Plant Gene and Traits 2024, Vol.15, No.4, 174-183 http://genbreedpublisher.com/index.php/pgt 175 This study comprehensively analyzes the genetic and molecular basis of the MlWE74 gene, with a particular focus on its role and signaling pathways in powdery mildew resistance. It discusses relevant research methods and experimental findings, while identifying the main challenges and future directions for the application of MlWE74 in wheat disease resistance breeding. The study aims to provide a thorough overview of the cloning, functional analysis, and breeding applications of the powdery mildew resistance gene MlWE74 inwheat. 2 Positional Cloning of the MlWE74Gene 2.1 Positional cloning strategy for the MlWE74gene The positional cloning of the MlWE74 gene, which confers resistance to powdery mildew in wheat, involves several strategic steps. Initially, the gene was identified in the hexaploid wheat line WE74, derived from wild emmer wheat (Triticum turgidumvar. dicoccoides). Genetic analysis revealed that the resistance is controlled by a single dominant gene, temporarily designated MlWE74. The strategy began with bulked segregant analysis (BSA) and molecular mapping, which localized MlWE74 to the terminal region of chromosome 2BS. This region was flanked by markers WGGBD412 and WGGBH346 within a genetic interval of 0.25 cM, corresponding to a 799.9 kb genomic region in the Zavitan reference sequence (Zhu et al., 2021). Further refinement of the positional cloning strategy involved the use of high-density molecular markers and comparative genomics. This approach has been successfully applied in other studies, such as the mapping of the Pm3b gene in hexaploid wheat, where the combined analysis of genomes from wheat species with different ploidy levels facilitated the establishment of a physical contig spanning the Pm3 locus (Yahiaoui et al., 2004). Similarly, the identification of closely linked markers and the construction of a genetic linkage map were crucial steps in the positional cloning of MlWE74. 2.2 Fine mapping based on high-density molecular markers Fine mapping of the MlWE74 gene was achieved through the use of high-density molecular markers. This process involved the development and utilization of markers that are closely linked to the resistance gene. In the case of MlWE74, the gene was delimited to a 799.9 kb genomic region on chromosome 2BS, flanked by markers WGGBD412 and WGGBH346 (Zhu et al., 2021). The identification of these markers was facilitated by bulked segregant analysis (BSA) and molecular mapping techniques. The importance of high-density molecular markers in fine mapping is underscored by other studies, such as the mapping of the MlIW39 gene, which was localized to a 460.3 kb genomic interval on wheat chromosome arm 2BS using molecular markers (Qiu et al., 2021). Similarly, the fine mapping of the Pm4b gene involved the development of SNP markers from transcriptome sequencing data, which were then used to construct a genetic linkage map (Wu et al., 2018). These examples highlight the critical role of high-density molecular markers in narrowing down the candidate region for the resistance gene and facilitating its eventual cloning. 2.3 Genome sequencing and genome-wide association study (GWAS) Genome sequencing and genome-wide association studies (GWAS) are powerful tools that complement the positional cloning and fine mapping efforts for the MlWE74 gene. Genome sequencing provides a comprehensive view of the genetic landscape, allowing for the identification of candidate genes within the mapped region. In the case of MlWE74, sequence annotation revealed several candidate genes, including two phosphoglycerate mutase-like genes, an alpha/beta-hydrolases gene, and five NBS-LRR disease resistance genes (Zhu et al., 2021). GWAS further enhances the identification of resistance genes by associating genetic variants with phenotypic traits across a diverse population. This approach has been successfully applied in the identification of other powdery mildew resistance genes, such as Pm5e, where a rare single nucleotide variant (SNV) within the C-terminal leucine-rich repeat (LRR) domain was found to confer resistance (Xie et al., 2020). Similarly, the use of bulked segregant RNA sequencing (BSR-Seq) in the mapping of the PmPBDH gene demonstrated the utility of combining genome sequencing with association studies to pinpoint resistance genes (Liang et al., 2022). 3 Functional Analysis of the MlWE74 Gene 3.1 Predicted function and encoded product of the MlWE74 gene The MlWE74 gene, derived from wild emmer wheat (Triticum turgidumvar. dicoccoides), has been identified as a significant contributor to powdery mildew resistance in wheat. Genetic analysis has shown that MlWE74 is a
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