Adult Plant Slow Rusting Genes Confer High Levels of Resistance to Rusts in Bread Wheat Cultivars From Mexico

Rust diseases continuously threaten global wheat production: stem rust, leaf rust, and yellow rust caused by Puccinia graminis f. sp. tritici, Puccinia triticina, and Puccinia striiformis f. sp. tritici, respectively. Recent studies indicated that the average losses from all these three rusts reached up to 15.04 million tons per year, which is equivalent to an annual average loss of around US $2.9 billion per year. The major focus of Mexican and worldwide breeding programs is the release of rust resistant cultivars, as this is considered the best option for controlling rust diseases. In Mexico, the emphasis has been placed on genes that confer partial resistance in the adult plant stage and against a broad spectrum of rust races since the 1970s. In this study, a set of the first-generation tall varieties developed and released in the 1940s and 1950s, the first semi-dwarfs, and other releases in Mexico, all of which showed different levels of rust resistance have been phenotyped for the three rust diseases and genotyped. Results of the molecular marker detection indicated that Lr34, Lr46, Lr67, and Lr68 alone or in different gene combinations were present among the wheat cultivars. Flag leaf tip necrosis was present in all cultivars and most were positive for brown necrosis or Pseudo Black Chaff associated with the Sr2 stem rust resistance complex. The phenotypic responses to the different rust infections indicate the presence of additional slow rusting and race-specific resistance genes. The study reveals the association of the slow rusting genes with durable resistance to the three rusts including Ug99 in cultivars bred before the green revolution such as Frontera, Supremo 211, Chapingo 48, Yaqui 50, Kentana 52, Bajio 52, Bajio 53, Yaqui 53, Chapingo 53, Yaktana Tardio 54, and Mayo 54 and their descendants after intercrossing and recombination. These slow rusting genes are the backbone of the resistance in the current Mexican germplasm.


INTRODUCTION
The three rust diseases continuously threaten global wheat production: stem rust, leaf rust, and yellow rust caused by Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and Puccinia striiformis f.sp. tritici (Pst), respectively. It is estimated that global annual losses to wheat rust pathogens range between US$ 4.3 to 5.0 billion (Philp Pardey University of Minnesota, unpublished;Figueroa et al., 2018). The deployment of rust resistant cultivars is considered the best option to control rust diseases and their development is the major focus of the breeding program at CIMMYT and worldwide.
Resistance to wheat rusts is generally categorized into two non-exclusive types, race-specific and race non-specific. Racespecific resistance is generally qualitative and usually shortlived due to the evolution of potentially virulent pathogens Wellings, 2011). In contrast, adequate levels of race non-specific resistance involve genes which might contribute from minor to intermediate effects.
Plants carrying this type of resistance are susceptible at the seedling stage but express resistance at the post-seedling stages of plant growth. This characteristic is called slow rusting and often associated with some forms of adult plant resistance (APR) .
In Mexico, emphasis has been placed on genes that confer partial resistance in the adult plant stage since the 1950s. The most important genes belonging to the category of "slow rusting" or APR include Lr34/Yr18//Sr57/Ltn1 , Lr46/ Yr29//Sr58/Ltn2  and Lr67/Yr46//Sr55/Ltn3 , which confer partial resistance to leaf rust, yellow rust, and stem rust. These genes are associated with flag leaf tip necrosis (LTN) a post-flowering morphological trait (William et al., 2003;Singh et al., 2011;Herrera-Foessel et al., 2014). Another important gene in this category is Lr68/Ltn4, which confers resistance to leaf rust , and apparently confers a certain degree of stem rust resistance not yet quantified and confirmed. Another example is the Sr2 gene for resistance to stem rust which provides useful protection, although at a lower level, than most of the race specific stem rust genes. It confers an "adult plant" type of resistance and became the backbone of the Mexican germplasm being effective since their introduction until now Singh et al., 2015). The Sr2 gene expresses as a "slow rusting" type of resistance in which the rate of epidemic development is considerable reduced. Sr2/Yr30 confers resistance to stem rust and yellow rust Singh et al., 2008;Mago et al., 2011b). This gene is derived from the cultivar Hope and had provided durable resistance to stem rust in the CIMMYT-Mexican spring wheat germplasm and can be identified by its linkage with the pseudo-black chaff or brown necrosis phenotype observed on the glumes and below the nodes (Borlaug et al., 1949).
The effects of these APR genes when alone, are moderate, however, they play an important role in gene combinations and interactions with other major genes and a range of minor QTLs that cause additive effects, resulting in high levels of durable resistance. In general, accumulating APR genes into a single cultivar can result in "near-immunity'' or a high level of resistance: three to four in the case of leaf rust, four to five in the case of yellow rust (Singh et al., 2000), but more than five for stem rust (Knott, 1988;Singh et al., 2015).
The objective of the present study was to investigate the presence and effectiveness of the known/unknown slow rusting resistance genes against the three rust diseases in selected tall and dwarf wheat cultivars released in Mexico

Plant Material
Fifty-one bread wheat cultivars ( Table 1) released in Mexico before and after the green revolution era were characterized in seedling tests in the greenhouse (GH) and in the field against stem rust, leaf rust, and yellow rust.

Stem Rust
Seedling evaluations against the stem rust RTR race were conducted at CIMMYT's GH facilities in El Batan, Mexico. Eight to 10 seeds per cultivar were sown per entry in plastic trays, as well as a set of both 20 differential from the Minnesota Cereal Disease Laboratory (CDL) (Jin et al., 2007) and the CIMMYT standard Pgt set (Singh, 1991). The seedling plants were inoculated 10 days after planting, when the seedlings had developed the second leaf, using urediniospore sprays of the RTR Pgt race ( Table 2) suspended in Soltrol 170 ® (Phillips Petroleum Company, Borger, TX) mineral oil (Singh, 1991) at a concentration of 5 mg/ml (Herrera-Foessel et al., 2007). Plants were placed inside a dew chamber overnight and then moved back to the GH.
A second set of the 51 cultivars and susceptible checks were tested against Pgt race TTKSK at the seedling stage at the United States Department of Agriculture -Agricultural Research Service (USDA-ARS) CDL, St. Paul, MN, following the procedure described by Rouse et al. (2011) andJin et al. (2007). Ten seeds of each cultivar were planted in trays. Eightday-old seedlings were inoculated with fresh urediniospores of Pgt race TTKSK.

Leaf Rust
Seedling tests were carried out at CIMMYT headquarters GH. Single spore races were used in the evaluations. Wheat seedlings were grown for 10 days under GH conditions (20°C/23°C night/ day temperatures) and inoculated with the rust in a Soltrol 170 spore suspension at a concentration of 5 mg/ml. The LR race used at seedling test was MBJ/SP described by Herrera-Foessel et al. (2012) (Table 2). After the inoculation, plants were kept in a humid chamber at 22°C for 16 h. Plants were then placed back in the GH for 2 more weeks and scored for IT on a 0-4 scale as described in Roelfs et al. (1992).

Yellow Rust
Nine to 12-day-old seedlings (two-leaf stage) were inoculated by using an atomizer to spray urediniospores suspended in lightweight mineral oil, Soltrol 170 at a concentration of 5 mg/ ml. The yellow rust race used in the study was MEX14.191 (Randhawa et al., 2018). The trays carrying inoculated seedlings were then placed in a mist chamber and incubated at 7-9°C for 24 h. Seedlings were then moved to a GH room maintained at 15/ 18°C night/day temperatures. The ITs were recorded 14 days post-inoculation using a 0-9 scale (McNeal et al., 1971).

Field Testing
The 51 bread wheat cultivars and the susceptible checks as follows: Sr RTR-Apav 1; TTKSK (UG99) Cacuke; Lr Avocet and Apav 1; Yr Avocet and Apav 1 were evaluated in field TABLE 1 | Mexican bread wheat cultivars, their parents, and year of release used in the study to determine the response to stem rust (SR), leaf rust (LR), and yellow rust (YR).
Spreader rows were planted on each side of the experimental area, as well as hill plots in the middle of the 30-cm pathway to the side of each experimental plot. The spreaders consisted of a mixture of susceptible cultivars (differing) for each rust disease at Cd. Obregon (Mexico). For the stem rust TTKSK race a mixture of the stem rust susceptible wheat cultivar Cacuke and six Sr24carrying cultivars were used. For leaf rust, a mixture of Avocet resistant to yellow rust was used, whereas in the case of YR, a mixture of wheat cultivars carrying Yr9+Yr27, Morocco, and Avocet near-isogeneic cultivars for gene Yr17 and Yr31 was used as spreader.

Spreader Inoculation
The stem rust and leaf rust spreaders at Cd. Obregon were inoculated with spores of the Mexican Pgt race RTR and Pt race MBJ/SP (Singh, 1991), respectively, suspended in Soltrol 170 at a concentration of 5 mg/ml 8 weeks after sowing. In Njoro, the spreaders were inoculated with a field collection of stem rust races including TTKSK by spraying with a mixture of urediniospores suspended in water at a concentration of 5 mg/ ml plus Tween 20 and performing needle inoculations using the same suspension .
For the yellow rust evaluations, the Mexican Pst race Mex14.191 suspended in Soltrol 170 at a concentration of 5 mg/ml was inoculated into YR spreaders (Ponce-Molina et al., 2018). The inoculation method was as described by Lan et al. (2014).

Rust Evaluation
Disease severity (DS) on the cultivars was scored at two to three times in each experiment using the modified Cobb's scale (Peterson et al., 1948) and host response to infection was determined according to Roelfs et al. (1992).

Leaf Tip Necrosis (LTN) and Pseudo Black Chaff (PBC) or Brown Necrosis
LTN and PBC morphological traits were scored as "+" when visible at all locations and years; if not clear or the presence of PBC was in doubt, we did not give the "+" score.

Genotyping Slow Rusting Genes via Molecular Markers
Gene-based and closely linked molecular markers were used to determine the presence of the resistance alleles of genes Lr34, Lr46, Lr67, Lr68, and Sr2 (Supplemental Table 2). For Lr34, a SNP marker designed around the 3bp indel in exon 11 was used . The Lr46 gene is not cloned yet, therefore functional markers were not available. Two SNP markers in the proximity of Lr46 were therefore deployed (Viccars, L., Chandramohan, S., and Lagudah, E. unpublished data). For Lr67, three diagnostic markers were applied, one sequence tag site (STS marker) and two SNP markers. Markers included the functional SNP observed in exon 2 of the gene (SNP_TM4) (Moore et al., 2015). A SNP marker derived from the linked CAPS marker, cs7BLNLRR, was used to determine the resistance allele for the gene Lr68. As with Lr46, Lr68, and Sr2 have not yet been cloned and hence no functional marker at 2 | Avirulence and virulence status of stem rust, stripe rust, and leaf rust and pathotypes used to inoculate at seedling in the greenhouse and adult plant in the field the 51 cultivars of bread wheat included in the study.

RESULTS
Cultivars under evaluation were separated into two groups, tall and dwarf in order to see the possible negative or positive effect of the dwarfing genes on the disease resistance. The grouping allowed us to estimate the frequency, i.e., presence/absence of the slow rusting resistance genes before and after the dwarfing genes were introduced and other relatively recent released materials.

Seedling Testing
At seedling stage, all tall cultivars tested (except Marroqui 588 and Lerma 50) were resistant to the RTR stem rust race; all dwarfs were also resistant to Pgt race RTR. Yaqui 50, Yaqui 53, Bajio 53, Chapingo 53, and Crespo 63 were resistant to the TTKSK (Ug99) stem rust race and the rest were susceptible. In the case of leaf rust, most cultivars tested were susceptible except Bajio 52, Toluca 53, and Bajio 53 among the tall ( Table 3) and Curinda M86 and Mochis T88 among the dwarfs ( Table 4). In the case of yellow rust, a behavior like that of leaf rust was observed, where most of the cultivars were susceptible as seedlings to race MEX14.191, except for Marroqui 588, Chapingo 48 and Kentana 48 among the tall, and Tobari F66, Orizaba 77, Huasteco M81, Huites F95, and Maya S2007 among the dwarfs.

Field Testing
Despite seedling resistance against Pgt race RTR of stem rust, different degrees of final disease severities were observed among the tall cultivars. These responses varied from near immune (NI) response in Candeal 48, Candeal 52, Verano Pelon, and Egipto101, to MSS in Lerma 50; and from NI in Saric F70, Guarina 85, Palmerin F2005 and Chapingo VF74, to MRMS in Curinda M87 among the dwarfs (Tables 3 and 4; Figures 1 and 2). The response of the cultivars to the TTKSK race was similar, but the number of resistant were less, and no NI were observed. Among the seedling susceptible (SS), Toluca 53 was resistant; the seedling resistant Crespo 63, Chapingo 53, Yaqui 50, and Yaqui 53 were classified as resistant in the field among the tall cultivars. Saric F70, Guarina 85, and Maya 74 were susceptible at seedling stage, but resistant in the field. In contrast, Kentana 54 among the tall cultivars, Mochis T88 and Yecorato F77 among the dwarfs showed the highest DS against stem rust race TTKSK.
In the case of leaf rust, most of the cultivars varied from near immunity to resistant in both Tall (Table 5) or in Orizaba 77, Huites F95, Bajio M67, Yecorato F77, Victoria M81, and Tepoca M89 among the dwarf cultivars ( Table 6).
All cultivars positive for Lr67 were tested several times including RL6077 in order to validate the marker(s) and  the methodologies for a new discovered-cloned leaf rust resistance gene. Among the dwarf cultivars (Table 6), the frequency of markers associated with Lr34 and Lr46 were the most common, followed by that of Lr68. Lr46 alone was identified in Guarina 85, Huites F95, Maya S2007, Batan F92, and Curinda M87. No cultivars positive for Lr67 alone or in combination were identified. No Lr34 or Lr68 alone were identified. The most common combinations were Lr34+Lr46, followed by the Lr34 +Lr68 in Chapingo VF74, Tobari F66, and Bajio M67. Victoria M81, Orizaba 77, and Mochis T88 were positive for the combination Lr34+Lr46+Lr68.

DISCUSSION
The concept of multiple disease resistance in cultivars containing the slow rusting genes and triple rust resistance were conceived by Dr. Borlaug and emphasized in his breeding schemes. Such concepts remain incorporated in the current breeding schemes at CIMMYT in the form of durable resistance genes which are effective against the actual rust races present worldwide. Bread wheat cultivars released in Mexico carried high levels of resistance to stem rust, leaf rust, and yellow rust. The molecular marker analysis revealed that most cultivars carry at least one or more of the following slow rusting resistance genes: Lr34/Yr18/Sr57, Lr46/Yr29/Sr58, Lr67/Yr46/ Sr55, Sr2/Yr30, and Lr68. Among the tall and dwarf cultivars evaluated, there is a considerable variation in their response to rust in the different locations and years. When the presence of a single gene-based on the molecular markers of a determined bread wheat cultivar-showed a higher level of resistance compared to a cultivar with the presence of more than one resistance factor based on the markers, the difference could be attributed to the presence of additional, not yet cataloged slow rusting resistance genes. There exists great variation in cultivar resistance (APR) to the three rusts. In some cases, levels of APR are similar between cultivars independently of whether the  High levels of resistance to leaf rust exist in all the cultivars tested except for Yaqui 48 among the tall cultivars and Chapingo VF74 among the dwarfs, but response to other rust diseases varied from highly resistant to susceptible. In the case of the yellow rust, cultivars such as Kentana 48 and Kentana 52 were highly resistant but their resistance to stem rust was intermediate.
Resistance in Supremo 211, Yaqui 53, Toluca 53, Bajio 53, Narinio 59, and Crespo 63 could be attributed to the gene combination Sr57+Sr58 and the presence of Sr2 indicated by brown necrosis. In Yaqui 53, however, the resistance could be attributed to the presence of Sr55 and Sr2. Supremo 211 clearly showed the necrosis in the glumes and internodes as noted by Dr. Borlaug since their release in Mexico in 1945 (Borlaug et al., 1949); however, the Sr2 marker used was negative in this variety. The same combination among the dwarfs Saric F70 and Sr58 +Sr2 in Guarina 85 and Maya S2007 confers high levels of resistance.
In the case of Ug99, Chapingo 48 was positive for Sr55 and Sr58 and showed moderately resistance; but Candeal 52 was positive for Sr2 and Sr58. Among the Dwarfs, Tobari 66, Orizaba 77, and Palmerin F2005 were MR to leaf rust, but moderate susceptible to Ug99 stem rust.  Gene combinations in Verano Pelon, Egypt 101, and Yaqui 50, while being effective against stem and leaf rust races, may not be enough against present yellow rust races with more virulence factors. The opposite can be found where gene combinations Sr57+Sr58 in Kentana 51 and Kentana 52, or Sr57+Sr58+Sr2 in Kentana 48 are highly effective against leaf and yellow rust, but moderately susceptible to RTR and Ug99 stem rust.
Mayo 54 and Marroqui 588, although resistant to leaf and yellow rust, showed marginal resistance to stem rust RTR and Ug99 races. Constitucion (Sr55/Lr67/Yr46), on the other hand, was resistant to stem and leaf rust, but moderately susceptible to Ug99 and yellow rust. Chapingo VF74 and Huites F95 were resistant to yellow and stem rust but moderately resistant to leaf rust and Ug99 stem rust. Other dwarfs, such as Jaral 66, Curinda M87, and Mochis T88 although showing an adequate level of resistance to leaf rust, demonstrated low resistance to stem rust RTR, Ug99 and yellow rust.
It is expected that, as the number of slow rusting resistance alleles increases in the cultivars, the levels of slow rusting resistance should increase (Singh et al., 2000), Near immunity is expected against leaf rust when two to three slow rusting genes are together (Singh et al., 2000). The same is expected when three to four genes are present against yellow rust and more than five in the case of stem rust (Knott, 1988). Although that has proven to be true in most cases, cultivars in our study showed near immunity response to stem rust race while associated with one or two markers as was observed in Candeal 48 and Candeal 52 with Sr58 or in Yaqui 53 with Sr2+Sr55 and Constitucion with Sr55, indicating that other nonidentified slow rusting genes are present in those cultivars. Another example is the combination Sr2+Sr57 +Sr58 which confers a NI response in Verano Pelon and Supremo 211 which was also found in the cultivars Kentana 54 and Lerma 50; but the different response of these cultivars to stem rust indicates the presence of other resistance genes in Verano Pelon and Supremo 211. One slow rusting gene alone conferring near immunity is unusual or never yet seen; therefore, more nonidentified or not yet cataloged genes must be present.
In the case of TTKSK (Ug99) stem rust, no NI groups were observed. Combination Sr57+Sr58+Sr2 in Crespo 63, Toluca 53, and Narinio 59 were grouped in the resistant category, but the same combination in Kentana 48, Lerma 50, and Kentana 54 and others resulted in their being grouped in the moderately susceptible category (up to 60% DS), again indicating the presence of other resistance genes besides those inferred by the molecular markers tested in the study.
New races with new virulences and adaptation to warmer temperatures are common in the yellow rust populations. Therefore, a single resistance gene is not going to be enough, as in the case of leaf rust, i.e., Yaktana Tardio (positive for Yr18) showed a maximum DS of 40%. Among the cultivars positive for the Yr29+Yr30, Candeal 48, Candeal 52, Bajio 53, and Yaqui 48 showed different degrees of resistance. Yaqui 53 (Yr30+Yr46) evidently carries additional alleles in order to be able to reduce the DS. The Yr18+Yr29 combination was very effective in Bajio 53, but less in Nayar, Lerma or Huamantla Rojo. Ten cultivars carried the Yr18+Yr29+Yr30 combination, which showed differing degrees of resistance, indicating that Narinio 59, Crespo 63, and Kentana 48 evidently carry additional resistance alleles.    Yr46 was detected alone, as in Mayo 54, being MR, but the Yr30+Yr46 combination displayed a range of rust responses varying from resistant in Chapingo 48 to MR in Yaqui 50 and Chapingo 53. Marroqui 588 carries the same gene combination but may also be carrying Yr67 (Li et al., 2009;Xu et al., 2014) which is effective in Mexico at the seedling stage and under field conditions as well.
Among the dwarf cultivars, Yr29+Yr30 keeps the yellow rust severity low in Maya S2007, but the same combination does not provide enough levels of resistance in Curinda M86 and Batan F92. In the cultivar Huites F95, where the presence of Yr30 was difficult to determine by the presence of brown necrosis or the molecular markers, the low DS could not be explained by the presence of Yr29 alone.
The Yr18+Yr29+Yr30 combination among the dwarfs grouped the cultivars positive for these markers into a range of resistant in Palmerin F2005, to moderately susceptible in Lerma Rojo 64. The level of disease provided by the Yr18+Yr29 combination in Yecorato F77, Tepoca M89, Victoria M81, and Mochis T88, were not the same as in Palmerin F2005, apparently due to the lack of the additional resistance provided by Yr30. The presence of Lr68 in Victoria M81 and Mochis T88 indicates that, under the conditions tested, this gene has no effect on yellow rust. The same could be true when the combination Yr18+Yr30 +Lr68, as in Chapingo VF74 and Tobari F66, was compared to Bajio 67 (Yr18+Lr68). Orizaba 77 was nearly immune to yellow rust with the Yr18+Yr29+Lr68 combination, but their resistance is more likely due to the presence of a race-specific resistance gene effective at all growth stages. Recently, a study was carried out (Muleta et al., 2017) to determine if Lr68 influenced yellow rust; the authors indicated that the presence of the marker could have a disease reducing effect.
The slow rusting APR genes Lr34/Yr18/Sr57, Lr46/Yr29/Sr58, Sr2/Yr30, and Lr68 were introduced into the Mexican germplasm in the first two cultivars released by Dr. Borlaug obtained by selection from crosses made by McFadden (Borlaug et al., 1949): Supremo 211 (Supresa//Hope/Mediterranean) and Frontera (Fronteira//Hope/Mediterranean), both sharing the same parents (Supresa = Polissu/Alfredo Chaves 6.21 and Fronteira = Polissu/Alfredo Chaves 6.21). The combination Lr46+Lr68+Sr2 was introduced through Egypt 101 (= Kenya governor). He also introduced Lr67/Sr55/Yr46 to the Mexican breeding program through Marroqui 588 (Florence/Aurore) in 1945 from Australia (Borlaug et al., 1949). Marroqui 588 is a cross made in 1922 in Australia and first released in Tunisia in 1925(Wenholz et al., 1939. An additional source of Lr34 came to Mexico through Mentana, introduced directly from Italy (Borlaug et al., 1949); it is found in the pedigree of Kentana crosses (Kenya/Mentana). The first crosses made by Dr. Borlaug in Mexico were Marroqui 588/ Newthatch (Florence/Aurore//Hope/*3 Thatcher), and Kenya/ Mentana in 1945 (Stakman et al., 1967). Using a shortcut of producing two generations per year and shuttle breeding between Chapingo and the Yaqui Valley, by 1949, Yaqui 48, Chapingo 48, Nazas 48, and Kentana 48 were multiplied and released. Lr67/ Yr46/Sr55/Ltn3, through Marroqui 588, added new sources of resistance to the already in use Sr2/Yr30 from Hope in Supremo 211 (Supresa//Hope/Med), Lr34+Lr46 in Frontera (Fronteira// Hope/Med) and Lr34+Lr46+Lr68 released in Mexico in 1945. Marroqui 588 was crossed with Thatcher and the cross was designated as C5 (Gutierres-Cruz, 1956). C5 was released in Mexico as Chapala. In our study, seed of this variety did not germinate, but DNA extracted from the seed indicated that Chapala was positive for the Lr67 marker. C5 also appears in the pedigree of Anahuac Barbon, Anahuac Pelon, Constitucion, and Leon 15, cultivars all positive for the Lr67 marker (data not shown) and was used intensively as a recurrent resistant parent (Gutierres-Cruz, 1956). The presence or absence of a resistance gene in a cultivar is the result of the presence of the gene in the parents; for example in the case of Lr67, the absence of the gene among the dwarfs can be explained by its absence in the parents rather than the effect of the dwarfing gene Rht-D1 located in the same chromosome (4DL), as has been suggested (Moore et al., 2015).
Lr34/Yr18/Sr57 and Lr67/Yr46/Sr55 molecular markers are undoubtedly linked to the resistance genes and we are confident that the cultivars mentioned indeed carry the gene(s) as is reflected by their levels of resistance. In contrast, despite molecular markers associated with Sr2/Yr30 and Lr46/Yr29/ Sr58 not being diagnostic, the presence of brown necrosis and LTN lends support for the inferred presence of Sr2 and Lr46, respectively, particularly when LTN is present, and cultivars are negative for the other diagnostic markers.
Lr34 was first described in Canada by Dyck (1977) in the cultivar Frontana, Lr46 in Mexico by Singh et al. (1998) in the variety Pavon F76, and Lr67 by Dyck and Samborski (1979) in the Pakistani accession PI250413. Because NILs RL6077 and RL6058 showed similar responses to leaf rust, we used them at CIMMYT as sources of Lr34 until diagnostic markers were developed for Lr34 that showed otherwise (Kolmer et al., 2008;Krattinger et al., 2009;Lagudah et al., 2009;Spielmeyer et al., 2013). Subsequently, the Lr67/Yr46 locus, which conferred resistance to leaf rust and yellow rust, was mapped to chromosome 4DL in two independent mapping studies (Hiebert et al., 2010;Herrera-Foessel et al., 2011). Herrera-Foessel et al. (2012) described Lr68 being present in a cultivar derived from the wheat cultivar Parula. All these adult plant slow rusting resistance genes have been used in the Mexican breeding program led by Dr. Borlaug since the release of Supremo 211 (Lr34+Lr46), Frontera (Lr34+Lr46 +Lr68) in 1945, and Chapingo 48 (Lr46+Lr67) in 1948 (Borlaug et al., 1949).
Lr34, Lr46, Lr67, and Lr68 are all associated with a trait expressed in the flag leaf after heading known as LTN (Singh, 1992;Navabi et al., 2005;Rosewarne et al., 2006;Herrera-Foessel et al., 2012;Herrera-Foessel et al., 2014). Wheat cultivars carrying LTN display a longer latency period for infection and fewer, smaller rust pustules when compared to a susceptible cultivar in the field or GH. In our study, all cultivars tested were positive for leaf tip necrosis, indicating that at least one slow rusting resistance gene was present. We did not observe an increased level of LTN as the number of slow rusting resistance genes increased. LTN could be an undesirable trait for some breeders due to the reduction of the photosynthetic area; however, the impact on yield is minimum compared to the protection offered by the slow rusting genes and the impact on rust epidemiology (Singh and Huerta, 1997) Another important durable APR gene that has provided effective resistance for many years is the Sr2 gene, which, in combination with other unknown minor genes, is referred to as the Sr2 complex (Rajaram et al., 1988;Singh et al., 2006). This gene, besides conferring resistance to stem rust, confers resistance to yellow rust (Singh et al., 2000;Singh et al., 2005;Mago et al., 2011b). Sr2 can be associated or identified by the presence of a morphological trait observed as a result of a black pigmentation called brown necrosis or Pseudo-black chaff which occurs around the glumes and the internodes of the stem after anthesis (McFadden, 1939;Borlaug et al., 1949). It has varying degrees of expression depending on the cultivar and environment . McFadden (1937) indicates that brown necrosis was the result of infection by stem rust; but Mishra et al. (2005) suggested that resistance was not always associated with brown necrosis; and Kota et al. (2006) reported that the two traits were inseparable by recombination which was subsequently confirmed by Juliana et al. (2015). The presence of necrosis can be noticed in the absence of the disease. The Sr2 gene was first introduced into the Mexican germplasm by Dr. Borlaug in the Cultivar Supremo 211 (a Hope-derived cross made by McFadden) (McFadden, 1930) and remains as the backbone of stem rust resistance and is associated with the brown necrosis trait (Borlaug et al., 1949).
The slow rusting genes Lr34, Lr46, Lr67, and Sr2 can be considered as backbone genes, which when present in combination with other major genes and/or with known or unknown small effect or minor genes (QTLs), have provided effective resistance over the years in wheat improvement (Ellis et al., 2014). Lr68 can be added to the backbone genes as a component of useful slow rusting genes that are important contributors to durable leaf rust resistance. The findings from the Mexican wheats lend further support to the significance of these backbone slow rusting APR genes, albeit in combination with unknown genes, in developing more durable rust resistance in wheat.

DATA AVAILABILITY STATEMENT
This article contains previously unpublished data. The name of the repository and accession number(s) are not available.

AUTHOR CONTRIBUTIONS
JH-E established the rust evaluation nurseries. JH-E, RS, LC-H, and EL took the phenotypic data and wrote the main manuscript. HV-M, and MR-G provided most of the germplasm and conducted rust evaluations. SD and DB-S extracted the DNA samples and run the molecular markers. EL developed and provided the molecular markers. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We are grateful to the Bill and Melinda Gates Foundation (OPPGD 1389) and the Department for International Development from the United Kingdom for supporting the CIMMYT's wheat breeding and research activities through the Delivering Genetic Gains in Wheat (DGGW) project.

SUPPLEMENTARY MATERIAL
The supplementary Material for this article can be found on line at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00824/ full#supplementary-material TABLE S1 | Wheat cultivars, their response to rust at seedling and adult plant in several locations and years. Leaf tip necrosis and pseudo Black Chaff and molecular markers associated to rust resistance and dwarfing genes.