Genome Size Determination And Chromosome Conformation Capture Of Huperzia Serrata Content

Huperzia serrata(Thunb.).(H.serrata),there are many studies on the synthesis of alkaloids,but there are few studies on the cell biology of H.serrata.Based on the study of genome size and chromosome conformation capture,the chromosome number of H.serrata was studied by different methods.The size of DNAC-value was analyzed by flow cytometry,and the chromosome conformation of H.serrata was captured by Hi-C technology.It can provide basic data for the whole genome sequencing and enrich the research content of cell biology of H.serrata.The main results are as follows.(1)Three methods were used to prepare chromosome plates,which were conventional squashing method,low permeability flame drying method and fluorescence in situ hybridization method.The chromosome of H.serrata(2n = 44)was obtained by conventional squashing method,pretreatment with ice water mixture for 24 h,acid hydrolysis with 1 mol /L HCl at 60 ℃ for 10 min,and enzymatic hydrolysis with 3% cellulase,3% hemicellulase and 2.5% pectinase for 4 h;The improved method used the method of wall removal and low permeability flame drying,pretreated with 0.2% colchicine and 0.002 mol / L8-hydroxyquinoline for 20 min,and treated with 2.5% cellulase and 2.5% pectinase mixture for 2 h.The chromosome morphology was clear and well dispersed.The average chromosome count of H.serrata was 2n = 134;Methods 3.The chromosome number of H.serrata was indirectly verified by fluorescence in situ hybridization(FISH).The telomere repeat probe was used to hybridize with the prepared metaphase chromosome sections.The results showed that the signal of fish fluctuated greatly,and the method needs to be further explored.(2)The DNAC-value of H.serrata was detected by flow cytometry.Four kinds of dissociation solution were screened by external standard method,among which m Gb dissociation solution had the best effect on H.serrata.Fresh young leaves and young leaves dried with silica gel for one week were selected.Corn was treated with m Gb dissociation solution as reference plant.The results of dry and fresh samples were 0.82 pg different by internal standard method.However,according to the comparison of CV value(the smaller the CV value,the more reliable the result),Fresh leaves were more suitable as materials,and the DNAC-value of H.serrata was 7.16 pg.(3)Statistical analysis of DNAC-values of pteridophytes.According to DNAC-value database and chromosome database,the data of pteridophytes were screened.Among them,the chromosome number of Equisetaceae was 2n = 216,and the distribution of DNAC-value was different.In Isoetes,the chromosome base was x = 11,with 11 ploid as high as 2n = 110,It is 1877 times of the minimum value of 0.08 pg in Selaginella.The chromosome number of Huperzia is also high,and the DNAC-value is higher than that of Lycopodiaceae.According to the flora,it can be divided into 10 orders,and the variance analysis of these 10 orders shows that there is no significant difference between Lycopodia,Equiseta,Lepidoptera,Marattiales and Eufilicales.The average value of C value of Pteridoptera is significantly higher than other orders,and the average value of C value is also significantly higher than other orders.(4)The spatial conformation of H.serrata chromosome was captured by Hi-C technique.From Hi-C data analysis,98.42% of the genome sequences were located on 72 chromosomes.Among the sequences located on the chromosome,the length of the sequence that can determine the sequence and direction is 2.98 Gb,accounting for 96.94% of the total length of the located chromosome sequence.The chromosome number of Huperzia serrata is 72,which is similar to the result obtained by the improved method in the first chapter.