Ploidy Identification And Genome Size Determination Of The Genus Aspidistra Based On Flow Cytometry

The genus Aspidistra,belonging to the family Asparagaceae,is a perennial herbaceous plant with a rich variety of species in the genus,with more than 200 species around the world,mainly distributed in tropical and subtropical regions of Asia.The distribution and differentiation center of the genus Aspidistra is mainly located in Guangxi,China and northern Vietnam.In this study,the genus Aspidistra were used as the research object.Flow cytometry was used to identify the ploidy and determine genome size of the genus Aspidistra,combined with geographical distribution analysis,in order to elucidate the origin and evolutionary relationship of the genus Aspidistra.The main research results were as fllows:1.Using the leaves of the genus Aspidistra as experimential materials,four different cell nucleus dissociation buffers,namely WPB,LB01,Galbraith’s and the modified Galbraith’s,were used to prepare cell nucleus suspensions.The analysis using flow cytometry showed that the modified Galbraith’s dissociation buffer works best for the genus Aspidistra plants samples.Therefore,this study chose the modified Galbraith’s dissociation buffer to prepare cell nucleus suspensions for the leaves of the genus Aspidistra plants.2.The ploidy of 51 species of the genus Aspidistra plants were identified by flow cytometry.The mature leaves of the genus Aspidistra plants were selected,and the nuclear suspension of the genus Aspidistra plants were prepared using the modified Galbraith’s dissociation buffer and propidium iodide(PI)as fluorescent dyes,and the peak DNA fluorescence intensity of the genus Aspidistra plants were determined,and the peak range was 1475846.92 ～ 6250430.20.Among them,the peak range of 49 diploid the genus Aspidistra plants was determined to be 1475846.92 ～3608070.35;the peak value of the tetraploid the genus Aspidistra plant was5347660.94;the peak value of hexaploidy the genus Aspidistra plant was6250430.20.A ploidy identification system based on flow cytometry for the genus Aspidistra plants was established.To verify the accuracy of flow cytometry ploidy identification,the chromosome number of 51 species of the genus Aspidistra plants was observed and counted by the conventional chromosome smear and counting method.The results were consistent with the ploidy identification results using flow cytometry.The chromosome number of 17 species of the genus Aspidistra plants was reported for the first time.3.Allium fistulosum with known genome size and complete genome sequencing as standard plant,the genome size of 25 species of the genus Aspidistra plants were determined by internal standard method,which was14.16 ～ 44.66 Gb.By comparing the genome size measured by internal and external standard methods,it was found that the results of the genome size of 25 species of the genus Aspidistra plants were similar.4.By analyzing the genome size and geographical distribution,it was found that Guangxi,China and northern Vietnam were the distribution and differentiation center of the genus Aspidistra plants.The genome size of the species in this region varied greatly,and widespread species were distributed throughout the center and edge of the distribution.The genome size was at the medium level.It can be seen that Guangxi,China and northern Vietnam were likely to be the center of origin of the genus Aspidistra plants and the center of species diffusion.In addition,intragenus species polyploidy occurs at the distribution marigin of genus or species.