Stripe rust resistance gene(s) postulation in wheat germplasm with the help of differentials and tagged molecular markers

Thirteen known Yr gene-associated markers pertaining to genes (Yr5, Yr10, Yr15, Yr24/Yr26) were used to identify the genes in selected wheat germplasm which were found resistant under field conditions at two locations in Punjab, India against stripe rust. In field evaluation, 38 genotypes exhibited highly resistant response, with a final rust severity (FRS) ranging from 0 to TR. Seven genotypes expressed a resistant to moderately resistant response with FRS ranging from 5MR–10S. In race-specific phenotying against most prevalent pathotypes of Puccinia striiformis tritici (46S119,110S119 &238S119) by seedling reaction test (SRT) 14 genotypes (29.2%) were found to be immune (IT = 0), 28 genotypes (58.3%) were resistant (IT = 1), and 3 genotypes (6.3%) were moderately resistant (IT = 2). Yr5 was detected in sixteen lines with the help of two markers Xwmc175 and Xgwm120 linked with Yr5. Yr10 was detected in ten lines with the marker Xpsp3000 and Yr15 was detected in fourteen lines with two linked markers; Xgwm413 and Xgwm273. Likewise, Yr24/26 was detected in 15 lines with two linked markers, namely Xbarc181 and Xbarc187. Based on the race specific phenotyping data and marker data, fourteen lines were found to carry a single gene, 16 showed the presence of two gene combinations, and seven genotypes were found to have a combination of three genes. Frequencies of Yr5, Yr15 and Yr26/Yr24 was high among test wheat germplasm in comparison to Yr10.

stripe rust throughout the crop season or at adult plant stage; we are left with only few effective Yr genes as many of them have been defeated due to the continuous and fast evolution of the pathogen in one or another part of the world. To broaden our gene pool for stripe rust resistance gene mining is a continuous process in the wheat breeding programs globally and further to achieve durability of resistance pyramiding is the need of the hour to keep pace with the ever-evolving pathogen. The known genes, namely Yr5, Yr10, Yr15, Yr36, Yr40, Yr47 etc. have been extensively exploited in our in house breeding program at Punjab Agricultural University. However, the climate change and the fungal evolution is always anticipated to create newer pathotypes/races of the pathogen. In view of this, the novel genes need to be streamlined and characterized genetically for its ready mobilization whenever required. Since, the germplasm collections from National Bureau of Plant Genetic Resources (NBPGR) constitute the diverse germplasm collected from variable sites, it holds the promise to harbour newer and unidentified resistance sources. So, in the present study efforts were made to evaluate the wheat germplasm against the most prevalent and virulent pathotypes of the stripe rust pathogen At seedling stage as well as at adult plant response against stripe rust under field conditions and further molecular markers were deployed to identify the already known stripe rust resistance genes in the resistant germplasm.

Materials and methods
Plant materials. Total of 45 wheat accessions from NBPGR which showed highly resistant to resistant reaction in the preliminary screening of ~ 1500 wheat germplasm accessions from national gene bank of India, ICAR-National Bureau of Plant Genetic Resources (Table 1) were selected for the present study i.e. to identify the stripe rust resistance gene(s) present in them. This included 22 indigenous accessions collected or derived in India and 23 exotic collections augmented from USA, Mexico and Australia. These lines were evaluated against the three most prevalent races (238S119, 110S119, and 46S119) of stripe rust pathogen both at seedling and adult plant stages. The highly susceptible cultivars PBW343, HD2967, and Agra Local, as well as the Avocet background near-isogenic lines (NILs), i.e., Avocet/Yr5, Avocet/Yr10, Avocet/Yr15, Avocet/Yr24, and Avocet/Yr26, were used as susceptible and resistant checks respectively in the study for comparison purpose.
Seedling reaction test. Wheat seedlings were raised in plastic germination trays (14 × 7 cups) filled with a mixture of soil having sandy loam soil cocopeet + Farm Yard Mannure and vermi compost. All the germplasm www.nature.com/scientificreports/ were sown in three sets for evaluation against stripe rust pathotypes 46S119, 238S119, and 110S119 separately. The leaves of 10-day-old seedlings were inoculated with uredospores of three different pathotypes (46S119, 110S119 & 238S119) separately and kept in separate poly-chambers. For a successful infection, the inoculated material was placed in a dew chamber for 48 h in the dark before being transported to a greenhouse and maintained with a photo period of 16 h light and 8 h of darkness. Regular irrigation was given to maintain the humidity. The host response for seedling infection types was recorded 14 days after inoculation using 0-4 scale by 25 (0 = immune = no uredinia or other macroscopic sign of infection, = nearly immune = no uredinia, but hypersensitive necrotic or chlorotic flecks present, 1 = highly resistant = small uredinia surrounded by necrosis, 2 = moderately resistant = small to medium uredinia surrounded by chlorosis or necrosis, green island may be surrounded by chlorotic or necrotic border, 3 = moderately susceptible = medium-sized uredinia that may be associated with chlorosis, 3+, 4 = susceptible = large uredinia without chlorosis). When the susceptible checks showed the highest infection score of 33+ or 4, the inoculations were considered successful.
Field evaluation. The germplasm was evaluated for three years (2018-2021) at PAU, Ludhiana, and at RRS Gurdaspur by following the standard protocol and relevant guidelines. Wheat germplasm lines were sown at both locations during the 2nd week of November, in a 1-m long pair rows of each genotype at a row-to-row distance of 20-22 cm. To ensure the uniform spread of rust inoculum, susceptible varieties like Agra Local, HD2967, A-9-30-1 and PBW343 were planted after every 20 rows of the test material, and infector rows were sown on the periphery of the experimental material. For comparison purposes and gene postulation, along with genotypes, NILs carrying known genes for stripe rust, susceptible and resistant checks were also sown in the field ( Table 2). The stripe rust epidemic was created under field conditions by repeated spray inoculations of experimental material with uredospores of Puccinia striiformis fsp tritici (Pst). Infected leaves of stripe rust susceptible varieties PBW343, Agra local, A-9-30-1, and HD2967 were immersed in water for extracting uredospores. The inoculum was prepared by suspending rust uredospores in 10 l of water using a few drops of Tween-20. The spray inoculations were done in the evening with an ultralow volume sprayer on alternate days beginning from the end of December till stripe rust appeared on the susceptible checks. Stripe rust was monitored regularly at weekly intervals starting from the second week of January to the first week of March using the modified Cobb's Scale 26 . The host responses were graded: S = susceptible, large uredia surrounded by necrotic tissues; MS = moderately susceptible, small uredia surrounded by necrotic tissues; MR = moderately resistant, small uredia surrounded by necrotic tissues; M = Moderately resistant to moderately susceptible, small to medium sized pustules surrounded by necrosis and chlorosis; R = resistant, very small uredia surrounded by necrotic tissues 27 . The area under disease progress curve (AUDPC), and coefficient of infection (CI) were calculated by using the formula's given below.
where, X i is the rust intensity on date i, t i is the time in days between i and date i + 1 and n is the number of dates on which disease was recorded.

Identification of stripe rust resistance genes by using tagged molecular markers. Under North
Indian conditions against the prevalent pathotype of the stripe rust pathogen five genes namely Yr5, Yr10, Yr15, Yr24, and Yr26 are highly effective so the tagged markers for these genes were used to identify the presence or absence of these genes in the tested germplasm. In total thirteen markers (SSR, EST-SSR, and STS) were used to profile the wheat genotypes. The Graingenes database (http:// wheat. pw. usda. gov/) was employed to retrieve the sequences of available markers (Table 3) along with their previously determined chromosomal positions. Integrated DNA Technologies, Inc., synthesized the primers.
DNA extraction. Genomic DNA was extracted from 100 mg of fresh leaves collected from individual lines using the modified CTAB extraction method [29][30][31] . To remove the contaminant RNA from the DNA extracted from fresh leaves, 10 μl/ml RNase was added and then incubated at 37 ºC for 45 min.
PCR amplification and electrophoretic separation of PCR products. After quality and quantity check the amplification using Polymerase Chain Reaction (PCR) was performed in an Eppendorf Master cycler to study the genetic polymorphism among the genotypes carrying known Yr genes. PCR analysis was carried out in the reaction volume of 11 μl containing the 3 μl template genomic DNA, 1.0 μl forward and reverse primers, 5 μl of 10× PCR buffer and 0.5 μl of BSA and PVP. Specific PCR amplification protocols were followed for each primer linked to different Yr genes. Protocol for Yr5 markers (Wmc175 and Xgwm120) was given by 32,33 , Yr10 (Xpsp3000) 34 , Yr15 (Xgwm413 and Xgwm273) 35,36 , Yr24/26 (Barc181) 37 and Barc187 38 . PCR products were separated through 6% PAGE using PAGE apparatus (Mega-Gel System) of CBS, Scientific, USA (C-DASG-400-50) and visualized under UV light in gel documentation (SYNGENE, G: Box, USA).

Postulation of Yr15. Yr15 gene diagnostics markers have been identified as Xbarc8, Xgwm273, and
Xgwm413. These three markers were used in the current study to detect the presence of Yr15 gene in wheat genotypes under study. The 3.5 cM proximal SSR locus Xgwm413 produced three types of alleles (90 bp, 95 bp, and 100 bp) among the wheat genotypes used in this study. The allelic profile of Xgwm413 showed variation. Fourteen genotypes amplified specific alleles of 90 bp, 18 genotypes amplified alleles of 95 bp, and 16 genotypes did not amplify any allele. (Fig. 4). The Xgwm273 marker is located 2.1 cM from Yr15 and amplified 5 different types of alleles (156 bp, 165 bp, 180, 200 bp, and 220 bp). 14 genotypes (29.2%) produced 156 bp bands specific for Yr15 (Fig. 5). Based on confirmation of Yr15 with both Xgwm413 and Xgwm273 markers, fifteen genotypes (31.3%) (IC534662, IC336645, IC530087, EC693621, EC635701, EC693322, IC445516, IC469420, EC0582305,    (Figs. 6, 7). The distribution of these five Yr resistance genes among 45 wheat accessions is illustrated in Fig. 8 and Table 7. Fourteen lines were found to carry a single gene, 16 lines showed the presence of two gene combinations, and seven accessions were found to have a combination of three genes. These 45 accessions exhibited strong resistance at seedling and adult plant stage.

Discussion
Many sources of wheat yellow rust genetic resistance have proven ineffective after only a short period of deployment 41 . Monoculture of wheat cultivar PBW343 in Punjab, India resulted in the emergence of a new Pst race, 78S84, and a decrease in the predominance of Pst race 46S119. Because the new race 78S84 was virulent on Yr27 thus PBW343 became vulnerable. During 2008-2010, the race 78S84 was found in more than 80% of infected samples collected from North Western Plains Zone of India. However, as PBW343 was replaced by cultivars with R genes other than Yr27, 46S119 became prevalent once more, while 78S84 became the least common one (5%). Three new races, 238S119, 110S119 and 46S117, have also emerged in Punjab and their prevalence's are increasing with every passing year 42 . The knockdown of race-specific single genes is always a concern for plant breeders. The pyramiding of more than one R gene into a single cultivar by MAS is the best way to develop cultivars with durable resistance and slows down the process of pathogen evolution. against current prevalent and new emerging Pst races 43 . In fact, continuous search for novel source of donor genotype is warranted for identification of resistance genes and their deployment in new cultivars or cultivars with defeated genes and unexploited genebank material is the best option for such kind of study. Forty-five genotypes including indigenous (IC-) and exotic collections (EC-) were selected out of ~ 1500 diverse set of genebank germplasm accessions screened for yellow rust disease during 2014-15 and 2015-16. Subsequently, this study was conducted to postulate the Yr genes in selected and pre-screened germplasm accessions for yellow rust resistance. Therefore, this study was conducted to postulate the Yr genes in selected genotypes (45) using Yr gene-tagged markers (13)  www.nature.com/scientificreports/ and to assess the contribution of Yr genes to the current status of Pst resistance. In the current study, we have found three types (single, two, and three genes) combinations among the germplasm under study. Fourteen lines of wheat germplasm were found to carry a single gene, 16 lines showed the presence of two gene combinations, and seven genotypes were found to have a combination of three genes. These 45 genotypes lines exhibited strong resistance at seedling as well as at adult plant stage. Out of 45 germplasm lines, eight germplasm lines showed no amplification with any of the markers used, which means some other already known gene (s) or some new gene (s) may be present which confers a good level of resistance. In the rest of the 38 lines, the amplification was achieved with one/two/more of the tagged Yr gene markers, indicating the presence of single or multiple genes. Rani et al. 44 found two genotypes (VL 3002 and VL 1009) with 15 Yr genes, followed by 14 genes in HI8759 and VL3010. Sobia et al. 43 and Yuan et al. 45     www.nature.com/scientificreports/ both seedling and adult plant growth stages and is located on chromosome 2BL 52 . In our study Yr5 presence was detected in eighteen (40%) lines with two linked markers, i.e., Xwmc175 and Xgwm120. Naruoka et al. 32 found six lines out of 13 carrying lines carrying the resistance-linked allele using the Xwmc175 marker. STS-9/10, developed by Chen et al. 49 , co-segregates with the Yr5 locus and amplified fragments of 439 or 433 bp for resistant or susceptible plants, respectively. Using the S19M93 molecular marker, Ullah et al. 53 discovered an 89% polymorphism rate of the Yr5 gene in 99 Pakistan wheat lines. The Yr5 gene was not amplified in any of the 45 genotypes using STS9/10. In India, to date, the Yr5 gene is effective against all prevalent races and can be an effective source for stripe rust resistance when used singly or in combination with other resistant genes. The Yr10 gene was also found effective against all the Pst races prevalent in India, Iran, China, Pakistan, and the United States 54 . The marker Xpsp3000, located at the end of chromosome 1BS, is 1.2 cM away from the stripe rustresistant gene, Yr10 55 which is co-dominantly inherited and can be used to identify genotypes of individuals at any growth stage 56 and is linked with brown glume color (0.2 cM) in wheat genotypes PI 178,383 and Moro 57 , while T. spleta 415 and T. vavilovii AUS22498 have white glumes 34,58 . Resistant genotypes with brown glumes during the phenotypic evaluation were observed in nine genotypes, which was further confirmed with the tagged marker Xpsp3000 yielded a specific allele of 260 bp. Bariana et al. 34 described two Yr10 alleles, Yr10 and Yrvav, and stated Yr10-containing varieties amplified 258-260bps fragments, whereas Yrvav-containing varieties amplified 285 bp and 240 bp for varieties lacking the Yr10 gene. Similarly, Yr15 is currently the most common all-stage resistance gene used in breeding programs it is effective against all identified races in the United States 59 . Yr15, a dominant gene derived from Triticum dicoccoides, is found on chromosome 1BS 60 . Sun et al. 61 , Peng et al. 62 , and Murphy et al. 35 mapped the Yr15 gene to a 6.4 cM interval flanked by markers Xbarc8, located 3.9 cM to the distal side, Xgwm413 located 2.5 cM to the proximal side, and Xgwm273 located at 2.5 cM and 2.1 cM to the proximal side. On the basis of confirmation with two linked markers, i.e., Xgwm413 and Xgwm273, Yr15 was detected in fourteen lines accounting for (31%) of the total genotypes. Kokhmetova et al. 63 used Xbarc8 and Xgwm413 markers to confirm the presence of Yr15 in seven genotypes (10%) that carried the Yr15 gene. Barc8 did not work well under our conditions. Yr15 and its flanking SSR markers Xbarc8 and Xgwm413 are separated by 3.9 and 2.5 cM, respectively. The two markers that have been most frequently used to incorporate Yr15 into wheat cultivars are Xbarc8 and Xgwm413 35,64 , but in our conditions, Xbarc8 did not work. Yr24, discovered from the K733 accession of T. turgidum var. durum, confers stripe rust resistance at all stages, and the Yr26 stripe rust resistant gene was discovered on chromosome 1B in the T. turgidum durum line 65 . Due to their similar infection types against stripe rust isolates, Yr24 and Yr26 are thought to be identical genes 66 and further demonstrated by Clemence et al. 39 . Combined results with both the markers exhibited the presence of Yr24/Yr26 in 11 genotypes (22.9%), namely IC111691, IC535470, EC217835, EC339610, EC635750, IC529052, IC529094, EC0612506, EC0105966, EC0610955, and ICO598571. In EC0635701 Yr24/26 were detected by Barc181 whereas in EC693322, EC692262, and EC0635659 genotypes they were detected by XBarc187. The Yr5, Yr10, Yr15, Yr24/Yr26 genes, which are specific to races, are still effective against the present dominant races prevalent in north western plains zone of India. To increase their durability, they have to be use in combination with other genes.

Data availability
The datasets generated during the current study will be available from the corresponding author on reasonable request.