Cloning Of Turnip Mosaic Virus Resistance Related Genes And Development Of Trap Marker In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.) is an important root vegetable crop worldwide, especially in East Asia. The viral disease caused by TuMV is considered the most serious disease of radish, not only its world-wide distribution, but also the resulting severe yield loss. Attempts to control TuMV by insecticides are often ineffective and could cause environmental pollution due to chemical residues due to its wide host range and non-persistent transmission by aphids. Therefore, the deployment of virus-resistant crop varieties is likely to be the most effective, environmentally friendly and sustainable approach to control the virus. The reports about viral disease in radish were the inheritance of resistance to TuMV, however, the ditterence of the method and material, causing there is no consistent conclusions. Now, there is no report about the molecular mechanism of resistance to TuMV. In the present study, a rapid method of TuMV detection by RT-PCR was established; the related genes of resistance to TuMV were isolated by T-A clone; the development of TRAP marker and its application in the genetic diversity of radish (Raphanus sativus L.) based on RGAs. The aim of this study is to lay the foundation for the research of the radish disease resistance breeding. The main results were summarized as followed:(1) Sequences of TuMV in GenBank database were aligned by using DNAMAN6.0version software, with specific primers designed based on the conserved sequence. RT-PCR for detection of TuMV was established and PCR products were purified and sequenced. BLAST analysis showed that these PCR products were parts of TuMV sequences with homology of sequences well above95%, indicating that RT-PCR had a high specificity in detecting TuMV. And the results of the detection of different cultivars, different parts of susceptible radish cultivar and different cDNA dilution factor indicated that the infection of TuMV in radish was systemic, and RT-PCR had a high specificity and sensitivity.(2) Two eukaryotic translation initiation factors, RseIF4E and RseIF(iso)4E were isolated by TA clone. The DNA full-length of RseIF4E was1483bp, including a complete open reading frame (ORF) of696bp, encoding232amio acids. And the DNA full-length of RseIF(iso)4E was1205bp, including a complete open reading frame (ORF) of594bp, encoding198amio acids. The amino acids in the active sites of the mRNA cap and4G with the two genes were highly conserved by homology analysis. The analysis of the cDNA sequences and theamino acid sequences of the12radish cultivars were found that there were SNPs, InDel and fragment inserted. The two genes were hydrophilic and present in the cytoplasm by the bioinformatic analysis. The detection of qRT-PCR indicated that the expressions of the RseIF4E and RseIF(iso)4E were showed that higher levels of the two genes transcript were observed in the TuMV-infected leaves. These datas showed that the two genes may be involved in the interaction of host and virus.(3) TRAP is a novel molecular marker technique which has been effectively used in genetic diversity analysis of germplasm and genetic mapping. However, it has not yet been applied to radish. In this study, novel TRAP markers based on expressed sequence tag (EST) and resistance gene analog (RGAs) were developed and applied to the genetic diversity analysis of radish genotypes. With data-mining method, a total of50RGAs including35unigenes and15singletons were identified from the public sequence databases and employed to design the fixed primers of TRAP markers. Fifty-nine RGA-derived fixed primers were combined with five arbitrary primers, and these TRAP primer combinations were tested in two genotypes (’NAU-YH’and’NAU-DY’). Furthermore,65TRAP primer combinations were selected for genetic diversity analysis and385polymorphic fragments were produced among30radish genotypes. Dendrograms constructed by UPGMA method showed that these genotypes could be clustered into four groups. Interestingly these groups were in highly accordance with the results of resistance evaluation to Turnip Mosaic Virus (TuMV). A cultivar identification diagram (CID) was made manually to discriminate the30radish genotypes using four polymorphic TRAP primer combinations. The results indicated that TRAP is an efficient genetic marker system for radish will provide an effective tool for genetic mapping and for marker-assisted selection in radish breeding programs.