Introduction

Leaf rust caused by Puccinia triticina continues to be one of the most important wheat diseases in Mexico. One of the cheapest and environmentally appropriate control methods is the use of resistant varieties. Within the populations of this plant pathogen, the physiological races that preferentially attack durum wheat have evolved quicker than those that attack bread wheat. For example, in 2001, race BBG/BP (^{Singh et al., 2004}) was identified as virulent to the resistance gene Lr72 (^{Herrera-Foessel et al., 2014}a), which sebsequently evolved and gave rise to the race BBG/BP with virulence to Lr27+31 (Huerta- Espino et al., 2009) and eventually, the pathogen evolved to overcome the resistance gene found in the variety Cirno C2008 (^{Huerta Espino et al., 2017}, Huerta-Espino et al., 2024). However, among the races that preferentially attack bread wheats, evolution has been slow over the past two decades, partly due to the type of resistance used in the breeding process. Resistance is generally classified in two non-exclusive types: race-specific and race- non-specific (^{Caldwell, 1968}) and equivalent to the concept of vertical and horizontal resistance (^{Vanderplank, 1963}). Race-non-specific resistance is also identified as partial resistance to late blight in potato (Solanum tuberosum L.) (^{Niederhauser et al.,1954}), partial resistance to leaf rust in barley (Hordeum vulgare L.) (^{Parlevliet,1975}), slow rusting to wheat (Triticum aestivum L.) leaf rust (Caldwell, 1968), or durable resistance to yellow wheat rust (^{Johnson, 1978}). Race-specific resistance is generally qualitative and usually short-lived, due to the evolution of potentially virulent pathogens (Huerta-Espino et al., 2011). On the other hand, adequate levels of race-non-specific resistance involves genes with minor to intermediate effects, so that plants carrying this type of resistance are susceptible at the seedling stage but display resistance in later growth stages (adult plant). This characteristic is named slow-rusting resistance and is associated with certain forms of adult plant resistance (^{Lagudah, 2011}). It has been observed that near-immunity to fungi that cause rust can be achieved by combining minor genes with additive effects (Singh et al., 2000). The most important genes belonging to the category of "slow-rusting" or "adult plant resistance" (RPA) are: Lr34 (Singh et al., 2012), Lr46 (Singh et al., 2013), Lr67 (Herrera-Foessel et al., 2014b) and Lr68 (Herrera-Foessel et al., 2012). These genes are related to leaf tip necrosis (Ltn), a morphological trait that appears after flowering (Huerta- Espino et al., 2020). It has recently been discovered that the gene Sr2, which provides resistance to stem rust is related to a gene that provides resistance to leaf rust, temporarily named LrB1 (^{Ye et al., 2022}). In Mexico there has been emphasis on the genes that provide partial resistance to the adult plant since the 1950s (Huerta-Espino et al., 2020). Based on molecular markers, a recent study pointed out the presence of Lr34, Lr46, Lr67, Lr68 and Sr2/LrB1 genes, on their own or in different combinations in the improved wheat genotypes before the Green Revolution, such as Frontera, Supremo 211, Chapingo 48, Yaqui 50, Kentana 52, Bajío 52, Bajío 53, Yaqui 53, Chapingo 53, Mayo 54. These genes were also found in their offspring, the semi-dwarf wheats, developed after crosses and by the recombination of these parents, in recently released varieties (Huerta-Espino et al., 2020). Considering the importance of leaf rust and the availability of molecular markers associated with the genes for partial resistance, the aim of this study was to determine the

presence of these markers in advanced wheat lines and recently released varieties.

Materials and Methods

Genetic material. The wheat genotypes used in this study are shown in Table 1.

Resistance tests. The resistance test on seedlings was carried out in the Laboratorio Nacional de Royas y otras Enfermedades de Cereales (LANAREC). The leaf rust race MBJ/SP was used, the avirulence/virulence formula of which is: Lr2a, 2b, 2c, 3ka, 9, 16, 18, 19, 21, 24, 25, 26, 28, 29, 30, 32, 33, 36/1, 3, 3bg, 10, 11, 13, 15, 17, 20, 23, 27+31

(^{Herrera-Foessel et al., 2012}). For this assay, 8 to 10 seeds of each genotype were placed in plastic trays, using sterile soil as a substrate; the differential leaf rust lines were also included (^{Huerta-Espino et al., 2021}). Thirteen days after planting, the plants were inoculated with a suspension of uredioniospores of the race in mineral oil (Soltrol 170®) in a concentration of 5 mg of urediniospores per mL of oil (^{Randhawa et al., 2018}) and they were watered using a sprinkler connected to an electric compressor (^{Valdez-Rodríguez et al., 2020}).

In an adult plant, the evaluation was performed in the field under rainfed conditions and in which the 34 bread wheat genotypes were established (Table 1) during the 2022 spring- summer cycle in the Valle de México Experimental Field of the (INIFAP-CEVAMEX). In the evaluation in the field, the 34 genotypes were planted in two furrows, each being one meter in length, with two repetitions. The genotypes were directly inoculated with the race MBJ/SP 45 days after planting, preparing an urediniospore suspension in mineral oil (5 mg of urediniospores mL-1 of oil) (^{Randhawa et al., 2018}) and sprayed using a manual sprayer. The evaluations of severity on the flag leaf were carried out following a scale that ranged from 0 to 100% (^{Huerta-Espino et al., 2014}).

Molecular analysis. The 34 genotypes listed in Table 1 were planted following the same procedure than the seedling tests, including the respective controls for each resistance gene. The molecular analysis was carried out in the Biotechnology Laboratory of the Wheat Molecular Breeding Program in the International Maize and Wheat Improvement Center (CIMMYT). For the DNA extraction, leaf tissue samples were taken and placed into 1.1 mL tubes arranged in 96-well plates. The samples were then stored at -80 °C for 3 h, and then transferred to a lyophilizer and kept at -50 ºC under a vacuum level of 0.0 to 0.120 mbar for 48 h. The tissue was ground with 4 mm steel ball bearings for 2 or 3 minutes until it became a fine powder, using a Geno Grinder® (Metuchen, NJ, USA). The quantification and the valuation of the DNA quality were performed following the laboratory protocols for wheat followed by ^{Dreisigacker et al. (2016}) and ^{Valdez-Rodríguez et al. (2020}).

Molecular trial for the genotyping of slow-rusting genes. Molecular markers were used to determine the presence of resistance alleles of genes Lr34, Lr46, Lr67, Lr68 y Sr2/LrB1 (Table 2). For Lr34, an SNP marker was used, designed around the 3-bp indel in exon 11. Gene Lr46 has not yet been cloned, so no functional markers were available. Therefore, two SNP markers located near Lr46 were used. For Lr67, two diagnostic SNP markers were applied. The markers included the functional SNP found in exon 2 of the gene (SNP_TM4). An SNP marker derived from the linked CAPS marker cs7BLNLRR was used to determine the resistance allele for the Lr68 gene. Similar to Lr46 and Lr68, the Sr2/LrB1 gene has not been cloned and functional markers are not available. To determine the resistance allele for Sr2/LrB1 an SNP marker designed based on the linked CAPS marker csSr2 was applied.

The SNP polymorphisms were found using Kompetitive Allele Specific PCR (KASP) reagents (www.lgcgenomics.com) in reactions containing 2.5 μl of water, 2.5 μl reagent mixture 2×KASPar, 0.07 μl of reagent mixture and 50 ng of DNA. The marker for Sr2/LrB1 was added 2.5 μl MgCl2. The program for the PCR was an inicial denaturation at 94 °C for 15 min, followed by 20 cycles at 94 °C for 10 s, 57 °C for 5 s and 72 °C for 10 s, followed by 18 cycles at 94 °C for 10 s, 57 °C for 20 s and 72 °C for 40 s. Fluorescence was read as an endpoint read at 25 °C. A BMG Pherastar Plus fluorescent plate reader (BMG LABTECH, Ortenberg, Germany) was used to read the PCR products, and the genotypic data were subsequently viewed graphically using KlusterCallerTM Software (LGC Biosearch Technologies).

Results and Discussion

The results for seedlings in the greenhouse in response to race MBJ/SP helped identify susceptible genotypes and resistant genotypes. The resistant genotypes in seedlings maintained their resistance in adult plants in response to the same race, with the exception of Kronstand and Faisán S2016 (Table 3). The susceptible genotypes in seedlings had a differential behavior in the field from 1% of infection in Borlaug and Bacorehuis, up to high levels of 50% in Cortázar S94 and Bárcenas S2002, which are considered susceptible (Table 3).

Out of the five resistance genes analyzed with molecular markers, Lr34, Lr46, Lr68 and LrB1 were identified, but gene Lr67 was not identified in any of the genotypes (Table 3). The marker related to Lr34 was only identified in seven of the 34 genotypes, making it the least frequent. On the other hand, the marker related to Lr46 was found in all of the evaluated genotypes. It was found only in the Kronstad variety and in advanced line B, as well as in combination with the Lr34 marker in Texcoco F2016 and with the Lr68 marker in advanced line A (Table 3). In combination with LrB1, the marker related to Lr46 was identified in 26 of the genotypes, 13 of which carried the combination of Lr46, Lr68 and LrB1 (Table 3). Another three-gene combination (Lr34, Lr46 and Lr68) was only identified in Valles F2015, Urbina S2007 and advanced line D. In the variety Norteña F2007, markers associated with Lr34, Lr46, Lr68 and LrB1 were identified, making it the only genotype in which the presence of four of the five markers used in this study was found.

The genotypes evaluated in the seedling stage in the greenhouse against the P. triticina race MBJ/SP that were resistant maintained their resistance in the field against the same race, with the exception of Kronstand, which has the gene Lr16. This gene only confers a moderate resistance to P. triticina race TCB/TD and at the adult plant stage behaves as a partial resistance gene, with a final leaf rust severity of 70 to 80% when the gene is on its own (^{Singh and Huerta-Espino, 1995}). In other varieties, such as Francolin, the effect of Lr16 is lower and resistance levels increase in the presence of non-race-specific genes (^{Lan et al., 2014}), such as in the case of Kronstand in the presence of Lr46.

For Lr16, certain molecular markers have been used, including Lr16/Xgwm210 (^{Lan et al., 2014}). When this marker was used in this study (data not shown), it only coincided with the types of infection and the previous postulation of the gene Lr16 (^{Huerta-Espino et al., 2021}) in Nana F2007, Don Carlos M2015 and Luminaria F2012, although no association was found in the cases of Onavas F2009, Faisán S2016 and Cisne F2016, and the cases of Roelfs F2007, Borlaug 100 and Noreste F2018 were false positives. In the case of the genotypes susceptible to MBJ/SP in the seedling stage, its response in the adult plant can be explained by the association of the molecular markers with the resistance genes that include Lr34, Lr46 Lr68 and LrB1, alone or combined. However, in the genotypes under evaluation, a higher number of adult plant genes would be expected to lead to lower infection levels, but it was not the case of advanced line B (KACHU/DANPHE) with only Lr46, or in the case of Cortázar S94 and Bárcenas S2002, in which, alongside Lr46, the marker associated with LrB1 was identified and the assumption was only fulfilled in the case of advanced lines C = (ND643/2*WBLL1/4/WHEAR/KUKUNA/3/C80.1/3*BATAVIA//2*WBLL1), D = [(Lucía “S” ) = ZCT/SLM//CHAZ/ /3/KITE/BOW”S”//MEX/ROM], E = [PAMDOLY-

PABG (C7).3,] and F = [PAMDOLY-PABG (C7).1] and Norteña F2007. This would indicate that, along with the genes identified with the molecular markers used, these genotypes contain other resistance genes still uncatalogued and with possible additive effects due to the low infection levels displayed in the field in adult plants. Other studies with molecular markers associated to partial resistance or slow rusting have been reported, such as Lr46 in Guarina and Huites (^{Huerta-Espino et al., 2020}), and Lr46+Sr2/LrB1 in Jaral 66, Curinda and Batan (Huerta-Espino et al., 2020). Although Lr67, alongside Lr46 and Lr68 was common in varieties of the 1950s, it was not identified in the semi-dwarf varieties and has not yet been found in combination with Lr34 (Huerta-Espino et al., 2020), and not even identified in this study. The association of resistance gene Sr2, which gives stem rust partial resistance was also recently found to confer partial resistance to leaf rust (^{Diéguez et al., 2014}) (and designated as LrB1), yet to a lesser degree that the genes previously reported as Lr34, LR46, Lr67 and Lr68 (^{Ledesma-Ramírez et al., 2018} and Huerta-Espino et al., 2020). However, the QTL in chromosome 3BS, mapped to the genomic region Sr2/LrB1 (^{Ye et al., 2022}), has been reported as a unique locus that provides resistance to leaf rust in races found in the different locations and years in which mapping populations have been tested (^{Rauf et al., 2022}). It has been reported that, with the combination of LrB1 and Lr46, leaf rust infection levels decrease by up to 45% (Ye et al., 2022). Despite having proven that the strategy of clustering several partial resistance levels is the most effective to control wheat leaf rust (^{Singh et al., 2000}), in practice, only in Norteña F2007 variety have genes Lr34, Lr46, Lr68 and LrB1 been able to be clustered, (present study), with Lr67 still absent.

The results of the present study indicate a reduction in the frequency of Lr34 and a considerable increase in Lr46. With the availability of the molecular markers associated to the resistance genes, an increase in the number of rust-resistant genes was to be expected, or at least, a certain number of combinations should be maintained. However, breeding programs need to select and prioritize the genes to be implemented or the combinations are most likely to provide durable resistance against the fungus that causes leaf rust and thus selection pressure on the pathogen, therefore limiting the emergence of new races. The results of this study support the notion that, ideally, multiple minor adult plant resistance genes and seedling resistance genes should be combined to optimize both the resistance level and its durability in wheat varieties to be released. Even so, a rigorous evaluation of the agronomic effect of the resistance gene combinations on phenotype and yield potential will be required.

Conclusions

With the analysis of the genotypes in seedlings and adult plants, susceptible and resistant materials were identified, which helped select them for future studies. The molecular markers used helped identify genes Lr34, Lr46, Lr68 and LrB1 in the wheat genotypes identified. In general terms, it was observed that, the greater the number of these genes in the genotypes, the lower the leaf rust levels became. The usefulness of molecular markers associated with the identified resistance genes will allow the selection of superior wheat genotypes that can be included in future crossing schemes of genetic breeding programs.