Triticeae Genomics and Genetics, 2025, Vol.16, No.4, 184-194 http://cropscipublisher.com/index.php/tgg 187 al., 2020; Gupta et al., 2021). It is best if each gene has a corresponding molecular marker. This way, it can be more convenient to screen out during breeding. As long as genes are selected according to these standards, breeding work will be smoother and it will be easier to breed disease-resistant and high-yielding wheat varieties, which will be very helpful in achieving green agriculture and food security. 4 Molecular Tools and Strategies in Resistance Gene Pyramiding 4.1 Marker-assisted selection (MAS) and its application Marker-assisted selection (MAS) is a technology used to track disease resistance genes. It uses DNA markers that are very close to resistance genes to help breeders find target genes in breeding populations. With MAS, breeders can select homozygous materials containing two or more disease resistance genes at an early stage. This not only combines resistance genes such as powdery mildew, rust, and nematodes, but also retains good agronomic traits and greatly accelerates the breeding process (Barloy et al., 2007; Wang et al., 2022; Jin et al., 2022). For example, through MAS, researchers successfully stacked gene combinations such as Pm2+Pm4a and Pm2+Pm21 into wheat, improving resistance to powdery mildew. Rust resistance genes such as Lr19/Sr25 and Yr15 were also combined to obtain more resistant and durable varieties (Pal et al., 2020). 4.2 Genomic selection and high-throughput genotyping Genomic selection (GS) is a method of predicting traits using whole genome data. This method can predict the breeding value of multiple complex traits, including disease resistance. With the development of next-generation sequencing (NGS) technology and the application of high-density SNP chips, researchers can build more detailed genetic maps, conduct GWAS analysis, and find QTLs that control disease resistance traits. These tools can help us select multiple resistance genes at the same time and speed up the selection of excellent wheat materials (Athiyannan et al., 2022; Melson et al., 2023). In addition, high-throughput genotyping technology can also quickly analyze large populations, save time, and improve efficiency. It is an important means to achieve precise disease resistance gene aggregation (Babu et al., 2020; Bariana et al., 2022). 4.3 CRISPR/Cas and gene editing for resistance stacking CRISPR/Cas is a gene editing tool that can directly modify wheat genes. We can use it to insert new disease resistance genes, knock out genes that are sensitive to diseases, and even stack multiple resistance genes in one step. This method breaks through the limitations of traditional breeding and MAS, and can achieve the goal without backcrossing generations (Laroche et al., 2019; Luo et al., 2023). It can also quickly use disease resistance genes from wild relatives or newly discovered disease resistance genes in breeding, thereby expanding the source of wheat resistance. These gene editing tools allow us to modify wheat more specifically, improve its disease resistance, and make breeding faster and more precise, which is conducive to breeding stronger wheat varieties that are more suitable for sustainable agriculture. 5 Case Studies of Resistance Gene Pyramiding in Wheat 5.1 Pyramiding of Lr, Sr, andYr genes for rust resistance Combining resistance genes related to leaf rust (Lr), stem rust (Sr), and stripe rust (Yr) is an important method to improve wheat's resistance to rust. Many breeding projects have adopted a "pyramid" aggregation strategy. For example, the Indian wheat variety PBW343 initially introduced two leaf rust resistance genes, Lr24 and Lr28, through marker-assisted selection. Later, multiple stripe rust resistance genes were added, including Yr5, Yr10, Yr15, Yr17, and Yr70. Later, researchers introduced exogenous genes Lr37/Yr17/Sr38 and Lr76/Yr70 through backcrossing. The new variety PBW723 that was eventually bred showed stronger and more durable resistance to rust in the field (Figure 2) (Sivasamy et al., 2017; Gautam et al., 2020; Gupta et al., 2021; Sharma et al., 2021). There are also some typical examples, such as the combination of adult plant resistance genes Yr18, Yr28 and Yr36 in excellent wheat materials. These combinations enhance wheat's resistance to stripe rust throughout the growth period, further demonstrating the effect of multi-gene aggregation (Liu et al., 2020; Wang et al., 2022).
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