Predict Of Genome Size And Analysis Of Karyotype In Calycanthaceae

The Calycanthaceae(Angiospermae-Dicotyledoneae-Archichlamydeae-Ranales)is a worldwide famous family of ornamental flowering plant.Especially,Chimonanthus praecox is famous for its blooming in winter when there is almost no other flowers.So far,the study on the genome size of the Calycanthaceae has not been reported.Therefore,in this study,the genome size of the Calycanthaceae was measured by flow cytometry so that the data can provide evidence for the genomics research.At the same time,The Photoshop image processing software was used for the karyotype analysis,which provided new cytological evidence for the origin and evolution.The major conclusions are as follows:1.The protocol of Calycanthaceae nuclear suspension preparationBy setting the 4 kinds of nuclei lysis control tests,i.e.,Tris·MgCl2?WPB?LB01 and Partec,the experimental materials,lysis processing time,the choice of internal and external standard method,the choice of the fluorescent dye and the dyeing time involved in the flow cytometry were explored.It has been detected that the best result is under this circumstance,fresh leaves as experimental material,cracking liquid extracting 3 min in WPB dissociation solution,cold dark DAPI(50?g/ml)stain 10 min.The method has the advantages of little cell debris and clear main peak.2.Genome Size Study of Calycanthaceae plantIn this study,the genome size of 6 kinds of Calycanthaceae plant was tested by flow cytometry under using Glycine max ‘William82' and Oryza sativa ‘Nipponbare' as a external standard.It's the first time to identify the genome size for Chimonanthus praecox,Chimonanthus nitens,Chimonanthus salicifolius,Chimonanthus campanulatus,Calycanthus floridus and Calycanthus chinensis,and they respectively are 805.88Mb?673.57Mb?873.58Mb?790.46Mb?854.79Mb?838.38 Mb.3.Karyotype Analysis of Calycanthaceae plantFor the observation of chromosomes karyotype,fleshyoung leaves were pretreated in8-hydroxyquinoline(0.002 mol/L)in dark for 3.5h or 4.0h before fixing in Carnoy's solution for 20-24 h.Then the fixed material was macerated in 1mol/L HCl(hydrochloric acid)at 60? for 8 min,and stained in improved carbol fuchsin and squashed beforeobserving under an Olympus microscope.The results shows that basic chromosome number of Calycanthaceae was proved to be X=11.Chromosome number was proved to be 2n=22.There is one chromosome type in the Calycanthaceae: metacentric chromosome(m)and no satellite.Karyotype type were identified as 1A and 1B.