Germplasm Evaluation and Characterization of Slow Rusting Resistant Gene against Stripe Rust of Wheat

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India is a privileged country to attain and retain the status of being the second largest producer of wheat and registering the historic production of 93.50 million tonnes during 2015-16.Among the three rust, stripe or yellow rust caused by Puccinia striiformis Westend f. sp.tritici is devastating foliar disease and is considered of immense importance in successful cultivation of wheat (Singh et al., 2014).Year after year the susceptible wheat cultivars that suffer from stripe rust disease result in increased inoculum build up thus posing major threat to wheat cultivation.Although remarkable progress has been made in breeding for stripe rust resistant varieties but the subsequent evolution of pathogen races at much greater pace continues to challenge this breeding programme (Singh et al., 2011) and stripe rust remains a threat to wheat cultivation (Sareen et al., 2012).Although the timely application of fungicides against this obligate parasite can manage the disease to some extent but their use add to the production costs.Breeding for resistance remains the most effective and efficient management strategy as it does not add input costs to farmers and is environmentally safe (Yang and Liu, 2004).The identification and knowledge of the resistance genes in commonly used parental germplasm and released cultivars is very important for utilizing the genetic resistance to manage this rust in full potential.The long term and economical strategy could thus be resistance breeding through deployment of effective rust resistance genes over space and time (Pretorius et al., 1997;Zeng et al., 2014).The genes expressing at adult plant stage have special significance because the cultivars having such genes have shown partial resistance that has remained effective for longer durations (Singh and Rajaram, 1991;Khan and Saini, 2009).
The outbreak of stripe rust in temperate areas of Jammu and Kashmir State, India is a matter of great concern owing to the fact that the rust inoculum generated in these areas may act as a reservoir of inoculum for rust initiation in North-West Plain Zones of the country.Therefore there is urgent need to curtail the pathway of rust pathogen in Jammu and Kashmir State on priority basis so that the same may not spread to food bowl of the country thereby causing a great threat to the National Food Security Mission (NFSM).With this background information the present study was undertaken to screen the wheat germplasm for identifying stripe rust resistant genotypes and then validate resistance gene (Yr18) at molecular level.

MATERIALS AND METHODS
The present studies were carried out in the Division of Plant Pathology, Faculty of Agriculture, SKUAST Jammu, Chatha, during rabi 2014-2015.

Screening of wheat germplasm for slow rusting traits
Thirty

Screening of wheat germplasm for slow rusting traits
During the present studies thirty-one   The Yr18 locus confers partial and durable adult plant resistance against stripe rust fungus.In order to track Yr18, PCR was carried out with an allele specific primer cssfr2 which resulted in the amplification of 523 bp fragment, which exhibited the presence and absence of banding pattern.Nine wheat genotypes (Sonara 64, K 9351, HP 1633, Raj 4037, Sharbati Sonara, K 9533, K 8434, NP 823 and Ajanta) amplified a band of 523 bp fragments which indicated the presence of Yr18 gene (Table 3,

Assessment of losses caused by stripe rust
While assessing the losses while evaluating thirty one genotypes at tillering, flowering and dough phonological stages, though there was no loss at the initial stage of tillering, but the per cent losses at flowering stage ranged from 11.95 to 36.15.In case of Raj 4037, NIAW 301, PBW 12 and HUW 213 the per cent losses estimated were 11.95, whereas, the per cent losses estimated were 20.75 (HD 2643 and NP 825), 22.95 (PBW 65) and 36.15(Agra Local).Similarly, at the dough stage the per cent losses estimated in K 9351, NIAW 301, PBW 12 and HUW 213 was 14.15, whereas, in case of PBW 65 and Agra Local the losses recorded were 25.15 and 44.95 per cent, respectively.Chen (2005) have reported that the disease may cause 40-78 per cent losses under normal conditions, but if the conditions were favorable for disease spread the loss might rise to 84 per cent (Murray et al., 1994).The predicted losses may be less if the wheat varieties were resistant or slow rusting against rust diseases (Salman et al., 2006).Similarly, the wheat varieties which showed less area under disease progress curve normally bear less yield losses (Hussain et al., 1996).

M = Ladder Samples - 1 (Fig. 1 .
Fig. 1.PCR amplification of wheat germplasm by cssfr2 Fig., 1).Five allele-specific markers (cssfr1-cssfr5) were developed by Lagudah (2009) based on a 3 bp deletion in exon 11 of the Yr18-gene which were closely linked to the Lr34/Yr18/Ltn1/Pm38 locus and have been shown to provide a much wider diagnostic ability for this multi-pathogen resistance trait in diverse wheat cultivars.The validation of results are in accordance with the findings of Laugdah et al. (2009).

validation of stripe rust resistance gene in wheat germplasm
(Doyle and Doyle, 1987)le which gives combined scores for level of severity and infection type was adopted to record the disease severity.Observations for slow rusting traits such as Final rust severity (FRS), Area Under Rust Progress Curve(Milus and Line, 1986), Coefficient of infection and Infection Rate (Brorers, 1989) were recorded.Molecular Nineteen wheat genotypes (Sonara 64, K 9351, HP 1633, Raj 4037, Sharbati Sonara, K 9533, K 8434, NP 823, Ajanta, PBW 12, KRL, RW 346, HD 2643, HS 1097, NP 825, PBW 226, NIAW 301, PBW 343, and NI 179) were selected on the basis of low disease severity under field conditions for molecular validation of stripe rust resistance gene Yr18 by using allele-specific markers Cssfr2 (F=TTGATGAAACCAGTTTTTTTTCTA R=TATGCCATTTAACATAATCATGAA).The genomic DNA of selected genotypes was isolated by CTAB (cetyl-trimethyl ammonium bromide) method(Doyle and Doyle, 1987).Polymerase chain reaction (PCR) ampliûcation was conducted by Cssfr2 primer with temperature proûles as described by Laugdah et al.(2009).The ampliûcation products were separated on 3 per cent agarose gels containing ethidium bromide and 1× TBE buûer.The gels were visualized using gel documentation system for documentation of allele type in selected genotypes for resistance gene Yr18 using a standard molecular ladder of 100 bp.

Table 1 .
Evaluation of wheat germplasm for slow rusting parameters against Puccinia striiformis

Table 2 .
Response of wheat germplasm against stripe rust under field conditions

Table 3 .
Molecular validation of Yr18

Table 4 .
Assessment of losses in wheat germplasm due to stripe rust (Puccinia striiformis) of at different growth stages