Studies On The WMV-2Reisitance Genetic And Molecular Markers Of Pumpkin (Cucurbit Pepo)

In recent years, more and more people attached importance to the economic value of thepumpkin, and then pumpkin cultivation area was expanding. Pumpkin virus disease hazards wereincreasing and particularly serious, especially in the pathogenesis of high temperature and droughtyear, which lead to serious economic losses to the pumpkin production. Viral disease spread byaphids and mechanical.Chemical control of cost was high, results were poor, and fruit and theenvironment were contaminated. Therefore, cultivating disease-resistant varieties was consideredto be the most effective way to sustainable development and non-pollution environment. It isnecessary that understanding the genetic mechanism of the anti-viral disease genes thoroughly andidentifying genetic patterns and resistance genes could import fine varieties or screen resistantvarieties hybrid breed superior varieties. Molecular marker technology was one of the most usefultools for screening and identifying resistance genes.In this study, disease-resistant inbred line ’0504’ and susceptible inbred line ’0223’ werehybrided, and then a single F₁was self-pollinated to produce F₂segregating population.Bulkedsegregant analysis（BSA）was used to screen SSR markers linked to pumpkin virus diseaseresistance gene. The main results were as follows:（1） Identification of pumpkin virus disease pathogens by RT-PCR. Pumpkin virus coatprotein gene sequence had been reported according to the design and synthesis of primers. TotalRNA was extracted form infected leaves and healthy leaves, cDNA synthetized and PCR amplified.The sense diseased liavers amplified a specific fragment of about800bp, but the healthy leaves hadnot such amplification product. The amplified fragment was WMV-2viral genome-specificamplification product. The target-specific fragment was sequencing. Nucleic acid and amino acidsequence alignment were analyzed in Genbank and had86%homologous with watermelon mosaicvirus-2（WMV-2） coat protein gene sequence. Finally, this pumpkin virus was determined asWMV-2.（2） Artificial inoculation of parents, F₁and F₂through the routine immunization. Theresults showed that F₁were all affected by disease. For157F₂plants,36demonstrated diseaseresistance.121showed susceptible. Chi-square detection for F₂resistant isolates of the virus isbasically in line separation ratio of1:3. WMV-2resistance was speculated as a single recessivegene control. （3） Using the suitable pumpkin SSR molecular markers reaction system,114pairs from352pairs which were screened from pumpkin genomic SSR primer combinations showedpolymorphism between the parents and polymorphism was32.4%;5pairs of primers showed8%polymorphism between the parents from60pumpkin relatives cucumber SSR primer pairs.（4） Ten F₂plants were selected from the F₂segregating population; high resistance andhigh sense monoclonal each constituted anti-sense gene pool. Through SSR markers and BSAmethod CMTm158and CMTm157were found as WMV-2resistance gene-linked markers from119SSR primer pairs, which showed parental polymorphism. These two markers validated by F₂plant genotype to detect F₂resistant plants. Genotyping results: CMTm157identified17recombinantes of which14amplified heterozygous fragmants and3amplified susceptiblefragmants; CMTm158identified10recombinants, of which6amplified heterozygous fragmants,3susceptible fragmants and1missing. Genetic distances of these markers with WMV-2resistancegene were6.2cM and10.9cM respectively.