| Phytophthora blight is one of the most destructive diseases affecting pepper production, which is caused by the soil born Phytophthora capsici. Up to now, this disease has become world-wide. Exploring the resistance gene(s) and transferring them into cultivated lines are promising method to control the disease. A lot of researchers have been focusing on the resistance inheritance of pepper to P. capsici for many years and found that inheritance of P. capsici resistance in peppers were variable. The various pepper resources and P. capsici races used in different studies were responsible for the different results. In this study, the resistant line PI201234 with simpler resistance inheritance and P. capsici race 2 with moderate aggression were used. By means of genetic analysis, gene mapping and VIGS, we expected to map the resistance gene(s) and validated the function of candidate genes, which would provide a basis for cloning the resistance gene(s), revealing the resistance mechanism and cultivating new varieties.In this study, RNA-seq technology was used to analyze root transcriptome changes in the highly resistant PI201234 following P. capsici race 2 inoculation.1220 differentially expressed genes were detected, of which,480 were up-regulated and 740 were down-regulated, with 204 candidate genes found to be involved in defense responses based on the gene annotations. The expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qRT-PCR), and the expression patterns of them were found to be significantly different between the resistant PI201234 and susceptible Qiemen. The SSR marker identified from the transcripts could be used for gene mapping and other genetic research.Iln order to map the resistance gene(s), an F2 population was developed by crossing the resistant PI201234 and susceptible Shanghaiyuan. Disease evaluation was performed on 794 F2 individuals, and it turned out that the resistant and susceptible plants segregated as 3:1 ratio. This result suggested that the resistance to P. capsici race 2 in PI201234 was controlled by a single dominant gene, which was tentatively named as CaPhyto. This gene was finally mapped into a region, which was about 1.08 Mb, on chromosome 5. There were 11 genes identified within this region based on the annotation of Zunla-1 pepper genome, and 5 genes among them were found to be the potential disease-resistance genes. Genetic mapping suggested that ZL6726 and ZL6970 were closly linked to CaPhyto. Based on the validation through practical application, the accuracy of marker ZL6726 was higher and reached up to 97.1%, which proved that it is a reliable marker for marker-assisted selection.VIGS was used to validate the function of the five candidate genes preliminarily. The disease evaluation was performed after the candidate genes were silenced by VIGS. The results indicated that only some plants with Capana05g000764 silenced showed susceptible symptom, suggesting that Capana05g000764 was related to the resistance of PI201234 to P. capsici. Based on the prediction of conserved domains and clustering analysis, Capana05g000764 was predicted as a serine/threonine-protein kinase BRI1-like 2 (BRL2) gene. Previous reports suggested that BRL2 gene could encode a LRR-receptor kinase that regulated the differentiation of provascular cells. The expression analysis showed that Capana05g000764 expressed higher in root than that in stem and leaves. After the pathogen inoculation, the gene was up-regulated in PI201234 but down-regulated in Shanghaiyuan. Taking into consideration the plant symptom caused by the pathogen, we predicted that this gene might be involved in the protection of plant from P. capsici infection. Further examination on functional analysis is needed.In the present study, the transcriptome of PI201234 was analyzed by RNA-seq.204 defense-related genes were identified, and 4723 SSR markers were developed. The resistance gene was finally mapped into a region with 1.08 Mb in length on chromosome 5. The candidate gene Capana05g000764 was verified to be responsible for the pepper resistance to P. capsici race 2 by VIGS and its function was also predicted. The data and results obtained in this study will benefit the further gene cloning and functional analysis. The molecular markers ZL6726 will provide convenience for pepper breeding. |
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