| Dioscorea,a kind of perennial climber belongs to the family Dioscoreaceae,is highly economically important tuberous crop with about 600 species throughout the world.The accurate karyotype analysis is important to understand regulation of hereditary variation,developmental mechanism and evolution relationship of these species,which is significant for the further exploitation of germplasm resources.Positioning accurately homologous chromosomes in mitotic cells is an important trait for the establishment of the basic karyotypes.However,it is almost impossible for us to establish the accurate karyotypes of Dioscorea plants by traditional methods because of their large number and small size of the chromosomes and complicated ploidy.Fluorescence in situ hybridization(FISH)is now widely used for the localization of genomic DNA fragments,and the identification of chromosomes,which can help us to make the karyotype information more accurate.In this research,in order to overcome difficulties in chromosome preparation from root tip cells,the tender leaves from Dioscorea plants were used to prepare chromosome samples using the method of enzymatic hydrolysis and low permeability for cell wall removal.Some important factors during the preparation,such as leaf ages,pretreatment methods,enzymatic hydrolysis time,were discussed to optimize the chromosome preparation.Then a comprehensive karyotype analysis of nine materials from three Dioscorea species including D.alata,D.persimilis,D.bulbifera was performed based on 18S rDNA-FISH for the first time.The main results are as follows:(1)In order to provide a convenient and practical chromosome preparation method for Dioscorea molecular cytogenetic study,we established the metaphase chromosome preparation method from tender leaf cells of Dioscorea plants.The results of chromosome preparation study showed that using the immediately expanded tender leaves pretreated with the solution of 0.002 mol/L 8-hydroxyquinoline at 4 ℃ for 2 hours,and then enzymolysised with the mixture of 3.5% cellulase and 1.75% pectinase for 1 hour at 37 ℃were proved to be the best for the chromosome preparation.Meanwhile,metaphase chromosomes of nine materials of Dioscorea plants were obtained successfully.We counted the chromosome number of these Dioscorea plants germplasm resources and found that their somatic chromosome number ranged from 2n=40 to 2n=80,and the most common cytotype is 2n=40 chromosomes.(2)During karyotype analysis based on 18S rDNA-FISH,two 18S rDNA sites were observed in metaphase chromosome and interphase nucleu of seven materials with 2n=40chromosomes;three 18S rDNA sites were observed in metaphase chromosome and interphase nucleu of one material with 2n=60 chromosomes;four 18S rDNA sites were observed in metaphase chromosome and interphase nucleu of one material with 2n=80chromosomes.The basic chromosome number of these materials is x=20 and the ploidy levels are diploids(2n=40),triploids(2n=60)and tetraploids(2n=80),respectively;the absolute length of chromosome varied between 0.43 μm and 2.86 μm;the relative length of chromosome varied between 2.18% and 8.40%;the ratio between the longest and shortest chomosomes varied between 1.73 and 3.62;the index of relative length varied between0.44 and 1.68. |
