China is the largest potato producing country worldwide,with this crop representing the fourth largest staple food crop in China.However,the steady presence of Potato spindle tuber viroid(PSTVd) over the past five d...China is the largest potato producing country worldwide,with this crop representing the fourth largest staple food crop in China.However,the steady presence of Potato spindle tuber viroid(PSTVd) over the past five decades has a significant economic impact on potato production.To determine why PSTVd control measures have been ineffective in China,more than 1 000 seed potatoes collected between 2009 and 2014 were subjected to PSTVd detection at the Supervision and Testing Center for Virus-free Seed Potatoes Quality,Ministry of Agriculture,China.A high PSTVd infection rate(6.5%) was detected among these commercial seed potatoes.Some breeding lines of potato collected from 2012 to 2015 were also tested for PSTVd infection,revealing a high rate of PSTVd contamination in these potato propagation materials.Furthermore,comparison of the full-length sequences of 71 different Chinese PSTVd isolates revealed a total of 74 predominant PSTVd variants,which represented 42 different sequence variants of PSTVd.Comparative sequence analysis revealed 30 novel PSTVd sequence variants specific to China.Comprehensive phylogenetic analysis uncovered a close relationship between the Chinese PSTVd sequence variants and those isolated from Russia.It is worth noting that three intermediate strains and six mild strains were identified among these variants.These results have important implications for explaining the ineffective control of PSTVd in China and thus could serve as a basic reference for designing more effective measures to eliminate PSTVd from China in the future.展开更多
Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. ...Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 (34) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 : 1 : 2 : 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..展开更多
基金supported by the China Agriculture Research System(CARS-10-P14)the National Science Foundation for Post-doctoral Scientists of China(20110491125)+2 种基金the Heilongjiang Funds for Distinguished Young Scientist,China (JC 201018)the Harbin Application Technology Research and Development Projects,China(2013AE6AW059)the Young Scientists Science Foundation of Heilongjiang Province,China(QC2015026)
文摘China is the largest potato producing country worldwide,with this crop representing the fourth largest staple food crop in China.However,the steady presence of Potato spindle tuber viroid(PSTVd) over the past five decades has a significant economic impact on potato production.To determine why PSTVd control measures have been ineffective in China,more than 1 000 seed potatoes collected between 2009 and 2014 were subjected to PSTVd detection at the Supervision and Testing Center for Virus-free Seed Potatoes Quality,Ministry of Agriculture,China.A high PSTVd infection rate(6.5%) was detected among these commercial seed potatoes.Some breeding lines of potato collected from 2012 to 2015 were also tested for PSTVd infection,revealing a high rate of PSTVd contamination in these potato propagation materials.Furthermore,comparison of the full-length sequences of 71 different Chinese PSTVd isolates revealed a total of 74 predominant PSTVd variants,which represented 42 different sequence variants of PSTVd.Comparative sequence analysis revealed 30 novel PSTVd sequence variants specific to China.Comprehensive phylogenetic analysis uncovered a close relationship between the Chinese PSTVd sequence variants and those isolated from Russia.It is worth noting that three intermediate strains and six mild strains were identified among these variants.These results have important implications for explaining the ineffective control of PSTVd in China and thus could serve as a basic reference for designing more effective measures to eliminate PSTVd from China in the future.
基金Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08)
文摘Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 (34) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 : 1 : 2 : 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..