Wheat (Triticum aestivum. L.) is one of the world’s most important staple crops, serving as a primary food source for a large percentage of the global population (Kiani et al., 2021) and providing nearly 18% of the calories and 19% of the protein for human consumption (Erenstein et al., 2022). There will be a substantial increase in the demand for wheat as the world’s population grows. Despite such demand, wheat production is challenged by several potential constraints, among which biotic stresses are exceptionally severe (Figueroa et al., 2017). Among others, stripe rust, induced by Puccinia striiformis f. sp. Tritici (Pst), is believed to be the most devastating and has been frequently reported to affect the production of wheat dramatically. This disease can cause yield reduction ranging from 10% to 70% on susceptible cultivars, with possible losses reaching 100%, particularly during intense epidemic time (Chen, 2020; Kokhmetova et al., 2021). Owing to the genetic diversity and rapid adaptation of virulent Pst races, the disease annually affects regions where climatic conditions and cropping practices are favorable for stripe rust (Chen, 2005). Ground data suggested that it become widespread in 41 primary wheat-producing regions worldwide, including China and Pakistan (Ma et al., 2023).

The surveillance for stripe rust in China has shown that the southwest and northwest regions are more vulnerable to the pathogen throughout the summer (Zhao and Kang, 2023). Both regions serve as significant origins and hubs for the Pst pathogen, contributing to its spread (Xu et al., 2019; Zhao and Kang, 2023). In China, there has been a substantial yield reduction caused by stripe rust outbreaks in various years, resulting in losses of 3.2 million metric tons (Mmt) in 1964, 1.8 Mmt in 1990, 1.3 Mmt in 2002, and 1.5 Mmt in 2017 (Wan et al., 2007; Chen et al., 2023). In Pakistan, areas like the northern and central-western regions are more prone to stripe rust (Bahri et al., 2011; Habib et al., 2020; Mehmood et al., 2020). Previous reports suggested that approximately 5.8 million hectares, comprising 70% of the wheat-growing area, is susceptible to stripe rust (Kiani et al., 2021), which accounts for around 70%–75% of Pakistan’s wheat cultivation area (Sobia et al., 2010; Habib et al., 2020). To address the ongoing rapid development of stripe rust, especially in areas where outbreaks are common, such as China and Pakistan, the most economical and sustainable method is to develop resistant cultivars (Kalogiannidis et al., 2022; Rebouh et al., 2023).

To control stripe rust genetically, there are currently more than 83 known Yr resistance genes that are spread throughout 21 chromosomes (Maccaferri et al., 2015; Mcintosh et al., 2016). Only eight Yr genes—Yr36 (Fu et al., 2009), Yr18 (Lr34) (Lagudah et al., 2009), Yr46 (Lr67) (Hiebert et al., 2015), Yr15 (Klymiuk et al., 2018), Yr5/YrSp (Marchal et al., 2018), Yr79, YrAS2388, and YrU111—have been functionally validated, while Yr10 was being provisionally cloned (Ni et al., 2023). The Yr5 gene, derived from T. spelta Album, is responsible for conferring resistance to Pst (Macer, 1966). Nevertheless, it is noteworthy that only a limited number of genotypes capable of overcoming Yr5 resistance have been identified so far, specifically in Australia (Mclntosh et al., 1995), India (Prashar et al., 2015), Turkey (Tekin et al., 2021), and China (Liu et al., 2020). The Yr10 resistance gene is effective in providing resistance against stripe rust in Pakistan (Sobia et al., 2010), Iran (Sarfaraz et al., 2013), India (Rani et al., 2019), and China (Huang et al., 2020). The primary gene responsible for resistance to stripe rust, Yr15, was discovered in Triticum dicoccoides accession G-25 and is located on chromosome 1BS (Klymiuk et al., 2021). Breeders across the globe have made use of the Yr17 gene, which confers resistance to stripe rust in wheat (Helguera et al., 2003). The Yr18 genes have been employed in breeding efforts for a century, and thus far, no evidence of pathogen adaptation has been discovered (Lagudah et al., 2009).

Recent advancements in molecular technologies have revolutionized plant breeding through the integration of molecular markers to improve breeding precision and efficiency (Sun et al., 2024). In contrast to conventional approaches that emphasize phenotypic selection, the use of molecular markers allows for the precise identification and monitoring of specific genes and quantitative trait loci (QTLs), thereby enhancing the processes of marker-assisted and genomic selection (Hasan et al., 2021; Lema, 2018). The advancement of these tools has significantly enhanced the development of better crop varieties by offering thoughtful insights into genetic diversity and complex traits, thereby facilitating more precise and effective breeding strategies (Nadeem et al., 2017). Significant advancements have been achieved in the development of stripe rust-resistant cultivars through breeding in both countries. However, the breeding program has been significantly impeded by the rapid evolution of pathogen races, which remain a global hazard to wheat cultivation (Bhavani et al., 2021). The objectives of this study are to (1) evaluate stripe rust resistance in Pakistani and Chuanyu wheat genotypes, (2) illustrate the distribution of major Yr genes by using molecular markers in Pakistani and Chuanyu wheat genotypes, and (3) analyze the effectiveness of major Yr genes and their combinations. This study provides valuable information for an efficient Yr gene utilization strategy to improve Pst resistance in Pakistani and Chinese wheat genotypes.

The constant evolution of the stripe rust races has created challenges in finding effective wheat sources of resistance (Haider et al., 2023). This highlights the need to identify and validate new sources of resistance against the stripe rust pathogen to ensure successful use in breeding programs (Mu et al., 2019). The present study evaluated various genotypes from breeding and genetic programs focused on enhancing wheat resistance to stripe rust in China and Pakistan. In this study, we categorized Pakistani wheat germplasm into two distinct subgroups based on preliminary field evaluation of resistance. The first subgroup consists of genotypes that have significantly lost resistance over time. Conversely, the second subgroup includes genotypes released after 2015, which exhibited noticeably higher levels of resistance in field evaluations ( Figure 3 ). This grouping enables a more comprehensive understanding of resistance dynamics over time and assists in the identification of more durable sources of resistance within the recent germplasm.

A total of 349 genotypes from Southwest China and Pakistan were evaluated for stripe rust, and 13 known Yr genes were detected—Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, Yr29, Yr30, Yr36, Yr48, Yr65, and YrSp—using marker-assisted selection (MAS). When performing MAS, it is crucial to consider the credibility and accessibility of the markers (Collard and Mackill, 2007). In this study, functional and linked-based molecular markers were used to identify the corresponding genes. Functional markers and linked markers are effective tools that enable plant breeders to target genes controlling important agronomic traits (Andersen and Lübberstedt, 2003).

The findings from the field evaluation showed different levels of resistance across different wheat groups. Among historic lines of Pakistan, 20% showed resistance, while over 60% were susceptible. For recent cultivars, 45% showed resistance, and 40% to 45% were susceptible. Meanwhile, in Chuanyu wheat, more than 60% exhibited resistance, while 20% to 15% of genotypes showed susceptibility. Certain genotypes such as Pakhtunkhwa-15, Abbasen-21, Wardan 17, Khosha, Nishan, T-9, Dirk, C-228, C-217, Chuanyu 25, Chuanyu29, Chuanyu31, Chuanyu33-1, Chuanyu33-2, Chuanyu37, Chuanyu40, Chuanyu42, Chuanyu45-3, Chuanyu46, and Chuanyu47 exhibited strong resistance to stripe rust, indicating their potential as valuable sources of resistance.

Shahin et al. (2024) employed MAS to identify stripe rust resistance genes in 38 wheat genotypes, including Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, Yr29, Yr30, and Yr36. They classified these genotypes as resistant and slow-rusting, and they highlighted the significance of pyramiding slow-rusting genes, such as Yr18 and Yr29, to achieve sustainable resistance. The current study investigates the essential role of gene pyramiding in effective resistance; however, the presence of Yr5, Yr10, and Yr15 in Pakistani lines shows potential regional differences in the application and effectiveness of resistance genes. Notably, Shahin’s study did not detect Yr36, which aligns with findings from Chuanyu wheat, indicating local variations in the utilization of resistance genes and potential resistance on certain genes. The findings of Zheng et al. (2017) support the observations of the current study regarding responses to stripe rust in Chuanyu wheat genotypes. It was found that Yr18 was rare, and Yr36 was absent in the Chuanyu wheat genotypes. Chuanyu wheat genotypes have more ASR genes, and some of them were overused, like Yr17, YrSp, and Yr48. The current study suggested that we need to add more APR genes to improve resistance and reduce the overuse of certain Yr genes like Yr17 and Yr15. Growing crops with the same resistance genes may have less genetic variety, making them more susceptible to new races. A certain Pst race may damage a crop. Overuse of resistance genes can lead to pathogens developing Pst races that can overcome current resistance mechanisms (Salgotra and Chauhan, 2023). In historical lines of Pakistan, Yr15, Yr26, and YrSp demonstrated significant effectiveness, showing strong resistance to stripe rust. These findings are consistent with the results of Haider et al. (2023), who proposed the presence of Yr5, Yr10, Yr15, and Yr26 in Indian varieties. The present study also showed similar results, showing that Yr genes such as Yr5, Yr10, and Yr26 exhibited moderate effectiveness, whereas Yr29 was absent in historical lines.

Pyramiding of effective Yr genes increases durable resistance. Molecular markers are useful in identifying wheat genotypes with multiple genes as well as facilitating the pyramiding of resistant genes (Muhammad et al., 2023). The correlation analysis conducted in the current study supports the findings of Liu et al. (2020) on the relationship between the number of pyramided Yr genes and IT values. The current study also showed similar results by increasing the number of effective pyramided Yr genes. The IT values reduce and enhance resistance to stripe rust.

Moreover, gene combinations like Yr9+Yr18 and Yr30+Yr46 have shown significant effects when combined. The results of the current gene pyramiding study is consistent with the findings of Liu et al. (2020), indicating that combining multiple resistance genes can lead to higher levels of resistance than individual genes. This combination can contribute to durable resistance against various diseases—for instance, in Chuanyu wheat, the combination of Yr26+Yr48 and Yr30+Yr17 demonstrates a lower IT value (3.1) compared to the individual genes alone. This suggests that pyramiding effective Yr genes has a greater effect when used together rather than individually.

Similarly, in Pakistani historic lines and recent cultivars, the combination of Yr26+Yr48, Yr29+Yr5, and Yr30+Yr48 is marked as significant and suggests effective resistance. Wang et al. (2022) focused on integrating Yr18, Yr28, and Yr36 within a controlled environment, demonstrating additive effects for all-stage resistance. In contrast, the current study evaluated a broader spectrum of genes, including Yr9, Yr26, Yr30, and Yr48, across multiple genotypes. Both investigations emphasize that pyramiding multiple genes provides improved and sustained resistance compared to individual genes, with significant implications for advancing global wheat breeding programs. Significant effective genes, such as Yr15, Yr17, Yr26, Yr48, Yr65, and YrSp, will be extensively utilized in breeding programs.

Additionally, genes exhibiting limited effectiveness under current field conditions should still be considered for incorporation in gene pyramiding strategies. This is because a single gene, when used alone, can become susceptible due to genetic shifts in the pathogen (Liu et al., 2020). The current research findings, along with the identification of several quantitative trait loci (QTLs) associated with resistance in wheat, as reported by Rosewarne et al. (2005) and Lowe et al. (2011), suggested that gene pyramiding could greatly enhance the durability of rust resistance. To enhance durable resistance, it is recommended to incorporate APR genes in combination with ASR genes. Furthermore, the results suggest an epistatic effect between the pyramided resistance genes as observed in the combinations of Yr30+Yr48, Yr26+Yr48, Yr29+Yr5, and Yr30+Yr17. Using specific combinations of genes for deployment provides long-lasting and enhanced resistance compared to relying on a single gene.

The findings of the current study emphasize the importance of identifying and pyramiding effective Yr genes against stripe rust resistance in wheat genotypes. Specifically, the study highlights the prevalence and effectiveness of certain Yr genes, such as Yr15, Yr17, Yr26, Yr29, Yr48, Yr65, and YrSp. Current research highlights the significance of pyramiding of effective Yr genes and reducing the overuse of certain Yr genes. This underscores the significance of gene combination as a strategy to enhance long-lasting resistance to stripe rust. Pakistani breeding programs must incorporate more diverse ASR genes and reduce the overuse of some Yr genes like Yr48, Yr65, and Yr26. Chuanyu wheat breeding programs have more ASR genes, but they need to incorporate more APR genes, such as Yr18 and Yr36, to enhance durable resistance.