Karyotype Analysis And Establishment Of Fluorescence In Situ Hybridization Of Germplasm Resources Of Semperflorens Begonias

Begonia semperflorens is an important species of Begonia.It has significant commercial value and market potential because it keeps flowering and is highly resistant to adversity.At present,domestic and foreign researches on Begonia semperflorens mainly focus on cultivation physiology,and there is almost no research on genetic background.However,interspecific and intraspecific genetic penetration of Begonia is frequent for frequent artificial hybridization.It is important to clarify genetic background for breeding of excellent cultivars of Begonia.Fluorescence in situ hybridization（FISH）has been widely used in genetic diversity and gene localization in plants and animals.However,due to the small size（0.5-5μm）and large number of chromosomes in begonias,molecular cytology studies are difficult to carry out.In this study,B.schmidtiana was used as plant material and the key steps affecting the effect of fluorescence in situ hybridization experiment with 5S r DNA as probe were explored.The traditional means of root tip chromosome production was optimized,and fluorescent in situ hybridization for Begonia was established,which laid the foundation for the development of molecular cytology studies on Begonia.At the same time,5S r DNA localization studies were conducted on important wild begonias that form semperflorens begonias group and commercial cultivars,and their genetic relationships were initially explored at the cytological.The results are as follows:（1）The steps of pretreatment,enzymatic digestion time and staining and filming methods that affect the effect of chromosome filming were discussed using three species of begonias as test materials.The results indicate that the best producing conditions of different plants of begonia were similar.The optimum conditions were as follows:pretreatment with 0.002mol/L 8-hydroxyquinoline for 3 h,enzyme mixture（2.7%Cellulase R10 and 1.3%Pectolyase,p H 4.8）at 37℃for 15–35 min,and DAPI fluorescence staining.（2）Three important wild begonias that form semperflorens begonias group and two excellent commercial cultivars were used as experimental materials.There are clear chromosome pictures and karyotype analysis was performed.The chromosomes of the wild species were all 2n=2X=34,and the cultivar‘Big Series’was tetraploid with 2n=4X=68.（3）Established fluorescent in situ hybridization system of Begonia.Chromosome pretreatment:drying 6 h in 65°C.After treatment with 0.1 mg/ml RNAase for 1 h,5μg/ml pepsin for 10 min and 4%paraformaldehyde fixation for 10 min in sequence,the chromosomes were dehydrated with alcohol gradient.The hybridization mixture consisted 50%deionized formamide,10%DS solution,2×SSC,3-4μl probe DNA and dd H₂O in 25μl system.Chromosomes and probes were denatured separately,probes at 96°C for 10min and chromosomes at 70°C for 5min-10min.In situ hybridization was incubated at 37°C for 12-16h.Then DAPI was re-stained and examined microscopically under fluorescence microscope.（4）Information on the 5S r DNA signal distribution of three wild begonias and two excellent commercial cultivars of semperflorens begonias group was obtained.The signal distributions of wild begonias all appeared in pairs,and the evolutionary degree of wild begonias was inferred from the number of loci as Begonia cucullata var.cucullata>B.schmidtiana>B.subvillosa.The high number of unpaired signals on the two semperflorens begonia species,especially‘Big Series’,suggests that there was infiltration of other germplasm resources in addition to chromosome doubling during its formation.In this study,the fluorescent in situ hybridization system of Begonia was established,and karyotype information of some Begonia and the distribution of 5S r DNA on chromosomes were obtained.It laid the technical foundation for the subsequent cytological study of Begonia.