Multiple RT-PCR Detection Of Leaf Roll-associated Virus And SSCP Analysis Of Genetic Variations Of Wine Grapevine In Ningxia

Grapevine Leafroll associated virus was a typical grape virus diseases,which was wide distribution worldwide and resulted in a huge losses.It had alreadly become a serious problem in grape cultivation areas all over the world.Helan Mountain East Region of Ningxia was one of the best wine grape cultivation areas in domestic,a few of research was reported regarding infection of GLRaVs in Ningxia wine grape plant,GLRaVs strain variation of wine grape and its relationship with the molecular properties.A multiple RT-PCR technique system was established to analyze and the molecular variation and group structure of GLRaVs isolates by SSCP based on the investigation of the natural incidence of grape virus disease in Ningxia,for monitoring and controlling of the changes of the GLRaVs group structure in Ningxia laid a solid foundation.The main results were as follows:1.Eight wine grape planting areas in Helan Mountain East Region of Ningxia for grape virus diseases natural infection situation were investigated,by adopting RT-PCR method to test 40 samples of five kinds of grape leaf roll virus infection from GLRaV-1 to 5.The results showed that the major wine grape virus diseases in Helan Mountain East Region of Ningxia include grapevine leaf roll disease,grapevine fan leaf disease and grapevine corky bark,meanwhile grapevine leaf roll disease was particularly serious;There was a difference between different wine grape planting areas as well as wine grape varieties of grape leaf roll disease infection.And the varieties of leaf roll disease incidence in the old grape wine region were obviously higher than the new wine region;'Cabernet Gernischt' and 'Pinot Noir' were the main grape varieties with grape leaf roll-associated virus with incidence rate as high as 85.1%and 52.7%.Through RT-PCR detection for five kinds of grape leaf roll-associated virus primers,three types of viruses were diagnosed with GLRaV-3 acting out the highest detection rate at 87.5%.2.We used a RT-PCR method to detect of wine grape leaf roll-associated disease in Helan Mountain East Region of Ningxia.A multiplex RT-PCR based on single RT-PCR detection of GLRaVs was applied to detecting two Grapevine leaf roll-associated viruses(GLRaV-1,3).We were optimized the multiple RT-PCR template concentration,primer concentration,the annealing temperature and TaqDNA polymerase concentration,set up multiple RT-PCR technique system which was used to detect the two viruses at the same time.The results showed that template concentration,primer concentration and TaqDNA polymerase concentration all had considerable influence on multiple RT-PCR amplification and the influence of annealing temperature wasn't significant.The PCR products of two Grapevine leaf roll-associated viruses were cloned and sequenced,the sequence identity of amplified gene fragment compared with landed in GenBank was 96%-99%.The multiple RT-PCR was proved sensitive for detection of these 2 GLRaVs in field samples.3.Eight GLRaV-1 isolates and thirteen GLRaV-3 isolates from different regions were analyzed by SSCP for their genetic variations.The SSCP analysis shows that eight GLRaV-1 isolates had obvious differences and classified into three categories;thirteen GLRaV-3 isolates had obvious differences.GLRaV-3-CP and GLRaV-3-HSP70 isolates were classified into two categories;GLRaV-3-RdRp isolates were classified into three categories.The isolates showed difference in different planting and different varieties of the same area.4.The positive samples of GLRaV-CP/HSP70/RdRp were used for cloning and sequence analysis.The results showed that each complete sequence of GLRaV-1-CP gene of the two isolates was 232 bp long and each of the nucleotide sequence homology was 90%.The two isolates of the nucleotide sequence homology was 90%-99%,which compared to other separation.Each complete sequence of GLRaV-3-CP gene of the two isolates was 942 bp long and each of the nucleotide sequence homology was 40%.The two isolates of the nucleotide sequence homology was 40%-99%,which compared to other separation.Each complete sequence of GLRaV-3-RdRp gene of the three isolates was 683 bp long and all of the nucleotide sequence homology was 90%above.The three isolates of the nucleotide sequence homology was 90%-99%,which compared to other separation.Each complete sequence of GLRaV-3-HSP70 gene of the two isolates was 546 bp long and each of the nucleotide sequence homology was 96%.The two isolates of the nucleotide sequence homology was 96%-99%,which compared to other separation.