During the early 1990s, a new era of quantitative disease resistance had been evolved with the identification of QTLs for disease resistance using DNA-based molecular markers. The era began with the publication of Lander and Botstein's iconic papers on interval mapping. Interval mapping had been used to investigate the genetic regulation of various characters in plant systems (including wheat), and resistance to spot blotch is no exception. MQTL analysis combines the findings of various QTL studies, allowing for the clarification of QTL locations and the discovery of closely related MM for the traits of interest. Several QTLs for resistance to spot blotch have been identified on various chromosomes (Sharma et al. 2007a; Neupane et al. 2007; Kumar et al. 2009, 2010, 2015; Gonzalez-Hernandez et al. 2009; Adhikari et al. 2012; Zhuang et al. 2013; Lillemo et al. 2013; Lu et al. 2016 Gurung et al. 2014). Nevertheless, four important QTLs were identified have been well described: (Lillemo et al. 2013) Sb1 on 7D, (Kumar et al.2015) Sb2 on 5B, (Lu et al.2016) Sb3 on 3B, and (Zhang et al. 2020) Sb4 on 4BL. Previously, the genetic mechanism of resistance to spot blotch was identified as eight important QTLs, namely QSb.bhu-2A, QSb.bhu-2B, QSb.bhu-2D, QSb.bhu-3B, QSb.bhu-5B, QSb.bhu-6D, QSb.bhu-7B, and QSb.bhu-7D (Kumar et al. 2009, 2010). Sharma et al. (2007a) discovered three SSR markers (Xgwm570, Xgwm67 and Xgwm469) associated with resistance to spot blotch. Further research has designated the QTLs QSb.bhu-3B QSb.bhu-5B and QSb.bhu-7D, as genes Sb3, Sb1 and Sb2, and (Lu et al. 2016; Lillemo et al. 2013; Kumar et al. 2015) However, in the current study, we only found two major resistance genes within the region of MQTLs for spot blotch, Sb1 and Sb2, with QTLs shown QSb.bhu-5B (Kumar et al. 2009), QSb.pau-5B (Kaur et al. 2021), QSb.bhu-7D (Kumar et al. 2010b) and QTs-7D (Wang et al. 2014).
Twenty-nine different studies were found to be resistance related to spot blotch QTLs directly or indirectly. The identified QTLs were associated with SB resistance (Zhu et al.2014, Singh et al. 2018; Shi et al. 2021; Roy et al. 2021; Lv et al. 2020; Liu et al. 2016; Lillemo et al. 2013; Li et al. 2021; Lehmensiek et al. 2004; Kumar U et al. 2009,2010, Kumar S et al. 2015,2016, Kumar P et al. 2022,2021, Kaur et al. 2020; Ghatyari et al. 2021) and for SG (Kumar et al. 2010; Christopher et al. 2018), BP resistance (Liu et al. 2016; Li et al. 2021), GLAD (Shi et al. 2019), GLMNS (Shi et al. 2019), WFLS (Wang et al. 2014,Verma et al. 2003), PH (Singh et al. 2018a,b; He et al. 2020), green colour (G), days to heading (DH) (Singh et al. 2018b, He et al. 2020), MRS (Vijyalakshmi et al. 2010), TMRS (Vijyalakshmi et al. 2010), chlorophyll content (SPAD) (Vijyalakshmi et al. 2010), and chlorophyll florescence (FV/FM) (Vijyalakshmi et al. 2010) content.
In this study, 22 MQTLs for spot blotch resistance were identified, having 71 QTLs. For this purpose, after through bibliographic search of 29 QTLs analysis studies, 275 QTLs were selected. However, all the 275 QTLs were utilized in the BioMercator v4.2.3 software. The QTLs have already been found using interval mapping with several mapping populations (mainly RILs and DH). In previous studies, MQTLs were found for tan spot resistance 20 MQTLs (using 106 QTLs), 35 MQTLs (using 128 QTLs) for leaf rust resistance, 61 MQTLs (using 184 QTLs) for stripe rust resistance and 48 MQTLs (using 208 QTLs) for MDR in wheat (Saini et al. 2022).
In this current study, 73788 markers were employed for the development of the CM. B sub-genome has the maximum number of markers (30327), while D sub-genome has the lowest (13727) while A genome contain 28674. markers A sub-genome also included the highest number of QTLs. These QTLs were linked to 14 distinct characteristics. Wheat flag leaf senescence and spot blotch resistance, stay green trait, were leading the ranking. The range of LOD value in all the QTLs were < 3 to > 15. In which, the LOD value of maximum QTLs (84) were had 3 to 5 followed by < 3 in 52 QTLs and 51 QTLs had LOD value 7 to 9 out of all QTLs. Similarly, maximum QTLs (55) were had 5 to 10% PVE followed by < 5% PVE in 51 QTLs and 10 to 15 PVE in 39 QTLs. Some studies related to MQTL analysis in wheat are: FHB (Liu et al.2009), SR resistance (Yu et al.2014), TS resistance (Liu et al. 2020), LR resistance (Soriano and Royo 2015), SR resistance (Jan et al. 2021) and MDR (Saini et al. 2022).
The proportion of PVE percent by individual QTLs varied from 1 to 88 percent (average = 6%) (Jan et al. 2021). In some other studied, the PVE% varied from 2.2–51.2% with a mean of 15.2%; LOD score varied from 2.9–48.4 with an average of 7.42 (Saini et al. 2022). The LOD score in the study of MQTL for tan spot resistance was in the range of 3.9 to 17.0 and the range of PVE was 6.0 to 27.4% (Liu et al. 2020). In the study of M-QTLs for leaf rust resistance, the LOD score was observed between 1.0 to 77.7 and the range of PVE% was 0.01–0.97 in the selected QTLs (Soriano and Royo 2015)
In this present study, 22 M-QTLs were identified using 275 QTLs and this resulted in a thirteen-fold depletion in the number of QTLs that control spot blotch resistance. From the sub-genome A, only seven MQTL identified. Maximum M-QTLs were found in the sub-genome B and minimum MQTLs were carried by the sub-genome D. In the previous study conducted on the identification of MQTLs for the resistance to FHB disease, researchers discovered a five-fold decline, for tan spot resistance MQTLs, four-fold decline in QTLs (Liu et al. 2020) were observed. The non-availability of M-QTLs on the chromosomes 1D, 3D, 4A, 5D, 6B, and 6D is consistent with previous research reports (Liu et al. 2020). The absence of M-QTLs on the few chromosomes was not exceptional, since there were no MQTLs for resistance to leaf rust on five chromosomes (1D, 3D, 5A, 5D, and 6D) (Soriano and Royo 2015), none for tan spot resistance on eleven chromosomes (1D, 3D, 4B, 4D, 5D, 6B, 6D, 7B, and 7D) (Liu et al. 2020), and none for FHB disease on six chromosomes (Liu et al.2009). From all the above studies including our, not even single MQTL found on the chromosome 5D and 6D.
The maximum QTLs (130) were reported within the A genome, followed by B (95). The limited QTLs reported on the D sub-genome due to the low degree of polymorphism correlated with them. Only 50 (18.18%) of the 275 QTLs used in this study of MQTL analysis belonged to the D sub-genome, suggesting that there were less QTLs in the D sub-genome overall. In previous studies of stripe rust resistance and tan spot resistance, no MQTL were found on chromosome 7D, 4B, 4D and 6A. Although, we found more than 5 MQTLs on these chromosomes.
Sb1 gene is a major resistance gene of spot blotch present on the chromosome 7D at (Lillemo et al. 2013) within the region of flanking markers wpt-7654-gdm88. These markers were discovered in the current study between the 39.8-47.8cM of MQTL22. Lr34 and Lr46 are the APR genes associated with Sb1 gene and provide leaf rust resistance to pathogen. Both the genes are also provided resistance to SB pathogen. Whereas Lr34 gene is explained up to 55% PVE for resistance to SB disease (Lillemo et al. 2013).
Sb2 is a R gene of spot blotch and present within the flanking regions of the markers (Xgwm639-Xgwm1043) on chromosome 5B (Kumar et al. 2015). This gene was also observed within the genomic region of MQTL14 at the locus 32.40 cM. Additionally, the spot blotch sensitivity gene Tsn1 was also observed nearby the Sb2 gene at a locus (35.30 cM) in the MQTL14. The Sb2 gene on the chromosome 5B known to engage with the Tsn1 gene and confer a susceptible reaction to Septoria nodorum blotch and TS and also SB have been thoroughly studied (Lu et al. 2016; McDonald et al. 2018; Navathe et al. 2020). Both P. tritici-repentis, P. nodorum and B. sorokiniana which, respectively, confer susceptible reactions to tan spot, S. nodorum blotch and spot blotch carries the gene ToxA virulent to Tsn1 (Faris et al. 2010; McDonalds et al. 2018; Navathe et al. 2020; Singh et al. 2021).
Majority MQTLs discovered in this investigation are controlled the multiple traits. This showed either a close relationship between genes for diverse phenotypes or the presence of genes have several functions. This could possibly be owing due to a tendency caused by the utilization of associated characteristics for identifying spot blotch resistance, as previously documented in QTL analysis in other publications (Lillemo et al. 2013; Kumar et al. 2015; Lu et al. 2016).
Most of the QTLs are implicated in MQTL2 on 1B and MQTL13 on 5A, which display distinct features on the same locus. SG, WFLS, GLAD, GL MNS, and SB resistance are all features. All of these characteristics are linked together. Previous research has established the linkage between the SG trait, GLAD, WFLS and SB resistance. WFLS and GLMNS are connected directly to remain green trait and indirectly to SB resistance.
The SG trait in wheat is caused by the alternative allele if one allele is connected to senescence. SG has reportedly been linked to SB disease resistance (Joshi et al. 2007a; Rosyara et al. 2008). The significant region in this chromosome resistance to SB disease was also demonstrated by several independent researches (Joshi et al. 2007a; Rosyara et al. 2008; Bainsla et al. 2020).
An important criterion may be the association between MQTLs and the pathotypes of particular pathogen found in a specific wheat-growing zones. We must bear in mind that virulence can also take a quantitative form while doing this (Jan et al. 2021). A crucial requirement for achieving broad spectrum, race non-specific resistance may also be provided by MQTLs that exhibit resistance to multiple pathotypes. In a recent study of M-QTL analysis for resistance to TS in wheat, significant MQTLs demonstrating resistance against multiple pathogen races.
Spot blotch resistance is directly associated with SG trait and indirectly associated with WFLS, GLAD and GLMNS (Joshi et al. 2007). QTLs for the SB resistance and SG trait are co-linked in the region of MQTLs that shows the strong linkage between them. These QTLs (QGlnms15-1B-3, QGlnms20-1B-3, QGlnms-20-1B-4, QGlad31-1B-6, Q50%G-1B-2, QGlad28-1B-4R, Q75%G-1B-1, QGlnms25-1B-2 and QGlad31-1B-5) MQTL2 (QMrs- 2A-1,QGlnms10-2A-5,QMrs- 2A-2, QGlnms30-2A-1, QTo-2A, QGlad28-2A-2, QGlnms10-2A-4 and QGlnms15-2A-4) are related to stay green trait and present on the MQTL3.
The CGs of prospective MQTLs within the necessary physical intervals were found using the Ensembl Plants BioMart tool (https://plants.ensembl.org/biomart/martview). The MQTL peak's precise location was first calculated, and then the whole 2 Mb region around it 1 Mb to either of its left and right was examined to find out any potential CGs. For the other situations, when the physical interval was > 2 Mb, M-QTLs with a physical interval of less than 2 Mb were considered indirectly for the identification of accessible CGs. The procedures needed to determine the physical intervals of MQTL peaks can be found in other sources (Jan et al. 2021). The function descriptions of the discovered gene models were extracted using the InterPro database (https://www.ebi.ac.uk/interpro/).
The present investigation 2509 CGs were discovered during the current study in which 501 CGs were unique. These candidate genes encode multiple proteins, at least some of which are recognized to be related to disease resistance. Among 501 proteins, the role of 412 protein have already been established in resistance to several biotic stresses. In the study of Jan et al. 2021. total 1581 CGs were identified in MQTL regions. In the study of the identification of multiple diseases resistance, 874 Candidate genes were observed in the genomic regions of 39 MQTLs (Saini et al. 2022).
In present study 72 DEGs are chosen 501 CGs based on the various expressive proteins they possess on the basis of our own transcriptomic data of the same disease. Out of these 72 DEGs, 26 are up-regulated, 10 are down-regulated, and the remaining genes are neutral. Additionally, these DEGs coded for various proteins falling under the following groups: R-domain proteins, transcription factors with the NAC domain, proteins with the zinc finger binding domain AP2/ERF, SANT/Myb domain (iii) a variety of protein kinases, (iv) transporters such as the sugar/inositol transporter and SWEET sugar transporter, (v) genes involved in antioxidative defence, such as glutathione S-transferase, (vi) genes involved in oxidation-reduction reactions such as cytochrome P450 (vii) invertas. (viii) cupin superfamily proteins, such as the germin-like protein. The DEGs that have roles previously identified as essential for disease resistance were the most promising CGs for certain M-QTLs.
Maximum number of candidate genes were shown on chromosome 1B (MQTL2) and 2A (MQTL3, MQTL4), because the region of the QTLs between these flanking markers is large. In stripe rust resistance maximum number of candidate genes were found on MQTL25, MQTL48, MQTL55 on chromosome 3A, 5B and 6B. In tan spot resistance MQTL-2B, MQTL3B.1, MQTL-3B.2, and MQTL5B.1 and MQTL-5B.3 were had the maximum number of candidate genes.
In current study, observation for the DECGs was consistent with the result of some other studies (Aduragbemi and Soriano 2021; Jan et al. 2021). Additionally, the DEGs encoded a variety of proteins, at least some of which have been shown to confer resistance to various diseases in crop plants. These proteins include sugar transporters (Moore et al. 2015), PK (Meng and Zhang, 2013), transcription factors, R-domain containing proteins, genes for oxidation-reduction reactions, and antioxidative defence (Gunupuru et al. 2018). This study shows that various different gene families may be involved in the management of multiple diseases, and that the R genes are not only the gene family participating in disease resistance (Gullner et al. 2018).
In terms of important biological, molecular, and cellular processes like phosphorylation, proteolysis protein ubiquitination, oxidation–reduction processes, transmembrane transport etc., the differential expression of 501 CGs primarily affects these processes. Similar to this, significant GO terms in the category of molecular functions included those related to ATP and ADP binding, catalytic activity, protein and nucleotide binding, iron ion and DNA binding, heme-binding, calcium and metal I on binding, transmembrane transporter activity, polysaccharide binding oxidoreductase activity, protein kinase activity, hydrolase activity, signal transduction, etc. Online tool WEGO (wego.genomics.org.cn/, accessed 10 July 2021) was used to represent all the CGs in a graphical form with visualizing gene ontology annotation results. Total of 456 genes were visualized in the figure. In molecular functions, the highest number of genes involved in binding activity was 88 percent. In the catalytic activity 45 genes were involved. Genes may also play an important role in cellular components and 38 percent genes were involved in this category. Besides it, 34 percent genes were engaged with metabolic function.
CGs underlying MQTLs in wheat have previously been discovered for numerous traits such as resistance to TS, FHB resistance and drought tolerance. For example, in maximum previous studies, the entire physical interval flanking the M-QTL region was considered for identification of CGs. However, in this study, we used a 2 Mb region flanking the exact physical position.
Among 501 CGs, 72 CGs showed DE and encoded important Resistance proteins including S/TPK, Serine-threonine protein phosphatase, N-terminal, SANT/Myb domain, NAC domain, AP2/ERF, proteins with the protein kinases, zinc finger binding domain, Ankyrin repeat-containing domain, AAA ATPase, AAA + lid domain, Ubiquitin-like domain, WD40 repeat, NBS-LRR proteins, DnaJ domain, WRKY proteins, MAP kinases, transporters and UDP-glucosyltransferases etc.
Here we discussed some proteins analysis with our own expressive data with 72 CGs. These candidate genes that encode different proteins related to diseases resistance. In this study, CGs TraesCS1B02G019100, TraesCS1B02G122000, TraesCS1B02G147700, TraesCS1B02G152100 were coding the protein P-loop containing NTPase. TraesCS1B02G073600, TraesCS1B02G091100, TraesCS1B02G154100, TraesCS1B02G171100, and TraesCS4B02G235800 genes were coding protein F-box-like domain superfamily and TraesCS1B02G094300, and TraesCS4B02G197600 genes were coding for protein sugar phosphate transporter domain. Two genes TraesCS1B02G105200 and TraesCS4B02G197400 were coding WD40-repeat-containing domain superfamily. Other two genes such as TraesCS1B02G107100, TraesCS4B02G202400 were coding protein Ankyrin repeat-containing domain superfamily. TraesCS1B02G119100, and TraesCS4B02G213500 genes coding COI1 and F-box proteins. Wheat genes TraesCS1B02G128100, and TraesCS4B02G210800 were encoded serine-threonine protein phosphatase and N-terminal. Three genes such as TraesCS1B02G131400, TraesCS4B02G231300 and TraesCS4B02G240100 were coding protein- Myb domain. Another three genes (TraesCS1B02G139000, TraesCS4B02G234600, and TraesCS4B02G249200 were encoded protein of leucine-rich repeat domain superfamily. The tetratricopeptide repeat protein was encoded by two genes i.e TraesCS1B02G140100, and TraesCS1B02G148700.
Four genes such as TraesCS1B02G448300, TraesCS4B02G262900, TraesCS4B02G287800, and TraesCS7B02G189400) were coding the same protein (MFS transporter superfamily). The protein-AAA ATPase and AAA + lid domain was encoded by three different genes (TraesCS4B02G189200, TraesCS4B02G268200, TraesCS4B02G268400)
The papain-like cysteine peptidase superfamily protein were encoded by two genes (TraesCS4B02G267000, TraesCS4B02G281400). Another leftover CGs were encoded only by single protein.
Among the 72 DECGS encoding proteins, the role of some of the proteins has found in the biotic stress resistance in according to previous studies. For example, cytochrome P450s (TaCYP72A), a member of the CYP72A subfamily and cloned in wheat cultivar CM82036. This is a membrane-bound enzymes that may carry out a variety of oxidation-reduction processes. They also participate in secondary metabolite formation in the traditional xenobiotic detoxification route as well as in plant defence. The evidence for cytochrome P450s' involvement in the host response to infections is found in wheat (Gunupuru et al. 2018) and maize (Li et al. 2020). In our study, a gene TraesCS4B02G237600 controlled the same protein. The gene of this protein is upregulated in the early disease response for incompatible reaction and negatively regulated in susceptible parent at the 24hpi.
F-box protein was associated with several genes and highly upregulated in the compatible reaction and the role of this protein in wheat resistance to the infection by LR pathogen has been observed (Li et al. 2020)
The role of protein kinase, ATP binding site has been identified in disease resistance for example mitog en-activated protein kinases (MAPK). The role of this protein is crucial in the signal transduction pathways of plants (Nadarajah et al. 2009). In our study, the related genes of this protein were upregulated in the compatible reaction.
A multifunctional enzyme known as serine/threonine protein phosphatases is actively involved in the defence mechanisms and set off appropriate defence response development (Bajsa et al. 2011). The related genes of this protein were upregulated in the compatible reaction during the defence response of plants against spot blotch pathogen.
In plants, ubiquitination is becoming a frequent regulatory mechanism that manages a variety of cellular functions. Recent interesting findings from several laboratories show that ubiquitination may be crucial for plant disease resistance. As defence regulators, several putative ubiquitin ligases have been discovered. Evidence suggests that this well-known protein-modification pathway may regulate plant pathogen defences., even though ubiquitin ligase targets linked to disease resistance in plants have not yet been discovered (Devoto et al. 2003). The gene of this protein was highly downregulated in the susceptible parent against spot blotch disease.
The MYB TF family is a large and functionally significant protein family that regulates practically all biological processes in plants (Stracke et al. 2001; Dubos et al. 2010). MYB/SANT domain, R1 type MYB transcription factors are functionally vital because they play a significant role in overcoming abiotic and biotic stressors by regulating diverse defence mechanisms in many plant species (Erpen et al. 2018). The conserved MYB repeats/SANT domain is involved in DNA binding and protein interactions, whereas the variable region is important in protein activity control (Tiwari et al. 2020). But this protein was not significantly regulated in against the spot blotch pathogen.
LRR proteins participate in particular protein-protein interactions and are found mostly in eukaryotes. Plant LRR proteins were initially reported as receptor serine/threonine kinases and polygalacturonase-inhibiting proteins. LRR proteins play an important function in plant defence. LRR proteins are involved in resistance to a wide variety of pathogens, as well as nematodes, fungi, bacteria, and viruses, either as resistance proteins or as proteins necessary for resistance proteins to function (Jones et al. 1997). The gene of this protein was not significantly regulated in our transcriptomic data.
Tetratrico Peptide Repeats proteins are found in a significant number in nature (TPRs). TPR motifs are protein-protein interaction modules that are important in the control of several biological activities. TTL1 was recently discovered as a protein with TPR patterns that are essential for abscisic acid reactions and osmotic stress tolerance (Schapire et al. 2006). In the current study, the related gens of this protein were downregulated in the incompatible reaction at the early stage of defence.
Members of the MFS have been demonstrated to play a critical role in plants' fundamental cellular processes as well as stress responses including xenobiotic detoxification, hormone transport, or nutrient scavenging in nutrient deficiency. But the related genes of this protein were not expressed significantly.
To control several features of jasmonate-regulated plant developmental processes and defence responses, the F-box protein COI1 assembles into SCF(COI1) complexes and recruits its substrate JAZ proteins for ubiquitination and destruction (Zhou et al. 2013). This protein regulated genes were significantly upregulated in the compatible reaction of spot blotch infected tissues.
The last acylation step in the production of triacylglycerol is mediated by diacylglycerol (DAG) acyltransferase (TAG). It may be found in the majority of plant parts, including leaves, fruits, anthers, petals and developing seeds (Hobbs et al. 1999) and found upregulate in the compatible reaction.
Elongator, as well as its epigenetic significance in plant growth and response to abiotic and biotic stresses Elongator, a six-subunit protein complex, was discovered in plants after being isolated as an interactor of hyperphosphorylated RNA polymerase II in yeast (Ding et al. 2015) and the associated gene of this protein was downregulated in incompatible reaction.
The histidine phosphatase superfamily is a large functionally diverse group of proteins. They share a conserved catalytic core centred on a histidine which becomes phosphorylated during the course of the reaction. Even though the superfamily is overwhelmingly composed of phosphatases, the earliest known and arguably best-studied member is dPGM. The related genes of this protein were upregulated during the defence response of wheat plant against spot blotch disease during the compatible interaction.
The related genes of proteins like papain-like cysteine peptidase superfamily (Zhai et al. 2021), Chaperone J-domain superfamily (Kampinga et al. 2019), Sugar phosphate transporter domain, (Gangola et al. 2018), Zinc finger, RING-type, conserved site, Midasin AAA lid domain 5- (AAA)-type ATPases (Kressler et al. 2012), Bulb-type lectin domain superfamily (Peumans et al. 1995), AAA ATPase, AAA + lid domain (Zhang et al. 2020) was not significantly expressed in our own transcriptomic data.
These proteins either directly or indirectly contribute to the resistance to biotic stress. We were only able to discuss some of the proteins involved in this study since explaining all of them would take a very long time. Please refer to the annexure for further information. Some of the proteins were discovered in other earlier research as well, while others were first discovered in the present study, suggesting that they may also be important in disease resistance. Therefore, the current study offered materials that may be used in wheat breeding in the future and in basic research on spot blotch resistance.