Genetic Diversity And Enhancement Of Germplasm In Rubus

Rubus germplasm resources were taken as materials and RAPD system and SSR system of raspberry were established and optimized.Genetic diversity of the genus Rubus was analyzed by RAPD markers and SSR markers with the 101 accessions of the genus Rubus growing in Shenyang Agricultural University raspberry germplasm nursery.The in vitro leaf regeneration system of raspberry was established and the polyploid plants were inducted.The main results were as follows:1.The modified CTAB method(3%CTAB in the extraction buffer) and the TIANGEN DNAsecure Plant Kit were the suitable protocols for the extraction of total DNA of raspberry. The production of total DNA was lower with the modified CTAB method,but the purity was relatively high and OD₂₆₀/OD₂₈₀ value was from 1.729 to 1.911.The production and the purity of total DNA was both high with the TIANGEN DNAsecure Plant Kit,and OD₂₆₀/OD₂₈₀ value was from 1.727 and 1.901.RAPD and SSR amplifications were carried on using total DNA of raspberry with the modified CTAB method and the TIANGEN DNAsecure Plant Kit, and several clear bands with better polymorphism,stability and repeatability could be obtained.2.A number of factors affected the amplification results of RAPD were investigated such as kinds and concentration of dNTP,DNA concentration,kinds and concentration of Taq DNA polymerase,Mg²⁺ concentration,kinds of PCR buffer and Mg²⁺ and cycle numbers,etc.The optimized RAPD analysis system of the genus Rubus was established:reaction mixture contained 1×PCR buffer,2.0 mmol/L MgCl₂,0.2 mmol/L dNTPs,0.3μmol/L primer,0.5 U Taq enzyme and 50 ng total DNA of raspberry in a total volume of 20μl.The cycling conditions consisted of an initial denaturation step at 93℃for 2 min,followed by 42 cycles at 93℃for 1 min,35℃for 1 min and 72℃for 2 min,and then a final elongation step at 72℃for 5 min.Sixteen primers screened out from 100 RAPD primers were suitable for the genetic analysis of the genus Rubus based on the number,polymorphism and clarity of the bands.3.A number of factors affected the results of SSR were investigated such as concentration of Mg²⁺,dNTP,primer,Taq DNA polymerase and DNA,annealing temperature and cycle numbers,etc.The optimized SSR analysis system of the genus Rubus was established:reaction mixture contained 1×PCR buffer,0.9 mmol/L MgCl₂,0.25 mmol/L dNTP,0.2μmol/L of each primer,0.5 U Taq enzyme and 60 ng total DNA of raspberry in a total volume of 15μl.The cycling conditions consisted of an initial denaturation step at 95℃for 5 min,followed by 30 cycles at 95℃for 45 s,suitable annealing temperature 50～66.5℃for 1 min and 72℃for 45 s,and then a final elongation step at 72℃for 5 min.Thirty eight primers were screened out from 60 SSR primers were suitable for the genetic analysis of the genus Rubus based on the number,polymorphism and clarity of the bands.4.After 50 polymorphism bands of RAPD were recovered,cloned and sequenced,27 different genome sequences of raspberry were obtained.Based on the analysis of genome sequences of raspberry,specific primers were designed and SCAR conversions of 16 RAPD markers were done successfully.5.The genetic diversity of Rubus germplasm resources were analyzed with molecular marker techniques.Two hundreds and seven polymorphism loci were detected from the 101 accessions of the genus Rubus with 16 RAPD primers.The dissimilarity coefficients of different raspberry germplasm resources were from 0 to 0.73,and dendrogram with UPGMA method displayed that distance coefficients of different raspberry germplasm resources were from 0 to 0.63;241 loci were detected from the 101 accessions of the genus Rubus with 38 SSR primers.Shannon genetic diversity ranged from 0.0953 to 0.8591,with the average of 0.6324.The dissimilarity coefficients of different raspberry germplasm resources were from 0 to 0.86,and dendrogram with UPGMA method displayed that distance coefficients of different raspberry germplasm resources were from 0 to 0.74,which indicated that the genus Rubus had abundance genetic diversity.6.Dendrograms of the genus Rubus were drawn separately based on the RAPD marker and the SSR marker.Dendrogram based on the RAPD marker was nearly the same as that based on the SSR marker.The two markers were both suitable to study on the genetic diversity of intraspecies and interspecies of the genus Rubus.7.In vitro regeneration from leaves of raspberry was investigated in this study.The whole leaf of red raspberry cutivar'Autumn bliss' inoculated on MS medium containing 2.00 mg/L TDZ and 0.10 mg/L LAA,dark incubated for the first 2 to 3 weeks and then transferred to light was the most optimum for shoot regeneration,and the callus percentage,the shoot percentage and average shoots number per explants were 100.00%,95.83%and 5.57±0.27, respectively.Regenerated shoots were cut and transferred to 1/2 MS medium containing 0.10 mg/L IBA for rooting.After cultured for 35 days,the rooting percentage reached up to 100.00%.8.The leaves of'Autumn bliss' was disposed by the solid medium containing 45 mg/L colchicine for 8 d and transferred to common medium for 3 weeks.The regenerating buds were inoculated on MS medium containing 0.5 mg/L BA,0.2 mg/L IAA and 1.0 mg/L GA for 40 d to induce plant differentiation.The result of chromosome numbers indicated that 5 out of 20 tested plants were tetraploid and the variation rate reached 25%.