Development Of A Stable SCAR Marker For Rapid Identification Of The Root-Knot Nematode Resistance Gene Me3 In Pepper (Capsicum Annuum L.)

Root-knot nematodes（RKNs）are obligate parasitic pathogens that do harm to more than 5,500crops annually,such as peppers and tomatoes,causing huge economic losses all over the world.At present,chemical nematicides supplemented by biological control are widely used to control RKNs,which is harmful to the environment and public health.The publication of the Zunla-1 and CM334pepper genomes provides a new tool for pepper resistance gene analysis.Although many nematode resistant genes have been found in peppers,few of them have been fine-mapping and cloned.The Me3gene can not only contain broad-spectrum resistance to Meloidogyne incognita,M.arenaria and M.javanica,but also maintain activity under high-temperature conditions.Therefore,Me3 gene can be used as excellent genetic resource for the molecular breeding of pepper.In this experiment,according to the results of re-squencing genome data analysis,559 molecular markers were developed for the fine mapping of RKN-resistance genes.Molecular markers with significant differences were screened,and these markers were used to determine the candidate interval of the Me3 gene.Analysis of 2148 F₂ progenies revealed that Me3 gene was located between SSR381and Indel537.And Indel537 was successfully transformed to an SCAR marker.On the basis of the Zunla-1 genome data,the DNA and cDNA sequences of the Me3 candidate gene Capana09g000159gene were amplified.The main experimental results are as follows:1.Fine mapping were constructed to screen recombinant individuals.By screening the SSR and Indel markers in the 6-Mb interval on the P9 chromosome of Zunla-1,seven markers were closely linked to the Me3 gene.The results showed that the Me3 gene was located between Indel537 and SSR381.Among these markers,Indel537 was closest to the Me3 gene,with a genetic distance of 0.5cM.2.A codominant molecular marker,SCAR537,was developed to rapidly identify whether the pepper plants have root-knot nematode resistance gene Me3.After PCR amplification,the results can be detected on agarose gel electrophoresis,which is more convenient and quickly.The amplification results of SCAR537 and the resistance identification results in the 54 F₂ progenies of[HDA149×Qiemen]were completely consistent.Thus,the marker SCAR537 can be used to identify whether a pepper plant is resistant to RKNs.3.According to the Zunla-1 genome data,the DNA nd cDNA sequences of Capana09g000159gene were cloned in both parental lines.Sequence alignments revealed that there are several SNPs and insertion/deletion sites between the resistant and susceptible cultivars,and these mutations result in the amino acid sequence changes.4.Based on previous study,the TRV silencing vector of Capana09g000163 and Capana09g000164gene was reconstructed.The results indicated that the silencing of Capana09g000163 gene showed a little effect on reducing the resistance of HDA149.In conclusion,according to the Zunla-1 genome data,several molecular markers tightly linked to the Me3 gene are designed,and an SCAR marker closely linked to Me3 gene is developed.The result of this research not only accelerate molecular marker assistant breeding,also lay the foundation for the fine mapping and cloning of Me3 gene.