EXPLORING DURABLE GENETIC RESISTANCE AGAINST LEAF RUST THROUGH PHENOTYPIC CHARACTERIZATION AND LR 34 LINKED STS MARKER IN WHEAT GERMPLASM

Present study was aimed to screening the population of 25 wheat genotypes from Baluchistan region of Pakistan along with five commercial cultivars for leaf rust adult plant resistance (APR) through gene postulation using natural inoculation of Puccinia triticina Erikss local pathotype. Infection severity was recorded on scale in comparison with susceptible control “Morroco” cultivar. On the basis of phenotypic score, seven accessions and four varieties (Zardana-89, Sariab-92, Zarlashta-99 and Raskoh-05) with AUDPC values up to 20% were characterized as resistant genotypes. Coefficient of infection (CI) score ranged from 0-10 for some accessions and cultivars showing high level of adult plant resistance. Furthermore, bi-allelic STS marker csLV34 having close linkage with Lr34 (0.4cM). This marker amplified one gene specific allele of 150bp in 21 genotypes, including 19 accessions and two commercial varieties (Sariab-92 and Zarghoon-79) which confirmed presence of Lr34 gene conferring adult plant resistance against leaf rust. The rust pathogenicity scale varied for accessions from resistant to moderately susceptible. However, beside Lr34, phenotypic gene postulation, in combination with marker assisted selection for leaf rust resistance, has revealed presence of some other unknown resistance genes in local wheat germplasm which signified its use in wheat improvement programs both locally and abroad.


INTRODUCTION
Leaf rust is one of the major biotic stresses of wheat (Triticum aestivum L.), making difficult its production, worldwide.Leaf rust is probably the most destructive wheat disease due to its repeated occurrence, prolonged presence throughout crop life cycle, pathogenicity, and epidemic nature.It breaks out during anthesis period at the time of grain formation and lasts long to inflict heavy damages to the yield if moderate temperature and high humidity is available (RATTU et al., 2010).The dynamic and rapid adaptation of its causative fungal pathogen (Puccinia triticina Erikss) conduce to a constant challenge of genetic resistance of the wheat cultivars, what is typically dependent on the effectiveness of the genes during the whole developmental cycle of the plant (BIANCHIN et al., 2012).To confront the challenge of rust, genetic resistance is the most effective, economical, longterm and environment friendly way instead of using expensive hazardous chemical applications (PINK, 2002;SHAH et al., 2014).
More than 60 leaf rust resistance genes (Lr1 to Lr68) have been located on 20 of the total of 21 wheat chromosomes working under gene for gene principle to counter avirulence of many isolates of fungal pathogens (MCINTOSH et al., 1995;MUSTAFA et al., 2013).The presence of important loci in host plant conferring resistance based on hypersensitive response (HR) against specific races of the leaf rust pathogen has been found ineffective against ever adapting new pathotypes with increased and variable mechanism of pathogenicity (LAGUDAH, 2009).Thus, it is necessary explore the race non-specific "durable resistance" against rusts in wheat germplasm, which work could spend lot of years.The characterization of Lr34 gene linked to leaf rust resistance in wheat by DYCK (1987) was major breakthrough; as it was found to be responsible for conferring non-race specific resistance against disease over a long time.Studies have confirmed effectiveness of Lr34 gene (also known as Yr18) against other wheat rusts type and disease i.e. stripe or yellow rusts (Puccinia striiformis) (MCINTOSH, 1992;SINGH, 1992), powdery mildew (Blumeria graminis) (SPIELMEYER et al., 2005) and stem rust (Puccinia graminis) (DYCK, 1992).Therefore, worldwide efforts are on to utilize natural mode of durable rust resistance through modern agriculture approaches by identification and gene pyramiding of rust resistance genes in bread wheat accessions and commercial cultivars (KHAN, 1987;DAKOURI et al., 2013).
Gene postulation has remained preferred method of researchers for systematic evaluation of local wheat and other closely related cereal's genetic stocks to screen large populations for rust resistance genes (SINGH et al., 2001;MIRZA et al., 2000;RATTU et al., 2010;HUSSAIN et al., 2015).However, in recent years, application of molecular markers, i.e., SCAR, SSR, CAPS and STS linked to leaf rust resistance have established their importance as an alternative to traditional phenotype based methods in the field to characterize large populations (SINGH et al., 2001;MAGO et al., 2005;DAKOURI et al., 2013).
In the present study, in order to identify potential sources of resistance against leaf rust in previously underutilized genotypes, our goal was evaluated Pakistani commercial cultivars and local accessions of the Baluchistan province (near the proposed center of origin of wheat) for leaf rust resistance gene Lr34, through gene postulation under field conditions and employing Lr34 linked Sequence Tagged Sites (STS) markers, to further validate the postulation for use in rust resistant cultivar breeding programs.

MATERIALS AND METHODS
A total of 30 genotypes (Table 1) were characterized in the field of Ayub Agricultural Research Institute (AARI) Faisalabad (Pakistan), in November 2010 along with a susceptible control Morocco as previous study suggested this to be the most susceptible to rust among local wheat varieties (Afzal et al., 2008).The plant material included twenty five Pakistani wheat genotypes from Baluchistan province (near the proposed center of origin of wheat) which were obtained from the gene bank of Plant Genetic Resources Institute PGRI, National Agriculture Research Center Islamabad and five wheat varieties, i.e., which were previously released by Agriculture Research Institute Quetta (ARIQ) Baluchistan over the last years.In the augmented field design, each genotype was planted in a 2m long row with 30 cm distance between the rows.For natural inoculation, mixture of leaf rust (Puccinia triticina Erikss) susceptible "Morocco" was sown around the trial as a spreader row to make possible development of rust epidemic throughout the controlled field.The entire compulsory agronomic and crop husbandry practices were carried out during the field experiment.

Disease testing in the field
Infection types (IT) and disease severity data was recorded in the field at three stages: 1) during adult plant phase at Feekes stage 10, when the first spikelet was visible, 2) then at the Feekes 10.5.3 growth stage when pollination was complete and disease development was at its peak as the susceptible control Morocco reached a disease severity of 100S, and 3) at Feekes 11.3 stage of maturity when kernels were hardened.The severity of disease was noted as percentage of the rust infectivity on the plants with reference to the modified Cobb scale (PETERSON et al., 1948) incorporating both percentage of leaf area diseased and the host reaction.To assess host response and infection type a modified scale method of Mcneal et al. (1971) 2).Area under Disease Progress Curve (AUDPC) and relative AUDPC in relation to susceptible control "Morocco" with 100% AUDPC value was calculated using SHANER and FINNEY (1977) where Y i = disease severity at ith date and T= days of the assessment.Genotypes were classified as Resistant, moderately resistant moderately susceptible, moderately susceptible and susceptible having AUDPC range of 0-20%, 21-40%, 41-60% and above 60% respectively.Also Coefficient of Infection (CI) was calculated by combining disease severity and host response data as defined by Pathan and Park (2006).

Molecular analysis
DNA was extracted from fresh leaves of 2 weeks old seedlings using modified CTAB method (HAMEED et al., 2004).Lr34 specific STS marker csLV34 (Gene link NY, USA) as reported by LAGUDAH et al. (2006) was employed to confirm presence or absence of the gene in 30 genotypes including genotypes and cultivars along with negative control "Morocco".PCR reaction was carried out in a 20µl aliquot containing 80-90ng of template DNA, 2.5µl Mg-free 10X PCR Buffer (Fermentas), 0.5µl (5unit/µl) of Taq DNA polymerase (Fermentas), 25mM of MgCl 2 , 2.5mM dNTPs (Sigma Chemical Co., St. Louis, MO), and 0.4µM of each forward and reverse primer.PCR was performed in Biorad machine (DNA Engine ALS-1296, USA) which was programmed for 45 consecutive cycles each one consisting of 2 minutes at 94°C, 3 min at 55°C annealing, 2 min at 72°C followed by a 10 min extension step at 72°C.PCR product was resolved on 2% agarose gel along with molecular size standard and visualized under Gel doc apparatus.

Data analysis
For field data, frequency distribution was performed upon disease severity scoring data and genotypes were classified accordingly using MS Excel 2007.Likewise for STS banding pattern, all amplified bands were considered dominant markers (present=1, absent=0) and their binary data was used to generate unpaired group arithmetic mean method (UPGMA) based dendrogram using NTSys PC version 2.1 (Exeter software Inc.USA).

AUDPC value
Based on AUDPC values, the genotypes were classified into four distinct groups (Figure 1).showing AUDPC above 800 as compared to the reference lines were susceptible.
Cluster analysis distributed 9 genotypes with Lr34 marker in group I while remaining 21 accessions and varieties who failed to amplify 150bp marker clustered in group II (Figure 4

DISCUSSION
Leaf rust, a common wheat disease caused by Puccinia triticina Eriks & Henn, is a serious production hazard (MCINTOSH et al., 1995).Incorporation of more than one gene to cultivars for durable leaf rust resistance has remained the focus of the breeders to cope with the dynamic nature of the pathogen (ROELFS, 1988).To address this issue, gene postulation as well as molecular marker approach is being utilized for enhancing rust resistance mainly through identification of durable rust resistance gene and pyramiding different seedling and adult plant resistance genes.Considering this, wheat genotypes postulated to carry Lr34 gene were screened with microsatellites and STS marker (csLV34) which were found to be a useful as molecular marker and Lr34 gene were reported to be linked closely (LAGUDAH et al., 2006).
The current study was conducted to characterize selected wheat genotypes (5 varieties and 25 accessions) from Pakistan (Baluchistan) for race-nonspecific resistance to leaf rust through gene postulation and molecular characterization for durable rust resistance Lr34 gene.Rattu et al. (2010) postulated host material of Pakistani wheat for Lr family of leaf rust resistance genes including Lr34 and had confirmed its presence in local genetic stocks.Previous study of FAYYAZ et al. (2008) has also examined 39 isogenic wheat lines and 12 commercial cultivars from Pakistan at different locations and observed virulence of Lr34 at Karachi and Nawabshah.In present investigation, the field testing revealed that 40% (12 genotypes) of germplasm studied was moderately resistant to moderately susceptible at adult plant stage having moderate values of rAUDPC (31 to 50 %) and low values of CI (0%-10%) and are potential donors for durable resistance against leaf rust.Similarly, the classification of wheat varieties based on values of rAUDPC was made by KHAN et al. (2002) describing groups from susceptible to fully rust resistant individuals.ALI et al. ( 2009) also used AUDPC and coefficient of infection (CI) approach to study partial resistance against yellow rust in wheat and observed multiple level of disease resistance from highly susceptible to partial resistance.We used the same methodology for disease severity determination and observed more than half of genotypes to be susceptible to virulence of leaf rust pathogen.The higher rate of occurrence of rust disease in wheat was also confirmed by Ali et al. (2009) as they found 90% of the wheat lines used in their study as susceptible.Cultivars possessing slow rusting illustrated lower rAUDPC at adult stage have race-nonspecific resistance as also described by Sandoval-Islas et al. (1998) and Singh et al. (2005).Because the durable resistance, like slow rusting and High-Temperature Adult Plant resistance is polygenic (at least 2-3 controlling genes) as also described by Dehghani and Moghaddam (2004), therefore it remains successful for longer time, even if the pathogen under goes mutations.Hence, by our findings, lines showing low frequency of disease severity with lower AUDPC values could be considered as slow rusting lines carrying durable rust resistance against Puccinia triticina Eriks and could be utilized in breeding programs.
For its relative facility, specificity and efficiency, many authors have employed PCR-based DNA markers to verify presence of leaf rust resistance in wheat (CHERUKURI et al., 2003;CHERUKURI et al., 2005;PRABHU et al., 2004;OBERT et al., 2005;LAGUDAH et al., 2006;DAKOURI et al., 2013;MUSTAFA et al., 2013).Also, it requires no laborious means or to wait for particular plant stage to observe time bound gene expression controlling trait of interest, i.e., adult plant resistance.Therefore, for further justification of leaf rust resistance estimation, Lr34 linked sequence tagged sites (STS) molecular marker (csLV34) were used to mark the presence or absence of the gene in accessions and cultivars.The marker gave successful amplification in twenty one (70%) out of thirty genotypes in the form of 150bp amplicon which is considered to be linked to durable resistance due to close linkage (0.4 cM) between this locus and Lr34 (Lagudah et al., 2006).A relatively smaller size amplicon of less than 100 bp was amplified in remaining 9 genotypes which is considered a marker for susceptible allele as LAGUDAH et al. (2006) revealed that a known fragment of 79bp (insertion in intron) is found to be linked to leaf rust susceptibility in bread wheat.Therefore, these wheat lines were found to be devoid of Lr34 gene.However, when molecular data was compared with field study, we observed three sets of observations.First type included those genotypes whose molecular data for Lr34 presence corresponded well with the field data, i.e., performance of genotype in the field against virulence of Puccinia triticina Erikss local pathotype(s).The genotypes that showed the presence of 150bp band for Lr34 exhibited moderately resistant to moderately susceptibility as also described by SHAH et al. (2010) andPRIYUMVADA et al. (2009).Similarly, the genotypes which failed to show the presence of 150bp band for Lr34 gene and were susceptible to leaf rust in the field.Hence, the absence of this gene as revealed by marker data corresponded well with the expression data in the field as also mentioned by LAGUDHA et al. (2006).In the second category of observation both the data sets did not match with each other, i.e., some of the genotypes showed the presence of Lr34 in the molecular analysis but in the field they remained susceptible to leaf rust.This contrast in the experimental and field results may be due to random mutations, suppression or deletion (AWAN et al., 2007), or evolution of new pathotype could also be the possible reason of inability of the wheat lines to cope with the avirulences (DAKOURI et al., 2013).Similarly, some of the genotypes did not reveal the presence of Lr34 locus when screened through molecular marker, although in the field those lines exhibited moderate resistance to leaf rust.This type of discrepancies have been reported in the recent past as Mcintosh (1992) postulated Lr34 in Popular wheat cultivars "Cappelle Desprez" on the basis of observed genetic association of leaf and stripe rust resistance.But, in a stark contrast, Lagudah et al. (2009) observed the cultivar to be devoid of the Lr34 using resistance gene specific marker.Besides, presence of other rust resistant gene(s) could also be the possible reason of plant's resistance against disease.That was recently demonstrated by Spielmeyer et al. (2013).They confirmed a presence of new leaf rust resistant gene (Lr67) which is almost similar in many characteristics to Lr34.Also, could be that PCR failure to amplify the particular band during amplification may be another probability of inconsistency between field and molecular marker data, as reported by Mustafa et al. (2013).Determining the presence of Lr34 in current cultivars can be helpful to predict the field resistance and durability of these cultivars and to aid decisions in selecting parents for future breeding and development of new and improved cultivars with increased leaf rust resistance.

CONCLUSION
Phenotypic gene postulation as well as application of molecular markers linked to Lr34 has confirmed presence of one or more genes in Baluchistan's accessions conferring natural resistance against leaf rust.The resistant genotypes should be used as a contributory source of adult plant resistance in wheat improvement programs.
was used.The score on scale for hostpathogen relation ranged up to 8 classes: Immune= no disease, R= resistant, RMR= Resistant-Moderately Resistant, MR= Moderately resistant, MRMS= Moderately resistant-Moderately Susceptible, MS= Moderately Susceptible, MSS= Moderately Susceptible-Susceptible, and S= Susceptible (see table

Figure 1 .
Figure 1.Distribution of genotypes into different classes for leaf rust resistance and AUDPC score.

Figure 2 .
Figure 2. Classification of germplasm based on response to leaf rust infection.

Figure 4 .
Figure 4. UPGMA based dendrogram showing two distinct groups with Lr34 presence and absence.

Table 3 .
The Infection Type, AUDPC, coefficient of infection, and rAUDPC for leaf rust in selected Pakistan wheat accessions and varieties to leaf rust.

Table 4 .
A comparison between molecular marker csLV34 data, field observation and Coefficient of Infection.