Cloning And Analysis Of Root-knot Nematode Related Resistance Gene Analogs In Watermelon

Watermelon (Citrullus lanatus (Thunb.) Manf.) is a worldwidehorticultural crops, ranked fifth in the world’s top ten in fruits and plays a veryimportant role. However, cultivated watermelon is vulnerable to be infected by avariety of pathogens such as root-knot nematodes, fungus fusarium oxysporum, virus,etc.. With the expansion of cultivated area of greenhouse watermelon and thelimitation of continuous cropping system in recent years, watermelon was infected byroot-knot nematodes even more seriously. Disease which caused by root-knotnematode in watermelon is a worldwide disease. Currently the main method toprevent root-knot nematode disease in watermelon is still using chemical methods,which exists a huge potential threat to both soil environment and human health.Biological control and physical control is relatively safe, but they work slowly.Therefore, breeding of watermelon varieties or stocks which has resistance toroot-knot nematode is the most fundamental method to prevent the infection ofroot-knot nematodes safely. The traditional breeding method has some disadvantagessuch as long breeding period and low efficiency etc., using molecular technologyassisted breeding can greatly shorten breeding period and accelerate breeding process,cloning watermelon disease resistance genes has become the basis of using moleculartechnology assisted breeding.This test uses RGA (Resistance gene analogs) strategy, which designs primersaccording to the conserved domains of cloned plant disease resistance genes, usinggDNA and cDNA as template for PCR amplification to obtain plant resistance geneanalogs (Resistance gene analogs, RGA), and then analyze the relationship betweenRGA and resistance genes, identify candidate genes and finally find new resistancegenes.In this experiment, watermelon root-knot nematode resistant material "Hongzi"and susceptible material "ZDPI0006" and their F1hybrids are used for the main study.Using CuGenDB database, on one hand, to design primers based on watermelonnematode resistance gene analogs which are searched from cloned plant nematoderesistance genes, on the other hand, to design primers according to NBS fromCuGenDB database, and finally47primers were designed. Extraction of watermelon leaf genomic DNA with improved CTAB method and extraction of watermelon leaftotal RNA then reverse transcribed cDNA with kit, using watermelon gDNA andcDNA as template to amplified, using PAGE to detect different fragments, totally12fragments were obtained. After searching in the GenBank and comparison of analogs,results show that there is one non-specific amplified fragment,11target fragmentswhich come from the amplification of primer W6580F/W6580R、D3F/D3R、N4F/N4R and N23F/N23R.Using bioinformatics to analysis these11sequences, the results show that: thereis no difference of length polymorphism among the amplification products fromprimer W6580F/W6580R which belongs to watermelon gene Cla006580, but thereare three SNPs. The fragment W6580-CP1which were amplified from the susceptiblematerial has a single amino acid mutation in LRR domain caused L become F,prediction of protein function shows that the fragment contains a Hs1pro-1_C domainand has61%homology to the beet nematode resistance protein, this gene hasimportant value to the location and cloning of watermelon root-knot nematoderesistance gene; the amplification products of primer N4F/N4R belongs towatermelon gene Cla012424, this gene may be absent in wild watermelon germplasm;the amplification products of primer N23F/N23R which belongs to watermelon geneCla006813or Cla006803has82%homology to melon R-FOM-2; the amplificationproducts of primer D3F/D3R has88%homology to the predicted cucumber TMVresistance protein-like N-terminal. These five genes has an important value for thestudy of watermelon resistance genes, molecular markers closely linked to resistancegenes can be developed, then applied to the MAS, finally accelerate the breedingprocess.