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Article

Marker Development and Pyramiding of Fhb1 and Fhb7 for Enhanced Resistance to Fusarium Head Blight in Soft Red Winter Wheat

by
Binod Gyawali
1,
Steven R. Scofield
2 and
Mohsen Mohammadi
1,*
1
Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA
2
Crop Production and Pest Control Research Unit, USDA-Agricultural Research Service, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Crops 2023, 3(4), 320-332; https://0-doi-org.brum.beds.ac.uk/10.3390/crops3040028
Submission received: 27 September 2023 / Revised: 26 October 2023 / Accepted: 24 November 2023 / Published: 7 December 2023
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Abstract

:
Fusarium head blight (FHB) is a devastating fungal disease of hexaploid wheat (Triticum aestivum). Several genetic loci were previously identified that control FHB resistance in wheat, including Fhb1. Fhb7, a major QTL conferring resistance to FHB, controlling for mycotoxin deoxynivalenol (DON) production, has been introgressed into soft red winter wheat (SRWW). As an exotic QTL, Fhb7 is associated with linkage drag, affecting agronomic and end-use quality performance. This study outlines a breeding strategy for introducing and pyramiding Fhb7 into SRWW breeding populations that already possessed Fhb1 and harbored some additional disease-resistance genes. In addition to the Fhb1-Fhb7 pyramiding, we developed gene-based markers for both genes and examined them on 57 SRWW breeding lines. Our data showed that 15 out of 57 breeding lines possessed both Fhb1 and Fhb7 resistant alleles. Two years of phenotypic data from the inoculated and misted irrigation field showed that the combination of Fhb1-Fhb7 lowers mycotoxin DON accumulation in kernels, which provides protection for end-users and the milling industry. The Fhb gene-pyramided lines, with the additional regionally important disease resistance genes, produced in this breeding pipeline showed reasonable agronomic traits and can be used in crossing programs for the widespread introgression in elite wheat cultivars.
Keywords:
FHB; Fhb7; GST; QTL; Thinopyrum

1. Introduction

Fusarium head blight (FHB), caused by the fungal pathogen Fusarium graminearum (Fg), is a serious threat to wheat production worldwide [1], which substantially reduces the grain yield and quality. Since F. graminearum is a hemi-biotrophic fungus, it penetrates the plant as biotrophs but later becomes necrotrophic. Because of its necrotrophic nature, the plant’s effector triggered immunity (ETI) cannot provide complete defense against F. graminearum. Resistance to FHB in wheat is quantitative and is controlled by minor genes; thus, it is very difficult, if not impossible, to develop complete resistance against F. graminearum. Infection by Fg starts with the bleaching of spikelets, that further results in the sterility or production of discolored and shriveled kernels, also referred to as Fusarium-damaged kernels (FDKs) [2,3]. Deoxynivalenol (DON) and nivalenol (NIV) are the primary trichothecene mycotoxins produced by F. graminearum that promote the fungal spread in wheat. Both DON and NIV are poisonous to humans and animals [2,4,5], and thus cause devastating economic losses.
The US Food and Drug Administration (FDA) has established guidelines for DON content in food products. FDA specifies a maximum of 1 part per million (ppm) DON in finished wheat products, e.g., flour, bran, and germ, and a maximum of 5 ppm DON in grains and grain by-products destined for swine consumption. In addition to the grain contamination, yield is also reduced by the FHB infection. In other cases, the presence of higher levels of DON in wheat grains leads to discounts in wheat price, causing financial loss for wheat growers [3], or even worse, the scabby wheat grains will not be purchased by millers for flour [4]. Continued crop failures due to FHB have driven many Minnesota wheat and barley farmers into bankruptcy [6]. Various approaches such as resistant cultivars and management practices can be deployed to lower the disease severity and DON contamination. The introgression of genes for biotic/abiotic stress resistance from exotic sources has been a common practice in wheat breeding for many years [7]. However, the introgression of genes from other species is associated with linkage drag for yield and end-use quality [8]. While existing germplasm in the soft red winter wheat region harbors multiple FHB-resistance quantitative trait loci (QTL), they still are not sufficient to maintain DON at a very low level [9]
Among the many QTL associated with wheat responses to FHB, Fhb1- and Fhb7-controlling type II resistance, which is the resistance to disease spread within the spike, have been intensively studied [10,11,12,13]. The Fhb1 resistance allele [14] from the Chinese cultivar ‘Sumai 3’ has been widely introduced and incorporated in diverse genetic backgrounds, including common and durum wheats worldwide [10,11,15,16,17,18,19]. The wild type (WT) Fhb1 gene encoding a putative nuclear-localized, histidine-rich calcium-binding protein corresponds to the locus TraesCS3B02G01990 in the reference genome sequence of Chinese Spring (CS), which also carries a 786 bp open reading frame [20,21]. The mutant Fhb1 gene (designated as Fhb1-R in this study) previously characterized in the cultivars Sumai 3 and Wangshuibai, carries a 752 bp deletion from the WT Fhb1 gene [20,21]. Studies suggest that Fhb1-R probably evolved only recently in East Asian countries (China and Japan) [20,21,22] and its frequency in worldwide common wheat germplasm has been estimated to be 5.2% or 24.9% [20,21,22]. Rawat et al. [23] found that the wheat PFT gene, predicted to encode a putative pore-forming toxin-like chimeric lectin protein, was suggested to be responsible for Fhb1-mediated FHB resistance [23]. However, a recent study [20] cannot confirm the role of this gene as the many wheat lines with functional PFT gene are susceptible to Fg infection. Together, these findings highlight the complexities in the function of Fhb1, which requires further studies to be resolved [24].
As wheat relatives, Th. elongatum and Th. ponticum are important genetic resources that can be used to improve wheat FHB resistance [13]. Thinopyrum species have been used to improve tolerance against abiotic stresses [25] as well as biotic stresses [26,27]. Many wheat–Thinopyrum derivatives have been developed and used as bridge materials for transferring valuable genes from Thinopyrum into common wheat. The Fhb7 locus on the distal end of 7E chromosome from Thinopyrum ponticum was introgressed in the 7D chromosome of wheat [28] and was validated by several studies [12,29]. The QTL mapped on the distal region on long arm of chromosome 7E [12], which was translocated to chromosome 7D of wheat [30] resulted in resistance to FHB. The introgressed alien chromatin region is not able to recombine when crossed with wild-type chromatin due to the lack of homologous pairing [31], and consequently, the long non-recombinant stretch of DNA might cause yield drag or quality deterioration that are associated with the chromosome segment inherited from Thinopyrum [32]. A series of wheat–Th. elongatum substitution, ditelosomic, and addition lines were reported and used for locating a novel FHB resistance gene on chromosome 7EL [26,33].
A recent study [30] sequenced the genome of Th. elongatum and cloned the glutathione S-transferase-encoding Fhb7 by genetic mapping. That study indicated that the Fhb7 transfer was without yield drag. The Fhb7 gene encoding a glutathione S-transferase was also involved in the xenobiotic detoxification of the trichothecene compounds as observed in Fhb1 [30]. Research efforts have been made to integrate Fhb7 gene into the wheat D genome through 7D–7E translocation from Th. elongatum and Th. ponticum, respectively [12,33,34]. Also, Fhb7 resistance has been successfully introduced into the wheat A genome via 7A–7E translocation originating from Th. ponticum [35]. The gene’s polymorphic nature highlights its adaptability to diverse environmental conditions and its potential role in fine-tuning FHB resistance responses. Moreover, the distribution patterns of GST-Fhb7 across different Triticeae species provide insights into its evolutionary significance and potential applications in breeding for FHB resistance [36].
We previously performed genome-wide association studies (GWAS) and evaluated FHB resistance in a SRWW breeding population, which showed the presence of QTL on chromosome 7D that regulates the DON level [9]. That study showed that the resistance coming from Thinopyrum elongatum in some germplasm was ambiguously located in one of the two QTL regions namely Q7D.1, and Q7D.2 on chromosome 7D [9]. However, we were unable to determine which one was Fhb7 and therefore could not identify which breeding lines possess the Fhb7 gene. The primary objective of this study was to shed light on the ambiguity of previous germplasm screening and identify the breeding lines that harbor both Fhb1 and Fhb7, by designing reproducible gene-based markers. The secondary objective was to further screen the lines for the leaf and stem rust resistance block (Lr19/Sr25) as well as barley yellow dwarf virus resistance gene (Bdv3) in the lines that are positive for both Fhb1 and Fhb7.

2. Materials and Methods

Germplasm development. Two heterogeneous early-generation breeding populations (07469 and 07117) segregating for Qfhs.pur-7EL, later named Fhb7 [12] and a type II FHB resistance QTL [28,29] were crossed with ‘Wheater’ (Figure 1). The wheat line ‘Wheater’, which carries a 7EL-7DL translocation from tall wheatgrass (Th. ponticum), which harbors a linkage block Lr19/Sr25 [37] conferring leaf and stem rust resistance. The 07469 population also carried a second 7EL-7DL translocation from intermediate wheatgrass (Th. intermedium), which harbors the barley yellow dwarf virus resistance gene, Bdv3. The heterogenous F1 materials were crossed with six adapted breeding lines or varieties, ‘1026A’, ‘1065RA’, ‘P25R62′, ‘1070RA’, ‘106A’, and ‘Roane’, that possess the resistant allele at Fhb1 (Figure 1). The crosses underwent five successive generations of backcrossing with the adapted recurrent parent to rebuild the regional adaptation and to remove the potential linkage drags on grain yield.
Sequence analysis and development of gene-based markers. To design the gene-based marker for Fhb7, approximately 57.5 kb long sequence of single BAC clone of B3227-3 [30] containing Fhb7 region was retrieved and subjected to the NCBI open reading frame (ORF) Finder available at https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/orffinder/ (accessed on 15 December 2022) with default parameters. Among the ORFs predicted, we selected the 846 bp long ORF, which encodes glutathione S-transferase (GST)—the reported candidate gene for Fhb7 [30]. Primers were designed to amplify the 822 bp of nucleotide from the GST ORF (Figure 2A).
To design the gene-based marker for Fhb1, the previously cloned sequence of the susceptible allele TaHRC-S as 2650-bp sequence from NIL-S (GenBank accession MK450309) and the resistant allele TaHRC-R as 2041-bp sequence from NIL-R (GenBank accession MK450312) were retrieved from Su et al. [20]. Clustal Omega software [38] was used for sequence alignment. For the amplification of the resistant allele, forward primer was anchored starting from 1138 bp of the ‘Ning7840’, which had 8 bp nucleotides mismatched in the ‘Clark’ allele (Figure 2B(a)). For the amplification of the susceptible allele, forward primer was anchored to 1424 bp of the susceptible allele (Clark), which had a 1 bp similarity and 18 bp deletion in the Ning7840 (Figure 2B(b)). Reverse primer for both resistance (Ning7840) and susceptible (Clark) sequences was designed by selecting a region that is common in both resistance (Ning7840) and susceptible (Clark) sequences (Figure 2B). Primers for both the Fhb1 and Fhb7 alleles were designed using the Primer3 tool [39]. The location of the primer sequences of the resistant allele of Fhb7 are depicted in Figure 2A. In addition, primer sequences for resistant as well as susceptible alleles for Fhb1 are depicted in Figure 2B(a)and Figure 2B(b), respectively.
DNA extraction and PCR diagnosis. In this study, 57 advanced breeding lines that had ‘7D(E)’ or ‘KS24-2-2(275-4)’ in their parentage among 436 SRW wheat germplasm were tested for presence of Fhb7. The genomic DNA was extracted from leaves of 7-day-old seedlings following the CTAB method [40]. The isolated DNA was quantified using a NanoDrop-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and diluted to 20 ng/µL. Polymerase chain reactions (PCRs) were performed using a BioRad thermal cycler model 563BR (BioRad, Hercules, CA, USA). Each reaction contained 2 µL of 10X PCR buffer, 0.2 μL of 10 mM dNTPs, and 0.2 μL (1U) of DreamTaq DNA polymerase (Thermo Fisher Scientific), along with 0.4 μL (10 µM) each of the forward and reverse primers. The denaturation was at 95 °C for two minutes, followed by 35 cycles of 94 °C for 30 s, annealing, and 72 °C for extension. Annealing temperatures (Ta), extension time and expected band size of each primer set are shown in Table 1. Amplified PCR products were separated through slow (60 V) agarose gel (2%) electrophoresis in 1X Tris-Acetate-EDTA (pH~8.0) buffer (Thermo Scientific). Primers used for the genotyping of Fhb1 and Fhb7 that were listed in Table 1 were designed in the current study, and those used for diagnosis of Sr25 and Bdv3 were used by earlier studies [41,42]. The mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.
Agronomic and Fusarium head blight resistance data. Agronomic and FHB phenotypic data for the current study are only the reanalysis of data we reported earlier [43] by comparing the phenotypes of the pyramided Fhb1-Fhb7 lines with the mean of the entire breeding population, comprising 392 lines. We were unable to make these comparisons in the earlier study due to the uncertainty in the Fhb7 genotypes. Briefly, experiments were conducted in standard breeding trials with 12 ft length and 4 ft width (~3 m × 1 m) for field testing in the 2017–18 and 2018–19 seasons in West Lafayette, Indiana, USA (40.47° N, 86.99° W, elevation 185 m. Grain yield (YLD), expressed in tons per ha were collected from each plot after adjusting to 13% moisture. The field inoculation was conducted using lab prepared F. graminearum-infested corn spawn, which was treated with nine isolates from Indiana, Illinois, and Ohio, as described by Gilbert and Woods [44]. The F. graminearum-infested corn spawn was applied at a rate of 40 g/m2 for approximately 2 to 3 weeks before the heading stage [43]. Disease severity (SEV) data were measured 21 days after anthesis as the percentage of infected spikelets within a spike averaged from 10 random spikes in the plot. The deoxynivalenol (DON) concentration was quantified using the gas chromatography–mass spectrometry (GC/MS) [45] method at the University of Minnesota Mycotoxin laboratory. Analysis of variance (ANOVA) for the grain yield was calculated in the R Studio v4.2.2 [46]. Detailed experimental designs about these studies are available in Gaire et al. [43].

3. Results

The aim of this study was to develop the gene-based markers and precisely screen the adapted SRWW that were created with the exotic Fhb7 and the prevalent Fhb1 gene. Among the 392 lines tested by [9], only 57 lines had ‘7D(E)’ or ‘KS24-2-2(275-4)’ in their parentage in their pedigrees. These lines were tested by PCR using gene-based markers for Fhb7 and Fhb1. The PCR diagnosis test by using PCR primer pairs developed in this study showed that 15 out of 57 advanced lines possessed Fhb7 (Table 2). The re-analysis provided below is only about the 15 selected lines from a previous study [43]. Additionally, the two pairs of primer pairs for the diagnosis of the Fhb1 resistance allele (specific to Ning7840), and presence of Fhb1 susceptible allele (specific to Clark) were successfully tested (Figure 3).
The re-analysis of ANOVA for the selected 15 lines showed significant effect of lines for grain yield (F = 4.042, p < 0.005). Unlike the claim of no yield penalty made by Wang et al. [30], the lines identified as positive for Fhb7 in this study showed a substantially lower yield than the highest yielding lines in the 2018–19 trials. We further calculated the average value of plant’s group with Fhb7 (3.57 tons/hac) as well as average value of plant’s group without Fhb7 (4.43 tons/hac). When conducting the one-way ANOVA for yield, two groups were on the border of significant differences (F1 = 4.119, p = 0.05201). The best agronomic performance in the 2018–19 trials was 9 tons per ha [43]. The grain yields of the 15 Fhb7-carrying lines are listed in Table 2. For comparison, we used two lines, i.e., ‘PU99646-7′ and ‘PU PU96134-1’, as an Fhb7-check, with Fhb1 and without Fhb7, and compared their SEV with these 15 lines. Tukey comparison was only able to identify PU10461-3 as being a better accession than the Fhb7-checks for SEV (Table 2). However, when we compared lines for DON content, there were few lines that exhibited significantly smaller amount of DON compared with Fhb7-checks (Table 2). Since these numbers are only preliminary, multilocation trails are required to further explore the effects of Fhb7 on grain yield.
In addition, these 15 lines showed superior FHB trait performance (Table 2). The combined year analysis of severity and DON for the 15 Fhb7-positive lines along with susceptible lines ‘PU99646-7′ as well as ‘PU PU96134-1′ are summarized in Table 2. PU10535-2, with the maximum yield, contained 1.92 ppm DON when grown and inoculated in an FHB-misted irrigation farm. Severity, FDK, and DON values for these lines are also at the lower tail of distribution observed from the 392 lines in the combined-year data analysis. The individual year severity and DON as well as the combined-year FHB data for the 15 Fhb7-positive lines are summarized in Table 2. Among the Fhb7-positive lines, PU10534-2 had the best resistance, with an average DON value of 1.04 ppm followed by PU10535-2, with a DON value of 1.92 ppm. Mean comparisons of SEV showed that both susceptible checks were significantly different with the rest of the 15 tested lines, whereas for DON, only the check PU99646-7 showed significant difference with tested lines.
Furthermore, the presence of alien stem rust and leaf rust resistance gene block Sr25/Lr19 originally from wheat relative Th. ponticum [47] and inherited from the Australian spring wheat variety ‘Wheater’ was verified in 13 of these lines (Table 2, Figure 3). PCR tests showed all 15 lines were positive for the presence of the Bdv3 gene (Figure 3). In addition, 13 lines carried the Sr25 gene, which is an additional disease package offered by these lines.

4. Discussion

The United States is one of the top three wheat exporter countries, accounting for approximately 6–7% of the global wheat exports. The soft red winter wheat class is grown on 6.86 million acres in the eastern USA, with a total production of 337 million bushels [48] of grains. FHB is a serious wheat disease particularly in the eastern USA that reduces yield, lowers quality, deteriorates kernels, and results in the accumulation of the mycotoxin DON. From the year 2003 to 2014, the annual average levels of DON found in USA wheat delivered to milling facilities was close to 0.5 mg/kg (or ppm), while DON levels in individual samples even exceeded 2 mg/kg [49], while the FDA has set the upper limit of 1 ppm (1 mg/kg) for finished wheat products. In the United States, a total of USD 1.176 billion in economic losses caused by FHB were reported in 2015 and 2016 [50]. These losses are anticipated to escalate due to more frequent and severe FHB outbreaks caused by rising temperatures and humidity levels.
There has been significant progress in improving resistance to FHB in winter wheat [51] over a 20-year period (1998–2018). This suggests that ongoing investment in wheat breeding for FHB resistance is effective and will continue to be important for decreasing FHB levels for growers and DON levels for end-users. Genetic Trend analyses on breeding trial data are helpful to track the progress of breeding programs [52], including FHB resistance, and determine if the investments in breeding are producing the desired results. To further lower the impacts of FHB disease, researchers have been extensively studying new genes that provide resistance to FHB from wild relatives of wheat, to improve the diversity and effectiveness of resistance to FHB in wheat [30,53,54]. This is the first study to report the pyramiding of Fhb1 with Fhb7 genes in soft red winter wheat in the eastern United States. The expectation is that the stack of Fhb1-Fhb7 in our lines will provide a better resistance package against FHB. These lines are also unique as they are the only source of Fhb7 germplasm adapted to the SRWW region and breeding history.
Fhb1 (syn Qfhs.ndsu-3BS), the most significant quantitative trait locus (QTL), was shown to provide a reasonably high level of genetic resistance against FHB [11,55], which has been effective across various genetic backgrounds [56]. Another study [15] showed that the Fhb1 locus decreased the severity of the FHB disease by 23%. Both in greenhouse and field circumstances, the Fhb1 resistance allele had a consistent impact on lowering the severity of FHB, with most Fhb1-carrying lines displaying consistent resistance to FHB [11]. After introducing Fhb1 from ‘Ning 7840′ (a ‘Sumai 3′ derivative), into the susceptible SRWW line IL89-7978, the percentage of scabby spikelets reduced dramatically from 70–80% to 30–40% [16]. Zhang et al. [17] found that for backcross progenies carrying the Fhb1 resistance allele, the mean number of diseased spikelets was 8.1%, and the disease index was 28.4% lower than in the recurrent parent. While successful in many genetic backgrounds, not all Fhb1-R-carrying lines exhibit type II FHB resistance. Nearly 36% of them are susceptible to FHB disease, probably caused by inhibitor genes [24]. Previous studies [15,18] show that transferring Fhb1 into commercial wheat varieties does not always result in FHB resistance. Su et al. [20] reported that about 10.6% of common wheat lines carrying Fhb1-R allele did not show the expected type II FHB resistance, indicating the need for incorporating novel resistance genes in addition to Fhb1.
It was shown that the Fhb7 significantly reduced the amount of Fusarium biomass in blighted kernels resulting in the reduction in DON contamination by up to 85% as compared to susceptible sibs [35]. A previous study from our group also showed that the pyramiding of Fhb1 and Fhb7 loci had no additional benefit for the severity; however, it resulted in fewer symptoms of FDK and DON [9]. This study shows additional benefits on severity as the Fhb7 and Fhb1 pyramided lines were significantly different than those susceptible checks for severity. A study conducted by Wang et al. [30] indicated that Fhb7 transfer was without yield drag. Recently, by backcrossing the FHB-resistant lines with the main cultivar Jimai 22, three wheat–Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, were successfully applied in wheat breeding without yield penalty [13]. We observed small degrees of yield penalty in some of the lines that were Fhb7-positive, although yield in the group that had Fhb7 as well as the group that did not have Fhb7 were not significantly different. This yield drag can be partially, if not totally, minimized by multiple backcrosses [33]. Techniques such as ph1b mutants [57] and disomic substitution lines [58,59] mediated by the gene Ph1b/Ph2 has been frequently used to enable non-homologous recombination in situations like this [34,60]. However, given the agronomic status of the lines produced in this breeding program, producing large breeding populations by crossing these 15 lines to the highest yielding lines of the region should be the first line of action to offset the yield penalty.
Since the cloning of Fhb7 in 2020 [30], gene-based markers are not available for the rapid and easy screening of the lines suspected to carry Fhb7. In this study, we retrieve the coding sequences of GST and designed the marker within the ORF. The development of gene-based Fhb7 markers will aid in the deployment of Fhb7 in wheat breeding programs to improve FHB resistance as well as marker-assisted selection. Furthermore, for the efficient amplification and the screening of Fhb1, Su et al. [61] proposed the markers for both Fhb1-R and Fhb1-S, considering TaHRC as the candidate For Fhb1 [20,21], but those markers are hard to reproduce as they have their own technical difficulties. Because of the unavailability of the efficient and highly reproducible gene-based markers for TaHRC (Fhb1), we designed Fhb1 markers for both resistant and susceptible alleles. The designing of the markers for Fhb1 will further aid in the rapid screening of the germplasm as well as the marker-based selection in the wheat breeding programs to improve FHB resistance.
Barley yellow dwarf virus (BYDV) disease is caused by Luteovirus genus transmitted by aphids, Rhopalosiphum padi and is a globally important disease of wheat [62]. BYDV causes wheat plants to produce fewer tillers, fewer seeds per tiller, and lighter seeds [63,64]. Wheat yield losses have been found to range widely between 11 and 33 percent [65], as high as 40 to 50 percent [66], and sporadically as high as 80 percent [65]. A base level of BYDV infection that causes an unperceived loss of 1% to 5% appears to exist even in years where there is not a significant BYDV outbreak in the US [6]. Additionally, the Sr25 [37] gene was introduced into wheat from Thinopyrum ponticum, which possesses the resistant linkage block Lr19/Sr25 [37]. The presence of these disease resistance genes along with the Fhb1-Fhb7 stack should provide useful resistance to a suite of diseases that tend to reduce wheat yield. To appreciate the value of this level of disease resistance protection, it is worth noting that in the US, a 1% decrease in wheat yield could result in a loss of USD 123 million at the current price.

5. Conclusions

In this study, we successfully developed the gene-based markers for both Fhb1 and Fhb7, which are the key wheat genes for the resistance to the Fusarium head blight. These markers will help in the deployment of the Fhb1 as well as Fhb7 for the wheat breeding associated with the FHB management. We further screened our soft red winter wheat lines for the pyramiding of both genes and found that 15 of the lines had both genes. In addition, we also identified additional disease resistance packages having Bdv3 and Sr25 genes in our germplasm.

Author Contributions

Conceptualization, M.M.; methodology, M.M. and B.G.; software, M.M. and B.G.; validation, M.M., B.G. and S.R.S.; formal analysis, B.G. and M.M.; investigation, M.M. and B.G.; resources, M.M. and S.R.S.; data curation, B.G. and M.M.; writing—original draft preparation, B.G. and M.M.; writing—review and editing, M.M. and S.R.S.; visualization, M.M. and B.G.; supervision, M.M. and S.R.S.; project administration, M.M.; funding acquisition, M.M. and S.R.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the USWBSI agreement 59-0206-0-142 and USDA Hatch grant 1013073 via Purdue College of Agriculture; U.S. Wheat and Barley Scab Initiative grant number 17000549. Also, SRS is supported by USDA-ARS number: 5020-21220-014-000D. The USDA is an equal opportunity employer.

Data Availability Statement

Data are contained within the article.

Acknowledgments

Additional financial aid and research support through College of Agriculture, USDA Small Grains Genotyping Laboratory, Raleigh, NC and the University of Minnesota DON Testing Lab is greatly appreciated.

Conflicts of Interest

Authors have no conflict of interest to report.

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Figure 1. The breeding history of integration of multiple disease resistance genes in soft red winter wheat germplasm already harboring Fhb1.
Figure 1. The breeding history of integration of multiple disease resistance genes in soft red winter wheat germplasm already harboring Fhb1.
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Figure 2. Alignment of nucleotide sequences as well as primer designing for the Fhb7 and Fhb1. (A). GST ORF as Fhb7 gene and the location of the primer sequences. (B(a)). Primer designing and location of the TaHRC_R from Ning7840 and its comparison with Clark. (B(b)). Primer designing and location of the TaHRC_S from Clark and its comparison with Ning7840. Sequence of primers for each of the resistant as well as susceptible alleles are underlined within the given sequences. “*” is alignments means homology.
Figure 2. Alignment of nucleotide sequences as well as primer designing for the Fhb7 and Fhb1. (A). GST ORF as Fhb7 gene and the location of the primer sequences. (B(a)). Primer designing and location of the TaHRC_R from Ning7840 and its comparison with Clark. (B(b)). Primer designing and location of the TaHRC_S from Clark and its comparison with Ning7840. Sequence of primers for each of the resistant as well as susceptible alleles are underlined within the given sequences. “*” is alignments means homology.
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Figure 3. Gel electrophoresis images for the amplification of the five alleles (Fhb7, Fhb1_R, Fhb1_S as well as Bdv3 and Sr25) in our soft red winter wheat (SRWH) lines already harboring Fhb1-R allele. 15 tested lines are represented from number 1 to 15. The presence of the genes followed by gel electrophoresis is indicated by the (+) sign, while absence is indicated by the (−) sign. Water template was used as negative control (NC), while the wheat lines Apogee and Yekora were negative control for the Fhb7 as well as Fhb1-R, but the positive control for the Fhb1-S which is known to have a susceptible allele of the TaHRC.
Figure 3. Gel electrophoresis images for the amplification of the five alleles (Fhb7, Fhb1_R, Fhb1_S as well as Bdv3 and Sr25) in our soft red winter wheat (SRWH) lines already harboring Fhb1-R allele. 15 tested lines are represented from number 1 to 15. The presence of the genes followed by gel electrophoresis is indicated by the (+) sign, while absence is indicated by the (−) sign. Water template was used as negative control (NC), while the wheat lines Apogee and Yekora were negative control for the Fhb7 as well as Fhb1-R, but the positive control for the Fhb1-S which is known to have a susceptible allele of the TaHRC.
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Table 1. Primer oligo sequences and PCR parameters for loci studied.
Table 1. Primer oligo sequences and PCR parameters for loci studied.
LocusMarker NamePrimers (5’-3’)Expected Amplicon SizeTa (°C)Extension TimeReference
Fhb7GSTF-CACCTCCACCCCAATCATCT822 bp561 minThis study
R-ACCTCGGCATACTTGTCCAG
Fhb1TaHRC_RF-TTGCTGGGGAGAGGAAGAAA892 bp561 minThis study
R-TTCAGCAGAGTTCGCACGAT
Fhb1TaHRC_SF-GGTAGGATCTGAATGCTTAG1132 bp501 min 20 sThis study
R-GATCATGATGGTGATGGTG
BYDBdv3F-CTTAACTTCATTGTTGATCTTA164/206/288 bp5230 s[42]
R-CGACGAATTCCCAGCTAAACTAGACT
Sr25BF145935F-CTTCACCTCCAAGGAGTTCCAC198 bp5430 s[41]
R-GCGTACCTGATCACCACCTTGAAGG
Table 2. Presence (+) and absence (−) of Fhb1, Fhb7, Bdv3, and Sr25 in the breeding lines along with their two-year average yield, severity, and DON data.
Table 2. Presence (+) and absence (−) of Fhb1, Fhb7, Bdv3, and Sr25 in the breeding lines along with their two-year average yield, severity, and DON data.
Line ID Fhb1Fhb7Bdv3Sr25 Yield (tons/ha) SEV% DON (ppm)
PU10535-1++++4.16 ± 0.57 ab39.15 ± 6.11 ab3.75 ± 1.67 bc
PU10461-1++++3.21 ± 0.88 ab34.99 ± 0.97 ab4.28 ± 0.84 bc
PU10534-1++++4.22 ± 0.31 ab26.06 ± 4.96 ab2.84 ± 1.21 c
PU10535-2++++5.28 ± 0.39 a52.98 ± 1.55 ab1.92 ± 0.35 c
PU10534-2++++3.79 ± 0.58 ab38.79 ± 13.38 ab1.04 ± 0.54 c
PU10642-1++++2.77 ± 0.52 ab51.34 ± 04.6 ± 0 bc
PU10642-2++++3.37 ± 0.81 ab43.79 ± 11.62 ab6.32 ± 2.19 bc
PU10642-3++++2.27 ± 0.19 b35.56 ± 0 ab4.05 ± 0 c
PU10548-1+++−2.23 ± 0.50 b40.51 ± 0.58 ab3.31 ± 0.98 bc
PU10642-4−+++3.64 ± 0.82 ab38.2 ± 0 ab4.29 ± 0 bc
PU10534-3++++4.12 ± 0.39 ab45.37 ± 18.09 ab3.29 ± 0.78 bc
PU10461-2++++2.94 ± 0.66 ab33.51 ± 6.45 ab7.16 ± 0 abc
PU10461-3+++−3.96 ± 0.30 ab27.12 ± 9.4 b2.99 ± 1.19 c
PU10461-4++++5.45 ± 0.45 a30.58 ± 0.45 ab6.36 ± 2.19 abc
PU10535-5++++2.23 ± 0.31 b45.56 ± 0 ab4.89 ± 2.13 bc
PU99646-7+−NANA3.89 ± 0.43 ab56.58 ± 3.55 a12.78 ± 0.55 a
PU96134-1+−NANA5.62 ± 0.63 a63.89 ± 1.89 a10.21 ± 2.50 ab
Sr genes are stem rust resistance genes that are resistant against the TTKS Ug99 race of stem rust. Bdv3 is the resistance gene against the barley yellow dwarf virus of wheat. Grain yield is expressed in tons/ha. FHB severity is expressed in percentage. DON content is expressed in parts per million. Lines sharing same letters have no significant differences. Lines not sharing letters showed significant differences at p-value = 0.05.
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Gyawali, B.; Scofield, S.R.; Mohammadi, M. Marker Development and Pyramiding of Fhb1 and Fhb7 for Enhanced Resistance to Fusarium Head Blight in Soft Red Winter Wheat. Crops 2023, 3, 320-332. https://0-doi-org.brum.beds.ac.uk/10.3390/crops3040028

AMA Style

Gyawali B, Scofield SR, Mohammadi M. Marker Development and Pyramiding of Fhb1 and Fhb7 for Enhanced Resistance to Fusarium Head Blight in Soft Red Winter Wheat. Crops. 2023; 3(4):320-332. https://0-doi-org.brum.beds.ac.uk/10.3390/crops3040028

Chicago/Turabian Style

Gyawali, Binod, Steven R. Scofield, and Mohsen Mohammadi. 2023. "Marker Development and Pyramiding of Fhb1 and Fhb7 for Enhanced Resistance to Fusarium Head Blight in Soft Red Winter Wheat" Crops 3, no. 4: 320-332. https://0-doi-org.brum.beds.ac.uk/10.3390/crops3040028

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