EVALUATION OF SEEDLING RESISTANCE AND MARKER ASSISTED SELECTION FOR LEAF RUST ( PUCCINIA TRITICINA ) RESISTANCE IN PAKISTANI WHEAT LANDRACES, CULTIVARS AND ADVANCED LINES

Leaf rust is amongst major biotic constraints of wheat (Triticum aestivum L.) having ability to cause substantial yield reductions worldwide. A continuous exploration for novel sources of resistance is pre-requisite for its management. Objectives of study were to conduct resistance evaluation of 112 Pakistani landraces and 48 advanced lines/ cultivars at seedling stage with total 10 virulent pathotypes of leaf rust, 3 from Pakistan and 7 from U.S.A and to detect closely linked markers for Lr10, 16, 34 and 67 genes through marker-assisted selection (MAS). Findings revealed most of Pakistani landraces showed lack of resistance at seedling stage. Only 7 accessions of landraces and 11 advanced lines were found highly resistant against all pathotypes of Pakistan. Similarly, 10 advanced lines exhibited high resistance while variability in resistance was recorded for landraces against all pathotypes tested from USA. Marker-assisted selection revealed Lr genes i.e. Lr10, Lr16, Lr34 and Lr67 were present at various frequencies. Highest frequency was observed for Lr34 followed by Lr16 & Lr67 while lowest was recorded for Lr10. These genetic resources and lines identified effective against Pakistan and USA pathotypes are potential sources for improvement of leaf rust (LR) resistance and can be utilized as valuable material for breeding resistant wheat cultivars.


INTRODUCTION
Bread wheat (Triticum aestivum L.) is one of the most frequently cultivated, widely adapted (FAOSTAT and Nations, 2016) and second most after rice (Ortiz et al., 2008).It represents around 19% of essential global cereal grain crop production (Todorovska et al., 2009).The crop is currently prone to numerous challenges like biotic constraints which cause yield reductions (Gowda et al., 2014).Three rust species are amongst the biotic constraints including brown rust caused by Puccinia triticina; black rust caused by P. graminis whereas P. striiformis is causative agent of yellow rust adversely affect quality of wheat grain (Chen, 2005;Bariana et al., 2007;Ellis et al., 2014;Singh et al., 2015) and the final yield.These rust pathogens can escalate into major epidemics resulting extensive economic losses leading to crop failures, under favourable conditions.Occurrences of colossal economic and yield reductions have been documented since ancient times (Wellings, 2011;Yamin et al., 2021).Amongst these rust diseases, leaf rust has broad range occurrence in almost all wheat producing areas of the world (Huerta-Espino et al., 2011).It is well adapted to broader climatic conditions and responsible for substantial yield and economic losses (Wamishe and Milus, 2004) in South America, Europe, Central Asia (Roelfs, 1992), in South Asia including Pakistan (Nagarajan and Joshi, 1985) and can be very serious in Great Plains of North America (Kolmer and Hughes, 2013).Due to broader climatic adaptation of different virulence phenotypes of this pathogen (Roelfs, 1992), long-lasting resistance has been difficult to achieve in the United States and across the globe.The environmentally friendly and most efficient method for decreasing damage caused by the pathogen, is cultivation of resistant cultivars (Oliver, 2014;Channa et al., 2021).Leaf rust resistance may be divided into two broad ranks of resistance namely adult-plant resistance (APR) and all-stage resistance (ASR) or seedling resistance (Chen, 2005).Seedling resistance is generally race-specific also known as monogenic resistance or vertical resistance present at all stages of plant growth, extends a high level of resistance (Chen, 2013) however it is frequently overcome by virulence variation of virulent pathotypes (Jin et al., 2010;Kolmer et al., 2013).Race-specific resistance is identified through varied range of hypersensitive reactions and contribute to involve levels of higher resistance as demonstrated by (McIntosh et al., 1995).Conversely, adult plant resistance commonly known as horizontal resistance or partial resistance is more durable when deployed in combination (Singh et al., 2011) .Race-nonspecific which is effective at later plant growth stages and can provide resistance to various prevailing strains (Lagudah, 2011).To date, more than one hundred resistance genes of wheat leaf rust have been reported while 72 of them permanently catalogued (McIntosh et al., 2017).Of these, thirty-three genes have been shifted from other species into bread wheat (McIntosh et al., 2013).Most of them confer hyper sensitive reactions, are race-specific resistance genes which have short-lived nature (Kolmer, 2013;Serfling et al., 2011) often lose effectiveness and relatively few provide resistance (Lowe et al., 2011) to the recent populations of pathogen.Wheat breeders are required to concentrate on the cultivars development having durable resistance against large genetic variation in pathogen populations causing frequent breakdown of leaf rust resistant varieties (Kolmer, 2005).Hence, it is dire need to explore new resistance sources to manage significant diseases of wheat.To produce such cultivars equipped with new resistance sources, genetic resources with superior agronomic traits along disease resistance are required.Resistant genetic resources i.e.Landraces, synthetic hexaploid, elite, advanced lines, segregating population can be used to transfer superior agronomic traits and control rust diseases viz.leaf rust through breeding and biotechnological approaches.Among genetic resources wheat landraces have significant potential sources endowed with new resistance genes since comparatively few of them have been utilized in modern plant breeding (Reif et al., 2005).Various studies have confirmed that wheat landraces can be effective resistance source of stripe, stem, and leaf rust (Zurn et al., 2014).To validate combination of genes in potential donor and breeding material closely related molecular markers are great choice, modern swift and reliable approach.Wheat research has been revolutionized with development of next-generation sequencing and technologies of highthroughput genotyping (Trick et al., 2012).Because of their cost-effectiveness (Mammadov et al., 2012) highthroughput sequence-based markers like singlenucleotide polymorphism (SNP) (Wang et al., 2014) and Diversity Arrays Technology have become suitable marker system and have provided a quick enhance in the detection of markers closely related with resistance to the disease attributes (Randhawa et al., 2014).Hence, objectives of current work were to evaluate wheat landraces, advanced lines and cultivars for recognition of potential resistance at seedling stage against P. triticina and to identify leaf rust genes in Pakistani advanced lines/ cultivars, wheat landraces and cultivars through related molecular markers for breeding purpose.

Wheat germplasm
In total, a collection of 160 genotypes comprising of 112 Pakistani wheat landraces and 48 Pakistani wheat cultivars/ advanced lines (Supplementary

Experimental locations
These experiments were conducted under standard greenhouse conditions located at plant growth facility, Department of Plant Pathology, University of Minnesota, Saint Paul campus (USA).

Seedling test
For the seedling evaluation, three to four seeds of each plant material were grown in filled with plastic cones (5 x 18 cm; d x h) within 98 count racks (instead of peat pots) with 50:50 mix of steam sterilized field soil: Sunshine MVP potting mix (gypsum, Canadian sphagnum peat moss, nutrient charge, vermiculite, and dolomitic limestone) & (Sun Gro Horticulture, Quincy, Michigan).The cultivar Morocco considered as susceptible to all pathotypes of leaf rust was grown as a check along with a set of 24 near isogenic lines (NILs) deviated in single resistance gene of leaf rust.Infection types exhibited by near isogenic lines were utilized identity of the pathotypes and virulence and avirulence composition.Seedlings were planted rust-free environment at temperature cycle gradually change from 18 o C to 25 o C with 16 hour photoperiod.

Leaf rust pathotypes
A total of ten Puccinia triticina pathotypes, seven are currently prevalent pathotypes in the USA & three pathotypes were utilized in the seedling tests from Pakistan (Table 1).Temperatures were kept between 18-25 o C in the greenhouse.Provision of natural light of the day was carried out for twelve hours per day with 120 μ E.M -2 S -1 photo synthetically active radiations released by cool white fluorescent tubes fixed over plants.Inoculation process was carried out according to the procedures followed by (Browder, 1971).

Incubation
Plants were incubated in a growth chamber (18-20 °C with 16 hour photoperiod supplied by 400 W high pressure sodium vapor lamps releasing 300 μmol photon s-1 m-2) after the infection period.No light was supplemented to plants in the final phases of the infection period since pathogen can infect wheat without this treatment.After two additional hours, the chamber doors remained unlocked half-way to permit the surfaces of leaf to desiccate slowly before moving the plants back to the greenhouse conditions.Procedure of incubation was done as the method reported by (Parlevliet and Kuiper, 1977).

Disease assessment and scoring
Twelve to fourteen days after inoculation, the infection type (ITs) on plants were recorded using 0 to 4 scale developed by (DL and Kolmer, 1989).Accessions with infection types between 0 and 2 were documented resistant, while 3 and 4 scored recognized susceptible respectively (Table 2).

Genotyping of markers, PCR Assay and PCR amplification confirmation and denaturation
For molecular characterization of wheat genotypes and accessions, total 4 sequences flanking the linked SSR, SNP and STS microsatellite primer pairs were utilized (Table 3).SSR marker was amplified utilizing conditions reported by (Hiebert et al., 2010) and SNP genotyping (KASP-assays were developed for SNPs) was carried out as described by (Kassa et al., 2017) while the method discussed by (Schachermayr et al., 1997;Lagudah et al., 2006)  Gel documentation (Digecel) system was used for presence and absence of gene (Doyle and Doyle, 1990).

Data analysis
Data on presence and absence of markers was collected after visualizing gel and comparing with band size of given markers linked to leaf rust resistance genes.The data was then subjected to frequency distribution and graphs were plotted accordingly.

Leaf rust resistance tests on Pakistani wheat landraces and cultivars
A set of 160 genotypes consisting of 112 Pakistani wheat landraces and 48 Pakistani wheat cultivars were utilized for evaluation of seedling resistance against leaf rust (Puccinia triticina).Total 10 wheat leaf rust (Puccinia triticina) races (7 races from U.S. and three Pakistani races) were used for seedling screening under controlled greenhouse conditions.Frequency distribution of Pakistani wheat landraces response against US leaf rust pathotypes showed that fifteen (15) Pakistani wheat landraces were found highly susceptible against all (seven) tested US leaf rust pathotypes while sixty-two (62) landraces were susceptible against six pathotypes; twenty-eight landraces had susceptibility against five pathotypes; six landraces were found susceptible against four pathotypes and one landrace found highly susceptible and had high seedling infection types of 3+ against two pathotypes (Figure 1).Frequency distribution response of Pakistani wheat cultivars and advanced lines against US leaf rust pathotypes showed that six (6) Pakistani wheat advanced lines/cultivars were found highly susceptible against all (six) US leaf rust pathotypes tested while twelve (12) advanced lines/cultivars found susceptible against five pathotypes; five (5) advanced lines/cultivars showed susceptibility against four; five (5) advanced lines/cultivars had high seedling infection types against three and eight (8) genotypes showed susceptibility against two while just two (2) advanced lines/ cultivars were susceptible against single pathotype whereas just ten (10) wheat genotypes had resistance against all tested US leaf rust pathotypes (Figure 1).Frequency distribution response of Pakistani wheat landraces against Pakistani leaf rust pathotypes showed that ninety-four (94) of Pakistani wheat landraces are susceptible against all (three) pathotypes and eight landraces (8) were found susceptible against two pathotypes while three (3) landraces had (HITs of 3+) against one pathotype tested.Just seven (7) wheat landraces had resistance against all Pakistani leaf rust pathotypes tested (Figure 2).Frequency distribution response of Pakistani wheat genotypes against Pakistani leaf rust pathotypes showed that twenty-one (21) Pakistani advanced lines/ wheat cultivars were found highly susceptible against all (three) Pakistani leaf rust pathotypes tested; eight (8) advanced lines/cultivars found susceptible against two pathotypes whereas eight (8) advanced lines/cultivars showed susceptibility against one pathotype tested and had seedling (HITs of 3+).Just eleven (11) wheat advanced lines/ wheat cultivars had resistance against all Pakistani leaf rust pathotypes tested (Figure 2).Lr34 and Lr67 respectively.STS marker Lrk10D1 & Lrk10D2 is dominant marker that was assayed for absence/ presence of Lr10 gene.
Frequency distribution showed that 12.5% percent wheat landraces/genotypes (20) resulted in amplification of 282bp fragment for marker Lrk10D1 & Lrk10D2 which is associated with the presence of Lr10 gene (Figure 3).Result revealed that 87.5% landraces/genotypes showed no desired band indicating lack of Lr10 gene.Results of the marker Lrk10D1 & Lrk10D2 are presented in (Figure 3).Whereas SNP marker 2BS-5175914_ kwm847 was utilized for detecting the presence and absence of Lr16 gene.Frequency distribution showed that current marker amplified successfully (826bp) desired fragment with percentage of 30.6% wheat landraces/ genotypes (49) that are associated with the presence of Lr16 gene (Figure 4).Results of the marker 2BS-5175914_kwm847 are presented in (Figure 5).While STS marker csLV34 was used for detecting presence and absence of Lr34 gene.
Frequency distribution showed that 50% percent wheat landraces/genotypes (80) resulted in amplification of 150bp fragment for csLV34 that is associated with the presence of Lr34 gene.Result revealed that no desired fragment band was observed for rest of 50% landraces/genotypes indicating lack of Lr34 gene.Results of the marker csLV34 are presented in (Figure 6).Whereas SSR marker CFD23 was analyzed for detecting the presence and absence of Lr67 gene.Frequency distribution showed that CFD23 marker amplified successfully 211bp desired fragment with percentage of 27.5% wheat landraces/genotypes (44) that is associated with the presence of Lr67 gene (Figure 7).Result revealed that 27.5% of the landraces/genotypes showed no desired band indicating lack of Lr67 gene.
Results of the marker CFD23 are presented in (Figure 7).Total fourteen ( 14  Comparative study of marker and seedling results showed that among fourteen (14) entries amplified and described above except PI 270023 accession with (HITs) all other accessions and cultivars possibly containing all tested seedling resistance genes (1, 2a, 2c, 3a, 9, 16, 24, 26, 3ka, 11, 17, 30, B, 10, 14a, 18, 21, 28, 41, 42, 3bg, 14b, 20 and 23) as they had low infection types (ITs 0 to 2) to all tested pathotypes.Almost all of pathotypes used in this seedling test were found virulent Lr3a, 3bg, 14b and 20 while avirulent Lr42, 23 genes.Among pathotypes BBBD and MCTNB were found avirulent to Lr10 gene and presence of this gene and other three Lr16, 34 and 67 in tested germplasm was validated by particular molecular markers identified to be linked to resistant genes.Indeed, leaf rust pathotypes that were utilized in current study were not sufficient to identify all of leaf rust (seedling) genes which were existed in accessions and cultivars or breeding lines.To identify additional genes, further studies may be conducted with more diverse collection of pathotypes and with help of marker assisted selection the presence of those leaf rust genes may be confirmed/validated.

DISCUSSION
Wheat landraces generally comprise collections from distinct geographical regions, which perform as a great source of new rust resistant genes for creating novel and genetically distinct disease resistant germplasm (Sthapit et al., 2014).These were well recognized as valuable genetic resources offering resistance against leaf rust (Van Ginkel and Rajaram, 1993) and considered as an 1500 bp 211 bp 1500 bp essential genetic resource with well adaptation to numerous climatic conditions (Dotlačil et al., 2010) and before green revolution were cultivated around the entire world.Development of genetically diverse cultivars with resistance to leaf rust disease is an important step to cope with new virulence phenotypes produced by rust pathogens frequently overcome ASR genes.Current study was designed to screen seedling resistance in Pakistani wheat landraces and cultivars and to detect leaf rust resistance genes in Pakistani wheat landraces through molecular markers.Closely linked molecular markers can assist the designing of gene combinations in potential donor sources and breeding material.Functional markers (FMs) are the highly advantageous markers for wheat breeding strategies and high-throughput genotyping for FMs could offer a colossal opportunity to efficiently practice marker-assisted selection while breeding cultivars.Data indicated that pathotypes tested for exploring the resistance in the Pakistan wheat landraces and cultivars or advanced lines had a wide virulence spectrum.Hence, the seedling analysis of wheat landraces, cultivars and advanced lines exhibited lack of seedling resistance as the majority of the landraces and cultivars were recorded with susceptibility at the seedling stage.These genotypes displayed enormous potential for seedling resistance against the leaf rust pathogen under greenhouse conditions.However, result revealed that seven wheat landraces (PI 181087, PI 210900, PI 210903, PI 210904, PI 220072, PI 270042 and PI 572784) and eleven wheat cultivars and advanced lines Sarsabz,TW96018, were recognized with seedling resistance against all pathotypes tested from Pakistan.
Lr10 is a leaf rust resistance gene, derived from hexaploid wheat gene pool and is located on chromosome 1AS (Feuillet et al., 1997;McIntosh et al., 2003).It is found in most old Australian wheat cultivars, present in North American wheat cultivars and lines derived from the CIMMYT (International Maize and wheat Improvement Center) wheat breeding strategy.In addition, it is also postulated in Pakistani wheat cultivars (Mirza et al., 2000;Rattu et al., 2010) but high virulence to this gene is existing in Pakistan (Rizvi et al., 1984;Hussain et al., 1980).To detect this resistance gene in the wheat genome, functional markers were designed (Feuillet et al., 2003) (Hussain et al., 1998;Mirza et al., 2000;Khan et al., 2002).Marker assisted selection in landraces and cultivars showed that (30.6%) wheat landraces/genotypes contain Lr16 gene.
Lr34 is a leaf rust resistance gene which is detected on chromosome 7DS (Schnurbusch et al., 2004a;Schnurbusch et al., 2004b) was originally identified in spring wheat material at the CIMMYT (Singh and Rajaram, 1992;Singh, 1992) considered as key source of durable resistance (Roelfs, 1988).In addition, it is capable of acting synergistically with other (Lr) resistance genes (German and Kolmer, 1992) and pleiotropic effect on various diseases (Spielmeyer et al., 2003).Presence of this gene has been reported by researchers in different countries viz., South American, Italian, Chinese (Dyck & Samborski, 1970) and Egyptian wheat (Imbaby et al., 2014).Current study also confirmed the presence of Lr34 gene in 50% wheat landraces, cultivars and advance lines of Pakistan.Data analysis showed that STS marker primer csLV34 (for Lr34) produced highest (80) number of bands than any other marker and resulted in amplification of 150bp fragment.Lr67 is a resistance gene of leaf rust, originated from Triticum aestivum (SI and WM, 2015), successfully mapped to chromosome 4D (Hiebert et al., 2010) and is one of non-specific genes which are most frequently introduced genes in wheat globally (Haile and Rouml, 2013).To detect high levels of durable APR to brown rust and yellow rust in wheat, Lr67/Yr46 (slowrusting genes) can be used in combination with other genes conferring slow rusting.Marker CFD23 was utilized for Lr67 gene detection and the desired band (211bp) was successfully amplified with percentage of 27.5 wheat landraces/ genotypes (44), indicating presence of this gene in Pakistani wheat landraces/ genotypes.Evidences from results suggested that all closely linked markers tested exhibited strong association with Lr10, 16, 34 and 67 and demonstrated their utilization in marker-assisted selection.

CONCLUSION
Major resistance genes have numerous disadvantages (Ayliffe et al., 2008) and are still broadly utilized in wheat breeding for resistance.Marker-assisted selection can provide great facility for leaf rust gene transfer which is incumbent for resistance breeding.Markerassisted selection revealed that leaf rust resistance genes Lr10, Lr16, Lr34 and Lr67 were present at various frequencies and successfully amplified with four closely linked markers.Total 9 accessions of wheat cultivars/advanced lines (Shalakot-13, Faisalabad-08, Benazir-13, Sarsabz, Galaxy-13, Seher-06, AAS-11, TW96018 and Guard-C) were found highly resistant against all pathotypes tested from Pakistan and USA.While 7 accessions of landraces (PI181087, PI210900, PI210903, PI210904, PI220072, PI270042 and PI572784) showed resistance against all pathotypes tested from Pakistan.This is suggested that genes (Lr10, Lr16, Lr34 and Lr67) which have been detected in Pakistani landraces, advanced line/ wheat cultivars should be transferred through molecular breeding into modern varieties or susceptible bread wheat cultivars via conventional breeding approaches for the improvement of crop.There is necessity to broaden the genetic base of resistance by pyramiding multiple resistance genes of leaf rust.Further studies should be taken in near future on landraces for tracing other resistance genes that can be useful and deployed in Pakistani wheat cultivars for resistance against disease.

Figure 1 .
Figure 1.Frequency distribution of Pakistani wheat accessions and genotypes response against US leaf rust pathotypes.

Figure 2 .
Figure 2. Frequency distribution of Pakistani wheat accessions and genotypes response against all Pakistani leaf rust pathotypes.

Figure 4 .
Figure 4. Frequency distribution of leaf rust resistance genes detected through marker assisted selection.

Table 1 .
List of leaf rust isolates from U.S.A and Pakistan.
sprayed onto (7-day-old) seedlings.Post inoculation, plants were humidified with fine droplets of distilled water produced with an atomizer and kept for twentyfour hours in dew chamber at temperature 18-22 o C and 90% relative humidity.Upon removing from dew chamber, to prevent possible contamination plants were positioned in isolated compartments in a greenhouse.

Table 3 .
Molecular markers used for the marker-assisted selection of leaf rust resistance genes.
denaturation at 94 ℃ viz., two (for STS) and one (SSR) sequential cycles each comprising of 60 sec at 60 ℃, 60 sec at 50-60 ℃ (contingent on the specific primers), 30 sec at 73oC & followed by step of extension at 73 ℃ ofElectrophoresis & Visualization of gelThe PCR products were detected loading 10ul of the PCR product on 1.2% Agarose gels in 1X TBE buffer & UV light was applied for visualizing after ethidium bromide.