Cytogenetic Study On Larimichthys Crocea And Nibea Albiflora

In this study,we obtained the optimal procedure of chromosome preparation by testing a variety of factors.And fluorescence banding and fluorescence in situ hybridization?FISH?were carried out to characterize and identify the chromosomes of Larimichthys crocea and Nibea albiflora.The main results were as follows:1.The motic index?MI?of head kidney cells increased by three-injection method with a mixgure of 8mg dangshen and 20?g BSA per gram of fish body weight.The optimal conditions of other steps were a colchicine injection with dosage at 0.5?g per gram of fish body weight,a hypotonic treatment duration at 40 min with 0.075M KCl,transfer fresh carroy's fixtive?methanol:acetic acid=2:1?just before dropping,and keeping the target slide a little wet when dropping.Under these conditions,the area of the spread and the length of chromosomes would be improved.2.The fluorescence banding of chromosomes in L.crocea:PI staining was uniform;DPI staining displayed highlight at NOR region on the end of short arm of chromosome 10;DAPI staining showed vague bands on each chromosome;DDAPI staining showed obvious highlight on centromere regions and uneven staining but no reproducible bands on the arms.In additon,the homologous chromosomes of pair 10 showed different shape preliminarily.The fluorescence banding of chromosomes in N.albiflora:PI staining was uniform;DPI staining displayed highlight at NOR region on 1/3 of long armto the centromere of chromosome 1;DAPI staining showed one pair of negative bandsin consistent with the location of NOR;the highlight with DDAPI staining located on centromere regions and NOR on chromosome 1.3.The results of FISH on chromosomes in L.crocea:18S rDNA located on the end of short arm of chromosome 10;?TTAGGG?_n located on all telomeres and the interstitial area on a pair of chromosome;H-P3K clone located on centromere of each chromosome and short arms;with probes from genomic DNA of L.crocea,strong positive signal were observed on centeomeric regoins of all chromosomes and telomeric regions of some chromosomes;with probes from genomic DNA of N.albiflora,strong positive signal were observed on both centeomeric and telomeric regoins of all chromosomes..The results of FISH on chromosomes inN.albiflora:18S rDNA located on 1/3 of long arm to the centromere of chromosome 1;?TTAGGG?_n located on telomeres;H-P3K clone located on centromere of chromosome 1;using L.crocea'genomic DNA as probe,it was found that there are positive singal disperse on the arm of each chromosome and enhanced onthe centromeric region and telomeric region of all chromosomes,espetially on centromere of chromosome1;using N.albiflora'genomic DNA as probe,it was found that positive singal dispersed on the arm of all chromosomes and enhanced on both centromeres and centromeres,as well as on NOR of chromosome 1.4.The Fluorescence Pattern?FP?and 3D optical density histogram were obtained with IPP6.0 softwares based on DAPI banding of chromosomes in L.crocea and GISH resulted image of chromosomes in N.albiflora.The karyotype were analysed based on these data.The karyotype formula of L.crocea and N.albiflora were summirized as 2n=42t+4st+2sm and 2n=48t,respectively.In this study,abundant morphology markers and parameters of chromosomes were obtained in L.crocea and N.albiflora.Based on these data and image analysis,accurate identification and pairing of chromosome were achived for the first time in L.crocea and N.albiflora,which was the basis for futher study in molecular cytogenetics of these two species.The results also revealed the characteristics of chromosome structure of L.crocea and N.albiflora,including the location of of major rDNA,heterochromatin,AT/GC blcoks and so on.The diference in chromosome structure between L.crocea and N.albiflora were exhibited,which would be useful in analysis genetic structure of L.crocea and N.albiflora,nd provide clues to explore the chromosomes evolution in Sciaenidae.