Molecular Pathogens, 2025, Vol.16, No.4, 159-170 http://microbescipublisher.com/index.php/mp 165 In addition to ROS, a series of hydrolase enzymes in the defense enzyme system are also indispensable to combat diseases. When wheat is infected, proteins related to disease courses such as β-1,3-glucanase and chitinase will accumulate significantly. Sensitive wheat often lacks fast and high-level relevant enzyme activity, while disease-resistant varieties can express these genes earlier, weakening the infectious structure of pathogens. Defense enzymes and ROS often work together, such as peroxidases that are both involved in ROS production and crosslink cell wall proteins, are key enzymes that connect chemical and structural defenses (Cui et al., 2021). 6 Molecular Breeding and Genetic Engineering Strategies 6.1 Marker-assisted selection (MAS) and genome-wide association analysis (GWAS) The development of molecular breeding technology has greatly accelerated the cultivation of new wheat disease-resistant varieties. Among them, marker-assisted selection (MAS) has become a conventional means to track gene transmission through DNA markers closely linked to the target disease-resistant gene, allowing breeders to efficiently aggregate multiple disease-resistant genes. Many important wheat disease-resistant genes were discovered soon after the corresponding PCR markers were developed and applied to breeding. For example, specific molecular markers of the wheat anti-powder mildew broad spectrum gene Pm21 are widely used to assist in polymerizing the gene into different varieties, thus creating a series of new wheat varieties that are highly anti-powder mildew. Marker-assisted selection is especially suitable for diseases whose resistance levels are controlled by multiple genes or have difficulty in phenotype identification. For example, the resistance of wheat gibberellia is controlled by multiple QTLs and is affected by the environment. Through MAS, multiple resistant QTLs can be polymerized in the early generation and the disease resistance can be improved. In addition to MAS targeting single genes, methods such as high-throughput genotyping, genome-wide association analysis (GWAS) and genome-wide selection (GS) are also increasingly used in wheat disease-resistant breeding research. 6.2 Application of gene editing (CRISPR/Cas) in disease resistance improvement Due to its efficient and precise genetic modification capabilities, CRISPR/Cas gene editing technology is becoming increasingly powerful tool for improving crop disease resistance. In the field of antifungal diseases in wheat, the application of gene editing is mainly reflected in the following two aspects: First, knock out susceptibility genes (S genes) to enhance disease resistance; second, directed modification of disease resistance genes to optimize their function or expression. After continuous exploration, researchers created new allelic mutations such as TaMLO-R32 in 2022, successfully eliminating the contradiction between disease resistance and growth, so that wheat can both resist powdery mildew and grow normally (Zhang et al., 2025). In terms of rust prevention and control, NWAFU Kang Zhensheng and others constructed the TaPsIPK1 three-homology knockout mutant. The Datian experiment proved that it showed stable high resistance to stripe rust within two years and the agronomic traits were not adversely affected. CRISPR also has a useful place in terms of targeted improvement of disease-resistant genes. For example, for some useful but defective disease-resistant genes, functionally enhanced versions of alleles can be created by editing. One example is that the wheat anti-powder mildew gene Pm4a can only resist some strains, and the modification of its promoter or specific sites through gene editing is expected to broaden its resistance spectrum. In addition, emerging multigene editing can knock out multiple S genes at once or modify multiple disease-resistant genes, thereby cultivating modern molecularly designed "immune wheat" (Talakayala et al., 2022). 6.3 Introduction and polymerization strategies of disease-resistant genes The ultimate goal of wheat disease-resistant breeding is to create excellent varieties that are lastingly resistant to major diseases. This usually requires reasonable combination of multiple disease-resistant genes to build broad-spectrum complex resistance. Traditional hybrid breeding can polymerize multiple genes into a variety through repeated backcrossing and selection. However, in the face of the diversity of fungal diseases and the speed of pathogen mutation, new strategies for more efficient gene introduction and polymerization emerged. First, distant hybridization and chromosome engineering provide a way to introduce new anti-sources. Many wild relative species such as long-spiked squid and tufted wheat contain excellent disease-resistant genes and can be introduced through hybridization, embryo rescue and other means.
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