Genome-wide association mapping revealed syntenic loci QFhb-4AL and QFhb-5DL for Fusarium head blight resistance in common wheat (Triticum aestivum L.)

Background Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a major threat to wheat production and food security worldwide. Breeding stably and durably resistant cultivars is the most effective approach for managing and controlling the disease. The success of FHB resistance breeding relies on identification of an effective resistant germplasm. We conducted a genome-wide association study (GWAS) using the high-density wheat 90 K single nucleotide polymorphism (SNP) assays to better understand the genetic basis of FHB resistance in natural population and identify associated molecular markers. Results The resistance to FHB fungal spread along the rachis (Type II resistance) was evaluated on 171 wheat cultivars in the 2016–2017 (abbr. as 2017) and 2017–2018 (abbr. as 2018) growing seasons. Using Illumina Infinum iSelect 90 K SNP genotyping data, a genome-wide association study (GWAS) identified 26 loci (88 marker-trait associations), which explained 6.65–14.18% of the phenotypic variances. The associated loci distributed across all chromosomes except 2D, 6A, 6D and 7D, with those on chromosomes 1B, 4A, 5D and 7A being detected in both years. New loci for Type II resistance were found on syntenic genomic regions of chromsome 4AL (QFhb-4AL, 621.85–622.24 Mb) and chromosome 5DL (QFhb-5DL, 546.09–547.27 Mb) which showed high collinearity in gene content and order. SNP markers wsnp_JD_c4438_5568170 and wsnp_CAP11_c209_198467 of 5D, reported previously linked to a soil-borne wheat mosaic virus (SBWMV) resistance gene, were also associated with FHB resistance in this study. Conclusion The syntenic FHB resistant loci and associated SNP markers identified in this study are valuable for FHB resistance breeding via marker-assisted selection.


Background
Common wheat (Triticum aestivum L.) is one of the most important cereals in the world and is the raw material for breads, biscuits, noodles and cakes [1]. Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most destructive fungal diseases in wheat, which spreads considerably due to farming practices and climate changes [2]. FHB does not only reduce grain yield and quality but also leads to infected kernels with excessive deoxynivalenol (DON), resulting in severe harm to human and animal health [3]. China has the largest wheat production and consumption suffering from severe FHB damages, especially in the Middle and Lower Reaches of the Yangtze River with its warm, humid environment. In recent years, FHB has become more serious and expanded in the major wheat production area of the Yellow and Huai River Valleys [4].
The most effective way for wheat producers to manage and control FHB is by breeding resistant cultivars. Great efforts have been made to find FHB resistance genes and understand the genetic mechanism of the resistance [5][6][7][8][9]. The genetic mechanisms for FHB resistance are complex, and the genotype by environment interaction has very strong effects on trait expression [10,11]. Resistance to F. graminearum in wheat has been classified into five categories: (1) type I for resistance to initial infection by the pathogen, (2) type II for resistance to fungal spread along the rachis, (3) type III for resistance to kernel infection, (4) type IV for resistance to toxin accumulation, and (5) type V for tolerance [12,13]. Many quantitative trait loci (QTL) have been identified for multiple types of FHB resistance in wheat with different magnitudes of effects [14][15][16][17]. Major and stable QTL often have large effects in multiple environments and are more valuable for practical breeding than minor QTL. However, major and stable QTL are rare for FHB resistance. Fhb1, identified from Chinese wheat Wangshuibai and Sumai 3 and located on chromosome 3BS, is the best characterized FHB resistance locus with major effect and stable resistance. Fhb1 was reported as a pore-forming toxin-like gene (PFT) QTL [18]. However, recent studies revealed an histidine-rich calcium-binding protein (His) was responsible for the Fhb1 resistance [19,20]. A comprehensive discussion on the two studies has been performed, and the nature of Fhb1 resistance remains unclear [21]. In addition, Fhb1 has shown linkage with poor agronomic traits and single resistance gene has been proven to be a major limitation for FHB resistance breeding as it may not provide sufficient protection under severe FHB epidemics [15,22,23]. Pyramiding Fhb1 and other major FHB resistance QTL into elite cultivars using MAS could be crucial for breeding new wheat varieties with better resistance to FHB [4,24]. Several cultivars such as Yang-mai158, Yangmai11 Yangmai12, Yangmai16, and Yangmai23 in the Middle and Lower Yangtze River Valleys with moderate resistance to FHB have been approved to be released and become main cultivars [25]. Most of Yangmaiseries cultivars don't carry the Fhb1 locus [26], indicating that other FHB resistance genes may be present in these cultivars and can be more easily applied to breeding. Therefore, discovering more FHB-resistant germplasms and new FHB-resistant loci is essential for breeding wheat varieties with better FHB resistance.
Genome-wide association studies (GWAS), based on linkage disequilibrium (LD) has been widely used to discover various quantitative traits associated nucleotide polymorphisms in plants. For example, using a panel of 192 bread wheat cultivars from southwest China, 57, 27, 30, and 34 single nucleotide polymorphism (SNP) were identified for associations with plant height (PH), grain protein content (GPC), thousand kernel weight (TKW) and sodium dodecyl sulfate (SDS) content, respectively [27]. One hundred-twenty consistent loci were detected using SNP-GWAS and Haplotype-GWAS, and 78 were potentially new [28]. The recently released reference genome sequence of Chinese Spring [29] provides an elit platform for detecting genes significantly associated with linked markers with known physical positions in the genome and promoting the molecular breeding process [30]. In this study, we report a GWAS analysis of FHB resistance using a set of 171 common wheat cultivars with 90 K SNP genotyping and 2 year's phenotyping data. The aims of this study were to identify stable loci for FHB resistance using GWAS and better understand the genetic basis of FHB resistance in natural population.

Phenotypic variation
Continuous variation for percentage of symptomatic spikelets (PSS) was observed at the GWAS panel in both 2017 and 2018 growing seasons, from highly resistant (PSS < 25%) to highly susceptible (PSS > 75%) (Fig. 1). The disease symptom was more severe in 2018 growing season (Fig. 2a). Wheat cultivars from different provinces of China exhibited different levels of resistance to FHB (Fig. 2b). Cultivars from Hunan and Jiangsu provinces exhibited consistently highly resistant to FHB in two seasons, whereas cultivars from Shandong province showed the highest susceptibility.

Population structure analysis
To estimate the sub-populations of the 171 wheat cultivars, population structure analysis was performed using 1676 polymorphic SNP markers distributing on 21 wheat chromosomes with r 2 values > 0.2. The results indicated that the cultivars could be separated into two sub-populations (K = 2) (Fig. 3a, b). Subgroup 1 consists of 99 cultivars, mainly comprising varieties from Anhui, Jiangsu, Henan, Shaanxi and Hunan; subgroup 2 consists 72 cultivars (Additional file 1: Table S1), most of which were from Henan, Jiangsu, Shandong, Shanxi. Wheat cultivars from Anhui and Hunan were all clustered into subgroup 1.

Linkage disequilibrium (LD) analysis
The filtered markers from the 90 K SNP genotyping arrays were used to calculated LD decay for the A, B, and D subgenomes separately as well as the whole genome. 38.9% of all pairs of loci had significant LD (P < 0.001) with an average r 2 of 0.281 from 23,556 polymorphic SNPs which distributed at the genome-wide level. The B sub-genome contained the largest number of significant markers (50.0%), followed by A (39.7%) and D (24.0%) sub-genomes. The highest LD decay distance was present in the D sub-genome and the lowest was found in the B subgenome. The average LD decay distance was~10.5 Mb for the whole genome and 10, 9.5, and 12 Mb for A, B, and D sub-genomes, respectively (Fig. 3c).
Due to the high level of LD in wheat, the SNP clusters identified on chromosomes 4AL (QFhb-4AL) from 621.85 Mb to 622.24 Mb and 5DL (QFhb-5DL) from 546.09 Mb to 547.27 Mb most likely represented chromosome regions containing significant FHB associated loci, respectively. Haplotype analyses of the associated markers revealed three haplotype groups (Fig. 5a), Haplotype 1 consisted of 149 cultivars with an average PSS of 48.92% over 2 years, in which 24 were resistant, 55 were moderately resistant, and 70 were susceptible. Haplotype 2 consisted of 19 cultivars with an average PSS of 19.94% over 2 years, and 12 of them were resistant and 7 were moderately resistant. Haplotype 3 comprised three resistant cultivars with an average PSS of 11.52%. The results indicated that other resistant genes also existed in the cultivars of Haplotype 1 ( Table 2, Additional file 1: Table S3). Interestingly, each haplotype contains wheat cultivars with same associated SNPs on both QFhb-4AL and QFhb-5DL simultaneously (Fig. 5b).

FHB resistance loci identified by GWAS
QTL for Fusarium head blight resistance have been extensively reported using different mapping populations and mapping platforms. From more than 250 documented QTL conferring FHB resistance, only Fhb1-Fhb7 haved been proven to be major effects QTLs. Qfhs.nau-6B (Fhb2), Qfhi.nau-4B (Fhb4), and Qfhi.nau-5A (Fhb5) were fine mapped in the 2.2 cM, 0.14 cM, and 0.09 cM interval [16]. Fhb1 has been cloned recently [18][19][20]. In current study, four loci (28 MTAs) were identified on chromosomes 1B, 4A, 5D and 7A in two seasons. In comparison to the SNP GENE-0293_154 on chromosome 1B identified for type II FHB resistance in this study, a minor QTL for type II resistance was found in a     Table S4).
QFhb-4AL and QFhb-5DL are located on syntenic genomic regions We detected two loci significantly associated with FHB resistance on 4AL and 5DL at a physical intervals of 0.39 Mb and 1.18 Mb, respectively. LD of markers and FHB severity analysis indicated that each haplotype contains wheat cultivars with associated SNP on both QFhb-4AL and QFhb-5DL simultaneously. Gene annotations of the genomic intervals revealed homologous gene pairs between 4AL and 5DL. Highly collinearity in gene order and content were observed for the two FHB resistant QTL regions, even through large fragment insertions/deletions were also presented (Additional file 1: Table S5; Fig. 6). Wheat has experienced structural evolution involving chromosome translocation of 4A, 5A, and 7B. The 4AL/5AL translocation taken place at the diploid level and existed both in T. monococcum and T. aestivum, followed by a 4AL/7BS translocation, a pericentric inversion (4AS;4AL) and a paracentric inversion (4AL;4AL) that occurred in the tetraploid progenitor of hexaploid wheat [40]. Recently, Dvorak et al. [41] reassessed the evolution of wheat chromosomes 4A, 5A and 7B after sequence comparison of wild emmer wheat and Aegilops tauschii. They found that the 596.20-631.84 Mb genomic region of 4A pseudomolecule was derived from ancestral 5AL with nested inversion and is corresponding to the end of the Ae. tauschii arm 5DL. The two FHB associated loci on 4AL (621.81-622.49 Mb) and 5DL (546.45-546.92 Mb) are located on the syntenic block with sequence inversion (Fig. 6), providing further information of this structure rearrangement containing important genes for agronomic trait.
The hypothetical proteins were predicted for the 4AL and 5DL syntenic blocks ( Table 3). Two kinase proteins, homologous to PTI1-like tyrosine-protein kinase 1 and Putative receptor protein kinase ZmPK1, proved to be associated with plant disease resistance were annotated in the corresponding genomic regions (Additional file 1: Tables  S4 and S5). Protein kinases (PKs) are important for transmembrane signaling that regulates plant development and adaptation to diverse environmental conditions [42]. Several kinase proteins have been reported related to plant innate immunity. For example, the combination of a kinase and a putative START lipid-binding domain is necessary to confer wheat rust resistance of Yr36 [43]. Wheat stripe rust resistance gene Yr15 (WTK1) [44] and barley (Hordeum vulgare L.) stem rust (P. graminis f. sp. tritici) resistance gene Rpg1 [45] contain a structure with tandem kinase domains. A maize wall-associated kinase protein (ZmWAK) was reported to confer quantitative resistance to maize head smut [46] and the PTI1-like kinase (ZmPti1a) was known to play an important role in the signaling pathway that facilitates pollen performance and male fitness [47].
Kinase proteins were also found to be important in F. graminearum. A MAP kinase gene (MGV1) in F.
(See figure on previous page.) Fig. 5 Haplotype analysis results. a Frequency distributions of the mean FHB severities for 171 cultivars with different haplotypes on chromosomes 4A and 5D. Gray, orange and red represent haplotype 1, haplotype 2 and haplotype 3, respectively. The x-axis exhibits 1-4 scores based on FHB severity (resistant, 0 < PSS ≤25%; moderately resistant, 25% < PSS ≤50%; moderately susceptible, 50% < PSS ≤75% and susceptible, 75% < PSS ≤ 100%). The y-axis represents the number of cultivars (also numbered on the bar) showing the FHB severity in different haplotypes. b Haplotype analysis of significant SNPs on chromosomes 4A and 5D. Solid bar plot displays average FHB severity of each haplotype. Gray, orange and red represent haplotype 1, haplotype 2 and haplotype 3, respectively. Left: Haplotypes of the significant SNPs based on 4A among wheat lines; right: Haplotypes of the significant SNPs based on 5D among wheat lines graminearum was required for much more developmental processes linked to sexual reproduction, plant infection, and cell wall integrity [48]. The glycogen synthase kinase gene orthologous to mammalian GSK3 was an significant virulence factor and Fgk3 glycogen synthase kinase was also important for growth, pathogenesis, conidiogenesis, DON production and stress responses in F. graminearum [49]. Taken the potential importance of kinase proteins in FHB resistance synthetic loci identified on 4AL and 5DL, the wheat homologs of PTI1-like tyrosine-protein kinase 1 and putative receptor protein kinase ZmPK1 might be considered as candidates of FHB resistance and need further characterization.

Conclusions
In the present study, we identified 26 FHB resistance loci using the wheat 90 K SNP assay, and four stable loci were detected in both seasons. Two new FHB resistance loci on 4AL and 5DL were found to be located on syntenic genomic regions, indicating that these regions contain important genes valuable for future research and breeding application. The SNP markers significantly associated with the FHB resistance could be used to develop diagnostic markers for marker associated selection of FHB resistance breeding.

Plant materials
An association panel comprising 171 wheat cultivars was used for SNP genotyping and 2 years FHB resistance phenotyping. Among them, three cultivars were derived from Italy, Mexico and Japan, and the other 168 cultivars were collected from 8 provinces at winter wheat region in Northern China and 9 provinces from Southern China (Additional file 1: Table S1)    The disease nursery was mist-irrigated for 5 min every 30 min from 7:00 am to 6:00 pm each day to ensure the inoculated spikes fully infected under high humid conditions [50]. The number of infected spikelets and the total number of spikelets of every tagged spike were recorded 25 days after inoculation. The average percentage of symptomatic spikelets (PSS) was calculated as the measure of FHB severity. All tested accessions were classified into four classes based on FHB severity, resistant (0 < PSS ≤25%), moderately resistant (25% < PSS ≤50%), moderately susceptible (50% < PSS ≤75%) and susceptible (75% < PSS ≤ 100%) [51].

Genotyping and SNP calling
Genomic DNA was extracted from fresh leaves of field grown non-infected plants at seedling stage using the CTAB method [52]. The association mapping population was genotyped from the wheat Illumina 90 K iSelect array with 81,587 SNPs (Wang et al. 2014) at the Biotechnology Center, Department of Plant Sciences, University of California, USA, using the Illumina SNP genotyping platform and BeadArray Microbead Chip [53]. To avoid spurious marker-trait associations (MTAs), SNP markers with minor allele frequencies (MAF) < 0.05 and missing data > 10% were excluded from subsequent analyses. The physical positions of SNP markers were obtained from Chinese Spring reference genome sequences at the International Wheat Genome Sequencing Consortium website (IWGSC, http://www.wheatgenome.org/).

Population structure analysis and linkage disequilibrium
Population structure was estimated using Structure 2.3.4 with 1676 polymorphic SNP markers distributing on all 21 wheat chromosomes with r 2 < 0.2, based on the Bayesian cluster analysis [54]. Six runs of Structure were performed with a K between 1 and 11, using the admixture model with 100,000 replicates each for burn-in and MCMC. The optimal K-value was determined using the ΔK method [55].Linkage disequilibrium (LD) among markers was computed by the full matrix and sliding window options in Tassel v5.0 with the filtered SNP markers. The pairwise LD between the markers was calculated using squared allele frequency correlations r 2 , according to Liu et al. [56].

GWAS for FHB resistance
Associations between genotypic and phenotypic data were analyzed using the kinship matrix in a Mixed Linear Model (MLM) by Tassel v5.0 to control background variation and eliminate spurious MTAs. The kinship matrix (K matrix) was considered as a random effect factor and the subpopulation data (Q matrix) was considered as a fixed-effect factor in the MLM analysis [57]. The calculation of K matrix and Q matrix was performed using the software Tassel v5.0 and the program Structure v2.3.4. The R 2 showing the variation explained by the SNP were recorded [58]. SNPs with an adjusted -log 10 (P-value) ≥3.0 were regarded as significant associated with FHB resistance. Significant SNP markers within one LD on the same chromosome were considered to represent one locus. Haplotype analyses of the significant SNPs were performed with Haploview v.4.2 [59].

Identification of candidate genes
To identify the candidate genes linked to significant SNPs, the physical positions of the markers preceded by the chromosome name were taken to Ensembl (https://urgi. versailles.inra.fr/gb2/gbrowse/wheat_survey_sequence_annotation), and the genes in the same genetic positions were considered. The intervals were then explored for predicted genes and annotations. For genes that are unavailable from the IWGSC annotations, we evaluated orthologous genes (proteins) in related species with reported predicted functions using the comparative genomics tool in Ensembl. When the genes had less than 70% similar ortholog in the annotated genomes of related species in Ensembl, the sequence of the T. aestivum gene was taken to search highly similar sequences using NCBI and basic local alignment search tool (BLAST) (http://blast. ncbi.nlm.nih.gov/Blast.cgi).
Additional file 1: Table S1 171 wheat accessions used in the genomewide association study (GWAS) for FHB severities and their origins,  Author's contribution WJH carried out the experiments and wrote the paper. DRG and HYW participated in the field trials and assisted in revising the paper. JL, CMZ, JCW, ZNJ, YYL and DSL, participated in the field trials. YZ and CBL designed the experiment and assisted in analyzing the data and writing the paper. All authors read the final version of this manuscript and approved it for publication.

Availability of data and materials
The phenotypic data of the current study is available in the Additional file 1: Table S1. The data sets supporting the results of this research could be obtained within the article and its additional files. Any other datasets used and/or analyzed are available upon request.

Ethics approval and consent to participate
We declare that these experiments comply with the ethical standards and legislations in China, and all wheat varieties were collected in accordance with national guidelines.

Consent for publication
Not applicable.