Introduction
Stripe rust and powdery mildew caused by Puccinia striiformis and Blumeria graminis are two economically significant fungal foliar diseases of wheat which are a significant constraint for sustainable wheat production, hampering both yield and quality (Elkot et al., Reference Elkot, Chhuneja, Kaur, Saluja, Keller and Singh2015; Bariana et al., Reference Bariana, Forrest, Qureshi, Miah, Hayden and Bansal2016; Bansal et al., Reference Bansal, Kaur, Dhaliwal, Bains, Bariana, Chhuneja and Bansal2017; Lan et al., Reference Lan, Hale, Herrera-Foessel, Basnet, Randhawa, Huerta-Espino, Dubcovsky and Singh2017). In recorded history, diseases and pests have caused substantial wheat yield losses ranging from 50 to 100% under epidemic conditions (Figueroa et al., Reference Figueroa, Hammond-kosack and Peter2018). The long-term use of chemical pesticides has had a significant negative impact on the environment as well as on human health. Thus, a holistic approach of introduction of disease resistance genes is much more lucrative and sustainable. McIntosh et al. (Reference McIntosh, Dubcovsky, Rogers, Morris and Xia2017) have reported characterization and deployment of more than 70 genes for each of these diseases from elite wheat genotypes, landraces and crop wild relatives to counter the problem of biotic stress on wheat production.
In wheat, disease resistance has been characterized into two categories based on their action across the lifecycle of the plant (Chen, Reference Chen2005). Firstly, the all-stage resistance (ASR) caused by seedling stage resistance genes, which start acting from the seedling stage and remain active throughout the plant life. These genes are mostly race-specific. Secondly, the adult plant resistance (APR) caused by genes that are primarily responsible for moderate to complete resistance at the adult plant stage only. APRs may confer hypersensitive reaction or provide more durable resistance to plants by providing slow rusting type resistance (Venkata et al., Reference Venkata, Bullet, Bansal, bullet, Singh, Robert, Park, Harbans and Bariana2008; Niks et al., Reference Niks, Qi and Marcel2015). This type of durable resistance is crucial as it does not exert evolutionary stress on the causal organism by retarding the development of disease progression. Although APR is a durable form of resistance, only a small number of resistance genes have been known to confer this particular type of resistance (Kankwatsa et al., Reference Kankwatsa, Singh, Thomson, Babiker, Bonman, Newcomb and Park2017).
Several Aegilops species have been exploited to transfer genes for resistance to wheat, against various insect pests, powdery mildew and rust diseases that have been commercially used (Kaur et al., Reference Kaur, Jindal, Kaur and Chhuneja2018). Kishii (Reference Kishii2019) has compiled a list of genes identified or transferred from various Aegilops species, including Aegilops speltoides, which have been the sources of leaf rust resistance genes (Lr28, Lr35, Lr36, Lr37, Lr47, Lr51 and Lr66), stem rust resistance genes (Sr32, Sr39 and Sr47), powdery mildew resistance genes (Pm1d, Pm12, Pm32 and Pm53) and green bug resistance gene (Gb5). Apart from disease resistance, Aegilops species have been reported to possess resistance to abiotic stresses like heat, salinity and drought tolerance (Monneveux et al., Reference Monneveux, Zaharieva, Rekika, Royo, Nachit, Di Fonzo and Araus2000; Colmer et al., Reference Colmer, Flowers and Munns2006; Rawat et al., Reference Rawat, Tiwari, Singh, Randhawa, Singh, Chhuneja and Dhaliwal2008; Liu et al., Reference Liu, Wang, Deng, Li, Lu, Wang, Wei and Zheng2015; Awlachew et al., Reference Awlachew, Singh, Kaur, Bains and Chhuneja2016).
During field examination of various crop wild relatives of wheat (at the School of Agricultural Biotechnology, Punjab Agricultural University) for more than 20 years, selected accessions of Ae. speltoides, the putative wheat B genome donor (Zhang et al., Reference Zhang, Zhang, Zhu, Cao, Sun, Ma, Chao, Yan, Xu and Cai2018), were found to be resistant to most of the diseases prevailing in the field. In this study, Triticum durum–Ae. speltoides backcross introgression lines (DS-BILs), developed through limited backcrossing followed by selfing, were evaluated for resistance against the two major wheat diseases, stripe rust and powdery mildew, and quantitative trait locus (QTL)/genes were mapped using single-nucleotide polymorphisms (SNPs) developed through genotyping by sequencing (GBS).
Materials and methods
Plant material
Eighty-nine BC2F10 DS-BILs developed from durum wheat cultivar PDW274 and Ae. speltoides accession #pau3809 were used in the study. Details of the development of the DS-BILs can be found in Awlachew et al. (Reference Awlachew, Singh, Kaur, Bains and Chhuneja2016). Ae. speltoides acc. pau3809 was crossed with T. durum cv. PDW274 as female and F1 was backcrossed to T. durum. All the BC1 plants were backcrossed and BC2F1s selfed to generate homozygous BILs of Ae. speltoides in T. durum background. This set of T. durum–Ae. speltoides backcross introgression lines will be denoted as DS-BILs henceforth in this paper.
Seedling screening
For screening against stripe rust (YR), and powdery mildew (PM), 89 DS-BILs along with recurrent parent PDW274 and susceptible check WL711, a hexaploid wheat cultivar susceptible to both the diseases, were planted in bread boxes with 10 seeds of each genotype (with one row of control check each) and were kept in different temperature- and moisture-controlled glass houses maintained specifically for each disease. The first leaves of 7-day germinated seedlings were inoculated with respective disease spores. For YR, two sets of the same BILs were inoculated, one with P. striiformis (Pst) pathotype 78S84 and other with a mixture of pathotypes collected from open field. For PM, a mixture of B. graminis (Bg) pathotypes collected from open field was used for inoculation. The inoculated bread boxes for each disease were placed separately in water-filled trays covered with a black sheet for 24 h at 100% relative humidity. After the incubation, the bread boxes were maintained in separate glasshouses for disease development. For YR, disease scoring was done using Stakamans' scale (Visioni et al., Reference Visioni, Gyawali, Selvakumar, Gangwar, Shekhawat, Bhardwaj, Al-Abdallat, Kehel and Verma2018) after 14 days when susceptible control showed complete susceptibility for respective pathotypes. Similarly, for scoring disease in PM, a linear scale of 0–9 was used (Yang et al., Reference Yang, Zhang, Zhang, Wang, Luo, Yang, Wang, Xiang, Zeng, Yu, Fu and Rosewarne2017).
Molecular analysis
DNA extraction of 89 DS-BILs along with PDW274 and Ae. speltoides acc. pau3809 was done using the cetyl trimethylammonium bromide (CTAB) method (Saghai-Maroof et al., Reference Saghai-Maroof, Soliman, Jorgensen and Allard1984). DNA was genotyped using GBS. The raw reads generated by GBS were subjected to SNP calling using the TASSELGBSv2 pipeline in TASSELv5.2 (Glaubitz et al., Reference Glaubitz, Casstevens, Lu, Harriman, Elshire, Sun and Buckler2014). The SNPs were called against the A and B genomes of wheat reference genome refseqV1.0. The vcf file generated using the pipeline was filtered for depth at 3 (DP3) and converted to HapMap format. The TASSEL output was then filtered for homozygous SNPs for each parental line, and the polymorphic SNPs between the two parental lines were selected. Furthermore, the SNPs were filtered for 20% missing data, and remaining SNPs were used for mapping. The distribution of SNPs along 14 chromosomes is presented in online Supplementary Fig. S1.
QTL mapping
For mapping, the disease resistance, scoring of the diseases was converted into linear scale as per Yang et al. (Reference Yang, Zhang, Zhang, Wang, Luo, Yang, Wang, Xiang, Zeng, Yu, Fu and Rosewarne2017) and 1095 SNPs were used for mapping using the CSL functionality of QTL IciMapping V4.1.0.0 employing single marker analysis (SMA) and stepwise regression-based likelihood ratio test (RSTEP-LRT) (Wang et al., Reference Wang, Li, Zhang and Meng2016). QTLs detected at LOD (logarithm of odds) score ≥ 2.0 and PVE (phenotypic variation explained) > 9.0 were considered to be significant.
Introgression profile of DS-BILs
Introgression profiling of Ae. speltoides fragments in T. durum background of 89 DS-BILs were done using GGT2 (van Berloo, Reference van Berloo2008).
Postulation of candidate genes
The physical positions of the mapped SNPs in the introgressed segments were used to identify the candidate genes conferring resistance to diseases in the annotated wheat genome present at https://wheat-urgi.versailles.inra.fr/Seq-Repository/Annotations. Jbrowse functionality was used to manually hunt for the candidate genes in the designated regions of the annotated wheat genome. Gene names and functions were identified from https://web.persephonesoft.com/?data=genomes.
Results
Evaluation of rust and powdery mildew resistance
Recipient parent PDW274 was completely susceptible at the seedling stage against stripe rust pathotype Pst78S84 and mixture of pathotypes while donor Ae. speltoides acc. pau3809 showed complete resistance. PDW274 depicted moderate resistance against stripe rust at the adult plant stage under artificial epiphytotic conditions. PDW274 thus might carry an APR gene for stripe rust while Ae. speltoides acc. pau3809 harbours ASR gene(s). Seedling screening of the DS-BILs against Pst78S84 showed wide variation ranging from complete resistance to complete susceptibility (Figs. 1a and 2a) with most of the DS-BILs showing susceptible reaction. Similarly, when DS-BILs were tested at the seedling stage with a mixture of stripe rust pathotypes collected from open field, most genotypes were highly susceptible (YR score of 8) and 12 DS-BILs were moderately susceptible (YR score of 5.33) with only two DS-BILs (DS-BIL6 and DS-BIL16) as completely resistant (YR score of 2.67). However, the DS-BIL panel was completely resistant at the adult plant stage under artificial epiphytotic conditions in the field.
Disease reaction of tested genotypes for (a) stripe rust pathotypes, and (b) powdery mildew pathotypes. The first three leaves represent susceptible check T. aestivum cv WL711, T. durum cv. PDW274 and Ae. speltoides acc pau3809, serially. Succeeding leaves represent reactions of DS-BILs.
Frequency distribution of the disease score of T. durum–Ae. speltoides DS-BILs for (a) stripe rust pathotype Pst 78S84, (b) mixture of pathotypes of stripe rust and (c) mixture of pathotypes of powdery mildew. PDW274 was highly susceptible with reaction score of 8 for all the stripe rust and powdery mildew pathotypes. Ae. speltoides acc. pau3809 was highly resistant with reaction score of 0.
Screening for powdery mildew with a mixture of powdery mildew (Bg) pathotypes collected from open field, identified 40 DS-BILs to be completely to moderately resistant with a score ranging from 0 to 3.3 while rest of the DS-BILs were highly susceptible, whereas recipient parent PDW274 showed complete susceptibility while Ae. speltoides showed complete resistance with a score of 0 (Figs. 1b and 2c).
QTL mapping
The results of QTL mapping using RSTEP-LRT for the additive effect of markers and single-marker analysis (SMA) are presented in Table 1. For stripe rust against field mixture of pathotypes, two QTLs (QYrAs.pau-2A.1 and QYrAs.pau-7B) were mapped on chromosomes 2A and 7B with SMA (LOD score 3.8 with PVE 24.51% and LOD score 2.4 with PVE 10.06%) and RSTEP_LRT (LOD score 2.6 with PVE 13.83% and LOD score 2.4 with PVE 10.06%) with resistance allele contributed by Ae. speltoides. Also, for the same field mixture of pathotypes, two QTLs were mapped with only SMA, QTL QYrAs.pau-1B on chromosome 1B with LOD score 2.0 with PVE 11.64% and QTL QYrAs.pau-2B on chromosome 2B with LOD score of 2.2 and PVE 12.8%. These QTLs also had resistance allele contributed by Ae. speltoides. For stripe rust, two QTLs, QYrTd.pau-2A.2 and QYrTd.pau-3B were mapped against Pst 78S84 on chromosomes 2A and 3B with both algorithms SMA (LOD score 2.9 with PVE 14% and LOD score 4.1 with PVE 19.29%) and RSTEP-LRT (LOD score 2.1 with PVE 9.4% and LOD score 4.1 with PVE 19.29%). PDW274 contributed resistance alleles for both the QTLs. However, the mapping could not be conducted for adult plant data as the whole of the population was resistant.
Summary of the QTL mapping using single marker analysis (SMA) and RSTEP-LRT for additive effect of markers algorithms of QTL ICI mapping
Similarly, for powdery mildew against field pathotypes at the seedling stage, five QTLs located on chromosomes 2A, 2B, 3B and 6B were detected using both algorithms. With SMA, QTLs QPmAs.pau-2A.2, QPmAs.pau-2B and QPmAs.pau-6B at a LOD score of 2.02 with PVE 11.49%, LOD score of 2.3 with PVE 14.05% and LOD score of 3.2 with PVE 17.75% were detected, respectively. With RSTEP-LRT, QTLs QPmAs.pau-2A.1, QPmAs.pau-3B and QPmAs.pau-6B were detected, respectively at an LOD score of 2.0 with PVE 9.16%, LOD score of 2.4 with PVE 12.89% and LOD score of 3.2 with PVE 17.75%. Ae. speltoides contributed all the resistance alleles in these QTLs. Fig. 3 summarizes the mapped QTLs along with the linked markers on the physical map of the DS-BILs.
Summary of the mapped QTLs for stripe rust and powdery mildew in the T. durum–Ae. speltoides introgression lines. Mapped QTLs are represented as bars alongside the carrier chromosomes. The black lines represent the SNP positions along the length of the chromosomes. The coloured lines on chromosomes and bars alongside represent the positions of QTLs. QTL names, and SNPs linked to the QTLs are presented alongside the coloured bars.
Since both the parents contributed to resistance for these diseases, QTLs mapped on segments donated by respective parents were studied. Studying the introgression in the resistant lines and the resistance donor fragments, 11 lines carrying multiple QTLs, mapped during this study, were identified. These lines are summarized in Table 2. Out of these six lines, namely DS-BIL6, DS-BIL8, DS-BIL16, DS-BIL18, DS-BIL20 and DS-BIL53 had three or four loci, for YR and PM, whereas other lines had two loci each. DS-BIL6 and DS-BIL16 had all the four loci (QYrAs.pau-1B, QYrAs.pau-2A.1, QYrAs.pau-2B and QYrAs.pau-7B) mapped for YR using field mixture of pathotypes and were completely resistant to stripe rust. However, lines with two or three loci showed moderate susceptibility. For PM, QPmAs.pau-2A.1, QPmAs.pau-2B and QPmAs.pau-3B provided complete resistance individually. The other two PM QTLs conferred moderate resistance only in combination.
Summary of the resistant DS-BILs along with QTLs present in these DS-BILs
Introgression profile of DS-BILs
Introgression profile of DS-BILs was studied to visually identify the regions of introgression harbouring QTLs which would finally help in the selection of BILs as a pre-breeding material for rust resistance. The introgression profile is given in online Supplementary Fig. S2. Since Ae. speltoides is known to carry genes epistatic to Ph1 locus of wheat (Millet, Reference Millet2007; Colas et al., Reference Colas, Shaw, Prieto, Wanous, Spielmeyer, Mago and Moore2008; King et al., Reference King, Grewal, Yang, Hubbart Edwards, Scholefield, Ashling, Harper, Allen, Edwards, Burridge and King2018), called Ph suppressors, which lead to homoeologous recombination of the alien genome with wheat chromosomes and are responsible for introgression of Ae. speltoides segments to both A and B genomes of T. durum, as seen in online Supplementary Fig. S2.
Postulation of candidate genes
The regions of 50 kb on both sides of the linked markers with respective QTLs were scanned to identify the candidate genes. The identified genes for each of the QTL mapped in this study are listed in Table 3. All the genes identified were high confidence genes as per annotation v1.1. For each target locus, genes known to be involved in different pathways of pathogen–host interactions and pathogenesis were identified and will be validated by developing mapping populations from the selected DS-BILs. No gene was detected in 50 kb region harbouring QTL QPmAs.pau-3B, hence a region of 500 kb was scanned on both sides of the linked SNP and candidate genes around the target loci were identified (Table 3).
Postulation of genes present in the survey sequence of wheat genome refseqV1.0
Distance from SNP represents the distance of start site of the gene to SNP linked with QTL, where + sign represents the gene was found downstream of the SNP and − sign gene was found upstream, 0 represents the SNP was present inside the gene, and all distances are in kilobases.
Discussion
Stripe rust and powdery mildew are major constraints to wheat production worldwide. Wheat breeding programmes, to counter these hindrances, need continuous identification and introgression of new disease resistance genes from diverse sources including crop wild relatives. The work in hand describes the transfer and mapping of new genes/QTL for stripe rust and powdery mildew resistance from Ae. speltoides. During this study on a set of T. durum–Ae. speltoides introgression lines, two major genic loci effective against stripe rust pathotype Pst 78S84 were identified. Five genic loci were identified against field mixture of Bg pathotypes. Four loci were identified against field mixture of Pst pathotypes collected from the field. These QTLs were present on the terminal ends of the respective chromosomes (Fig. 3) which have been reported to have recombination hot spots of the chromosomes. The phenotypic data of the DS-BILs showed that a large number of loci might be responsible for resistance reaction as the variability of different reaction types shows the involvement of additive effects of multiple loci. Despite this, 11 loci were identified to provide resistance when mapping was done using RSTEP-LRT for the additive effect of markers and SMA. Since there were few sites on different chromosomes where there was low coverage/density of SNPs, and this hindered identifying QTLs (if present) in these locations. All the QTLs detected in the current study had good marker density (one SNP per 2.03 Mb against genomic average of one SNP per 8.60 Mb) in the genomic regions harbouring target QTL. To explain the residual resistance, which was not mapped in this study, a higher density of SNPs providing more coverage of the genome and a bigger set of the DS-BILs can be used. All the selected DS-BILs with different QTL combinations can be used in breeding programmes for marker-assisted transfer of the respective QTLs to the hexaploid backgrounds providing a good source of resistance.
Various designated genes, temporarily designated genes and QTLs for YR and PM reported from various studies have been compiled in online Supplementary Fig. S3 (source: www.wheat.pw.usda.gov). QTL QYrAs.pau-1B reported herein has been found in the vicinity of designated genes Yr29/Lr46 and QTL QYr.cim-1BL (Lan et al., Reference Lan, Rosewarne, Singh, Herrera-Foessel, Huerta-Espino, Basnet, Zhang and Yang2014). Similarly, QTL QYrAs.pau-2A.1 was found in the vicinity of Yr56 where some QTLs for YR have also been reported like QYr.sun-2A_Wollaroi, QYr.tam-2AS_TAM111, QYr.ucw-2A.2(IWA422), QYr.ufs-2A_Cappelle-Desprez_Yr16 and QYr.inra_2AS.1_Recital (Maccaferri et al., Reference Maccaferri, Zhang, Bulli, Abate, Chao, Cantu, Bossolini, Chen, Pumphrey and Dubcovsky2015). QTL QYrAs.pau-7B has been physically located in the vicinity of stripe rust resistance genes Yr52 and Yr59 (McIntosh et al., Reference McIntosh, Dubcovsky, Rogers, Morris and Xia2017). Interestingly, QTL QYrTd.pau-3B mapped from tetraploid donor PDW274 in the current study was found in the same genomic region as QTL QYrEDWL.par-3BL which was mapped in the tetraploid background of Ethiopian spring wheat by Liu et al. (Reference Liu, Maccaferri, Rynearson, Letta, Zegeye, Tuberosa, Chen and Pumphrey2017). For PM, QTL QPmAs.pau-6B is located in the region harbouring QTL QPm#66-2B (Ben-David et al., Reference Ben-David, Peleg, Dinoor, Saranga, Korol and Fahima2014). Other QTLs reported in this study could also be traced to the same arm, or close vicinity of genes/QTLs reported in the literature, as is depicted in online Supplementary Fig. S3. It was not possible to compare exact locations of the other QTLs mapped in this study with reported genes or QTL as most of the reported genes or QTLs have been mapped on the based-on linkage; whereas in the current study, genes/QTLs have been physically mapped to specific chromosome regions.
Studying the annotated reference of wheat genome refseqV1.0 showed the genes present in the genomic regions of mapped QTLs. Various categories of these genes are NBS-LRR protein family responsible for disease resistance, receptor or receptor-like proteins/motifs like Zinc finger, ubiquitin pathway proteins like ubiquitin regulator units and glutathione S transferase (GST), antioxidant pathway enzymes like L-gulonolactone oxidase, various kinases, auxin response factors (ARFs) and mitochondrial transcription termination factor-like (mTERF) proteins (Table 3). All these are known for their action in pathogen recognition, reactions, being involved in various biotic and abiotic stresses or plant-pathogenesis pathways. A complete list of genes is given in online Supplementary Table S1. The role of other genes/proteins in plant pathogenesis is either yet not reported or not well documented.
QYrAs.pau-1B and QPmAs.pau-2A.1 loci harboured genes with NBS-LRR and zinc finger motifs. NBS-LRR genes are from the most abundant disease resistance gene family in plant genomes, and zinc finger motifs have been reported to be major motifs linked with the response of plants to various biotic and abiotic stresses (DeYoung and Innes, Reference DeYoung and Innes2006; McHale et al., Reference McHale, Tan, Koehl and Michelmore2006; Lee and Yeom, Reference Lee and Yeom2015; Dubey and Singh, Reference Dubey, Singh, Singh and Singh2018). Zinc finger motif was also found in the region of QTL QYrAs.pau-2B and QYrAs.pau-7B. PM QTL QPmAs.pau-3B was found to be located in the genomic region having the gene coding for protein-enhanced disease resistance 2 which induces resistance by negative regulation of salicylic acid in biotrophic pathogens like PM (Tang et al., Reference Tang, Ade, Frye and Innes2005). Zhang et al. (Reference Zhang, Mao, Wang, Zhang, Wang, Lv, Liu, Wang and Ji2019) suggested that in wheat, pathogen resistance genes can be activated by alternate splicing regulators in salicylic pathways, down-regulating its synthesis. L-gulonolactone oxidase (in the region of QTLs QYrAs.pau-2A.1) is a key enzyme in the formation of ascorbate. Thus, the regulation of this enzyme is essential in the regulation of ascorbate formation in plants (Gullner and Kômíves, Reference Gullner and Kômíves2007). Ascorbate is one of the major antioxidants of plants (Potters et al., Reference Potters, Horemans and Jansen2010; Paciolla et al., Reference Paciolla, Paradiso, De Pinto, Gupta, Palma and Corpas2016) and second, being glutathione, both act against reactive oxygen species (ROS) produced under biotic stresses (Kuźniak, Reference Kuźniak, Anjum, Umar and Chan2010). Being part of the ascorbate–glutathione cycle, it takes part in signal transduction in biotic stress besides regulating the expression of nuclear genes as a response to invading pathogen providing both local and systematic defence (Sarowar et al., Reference Sarowar, Eui, Young, Sung, Ki, Byung and Jeong2005; Kuźniak, Reference Kuźniak, Anjum, Umar and Chan2010).
QTL QPmAs.pau-2A.1 region also annotated gene for regulatory subunit of 26S proteasome subunit. Proteasome, which is a part of the ubiquitin-proteasome system (UPS), functions by removal of misfolded and defective proteins along with eliminating short-lived proteins (Vierstra, Reference Vierstra2009). Along with this, various pathways are controlled by UPS which include response to biotic and abiotic stresses (Sadanandom et al., Reference Sadanandom, Bailey, Ewan, Lee and Nelis2012), and acts as one of the major systems in plant immunity (Üstün et al., Reference Üstün, Sheikh, Gimenez-Ibanez, Jones, Ntoukakis and Börnke2016). Besides immunity, their role in defence responses by the production of ROS and forming hypersensitive reactions were reported (Marino et al., Reference Marino, Peeters and Rivas2012). Üstün et al. (Reference Üstün, Sheikh, Gimenez-Ibanez, Jones, Ntoukakis and Börnke2016) showed that proteasome mutants impaired/reduced systematic acquired resistance (SAR) on secondary infection and concluded that proteasome is essential for the pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and SAR. In a study involving Arabidopsis with loss of function mutants, Yao et al. (Reference Yao, Wu, Nie and Tang2012) reported that 26S regulatory subunit of proteasome, RPN1a, is essential for resistance. It induced cell death when Arabidopsis was infected by powdery mildew, concluding its effect on basal defence and resistance protein-mediated defence. Dielen et al. (Reference Dielen, Badaoui, Candresse and German-Retana2010) in a review on UPS (26S) highlighted the involvement of the system in defence mechanisms regardless of pathogen type.
ARF (in the region of QTL QYrTd.pau-2A.2) in various studies has been explained as a mediator of auxin to biotic and abiotic stresses (Ghanashyam and Jain, Reference Ghanashyam and Jain2009; Fu and Wang, Reference Fu and Wang2011; Bouzroud et al., Reference Bouzroud, Gouiaa, Hu, Bernadac, Mila, Bendaou, Smouni, Bouzayen and Zouine2018). Bouzroud et al. (Reference Bouzroud, Gouiaa, Hu, Bernadac, Mila, Bendaou, Smouni, Bouzayen and Zouine2018) reported that ARFs have a vital role in alteration (activation or repression) of the rate of transcription of auxin-responsive genes. Both biotic and abiotic stress-responsive genes are enriched in cis-elements of 5′-regulatory units in ARFs. They showed that under stress conditions, ARFs are actively regulated at the post-transcriptional level. Besides this, Fu and Wang (Reference Fu and Wang2011) reported that pathogen produced indole acetic acid (IAA) by the action of ARFs can cause either resistance to a necrotrophic pathogen (through ethylene signalling or camalexin biosynthesis), susceptibility by cell wall expansion or stomatal opening (through host IAA biosynthesis or IAA conjugation), basal resistance by IAA conjugation (differential regulation) or resistance to biotrophic pathogen (through indole glucosinolate biosynthesis and/or salicylic acid signalling).
High inducibility of GST in biotic stress in response to bacterial, fungal or viral infection by up-regulation of key defence enzymes has been reported in various studies (Gullner and Kômíves, Reference Gullner and Kômíves2007; Taylor et al., Reference Taylor, Street, Wt, Signaling, Dubreuil-maurizi, Poinssot, Dubreuil-maurizi and Poinssot2012; Gullner et al., Reference Gullner, Komives, Király and Schröder2018). In our study, QTLs QPmAs.pau-2A.2, QYrTd.pau-3B and QPmAs.pau-3B were found to be linked to GST gene/s. These authors have reported that besides its role in detoxification of various toxic substances and as antioxidative in reaction in infected cells, it also regulates the expression of various protective genes. Changes in expression of GSTs are reported to be modifying symptoms of a disease and sometimes the rate of multiplication of pathogens. Some GSTs with peroxidase activity are also known to detoxify lipid hydroperoxidases.
QTL QPmAs.pau-6B region was found to carry three genes, one of receptor kinase 1 and two of mTERF gene. Receptor kinases are known to be modulating plant defence responses. Receptor-like kinases (RLKs) and receptor-like proteins (RLPs) act as pattern recognition receptors (PRRs) (Tang et al., Reference Tang, Wang and Zhou2017) and thus lead to first defence response. Multi-protein immune complexes of PRRs and other RLKs are formed at the surface of interaction. The two broad classes of receptors are, one in the cytoplasm with NB-LRR and the other on the cell surface with RLKs and RLPs (Jones and Dangl, Reference Jones and Dangl2006; Jones et al., Reference Jones, Vance and Dangl2016). In wheat, TaRLK-R1,2,3 (Zhou et al., Reference Zhou, Li, Deng, Wang, Chen, Zhang, Chen, Ling, Zhang, Wang and Zhang2007) and LRK10 (Feuillet et al., Reference Feuillet, Schachermayr and Keller1997) have been involved in plant immunity where TaRLK-R1 has also been cloned (Qin et al., Reference Qin, Chen, Cao, Wang, Xing, Ling, Wang, Yu, Xiao, Ji, Chen, Chen, Liu and Wang2012). Wang and Bouwmeester (Reference Wang and Bouwmeester2017) suggested that PRRs recognize not only the invading organism's surface effectors but also damage-associated molecular patterns. PTI acts as a primary defence, and ETI acts as a secondary defence by recognition of by-products of effector specific resistance genes (Shi et al., Reference Shi, Zhang, Friesen, Raats, Fahima, Brueggeman, Lu, Trick, Liu, Chao, Frenkel, Xu, Rasmussen and Faris2016). Thus, they both result in biotrophic pathogens' growth reduction. While mTERFs are best known to act against abiotic stresses and since only eight plant mTERFs are known to be characterized, very little is known about their action against biotic stresses (Babiychuk et al., Reference Babiychuk, Vandepoele, Wissing, Garcia-Diaz, De Rycke, Akbari, Joubès, Beeckman, Jänsch, Frentzen, Van Montagu and Kushnir2011; Vardhan and Kousar, Reference Vardhan, Kousar, Barh, Khan and Davies2015; Chen et al., Reference Chen, Feng, Qi, Xu, Yao, Wang and Song2017; Pan et al., Reference Pan, Ren, Zhao, Liu, Tan and Qiu2019). However, mTERFs are known to show changed nuclear gene expression, which could support their role in various stresses.
The inference from this outcome requires studying the functions, activation, deactivation or alteration in the rate of expression of these loci in the process of development of resistance to specific diseases. However, regions having Ae. speltoides specific introgression may carry novel genes. In either case, there is a need to study the regions countering resistance at the transcriptional level to evaluate the actual cause of resistance in DS-BILs which would further help in the identification of unique pathways of development of disease resistance genotypes.
While several lines did contain large segment substitutions from Ae. speltoides, it is difficult to detect the QTLs that are close to each other with opposite effects. Hence, transferring them to different backgrounds can identify some additional genes or QTL. To conclude, despite the selected 11 DS-BILs being a good source of resistance to YR and PM, only the functional study of the regions could elaborate on the effect of these loci/QTLs in providing disease resistance.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262120000222.
Acknowledgements
The financial support provided by the Department of Biotechnology, Ministry of Science and Technology, Government of India in the form of DBT Programme Support (grant no. BT/PR/5468/AGR/02/851/2012 dated 15.5.2012) is gratefully acknowledged. The supply of the rust inoculum by the Indian Institute of Wheat and Barley Research Station, Flowerdale, Shimla, India is also gratefully acknowledged.
Author contribution
GSD conducted disease screening, QTL mapping and manuscript writing; SK, JK and RS helped in the development of material and disease screening; ND supervised the study, helped in writing the manuscript; JP conducted genotyping by sequencing; PC designed the study, provided the basic genetic material, supervised the study and finalised the manuscript. All the authors have read the manuscript and approved it.
Conflict of interest
The authors declare that they have no conflict of interest.
