zhuye Yin','Duolie Yin','Wuban Yin','Yin Shizi','Yuanban Zi Jin','Qiu Cheng'and'Yin Dan'from Wuxi Plum Garden, were different not only on morphological level but also on gene level compared with other cultivars, so they could be further classified as new cultivars.4. Primary Phylogeny Study of O. fragrans Based on nrDNA ITS and cpDNA trnL-F SequencesThe ITS and trnL-F sequences of 4 cultivars representing 4 groups, wild species of O. fragrans were measured by using direct sequencing of PCR product, and sequences of 6 cultivars from Albus group were compared based on the measured sequences. The sequences of ITS and trnL-F were analyzed with O. heteropyllus and O. cooperi as outgroups, and genetic relationships of the 4 groups of O. fragrans was discussed primarily by setting up UPGMA tree based on the combination of two sequences. The length of ITS sequence of all tested materials was 620-650bp and an regular 617bp sequence was obtained by sequence comparison and sequence cutting on both ends. There were 17 variable sites, out of which 2 were parsim-info ones, accounting for 2.76% and 0.32% of the total length. The length of trnL-F sequence of all tested materials was 860-900bp and an regular 846 bp sequence was obtained by sequence comparison and sequence cutting on both ends. There were 11 variable sites, out of which 4 were parsim-info ones, accounting for 1.28% and 0.47% of the total length. ITS sequences of 6 cultivars from Albus Group varied slightly, so it was deduced that genetic relationships of cultivars in the same group can not be revealed by their ITS sequences.Genetic relationships of the 4 groups of O. fragrans could be revealed to some extent based on the combination analysis of two sequences, with O. heterophyllus and O. cooperi as outgroups. Among autumn flowering cultivars, the genetic relationships between Aurantiacus Group and Asiaticus Group was the farthest, the genetic relationships between Aurantiacus Group and Luteus Group was the nearest. Among the 4 groups, the genetic relationships between Aurantiacus Group and wild species of O. fragrans was the farthest, which agreed with the conclusion drawn by several scholars that Aurantiacus Group is the most developed among autumn flowering cultivars based on morphological classification. Both ITS and trnL-F sequences had some variable sites that could be used for distinguishing O. heterophyllus and O. cooperi with O. fragrans (wild species and cultivars), or wild species with some cultivars of O. fragrans.5. Cultivar Identification of O. fragrans Based on Morphology and DNA Fingerprinting Maps Based on Molecular MarkersKeys of O. fragrans cultivars based on morphology and DNA fingerprinting maps based on molecular markers can be used as morphological and molecular traits respectively for identification of O. fragrans germplasm.Different cultivars of O. fragrans vary distinctively in morphology, in aspects like flowering season, flower character (inflorescence type, flower type, flower size, flower color, flower smell, stamen, pistil and fertility), fruit and seed, stem, bark, branch, leaf, etc., Some of these morphological characteristics are stable enough to serve as important taxonomic traits of morphology. The 88 cultivars belong to 4 groups, based on different flowering seasons, inflorescence types and flower colors. They are O. fragrans Asiaticus Group, O. fragrans Albus Group, O. fragrans Luteus Group and O. fragrans Aurantiacus Group. The keys to 88 cultivars were made based on their major morphological characteristics.Special SRAP and ISSR markers of 88 cultivars, wild species of O. fragrans were obtained by 18 SRAP primer pairs and 28 ISSR primers, with O. heterophyllus and O. cooperi as contrast species. SRAP fingerprinting map in DNA of O. fragrans was constructed by two primer combinations pm17-em10 and sa15-em10; ISSR fingerprint map in DNA of O. fragrans was constructed by the combination of two primers ISSR12 and ISSR27. The result showed that 76 cultivars, wild species of O.fragrans, O. heterophyllus and O. cooperi had special SRAP bands, whereas 64 cultivars, wild species of O. fragrans, O. heterophyllus and O. cooperi had special ISSR bands, and these bands could serve as special SRAP and ISSR markers for molecular identification of O. fragrans germplasm. The SRAP and ISSR fingerprinting maps of O.fragrans constructed both can identify each of 91 germplasm and can be used as molecular marker traits for identification of O. fragrans germplasm. Identification of O. fragrans germplasm can be conducted more effectively and accurately by combining keys of O.fragrans cultivars based on morphology with DNA fingerprinting maps based on molecular markers.The results from morphology, DNA markers and DNA sequences showed that O. fragrans germplasm is rich in genetic biodiversity. Different cultivars and groups vary not only on morphological level but also on molecular level. Genetic relationships between Aurantiacus Group and Asiaticus Group is the farthest, while the genetic relationships between Aurantiacus Group and Luteus Group is the nearest. Cluster results based on SRAP and ISSR markers showed that it is rational to divide cultivars of O. fragrans into four groups (Asiaticus Group, Albus Group, Luteus Group and Aurantiacus Group), but flower color is only one criterion, other characteristics also should be considered.Analysis based on three aspects showed that cultivar classification, group evolution and genetic relationships of O.fragrans is rather complicated, and different methods might draw different conclusions. In order to solve above problems more scientifically, more methods like anatomy, palynology, quantity research, DNA molecular markers and DNA sequences should be combined and verified with each other, with sufficient and reliable materials.