QRT-PCR Detection And Phylogenetic Analysis Of Potato Spindle Tuber Viroid

AbstractPotato spindle tuber viroid(PSTVd) is an important disease that threatens potato production in China. PSTVd can cause potato reduction in plant size, uprightness and tubers reduced in size, misshapen, spindle or dumbbell-shaped and production decline. It can be transmitted by mechanical touch, asexual reproduction and true potato seed(TPS). Right now, many potato breeding materials(germplasm resources and breeding lines) have been infected with PSTVd. This disease imposed great difficulties to potato breeding work in China and caused latent danger for the breeding efforts because it is very difficult to eliminate it from infected ones. Producing viroid-free seed potatoes is the most powerful method to control it. In seed potato production process, implementation of the field, warehouse and laboratory detections for seed potato or seedling is the most basic and important way. At present, there are some problems in PSTVd research:(1) PSTVd detection system is not perfect;(2) PSTVd occurrence, virulence and phylogenesis in China are not very clear; and(3) the symptoms of different potato varieties infected with PSTVd are complex and uncertain. In order to solve these problems, a series of studies were carried out and the results were as followings:(1)A PSTVd q RT-PCR detection system was established. The primer pair ‘PSTV234F, PSTV356R’ was selected from three pairs of primers. By use of the primer pair of ‘PSTV234F, PSTV356R’ and Taqman probe ‘PSTV 251T’, a q RT-PCR detection system for PSTVd was established. The sensitivity of this system is 31.1 copies/μL(101.17 ag/μL). Stability test analysis showed that this system has a very good stability. Moreover, potato samples were successfully detected by use of this system. The establishment of this system enriches the PSTVd detection technology and could further improve the accuracy of it.(2)Survey of PSTVd occurrence in China and sequence analysis of the Chinese PSTVd isolates were conducted. Between 2009 and 2014, more than 1, 000 of seed potato / seedling samples were investigated for PSTVd. The results showed that the PSTVd occurred every year during 2009-2014 and the average infection rate is 6.5%. Besides, 71 PSTVd isolates collected from Heilongjiang, Jilin, Liaoning, Shandong, Inner Mongolia and Shaanxi provinces from 2009 to 2014 were sequenced. Seventy four prominent sequences were acquired and they contained 42 different PSTVd variants(accession numbers: KR611334~KR611376). BLAST results showed that there were 12 variants which have the same sequences as those deposited in Gen Bank and 30 novel PSTVd variants specific to China. These sequences lay the foundation for understanding the introduction, epidemic and genetic variation of PSTVd. Firstly, Chinese prevalent PSTVd sequence variants were analyzed and the results showed that Chinese PSTVd isolates have high sequence similarity with PSTVd variants from other countries. They were similarity or identical. Secondly, except mild strains in China, there still have three intermediate strains. This means that Chinese PSTVd isolates have different virulences. Besides, a comprehensive phylogenetic analysis was performed using the sequences from the 42 PSTVd variants and 165 naturally occurring PSTVd variant sequences deposited in Gen Bank. As a whole, PSTVd variants were clearly clustered into a small group(I) and a large group(II). The group II was further divided into three subgroups(II A–C). All PSTVd variants from China and Russia belong to group II. This show ed that the PSTVd of China and Russia may have the same origin of PSTVd. At last, the mutations of 42 Chinese PSTVd variants were systematically analyzed and most of the variants were linked to each other with only one mutation. This result suggests that the mutation may be the main driving force of evolution of Chinese PSTVd isolates.(3)Pathogenicity test of Chinese PSTVd isolates was made and symptoms of different potato varieties infected with PSTVd were investigated. Four potato varieties(‘Helan 15’, ‘Shepody’, ‘Kexin 18’ and ‘Youjin’) were inoculated with PSTVd and the plant height, foliage, tuber yield and tuber appearance were studied after inoculation. The results showed thatall of the four potato varieties expressed some symptoms, such as dwarf plants, wrinkled leaves, smaller tubers, yield decline and tuber deformity. These results showed that all of the four potato varieties are sensitive to PSTVd. Statistics showed that compared with the control, the average height of infected plants reduced by 30.1% and average yield per plant declined by 40.2%. The above results showed that although Chinese PSTVd isolate may be mild strain, it still has a serious effect on the potato production. In addition, more attentions should be paid for different varieties during field and warehouse detection because different varieties show different symptoms.Generally, a PSTVd q RT-PCR detection system was established which improved the sensitivity and accuracy of PSTVd detection technique. Through investigation and analysis, the current occurrence of PSTVd in northern China was clear. Chinese PSTVd isolates were analysed systematically by phylogenetic analysis and the evolutionary and phylogenetic relationship of them were acquired. The virulence of Chinese PSTVd isolates was conjectured by inoculating PSTVd to different potato varieties and the symptoms of plants and tubers can provide helps to field and storehouse detection. These results lay the foundation for PSTVd control and risk assessment, and also provide some technical means against PSTVd’s impacts on potato breeding.