| As one of the most attractive ornamental flower, Dendrobium has exhibited its promising market potentiality. At present, there are some the problem to urgent need solve in the dendrobium industry, One of the most critical problems is lack of native varieties with independent intellectual property rights and market competitiveness. In turn, it has become the bottleneck that restrains the development of dendrobium industry in China. And evaluation and innovation of germplasm resources is the basis of breeding work, genetic linkage map can enhance the breeding efficiency by shortening the period. Nowadays, evaluation, innovation and genetic linkage maps of Dendrobium were rarely reported, Restricts the breeding and the sustainable development of the dendrobium industry. In view of this, In this study, Method of botany, cytology and molecular biology was used to evaluate Dendrobium germplasm resources collected for us, include on main cultivars at home and abroad. Germplasm innovation and construction of Genetic linkage map were studied on Dendrobium based on comprehensive evaluation.This work will lay a foundation for the evaluation and breeding of Dendrobium.The research result obtained as follows:1. The botany evaluationAccording to the standard of<Descriptors and data quality control for Dendrobium>,11 botanical characters of 39 Dendrobium Cultivars were evaluated. The results showed that the plant height, flower number, flower length and breadth of these Dendrobium are above the medium level, the inflorescence length is on the medium level; The flowering length of most varieties is between 25～40 days, the shortest is 18 days, and the longest is 64 days; According to the colour of the petal, the 39 Dendrobium Cultivars can be divided into red line(16), green line(5), purple line(4), white line(7) and compound colour line(7).We analysis the 11 botanical characters of 39 Dendrobium Cultivars base on the Eudidean distance cluster analysis, the result showed that these Dendrobium Cultivars can be divided into two groups, the first group includes 8 varieties, most is the varieties that has long flowering length and higher plant height; the second group include 31 varieties, this group divided into two subgroups, the first subgroup is main include the varieties with red colour and white colour flower, and the second subgroup is main on the varieties with red colour and green colour flower.2. The cytological evaluationThe number of chromosomes in six Dendrobium cultivars were investigated. It was found that five cultivars(Den.Mangosteen, Den.Burana Pink No.2, Den. Burana Star Blue, Den.Sunrise Crame, Den.Buranan Beauty×D.Toraohash) were tetraploid (2n=4x=76), one cultivars(Den. Copper King) was diploid(2n=2x=40).3. The molecular biology evaluationIn order to clarify the genetic diversity and genetic relationship of Dendrobium germplasm resources, based on the Nei & Li similarity coefficient, we analysis the Dendrobium resources used UPGMA method to clustering, this research result will provided the basis for parent selection for Dendrobium hybridization.The genetic diversity of 33 Dendrobium native species was analysed by RSAP, the analysis result showed that the similarity coefficient is 0.081～0.442, the 33 Dendrobium native species can be devided into 6 classes at the 0.181 similarity coefficient, this result is in according with the Flora of China, this result reveal that the Dendrobium native species has a rich genetic diversity.The genetic relationship of 42 Dendrobium Cultivars was analysed by SRAP, clustering analysis result showed that, at the 0.332-0.936 similarity coefficient, the nearest genetic relationship is Den.caesar and Den,Sunrise Crame, and the longest is Den. White Antelope and Den.Burana Twist No 17. The 42 Dendrobium Cultivars can be divided into 5 classes at 0.667 similarity coefficient, the clustering is showed a close relationship to flower shape and colour, this result is different from clustering analysis result of botanical characters.4. The innovation of Dendrobium germplasm resourceHybridization method:According to the research result of botany, cytological, molecular biology evaluation and the breeding aim, we make set 80 hybridized combinations, among the 80 hybridized combinations,29 hybridized combinations bear fruit, and 10 hybridized combinations germinate. The hybridization result showed that the arrange base on the combination bear fruit rate is:hybridized combination within group of variety> hybridized combination between-group> hybridized combination between native species> hybridized combination between native species and variety. The bear fruit rate exist difference of reciprocal cross hybridization combinations.New Dendrobium germplasm resources are obtained:RY1024 and RY1132 have the flowering plant; RY4-7, RY7-10 and RY6-5 have plantlet.Mutagenesis method:We treated three Dendrobium varieties plantlet(088,168, 166) with different irradiation doses(10,20,30,40 Gy)60Coy ray, the results showed the half lethal dose were different among the varieties, the half lethal dose of 168 is 35.44 Gy, and determine 35 Gy as the optimal radiation dose of 168; the half lethal dose of 166 is between 30-40 Gy, and the the half lethal dose of 088 is above 40 Gy.We analysis of irradiation affect to rate of survival, plant height, pseudobulb length, leaf length and breadth, the result showed that all the radiation dose has inhibition for plant development,30Gy and 40Gy has a obvious inhibition for plant height, pseudobulb length and leaf length, and have a difference between the varieties, but there is no obvious affect to leaf breadth for difference radiation dose.5. construction of genetic linkage mapBased on the results of the above evaluation, we selected Den.Mangosteen as female parent, and Den.Burana Pink No.2 as male parent,which there are relatively larger genetic differences between them. An integrated genetic linkage map was constructed from the Fi population with 190 individuals by SSR, SRAP, RSAP and ISSR markers by"double pseudo-testcross" strategy.25 SSR segregation loci,346. SRAP segregation loci,31 RSAP segregation loci and 51 ISSR segregation loci were identified and used for genetic linkage analysis. The genetic linkage map for Den.Mangosteen and Den.Burana Pink No.2 was successfully constructed. For the map,216 loci were arranged into 29 major linkage groups (LGs), each containing a minimum of four markers, and a further 58 markers were distributed to 14 triplets and 6 doublets. The map covered a total distance of 1421 cM, the average distance of the map being 9.56 cM between adjacent markers. The length of the linkage groups ranged from 8 to 101 cM, and the number of markers per group varied from 4 to 18. the largest number of markers is Linkage group LG1(18), The longest is Linkage group LG3(101 cM), On average, each LG contained 7.45 markers.The longest gap of 33cm was found on LG21. Total length of linkage group is 1842 cM. Frame map coverage and total linkage group coverage in this study is 71.9% and 93.2%, respectively. Construction of the maps will providing an basis for the application of marker-assisted selection in Dendrobium breeding program. |
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