Establishment And Optimization Of FISH In Rubus

Rubus L., known as raspberry which is perennial small shrubs deciduous fruit trees. There are variety of raspberry plants in China, mostly in the wild, and the genetic background is complexity. Raspberry plant resources mainly concentrated in the morphological identification, pollen and cytological identification, molecular biology and other means relatively small. Through select the process of FISH, establishment a suitable system of FISH to wild Rubus plants. Then Use the 45S rDNA probe in R. ellipticus var. obcordatus of FISH. The main results showed as follows:(1) 45S rDNA probe in the chromosome location main in the short chromosome, long chromosome centromere regions and short chromosome centromere regions to the short arm area. The phenomenon of 45S rDNA probe appeared less that Arm ratio)4:1, only number 2 chromosome centromere regions emergence a very weak signal,45S rDNA in centromere regions to the short arm region about 50%. Karyotype formula is 2n=12sm+2t.(2) The suitable chromosome for FISH is this:when the root tip reach to 1～2cm, the ambient temperature at 17～23℃, use 0.002mol/L 8-hydroxyquinoline treatment at 4℃for 24h, Carlo's fixativeⅠ(ethanol:acetic acid=3:1)at 4℃for 24h. Then the root tip deal with 1mol/L HC1 at 60℃for 30～40 seconds, The meristematic cells were macerated in a drop of 45% acetic acid and squashed gently under a coverslip which was then removed following nitrogen freezing. The chromosomes were fully applicable to the FISH.(3) Use the 45S rDNA nucleotide sequence of R.parkeri (Gene Bank:FJ472913) designed the PCR primers, deal with the concentration of primers, Mg2+ and other factors by orthogonal design and optimization, in 16 combinations, the 1,2,3,4,12,13 and 14 bands were clear,7 to 10 bands is weak, the 5,6,11 and 16 bands does not appear. We analysis found that the combination of clear bands of its primers are 5ng, the rest of the factors has little effect on the results. Therefore, the research select No.1 as the PCR amplification system. The PCR amplification system as follows:5ng Rubus DNA template,0.5μM each Primer,2mM dNTP,12.5mM Mg2+,0.75M Taq DNA enzyme, 2μL PCR Buffer.(4) For FISH, slides were treated with 100μg/mL RNAse in 2×SSC at 37℃for 1 hour, and wash in 2×SSC for 2×5min. Chromosomes were stabilized in 4% solution of freshly depolymerized paraformaldehyde in water (pH=7.5) at room temperature for 10min, washed in 2×SSC, dehydrated in an ethanol series(70%,95%,100%;5min each), and air dried. Chrommosomal DNA on the slide was denatured in 70% formamide in 0.2×SSC at 70℃for 3min, rapidly dehydrated in an ethanol series(70%,95%,100%; 5min each) at -20℃, and air dried. The hybridization mixture consisted of 5ng/μL labeled probe DNA,5×SSC,50% deionized formamide,10%DS,0.1%(w/v)SDS, and 3ng/mL of sonicated, sheared salmon sperm DNA. The mixture was denatured for 5 min in boiling water and chilled on ice for 10min. Then,20μL of the probe mixture were loaded onto each slide, and covered with a coverslip. Hybridization was carried out overnight at 37℃in a moist chamber. After hybridization, the cover slips were loosened and the slides were immersed in 2×SSC. They were washed by immersion in 20%(v/v) deionized formamide in 0.1×SSC for 30min at 42℃, in 2×SSC for 3×5min at room temperature, in 2×SSC with 0.2% Tween-20 for 3min, and in distilled water, and then air dried. Then, slides were treated with 5μg/mL Avidin-FITC at 37℃for 1h, slides were stained with 1μg/mL DAPI, The slides were stored in the dark for 30min, and examination with the Olympus BX-51.