Identication of a Novel Genomic Region Associated With Resistance to Fusarium Head Blight in Chinese Winter Wheat

Background: Fusarium head blight (FHB) is a disease affecting wheat spikes caused by Fusarium species, which leads to cases of severe yield reduction and seed contamination. Therefore, identifying resistance genes from various sources is always of importance to wheat breeders. In this study, a genome-wide association study (GWAS) focusing on FHB using a high-density genetic map constructed with 90K single nucleotide polymorphism (SNP) arrays in a panel of 205 elite winter wheat accessions, was conducted in 3 environments. Results: Sixty-six signicant marker–trait associations (MTAs) were identied (P<0.001) on fteen chromosomes explaining 5.4–11.2% of the phenotypic variation therein. Some important new genomic regions involving FHB resistance were found on chromosomes 2A, 3B, 5B, 6A, and 7B. On chromosome 7B, 6 MTAs at 92 genetic positions were found in 2 environments. Moreover, there were 11 MTAs consistently associated with diseased spikelet rate and diseased rachis rate as pleiotropic effect loci. Eight new candidate genes of FHB resistance were predicated in wheat. Of which, three genes: TraesCS5D01G006700, TraesCS6A02G013600, and TraesCS7B02G370700 on chromosome 5DS, 6AS, and 7BL, respectively, were important in defending against FHB by regulating chitinase activity, calcium ion binding, intramolecular transferase activity, and UDP-glycosyltransferase activity in wheat. In addition, a total of six excellent alleles associated with wheat scab resistance were discovered. Conclusion: These results provide important genes/loci for enhancing FHB resistance in wheat breeding populations by marker-assisted selection.


Background
Wheat (Triticum aestivum L.), one of the three major food crops, is grown worldwide as an important source of food and fodder. Therefore, ensuring stable wheat production has become a common focus of agricultural scientists around the world. However, wheat has a long growth period and is vulnerable to biotic (diseases, insect pests, etc.) and abiotic (drought, freeze damage, etc.) stress. Fusarium head blight (FHB), or scab, is a disease affecting wheat spikes caused by Fusarium species. This disease not only causes severe wheat yield reduction but also contaminates wheat seeds with DON toxins [1]. In China, FHB not only severely affects the wheat grown in the middle and lower Yangtze River Valley region but its incidence has increased in the Yellow and Huai River Valley regions over the last two decades due to the change in climate and tillage systems. Moreover, it has become the most destructive wheat disease in the world because no completely immune varieties have been found so far, which seriously threatens food production and food security. Breeding resistant varieties and discovering resistant genes are the most effective ways to solve the problem of FHB.
FHB is a complicated trait which is affected by genotype and environment, making it a typical quantitative trait. Previous studies have shown that plant height, heading date, owering time, anther extrusion, etc. in uence FHB resistance. At owering time, the weather condition (rainy or sunny) is very important for the development of this disease. FHB resistance has been studied extensively in wheat using genetic linkage analysis, and many QTLs(Quantitative trait locus) (more than 400 distributed on 21 chromosomes) associated with FHB resistance were reported. At present, FHB resistance is classi ed into ve types, namely: resistance to initial infection of spike (Type I), resistance to spread infection of spike (Type II), resistance to accumulate mycotoxins (Type III), resistance to kernel infection (Type IV), and resistance to yield reduction (Type V). Of these, Type I and Type II have been studied the most in previous studies. Seven genes (Fhb1 to Fhb7) for FHB resistance have been found, and Fhb1, Fhb2, Fhb4, and Fhb5 were on chromosomes 3BS, 6BS, 4BL, and 5A, respectively in common wheat; however, the remaining genes were derived from wheat relative species. The Fhb1 gene has been widely dissected and sequenced to nd a poreforming toxin-like (PFT) gene that is responsible for FHB resistance [2]. Nevertheless, another new gene was later discovered, encoding a putative histidine-rich calcium-binding protein (His or TaHRC) that was adjacent to PFT [3,4]. Based on these studies, function markers have been developed for Fhb1 and used in molecular breeding for enhancing scab resistance. However, the mechanism of the resistance imparted by these two genes (His or TaHRC) appears to be different. As a result, this gene still needs to be further studied in order to dissect its molecular identity [3,4]. Recently the candidate gene for Fhb7 was determined and cloned, which revealed that it encodes a glutathione (GST) that can detoxify trichothecene toxins [5]. Its resistance depends on a reduction of pathogen growth in spikes, which is different from the resistance of Fhb1. However, the other ve FHB genes have not been cloned until now.
Besides these seven FHB genes, some important loci for resistance have been found. For example, QFhb.mgb-2A was identi ed as a WAK2 gene [6], whose function has been clari ed [7]. Another important locus on chromosome 2DL was thought to be transcription factor TaWRKY70, which regulates the expression of metabolite biosynthetic genes including TaACT, TaDGK, and TaGLI to in uence FHB resistance [8,9]. Using two Recombinant Inbred Lines (RILs) populations with one common parent, named AC Barrie, from Canadian spring wheat, QFhb.mcb-3B, QFhb.mcb-6B, and QFhb.mcb-5A.1 were mapped to the expected location of Fhb1, Fhb2, and Fhb5, respectively [10]. On chromosome 5B, the prominent resistance gene, QFhb.mbr-5B was found to explain up to 36% of the phenotypic variation [10].
With the development of genomics, the genetic basis of complex traits such as grain yield, starch content [11], and agronomic traits [12] in wheat has been dissected using genome-wide association study (GWAS). However, very few studies on FHB resistance have been reported using GWAS. Furthermore, in doing so, different researchers have obtained various results. For example, using European winter wheat, Kollers found the signi cant association loci involving all chromosomes of wheat except 6B. Additionally, in US winter wheat, 10 SNP loci associated with FHB resistance on chromosomes 4A, 6A, 7A, 1D, 4D, and 7D were detected; moreover, there seem to be multiple loci on chromosome 3B associated with Fhb1 involving FHB index, DON content, FHB severity, and incidence. Further, using spring wheat lines, QTLs for FHB resistance were found on six chromosomes including 1B, 2B, 4B, 5A, 5B, and 6A [13]. Zhu et al. (2020) identi ed ve QTLs on ve chromosomes, namely 1AS, 2DL, 5AS, 5AL, and 7DS, using a mixed linear model, and developed molecular markers based on SNP loci on 1AS, 5AS, and 5AL. However, little is understood about the genetic basis of Type II resistance using GWAS in Chinese elite winter wheat varieties (lines) [14].
Therefore, the objective of this study was to identify some important novel genomic region associated with the Type II resistance of wheat in different environments, and to predict candidate genes for loci associated with these traits to improve wheat FHB resistance by breeding.
The genome-wide association analysis based on SNP markers was used to analyse the FHB resistance of wheat, and the associated major locus was used for molecular marker-assisted selection (MAS). It has become an important method of improving FHB resistance by breeding. In this study, 205 natural populations of wheat (including Chinese winter wheat cultivars and breeding backbone parents) were used as materials to identify the phenotype of wheat FHB resistance in 3 different environments. The genome-wide association analysis was conducted with the 90K wheat gene chip and the SNP markers.
Candidate genes signi cantly related to FHB resistance were obtained through the identi cation of excellent alleles and the prediction of candidate genes, so as to lay a foundation for homologous gene cloning and MAS.

Plant materials
The association mapping panel of 205 wheat genotypes for GWAS comprised 77 released cultivars, 55 founder parents including 2 lines from Mexico and France, and 73 breeding lines from 10 provinces that represent the major winter wheat production regions in China [12]. Sumai 3 was selected as an FHB resistant check, Shannong 102 as a moderately resistant check, and Ningmaizi 22 as a moderately susceptible check.

Inoculum preparation
In this study, the mixed conidiospore suspension of 7136, F301, F609, and F15 virulent strains of F. graminearum used, was obtained with the courtesy of Nanjing Agricultural University. The pathogen was inoculated in a mung bean medium and vortexed at 1500 rpm under 25 ℃ for 5-7 days. After culturing and ltering, the mass of conidia was examined under a microscope; then, the four pathogen strains were mixed equally and stored at 4 ℃ for later use. In the greenhouse, wheat was inoculated with 10 μl of the F. graminearum conidia suspension (50,000 spores/mL) applied to the middle and lower part of the spikes during owering. Ten spikes were inoculated per line. In the eld, wheat was sprayed with the conidia suspension and 10 spikes were inoculated in each line. The whole wheat spike was then covered with a self-sealing bag to retain moisture, sprayed with water 1-2 times a day, and had the self-sealing bag removed after 3 days. The disease symptoms were investigated on day 21 after inoculation, and the diseased spikelet rate (DSR), diseased spike rachis rate, and disease index (DI) were calculated. All spikes were classi ed into ve classes of disease severity according to the diseased spikelet rate ( Genome-wide association analysis SNP markers, genotyping, and the population structure of the samples have been previously reported [12]. Based on this information, signi cant marker-trait associations (MTAs) were identi ed using a mixed linear model (MLM) in TASSEL3.0. The P-value was used to determine whether a QTL was associated with a marker, while the R 2 value was used to evaluate the magnitude of the MTA effects. SNPs with P-values ≤0.001 were considered to be signi cantly associated with phenotypic traits. Further, when the marker was detected in two or more environments at the same time, it was considered a stable association site.

Statistical analysis
Analysis of variance (ANOVA) and correlations among phenotypic traits were carried out using the statistical software SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).
Forecasting candidate genes for FHB resistance A BLAST (Basic Local Alignment Search Tool) search was performed on the International Wheat Genome Sequencing Consortium database (IWGSC; http://www.wheatgenome.org/) using the sequence of the signi cant SNP markers identi ed by GWAS. When an SNP marker sequence from the IWGSC was 100% identical to any wheat contig, the sequence was extended by 2Mb for each marker using the IWGSC BLAST results. Afterwards, the extended sequence was used to run a BLAST search on the National Center for Biotechnology Information (NCBI) database (http: //www.ncbi.nlm.nih.gov) and on Ensembl Plants (http://plants.ensembl.org/Triticum_aestivum/ Tools/Blast) to con rm possible candidate genes and functions.

Phenotypic variation analysis of wheat FHB resistance
The variation coe cient of diseased wheat spikelet rate (DSR) was the highest (52.96%) in E2, followed by that in E3 (44.30%), and E1 (36.55%) ( Table 1); thus, the genetic variation of DSR was abundant. The variance analysis of FHB resistance of the spikelet and spike rachis indicated that signi cant differences were present between cultivars and environments, and their interactions ( Table 2). This illustrated that the FHB resistance was a quantitative trait affected not only by genotype but also by environment. Furthermore, there were signi cant positive correlation coe cients between spikelet and spike rachis, spikelet and spikelet, and spike rachis and spike rachis in the three environments; thereby, indicating that the development trend of FHB was consistent between spikelet and spike rachis ( Table 3).    on chromosomes 1A, 1B, 2A, 2B, 2D, 3B, 3D, 4A, 5A, 5B, 5D, 6A, 6B, 7A, and 7B (Table   4; Fig. 1). The contribution (R 2 ) of MTA loci to FHB resistance ranged from 5.45% to 11.20%, of which, 11 MTA loci were detected in both spikelet and spike rachis. On chromosome 7B, a novel genomic region from genetic position 92 to 103, signi cantly associated with FHB resistance, was detected in all three environments. Moreover, there was one major locus at genetic position 92 of chromosome 7B accounting for 11.20% of the phenotypic variation in the spikelets, namely locus BS00025286_51, which can also be detected for the spike rachis, explaining 7.07% its phenotypic variation. In E3, four loci on chromosome 7B were found to be associated with both diseased spikelet rate and diseased spike rachis rate. In addition, there were some genomic regions associated with FHB resistance on chromosomes 5B, 6A, 2A, and 3B, but they were found only in a single environment. The other six loci, including D_contig74317_533 on chromosome 5D, Kukri_c14239_1995 on chromosome 1B, Kukri_c7087_896 on chromosome 3B, RAC875_c35801_905 on chromosome 3D, BS00099729_51 on chromosome 5B, and RAC875_c68525_284 on chromosome 6B, were also identi ed to be associated with both the diseased spikelet rate and the diseased spike rachis rate. The remaining MTA loci were detected only for a single trait in a single environment.  E1, E2 and E3 were the same as the Table 1.

Allelic variation analysis of MTA loci
The allelic variation of 10 MTA loci was analysed (Table 5). Alleles T and C of the marker, Kukri_c14239_1995 on chromosome 1B were associated with the largest phenotypic difference (0.2297). Speci cally, the phenotypic value of diseased spikelet rate associated with Kukri_c14239_1995-T was signi cantly higher than that associated with Kukri_c14239_1995-C, indicating that Kukri_c14239_1995-C was better than Kukri_c14239_1995-T for FHB resistance (Table  5). Furthermore, because allele C of D_contig74317_533 showed a signi cantly higher diseased spikelet rate than D_contig74317_533-T, allele T was deemed to be better for improving FHB resistance. On chromosome 7B, allele C of BS00025286_51 had a higher diseased spikelet rate than allele T; thus, allele T for this locus was favourable for FHB resistance. Nevertheless, for the other four loci on this chromosome, signi cant differences between the two alleles for diseased spikelet rate seemed to be at 5%. The least difference for diseased spikelet resistance was observed between Kukri_c7087_896-G and Kukri_c7087_896-T, which indicated that this locus affected FHB resistance to a smaller degree. Moreover, on chromosome 3D, RAC875_c35801_905-G yielded better results than RAC875_c35801_905-A for FHB resistance. Markers were selected for prediction from loci that were signi cantly associated with diseased spikelet rate and diseased spike rachis rate (Table S1). From this, some important loci were identi ed.
The candidate gene, TraesCS3D02G326700 located on chromosome 3D is associated with actin binding in wheat. The biological processes of Os05g0500500, Os08g0436400 (OsSAP12), and ORUFI08G17770 involve stress response; while that of Os01g0888600 (OsMLO5) involves defence response and response to biotic stimulus. In addition, the candidate genes are known to be related to UDP-glycosyltransferase activity in Oryza sativa Indica, Oryza sativa Japonica, and Oryza ru pogon.
The candidate gene, TraesCS5D01G006700 of the marker, D_contig74317_533 on 5DS was predicated in wheat, whose function was intra-molecular transferase activity. Homologous genes of TraesCS5D01G006700 were found in Hordeum vulgare, Arabidopsis, Indica rice, Japonica rice, and Oryza ru pogon, and most of their functions were involved in defence response to fungi; thus, this locus should be further studied.
The candidate genes TraesCS6A02G013700 and TraesCS6A02G013800, predicated by IAAV9150, participate in the ubiquitin-dependent ER-associated degradation (ERAD pathway in wheat. Its homologous genes, AT1G24010 and AT5G43590 in Arabidopsis thaliana are involved in the defence response of the biological process, while the AT1G22870, BGIOSGA008772, and BGIOSGA037437 are involved in ATP binding and protein kinase activity.
The candidate gene, TraesCS6A02G013600 of Excalibur_c20597_509 has the function of GTP binding in wheat, and some homologous genes of this candidate gene are involved in defence response and the cell surface signalling pathway in Arabidopsis thaliana and Japonica rice. Their molecular functions were mainly related to ATP binding, protein kinase activity, and calcium ion binding.
On chromosome 7BL, there is one important candidate gene in wheat, TraesCS7B02G370700 of BS00025286_51, which is involved in the biological process of defence response to fungi. Its function is chitinase activity. Notably, a total of ve homologous genes in Hordeum vulgare, Oryza sativa Indica, and Oryza sativa Japonica, also participate in defence response to fungi. Therefore, this candidate gene can be further studied as a new important gene for FHB resistance. The three loci, RAC875_c18043_369, RAC875_c18043_411, and Kukri_c4143_1055, on chromosome 7BL are all in the same physical location, and the candidate gene, TraesCS7B02G340200 in wheat was identi ed. Six homologous genes in Hordeum vulgare were involved in the biological process of defence response to fungi, as were three homologous genes in Arabidopsis. The candidate gene, TraesCS7B02G340100 of RAC875_c5646_774 is associated with the carbohydrate metabolic process in Triticum aestivum, but its homologous genes, ORUFI12G13460 and ORUFI01G37780 in Oryza ru pogon, are involved in defence responses.

Analysis of marker haplotype and resistance
Among the alleles at the associated site, the alleles with decreasing spike rate were assumed to be the resistance alleles at this site. This led to the collection of six excellent alleles of resistance. Among them, B202 and B34 contain ve excellent alleles, and the haploid types were TCTACA and TTTGCA, respectively.
Among the four excellent alleles, the haploid types of B16 and B68 were CTTGCA, the haploid types of B46 and B179 were TTTACA, and the haploid types of B131 and B200 were TTCGCA. In addition, the haploid type of the material with three excellent alleles was CTTACA, TCCGAG, TTCACA, and CCTGAG. By combining with the resistance phenotype of natural population materials, it was found that in the materials with multiple excellent alleles, the resistance expression was better (Table 6). This indicates that multiple haploids of the materials played an important role in the screening of anti-scab materials. For example, B34 and B16 were only different in D_contig74317_533, and their disease index was 44.67% and 46% respectively.

Discussion
Fusarium head blight (FHB) is affected by both the environment and genetics [15,16], and is a quantitative trait controlled by multiple genes [1]. This disease has become an important disease in the Yellow and Huai River Valleys [17], and seriously threatens wheat production and processing. In order to improve the resistance of FHB in wheat cultivars, the majority of previous researchers paid attention to identifying the important genes/loci of FHB by QTL mapping or association mapping [18,19,20]. The previous results indicated that while almost all the chromosomes of wheat were involved [21,22], the chromosomes 3B, 4B, 5A, and 6B seemed to be important because of the Fhb1, Fhb2, Fhb4, and Fhb5 genes [16,23]. By GWAS analysis, ve QTLs were identi ed on chromosome arms 1AS, 2DL, 5AS, 5AL, and 7DS [14]. Wang et al. (2017) identi ed consistent and signi cant single nucleotide polymorphisms associated with multiple traits on 1B, 2B, 4B, 5A, 5B, and 6A chromosomes using spring wheat lines. Of which, the locus for reducing the content of deoxynivalenol on chromosome 5B may be novel. However, in this study, fteen chromosomes were involved in the MTA loci, and some important genomic regions involving FHB resistance were found on chromosomes 2A, 3B, 5B, 6A, and 7B. This result further con rmed the importance of chromosome 3B for FHB resistance [24]. Additionally, on chromosome 7B, six MTAs at 92 genetic positions were found in two environments. Of which, ve MTAs consistently associated with diseased spikelet rate and diseased rachis rate as pleiotropic effect loci, and the BS00025286_51 locus (P<0.0001) could explain 11.20% of the phenotypic variation. These MTA loci were novel for FHB resistance, as was D_contig74317_533 on chromosome 5D, Kukri_c14239_1995 on chromosome 1B, Kukri_c7087_896 on chromosome 3B, RAC875_c35801_905 on chromosome 3D, BS00099729_51 on chromosome 5B, and RAC875_c68525_284 on chromosome 6B.
Plants and pathogens interact and in uence each other in the long-term evolutionary process. Consequently, plant disease resistance is a complex molecular process controlled by genes [22]. Although researchers have located hundreds of QTLs distributed on wheat, including 21 chromosomes from common wheat varieties or related species through linkage and association analysis [25,26], there were few major genes. Nevertheless, with the development of molecular technology and wheat genome sequence, some genes (including major and minor genes) seemed to be easier to discover than before. Moreover, the isolation and functional veri cation of FHB resistance genes are bene cial to understanding the pathogenesis and resistance mechanism of wheat FHB at the molecular level [16]. Previous studies have shown that the mechanism of the genes/loci identi ed in FHB resistance could be involved in the complex signal transduction pathway and associated with the synergistic effect of many protein factors [23,27,28]. For example, the genes encoding a 12-oxophytodienoate reductase-like protein identi ed in the region of QFh.hbaas-1AS may be related to the biosynthesis or metabolism of signalling molecules, oxylipins, such as jasmonic acid (JA) [30,31]. The proteins encoded by these identi ed genes had receptor-like kinase, UDP-glycosyltransferase, pathogenesis-related protein 1(PR1), and glucan endo-1,3-beta-glucosidase (PR2), to name a few [22,31,32].
In this study, the predicted genes on chromosome 3D encoded UDP-glycosyltransferase activity and were related to the defence response to biotic stimulus.
This indicated that this gene could enhance resistance to FHB because this protein could detoxify both DON and NIV produced by F. graminearum [14,33,34]. By performing homologous gene detection on the D_contig74317_533 locus of chromosome 5D, the genes were found to have homology in nucleic acid binding and defence function in barley, Arabidopsis, Indica rice, Japonica rice, and wild rice. Of these, the AT2G39510 gene is related to the activity of glutamine transmembrane transporter protein. Studies have shown that the glutamine-gated ion channel is related to Fhb5 and can control Ca 2+ in ux [35,36].
It was also found that Ca 2+ was involved in early signalling defence to FHB [28]. In recent years, some studies have shown that wall-associated kinase (WAK) is a kind of receptor like protein kinase with multiple functions, which is involved in signal transduction and the defence response of plants [37,38]. In this study, the gene, Traescs6A02g013600 has homologous genes in Arabidopsis and Japonica rice, which were found to be related to the cell surface receptor signalling pathway, and some of these genes encoded the receptor-like cytoplasmic kinase (RLCK) and serine carboxypeptidase (SCP) families. Previous studies have shown that chitinase, as a pathogenesis-related protein (PR), participates in the plant's basic defence response and begins to accumulate during pathogen infection [21]. The cytochemical localisation of chitinase showed that in the cell walls of lemmas, ovaries, and cobs infected with pathogens, the concentration of chitinase in resistant varieties was signi cantly higher than that in susceptible varieties [39]. Fortunately, the gene, TraesCS7B02G370700 of BS00025286_51 on the 7BL chromosome was also found to be related to the chitinase activity and the defence response for fungi in our study. In addition, the seven candidate genes identi ed were associated with either calcium ion binding or GTP binding, which has been shown to be involved in the early response of wheat to F. graminearum infection by salicylic acid (SA) and Ca 2+ signals [29]. As a study found that Ca 2+ signal transduction was essential for transcriptional reprogramming of innate plant immunity [40], and the change of cytoplasmic Ca 2+ concentration during pathogen infection was found to mediate the signalling process, we can surmise that these genes identi ed with Ca 2+ signals perhaps played important roles in defending against FHB.

Conclusions
In this study, 66 signi cant MTAs were identi ed (P<0.001) on 15 chromosomes explaining 5.4-11.2% of the phenotypic variation. Some important new genomic regions involving FHB resistance were found on chromosomes 2A, 3B, 5B, 6A, and 7B. There were eleven MTAs consistently associated with diseased spikelet rate and diseased rachis rate as pleiotropic effect loci, and eight new candidate genes of FHB resistance were predicated in wheat. Of these, three genes: TraesCS5D01G006700, TraesCS6A02G013600, and TraesCS7B02G370700 on chromosome 5DS, 6AS, and 7BL, respectively, were important in defending against FHB by regulating chitinase activity, calcium ion binding, intra-molecular transferase activity, and UDP-glycosyltransferase activity in wheat.
In addition, a total of six excellent alleles associated with wheat scab resistance were discovered in this study and the disease index was lower in the materials with multiple excellent alleles. Whole genome association analysis QQ map (left) and Manhattan plot of diseased spikelet rate (right) E1, E2 and E3 were the same as the Table 1.