Molecular Microbiology Research, 2025, Vol.15, No.2, 82-92 http://microbescipublisher.com/index.php/mmr 88 Early MAS used RAPD and AFLP markers. With the development of high-throughput sequencing, SNP and InDel markers are now widely used. For example, core resistance genes against rice blast such as Pi9, Pigm, and Pita have functional markers developed for quick identification (Xiao et al., 2019). Similarly, in bacterial blight, genes like Xa21, Xa13, andXa23 now have KASP and CAPS markers for high-throughput detection (Li et al., 2025). One major use of MAS is gene pyramiding—combining several resistance genes in the same plant. For example, the variety "Huakangdao 1" was developed by combining Pi1, Pita, and Pik, showing broad resistance to rice blast (Zhang et al., 2021). This method expands the resistance range and delays resistance breakdown. MAS can be more effective when combined with field resistance evaluation. In large populations, MAS helps discard plants lacking target genes early, while field tests confirm the final resistance performance. However, MAS still faces challenges such as background dependence and possible recombination near the marker site. In the future, combining MAS with genomic selection (GS) and precise phenotyping will make it more powerful. 5.2 Gene editing and precision breeding 5.2.1 Application cases of CRISPR/Cas system in resistance improvement In recent years, CRISPR/Cas system has become one of the most promising molecular tools in rice disease resistance breeding. The five main application strategies of this system in rice resistance improvement, from knockout of susceptible genes, activation of resistance genes, to transcription factor regulation and precise editing of promoters, comprehensively demonstrate the multi-dimensional expansion path of CRISPR system in rice immune regulation (Figure 2) (Huang et al., 2024). First, targeted knockout of susceptible genes (S genes) is one of the most common strategies at present. Some rice genes (such as SWEET gene family) are abnormally activated after being acted upon by pathogen effectors, thereby providing carbon sources for pathogens. Precise knockout of these S genes or their promoters through CRISPR/Cas system can effectively block the nutrient hijacking behavior of pathogens and improve natural resistance to bacterial diseases such as bacterial blight. The "Knock-out of S genes" part in the figure shows the core logic and technical links of this strategy. Secondly, activating the expression of disease resistance genes (R genes) is also an important direction. Some resistance genes are expressed at low levels under normal conditions and cannot play a full immune role. With the help of the CRISPRa system, the expression level of target genes can be upregulated by guiding the activation complex to enhance the response to diseases such as rice blast and bacterial leaf streak. Third, targeting transcription factors (TFs) regulatory factors has become a key strategy to bridge broad-spectrum resistance and plant development coordination. Transcription factors such as WRKY and NAC in rice are often involved in immune signal integration and expression regulation. As shown in the "TFs" strategy in Figure 2, this approach focuses on the transformation of the regulatory level, reflecting stronger "mildness" and controllability. Fourth, precise reconstruction of the promoter region is one of the current research hotspots. Some major disease resistance genes, such as xa13, have normal coding regions but the promoter is easily recognized by pathogenic TAL effectors. This activation can be avoided through promoter editing to achieve the purpose of "turning off the pathogen switch", while avoiding the insertion of exogenous sequences and improving social acceptance. Finally, multiplex editing integrates the above strategies and becomes an effective means to breed broad-spectrum composite resistance varieties. By designing multiple sgRNA sequences and editing multiple gene loci simultaneously in one transformation, synergistic resistance to multiple pathogens can be achieved. The study by Huang et al. (2024) not only intuitively presents the five core paths of the CRISPR system in rice resistance improvement, but also reflects the current trend of gene editing breeding from single gene manipulation to regulatory integration and phenotypic precision evolution. These strategies provide an operational framework
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