The field trials were conducted at BISA research farm, Pusa, in India for three consecutive years from 2016-17 to 2018-19 and BARI farm, Jamalpur in Bangladesh during 2016-17 crop season. Both the locations fall under the non-traditional, warmer wheat-growing regions belonging to Mega-environment 5 characterized by hot, humid conditions as per CIMMYT’s system for classifying wheat-growing environments in developing countries . The average temperature during the wheat plant reproductive phase at Jamalpur and Pusa is higher than 190C with a high relative humidity  (Table S3).
The spot blotch disease incidence was captured as percentage of infected leaf area at three different growth stages to minimize the chances of disease escape due to environmental factors. However, the scoring date showing highest disease pressure (usually the second one) was used in the analysis. Since the susceptible parent displayed highest disease severity at growth stage 77 (GS77) on Zadoks scale , to make better judgment about the level of resistance, disease severity was recorded at this stage (usually second scoring) was used to differentiate each line.
The nearly continuous distribution of lines in all the environments show quantitative nature of resistance. The same has been supported by earlier findings where more than two genes [35, 12, 13, 36, 37] and multiple alleles with minor effect [30, 21] to control spot blotch resistance is reported. It was found that the log transformation improved the data normality which was also reflected by the improved consistency in the GWAS results across locations. We observed significant genetic variation for disease susceptibility in the population. The genetic variances and moderate to high heritabilities for spot blotch were comparable with earlier findings in wheat [37, 38, 36]. Despite significant genotype × environments interactions, we observed moderate to high heritabilities within environments (Table 2). The environmental interactions might ascribe to difference in the pathogen isolates prevalent in NEPZ of India and Bangladesh in case of locations and weather conditions mainly within location. For example, the maximum mean disease severity of the susceptible lines were up to 43% in Env3 while it was 70% in Env1 (Table 1).
The linkage analysis was based on 18637 filtered SNP markers covering all chromosomes. The redundant SNPs with 0 cM distance and with same gene annotation were removed from the linkage mapping as no additional information is expected. After GWAS analysis, 14 chromosomes harboring significant QTL regions forming 23 linkage groups were used for further analysis and graphical representations. The SNP lies more than 10 cm apart based on linkage mapping, were placed in a separate linkage group. (Fig. 2).
We used genotyping information for the PCA where most of the groups were based on the proportion of genome shared by the parental pool except few exceptions. For example, the subgroup (G-VIII) consisted common parent TRCH/SRTU//KACH while the largest group (G-VII) consists lines with mixed pedigrees dominated by SAUAL, WBLL#1, Kachu #1, BAV92//IRENA/KAUZ, FRANCOLIN#1, MUCUY and PBW343.
Several spot blotch resistance QTLs have been reported on different chromosomes [39, 16, 40, 12, 13, 41, 42, 17, 43, 20, 18, 44, 45]. However, only three major QTLs designated as Sb1 on 7D , Sb2 on 5B , and Sb3 on 3B  are well described. We also observed consistent chromosome regions on 2B and 5B, appeared in more than 2 or all the environments (Table 3). The QTL on 5B, named as Sb2 gene earlier have been studies in detail . The Sb2 gene is known to interact with Tsn1 gene, conferring susceptible reaction to tan spot and Septoria nodorum blotch . The gene ToxA virulent to Tsn1 exists in both Pyrenophora tritici-repentis and Parastagonospora nodorum confer susceptible reaction to tan spot and Septoria nodorum blotch respectively . Friesen et al. 2018  demonstrated major effects of the Tsn1 locus on chromosome 5B. However, the importance of Tsn1 in spot blotch disease resistance under field condition is not known. The QTL on 7D was the first one studied in detail and reported to be associated with Lr46 , Lr34 and leaf tip necrosis . Based on the fine mapping studies, it was named as Sb2 gene . It is interesting to note that Ayana et al. 2018  identified six potential QTLs (QSb.sdsu-2D.1, QSb.sdsu-3A.1, QSb.sdsu- 4A.1, QSb.sdsu-4B.1, QSb.sdsu-5A.1, QSb.sdsu-7B.1) in hard winter wheat using the isolate, SD40 in greenhouse conditions. The chromosome regions on 4A (Env2 and Env4) and 4B (Env4) and 7B (Env1 and Env3) were consistent with the results of . Similarly, four chromosome regions on 1B, 3B, 4B and 5B are validating the finding of  which were based on testing in the field condition.
Regardless of % phenotypic variance explained by an allele, almost all wheat chromosomes except 3D and 5D reported to have contributed for spot blotch disease resistance depending on, spot blotch isolate, the breeding material or parents in case of bi-parental population [16, 12, 13, 42, 17, 20, 18, 19, 30, 21]. The minor QTL were reported on, 1BS, 1D, 2D and 3A, 4DS and 6D contributed by ‘CIANO T79’, ‘WUYA’ and ‘BARTAI’ . The broad range of environmental conditions at our field sites allowed us to capture considerable genetic variation underlying spot blotch resistance. We identified 23 QTL regions on 14 chromosomes validating previous results. The new genomic region detected on chromosome 3D associated with the SNP marker S3D_610628298 explained up to 6.94% of the phenotypic variance but detected in Env2 only. Similarly, the SNP (S2B_90662917) on chromosome 2B was most significant, explained only up to 10% of phenotypic variance, while the SNP on 5B explained largest phenotypic variance in Env1 (Table S1). Out of 23, 9 chromosome regions on seven chromosomes (1A, 1B, 2A, 5A, 5B, 7B, 7D) were already mapped in independent studies earlier [16, 13, 42, 17, 20, 18, 19, 30, 21].
So far, based on the consistency in independent QTL mapping studies using different source of resistance, it seems that there is not much genetic variability in spot blotch pathogen across the continent. However, several studies described clear grouping among spot blotch isolates based on the fungal hyphae color, aggressiveness and DNA fingerprinting [48, 49, 50, 51]. Four chromosomal regions on 1B, 2B, 4A and 6B are consistent between Pusa India and Jamalpur Bangladesh. This may be due to prevalence of most aggressive isolate of spot blotch pathogen (isolate No. ICMP 13584, Auckland, New Zealand) common in South Asia .
To study the importance of significant SNPs in disease resistance, we annotated all SNPs using wheat genome assembly annotation (IWGSC Ref Seq v1.0) and traced the protein synthesized by the annotated gene. The literature was mined to look for the putative functions of those proteins. We found that several genes functional annotation strongly associated with disease resistance and observed across the year and environments (Table 3). For example, seven SNPs (S2B_13814702, S2B_533178164, S2B_14809954, S2B_14963432, S2B_15129248, S2B_504717, S2B_78065) on chromosome 2B associated with eight geneIDs, TraesCS2B01G030500, TraesCS2B01G373900, TraesCS2B01G031700, TraesCS2B01G031900, TraesCS2B01G032000, TraesCS2B01G032100, TraesCS2B01G001100 and TraesCS2B01G000400 involved in synthesis of Cytochromosome P450 family protein. The role of Cytochrome P450 family protein in plant defense, secondary metabolite biosynthesis in the classical xenobiotic detoxification pathway is well established by Thapa et al. 2018 . It is involved in resistance to DON which is a trichothecene mycotoxin produced by Fusarium species and increase yield. The Cytochrome P450 family protein may not involve directly in yield increase but to enhanced Fusarium head blight disease resistance.
The SNP (S2B_28592818) detected in Env4 on same chromosome (2B) but at different region synthesizes NBS-LRR disease resistance protein family contribute for disease resistance [54, 55]. Similarly, the SNP S2B_8311062 and S5B_683352145 also associated with the gene synthesize NBS-LRR disease resistance protein family and contribute for fungal disease resistance. The role of NBS-LRR disease resistance protein is disease resistance mechanism is well established [54, 55]. One of the significant SNP located on chromosome 2B, S2B_15129248 is linked to two geneIDs, namely, TraesCS2B01G032100 (synthesize Cytochrome P450 family proteins) and TraesCS2B01G032200 (involved in GRF zinc finger family protein). Both the proteins play an important role in plant disease resistance [53, 56].
It is interesting to note that the most important SNP S5A_595393566 detected in Env1on chromosome 5A belongs to the gene TraesCS5A01G402800 which mediates spot blotch resistance in wheat. This gene is involved in the synthesis of Myb family transcription factor-like protein, found to mediate host resistance to Bipolaris sorokiniana in wheat . The same region has been reported in other independent studies as well [18, 20, 30, 21]. Similarly, the SNP S3A_67065083 associated with geneID TraesCS3A01G103500 involved in synthesis of 1R-MYB Transcription factor which plays an important role in disease resistance against stripe rust fungus and ear head disease in wheat .
The key SNPs on chromosome 1A (MAPK module FgSte50-Ste11-Ste7 in F. graminearum), 1B (stripe rust & powdery mildew), 1D (Serine/threonine-protein kinase), 2B (RPP13, Avr9/Cf-9 rapidly elicited protein, NBS-LRR protein, F-box family protein, pentatricopeptide repeat-containing protein, Peptidylprolyl isomerase, Uroporphyrinogen decarboxylase and resistance to DON), 3A and 3B (1R-MYB TF, wheat NAC protein and interaction with an orphan protein), 4B (Uroporphyrinogen decarboxylase), 5A (Myb family transcription factor-like, Serine/arginine repetitive matrix, NBS-LRR & transmembrane protein), 5B (B3 domain-containing, Mannitol transporter & NBS-LRR family-1 protein) and 7D (implicated in the defense through cell wall modification, degradation, carbohydrate metabolic processes) annotated and found to synthesize different proteins involved in fungal defense mechanism (Table 3) [58, 59, 60, 55, 61, 54, 62, 63, 64, 65, 32, 66, 67, 68, 69, 57, 70, 71]. The consistency in identification of key SNPs involved in resistance mechanism through protein annotation was confirmed where same protein family was identified independently in different environments (Fig. 4).
Maximum number of known proteins involved in fungal defense were based on 14 SNPs on chromosome 2B showing the importance of this chromosome in disease resistance. The earlier independent findings also describe the importance of chromosome 2B in spot blotch disease resistance [12, 19]. Interestingly QTL found in the present study on 1B in Env1, the proteins involved are Pentatricopeptide repeat-containing protein (TraesCS1B01G424000) mRNAs renders more susceptible to pathogenic bacteria and fungi in Arabidopsis thaliana  and Homeobox protein (TraesCS1B01G424100) associated with reaction to stripe rust and powdery mildew in common wheat . The SNPs (S1B_646895451 and S1B_647195634) detected in two environments located on chromosome 1B are 18.97 cM apart on genetic linkage map while those belong to the same geneID TraesCS1B01G424000. The gene annotation results indicate role in plant disease resistance [69, 65, 72]. This information obtained from gene annotation could potentially be used in fine mapping and map-based cloning to further characterize the mechanisms of spot blotch disease resistance. The markers with lowest P-values may be converted in to diagnostic markers to validate the SNPs and used in identification of lines with desired alleles in early generations.