Research Of RAPD Markers And Cytogenetics On Trichobitacin Of Different Genotypes

In this experiment,molecular technique and chromosome karyotype analysis were used to analyze the genetic relationship of nine cultivars and nine wild stirps of Trichosanthes kirilowii Maxim which were from different regions of china,so that we can establish a set of rapid,sensitive and accurate systems of seed identification,the healthy development of Trichosanthes kirilowii Maxim will be promoted and the sustainable utilization of Trichosanthes kirilowii Maxim resources will be realized.Results were as follows:1.In the study,the genomic DNA extracted by advanced SDS method could be fully used for RAPD analysis.The extraction method is simple,economic and quick.2.RAPD technique characterized with no strict limitation to primer,strong polymorphy and sensitive,but it is not good in stability and repeatability,so there is a necessity to find a RAPD reaction system suitable for the experiment in order to promote stability and repeatability.The influencing factors and experimental parameters on RAPD were studied in the items,such as template,dNTPs,Taq DNA polymerase,primer,Mg²⁺ and denaturation temperature,annealing temperature and time,the elongation time and cycle numbers.As a result,optimal PCR amplification system included 1×PCR buffer,1.5mmol/L Mgcl2,0.2mmol/L dNTPs,0.4μmol/L random primers,75ng DNA template,1U TaqDNA polymerase.The optimal amplification program was as follows:5min at 94℃, followed by 94℃for 30sec,38℃for 80sec,72℃for 120sec,40 cycles and final extension time of 72℃for 10min.3.A total of 259 products were amplified by 30 selected primers,of which 241(93.1%) were found to be polymorphic among the 18 genotypes,random primers S460 and S1114 had the most products up to 13 bands,while S497 and S2032 had only 4 bands.The length of most amplified fragments ranged from 100 to 2000bp.It was concluded that eighteen breeds which had been tested in the research could be classified into seven groups.4.RAPD analysis can examine lots of variations.For example,Changxing Trichosanthes which was amplified by primer S385 can produce specific marker S385—720bp,Sichuan Trichosanthes which were amplified by primer S1134 can produce S1134-300bp band deletion.Shandongxiaopian can produce S1137bp-300bp band deletion and specific band of S460—200bp respectively amplified with primer S1137 and S460.These specific bands were important characterization for distinguishing Trichosanthes kirilowii Maxim cultivars.5.In studies of cytological markers,cells of chromosomes scattered,flatted,more split phases were obtained using modified F-BSG method.The results of study for karyotype showed that six breeds had similar karyotypes,but they also had some differences among them,such as size and number of satellite.According to those we could identify them. Therefore this cytological research can be cytological markers used to analyze the relations and the breeds distinction of various genotypes of Trichosanthes kirilowii Maxim.