Effectiveness of multigenerational transfer of Sumai 3 Fusarium head blight resistance in hard red spring wheat breeding populations

Abstract: Several quantitative trait loci (QTL) have been identified for Fusarium head blight (FHB) resistance in the cultivar Sumai 3. Wheat breeders need to know which Sumai 3 loci are present in derived lines used as parents for effective marker-assisted selection for genetic improvement. This study was conducted to identify the loci in Sumai 3 derived parents that contribute FHB resistance in breeding populations. Three doubled haploid (DH) populations utilizing Sumai 3 derived parents, ND3085, ND744, and Alsen, were evaluated during 2007 and 2008 in FHB nurseries near Carman, MB, Ottawa, ON and Charlottetown, PE. The percentage of incidence, severity, Fusarium-damaged kernels (FDK), and deoxynivalenol (DON) accumulation were measured, and FHB index calculated. DNA markers at six FHB resistance loci detected in Sumai 3 were evaluated on the populations. For each trait, a t test was applied to means of observations pooled by parental type of each marker to determine which loci contributed to resistance. The alleles at 3BS and 5AS most frequently contributed to Type I and Type II FHB resistance, as well as to reduced FDK and DON in all three populations. Markers revealed resistance on 3BS and 5AS in Alsen, ND3085, and ND744, on 3BSc, 4D, and 6BS in ND744, on 4D in ND3085, and on 6BS in Alsen. In some environments, the susceptible parent Infinity contributed minor QTL on 2D, 3BSc, and 6BS. Likewise, Helios contributed minor QTL on 5AS and 6BS.


Introduction
Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe [teleomorph: Gibberella zeae (Schwein.) Petch] is one of the most significant limitations to wheat production in Canada and the world (Gilbert and Tekauz 2000). While the disease causes severe yield reduction and grain quality losses the biggest threat to both humans and livestock is the mycotoxin deoxynivalenol (DON); a toxic compound that may be found in Fusarium-infected grain (Charmley et al. 1994). Using resistant varieties is considered the most effective component of an integrated control strategy for the long-term management of FHB (Buerstmayr et al. 1999).
Resistance to FHB is the result of a complex relationship among several resistance components (Ban 2000). Resistance to initial infection (Type I), resistance to the spread of infection within the spike (Type II), and resistance involving mycotoxin degradation (Type III) have drawn most of the attention of breeders. Therefore, considerable effort has been extended by breeders to identify appropriate cultivars carrying these FHB resistance components. FHB Type I and II resistance were identified in Asian spring wheat sources such as Ning 894037, Sumai 3, Wangshuibai, and Wuhan 1, and Type III resistance was identified in the South American spring wheat cultivar Frontana (Buerstmayr et al. 1996;Shen et al. 2003a;Somers et al. 2003;Ma et al. 2006).
Among the highest levels of resistance to FHB occur in the Chinese cultivar Sumai 3 (Zhou et al. 2010). Many studies confirm the existence of FHB resistance quantitative trait loci (QTL) in Sumai 3 (Bai et al. 1999;Anderson et al. 2001;Buerstmayr et al. 2002;Zhou et al. 2002). Sumai 3 has been extensively used, along with its derivatives, as a major FHB resistance source in breeding programs globally, because its Type II resistance is among the highest and most stable (Li and Yen 2008).
Improving resistance to FHB in wheat is challenging to breeders because it is quantitatively inherited, being controlled by multiple genes that are environmentally dependent, thus demonstrating inconsistent expression across environments. Over 40 QTL mapping studies have been conducted for FHB resistance that have identified over 200 QTL that are distributed across every chromosome (Buerstmayr et al. 2009;Liu et al. 2009). Despite the large number of QTL identified and the mapping work reported on FHB resistance, little work has been done on the transferability of Sumai 3 derived resistance loci and assessment of the impact on cultivar development has been minimal. The validation of the presence and expression of FHB resistance loci is a prerequisite to marker-assisted selection (MAS). The identification and assembly of validated resistance loci enables pyramiding of suitable alleles in targeted breeder germplasm with which to enhance resistance in elite cultivars.
A second major FHB resistance QTL was identified by Buerstmayr et al. (2002) in the distal region of chromosome 5A, Qfhs.ifa-5A, in CM82036 (a Sumai 3-derived line) in an interval flanked by simple sequence repeat (SSR) markers Xgwm293 and Xgwm304. This QTL on 5A was reported to be associated mainly with Type I, but also with Type II and Type III resistance in diverse sources, including the Sumai 3 related lines DH181 and W14, the Japanese landrace NyuBai, the Brazilian cultivar Frontana, and the soft red winter wheat cultivar Ernie (Buerstmayr et al. 2003;Somers et al. 2003;McKendry et al. 2004;Steiner et al. 2004;Yang et al. 2005;Chen et al. 2006).
The third major QTL for FHB resistance was located on chromosome 6BS. It was reported to be associated with Type I and Type II resistance in Chinese sources including Sumai 3 and also in Frontana (Waldron et al. 1999;Anderson et al. 2001;Shen et al. 2003b;Lin et al. 2004;Yang et al. 2005). This QTL flanked by markers Xgwm133 and Xgwm644 was mapped qualitatively and renamed Fhb2 (Cuthbert et al. 2007).
While the QTL on 3BS, 6BS, and 5AS have been the most effective and the most characterized, FHB resistance QTL on 2D and 4D have also been identified and appear to have different effects on FHB resistance. He et al. (2016) reported in the Soru#1/Naxos cross a major QTL on 2DL (centromeric region) conditioning resistance to initial infection, and a minor QTL on 2DL (distal region) and 4DS significant for DON and Fusariumdamaged kernels (FDK) reduction. McCartney et al. (2016) also identified FHB resistance QTL located on both short and long arms of chromosomes 2D and on 4D in Kenyon/86ISMN 2137. In a Sumai 3 derived doubled haploid (DH) population, Suzuki et al. (2012) reported the effect of a Sumai 3 allele in the 2DL chromosome arm on decreasing FHB severity. Yang et al. (2005) showed a resistance effect in the 2DS genomic region near the centromere from a cross involving a line designated DH181, which is derived from Sumai 3. In the region of the SSR marker Xgwm539, a significant contribution to Type I resistance was observed, and in the region of the SSR marker Xwmc144, a significant contribution to Type II resistance was observed. The same 2D locus is involved in epistasis for resistance to initial infection (Yang et al. 2005). The DH181 line also contributed a resistance allele at the 2DL genomic region, which reduced FDK, and at the 4DL genomic region, which reduced FHB initial infection and FDK (Yang et al. 2005). In contrast, through the development of substitution lines for specific chromosomes using Sumai 3 as the FHB resistance donor, Zhou et al. (2002) reported that chromosomes 2DL and 4DL from Sumai 3 increased FHB susceptibility and, more precisely, DON concentration. A major QTL on 2DS restricting FHB spread was identified from the populations of Sumai 3/Gamenya and Nobeokabouzu-komugi/Sumai 3 in which resistant 2DS alleles were derived from the susceptible parents Gamenya and Nobeokabouzu-komugi and not from Sumai 3 (Xu et al. 2001;Shen et al. 2003a;Handa et al. 2008). Handa et al. (2008) explained that Sumai 3 possesses a susceptible allele at the 2DS QTL, coding for a multidrug resistance-associated protein TaMRP-D1, which is reported to be involved in DON sequestration. On the other hand, from the same cross, Sumai 3/Gamenya, Handa et al. (2008) also identified a field response FHB QTL, including Type I and Type II resistance, associated with Xgwm539 on chromosome 2DL. More recently, Basnet et al. (2012) further showed that Sumai 3 contains a QTL for susceptibility on chromosome 2DS and that selection against this QTL may potentially increase resistance levels among Sumai 3 derived genotypes.
On the short arm of chromosome 3B proximal to the centromere (3BSc), a minor FHB resistance QTL was reported (Buerstmayr et al. 2009) in several studies of diverse sources of Asian spring wheat (Sumai 3, Wangshuibai, and Nyu Bai), as well as in soft red winter wheat from the United States (Ernie) ). This QTL is also likely stable for Type II resistance, but with a much smaller effect than Fhb1 (Zhang et al. 2012).
Efforts were made to develop Fusarium resistant cultivars at North Dakota State University (NDSU) by transferring FHB resistance from Sumai 3. The hard red spring wheat (HRSW) cultivars Alsen (Frohberg et al. 2006), ND3085 (Garvin 2002) and ND744 (Mergoum et al. 2005) were derived from crosses involving Sumai 3 or resistant derivatives. Alsen was the first NDSU spring wheat cultivar released with good FHB resistance, especially the Type II resistance probably inherited from Sumai 3 (Mergoum et al. 2005). ND744 and ND3085 combine a high level of FHB resistance with other desirable traits. They showed great potential as cultivars and have been of interest in different breeding programs worldwide where FHB is a concern.
Despite the genetic gain and the improved FHB resistance in derived cultivars, Sumai 3 remains a more resistant cultivar. Indeed, the introgression and expression of resistance loci is challenging due to the complex, multigenic, and quantitative inheritance of FHB resistance that includes the influence of the genetic background of the recipient genotype. When multiple genetic loci are involved in resistance, it is possible that the genes will be broken up and not all recombined in progeny within a desirable adapted background (Knox et al. 2008). The way to know which loci were passed from the resistant source to the progeny is to track their transfer and test for the presence and expression of resistance alleles at contributing loci. For molecular markers to be effective and useful for breeding, the loci they track must be validated by examining their segregation with FHB resistance within different genetic backgrounds of a breeding program's germplasm pool. This study was conducted to evaluate the effectiveness of the transfer and expression of FHB resistance loci from Sumai 3 through the derived parents, ND3085, ND744, and Alsen, in three breeding populations. We studied the effectiveness of the transfer of six Sumai 3 FHB resistance QTL using nine molecular markers by partitioning FHB incidence, severity, and index, FDK, and DON accumulation effects in the progeny of crosses involving three Sumai 3 derived cultivars.

Plant material
The transmission of FHB resistance QTL was assessed using three DH populations of wheat (Triticum aestivum L.) developed from crosses Infinity/ND3085, Infinity/ND744, and Alsen/Helios using the wheat × maize hybridization method (Knox et al. 2000). Alsen is a NDSU HRSW cultivar released with good FHB resistance (Frohberg et al. 2006). Alsen was derived from the three-way cross ND674// ND2710/ND688, where the line ND2710 is derived from a cross involving Sumai 3. ND3085 is derived from the cross ND2891/BW349 (Garvin 2002) where ND2891 is derived from a cross involving Sumai3. ND744 is a HRSW developed at NDSU with good FHB resistance (Mergoum et al. 2005). ND744 is derived from a threeway cross, ND2831/Parshall//ND706. Sumai 3 is in the background of ND2831. Infinity is a HRSW developed at the Swift Current Research and Development Centre (SCRDC), Agriculture and Agri-Food Canada (AAFC), Swift Current, SK (DePauw et al. 2006), and is susceptible to FHB. Helios is a HRSW released from SCRDC ) that has an intermediate resistant reaction to FHB.

FHB assessment
Eighty lines per population and three sets of each parental check cultivar were evaluated for incidence (Type I) and severity (Type II) FHB resistance in a randomized complete block design. Trials were grown in two FHB screening nurseries near Carman, MB [AAFC and the University of Manitoba (U of M)], a nursery in Ottawa, ON, and a nursery near Charlottetown, PE, in 2007. In 2008, trials were grown in the same locations except AAFC Carman. The experiments were replicated twice in each location except Charlottetown, where the experiments were replicated three times. Fifty seeds of each entry were planted in single-row plots.
At the Charlottetown nursery, a suspension of five F. graminearum isolates adjusted to 50 000 macroconidia mL −1 was applied to individual rows weekly for 3 wk following anthesis. To maintain high humidity after inoculation, the nursery was mist-irrigated using nozzles located 3 m apart in the row and 4.9 m between nozzle rows. About 650-700 L ha −1 were applied during these different spraying time intervals: every 30 min from 0700hr to 1000hr, every 15 min from 1000hr to 1900hr, every 30 min from 1900hr to 2200hr, and every hour 2200hr to 0700hr.
At the Ottawa nursery, 50 g of barley and corn kernels colonized by a mixture of three isolates of F. graminearum (DAOM232369, DAOM 212678, and DAOM 178146) were scattered evenly by hand between alternating rows, 3 and 2 wk before anthesis. Sprinkle irrigation was applied for about 30 min each morning and afternoon (excluding days with rain), starting at the first inoculation with infested kernels and continuing until about 3 wk after anthesis, when plants were at the soft dough stage.
Fusarium head blight incidence (Type I) and severity (Type II) were evaluated 18-21 d after inoculation. The incidence was calculated as the percentage of infected spikes in the row. Severity was calculated by estimating the percentage of infected spikelets in the infected spikes. The FHB index was calculated using the formula (incidence × severity)/100. Fusarium-damaged kernels for each line were determined as the mass proportion of visually infected kernels in a 15 g sample from handthreshed and cleaned spikes. For each location (except AAFC Carman in 2007), DON accumulation was measured on each line from a bulk subsample of the replicates by using the immunochemical enzyme-linked immunosorbent assay test.

Genotyping
Genomic DNA was extracted from parental and DH lines as described by Knox et al. (2014). The DH lines were genotyped in the chromosomal regions defining reported FHB resistance QTL on chromosomes 2DL, 3BS, 3BSc, 4DL, 5AS, and 6BS with SSR and sequence-tagged site (STS) markers previously reported as associated with Sumai 3 FHB resistance (Liu and Anderson 2003;Somers et al. 2004;Cuthbert et al. 2007) or proximal to the reported markers selected based on map information , GrainGenes) (Supplementary Table S1 1 ). The markers were first evaluated on Infinity, ND3085, ND744, Alsen, and Helios for polymorphism, and the appropriate markers were applied to each population. The SSR marker Xgwm349 was used for genotyping the chromosome 2DL QTL region. This marker was selected based on its proximity (2 cM)  to Xgwm539, which was the marker previously mapped around the FHB resistance QTL identified on chromosome arm 2DL in Sumai 3. Five SSR markers, Xgwm285, Xgwm566, Xwmc471, Xwmc418, and Xwmc653, were used to genotype the 3BSc region. Four markers, Xgwm493, Xgwm533.2, Xwmc505a, and STS marker STS3B-66, were used to genotype the 3BS QTL region. One SSR marker, Xwmc48, for the chromosome arm 4DL QTL region was chosen based on map information for its proximity to Xwmc331, the marker previously reported by Yang et al. (2005) to be associated with FHB Type I resistance in a cross involving a Sumai 3 derived line. Two SSR markers, Xgwm293 and Xgwm304a, for the chromosome 5AS QTL region and two SSR markers, Xwmc397 and Xgwm608, for the chromosome 6BS QTL region were evaluated. The genotypic data was used to classify lines of each population by parental-type molecular variant.

Statistical analysis
Using the SAS program version 9.3 (SAS Institute Inc., Cary, NC) PROC MIXED procedure, the least squares (LS) means for each of the FHB measures was calculated for each population and the associations between individual markers and disease traits were tested with a t test. The effect on phenotype of loci identified as being associated to FHB resistance was determined by quantifying disease measures across the lines of each population for each molecular variant of each marker. Analysis of variance (ANOVA) was also carried out with the PROC MIXED procedure. The ANOVA results were used for calculating the heritability estimates, using the formula g stands for genetic variance, σ 2 g×y for genotype × year interaction, σ 2 g×y×l for genotype × year × location interaction, σ 2 e for residual variance, y for the number of years, l for the number of locations, and r for the number of replications. The heritability for DON was calculated using the formula h g×y =y þ σ 2 e =ylÞ, in which σ 2 g stands for genetic variance, σ 2 g×y for genotype × year interaction, σ 2 e for residual variance, y for the number of years, and l for the number of locations.

Results
The means for the different FHB measurements of parental and population lines and population ranges are presented in Tables 1 and 2. Infinity was the most susceptible parental cultivar. In at least one environment for each FHB response measured, Infinity was significantly (P ≤ 0.001) more susceptible than either ND3085 or ND744. For the U of M location in Carman in 2007 and 2008, Infinity was significantly higher than ND3085 and ND744 for all traits measured except incidence in the Infinity/ND744 test 2007 (Table 1). In each location for each measurement, Infinity usually had a higher rating than Helios. Helios was either similar to or somewhat more susceptible than Alsen, depending on the environment, being significantly higher than Alsen (P ≤ 0.05) for some traits in at least one environment (Table 1). Considering the population means, frequently a higher level of infection was observed in the Infinity/ ND744 population compared with the Infinity/ND3085 and Alsen/Helios populations ( Table 2). The different environmental conditions among and between years and locations generated differences in FHB response of the parents and population lines. For example, in 2007 the FHB-severity population mean for Infinity/ND3085 was 10.9% in the U of M Carman trial and 57.3% in AAFC Carman (Table 2), and the percentage of FDK in Infinity/ ND744 varied from 9.5% in 2007 to 32.5% in 2008 in U of M Carman (Table 2).
In each environment, the measured FHB components (incidence, severity, index, FDK, and DON) showed a similar trend. A resistant reaction is indicated by low levels of infection and a susceptible reaction is indicated by high levels of infections for all traits. Disease index reflected both incidence and severity, while DON content was more related to the percentage of FDK.
Analysis of variance revealed highly significant genotypic variation in the three populations for FHB incidence, severity, index, FDK, and DON. Genotype × location and genotype × year × location interactions were also significant for FHB incidence, severity, index, and FDK (Tables 3 and 4). Heritabilities were estimated on the three populations for all FHB components. Moderate to high heritability estimates were obtained for the different FHB traits in the different populations (Tables 3 and 4).
Significant t test values for the differences in disease reaction between the lines carrying the marker molecular variant of the more resistant parent and the lines carrying the marker molecular variant of the more susceptible parent for each QTL were observed (Tables 5-10). Significant effects were revealed at six loci in the Infinity/ND3085, five loci in the Infinity/ND744, and three loci in the Alsen/Helios populations. The presence of resistance alleles that significantly lowered disease levels were contributed by the resistant parents, but also by the most susceptible parent Infinity.

3BS genomic region
Two markers, gwm493 and wmc505a, were polymorphic in the Infinity/ND3085 and Infinity/ND744 populations, and two markers, gwm533.2 and STS3B-66, in the Alsen/Helios population. ND744 variants for both 3BS markers contributed to FHB resistance in the Infinity/ ND744 population. In 2007, the most consistent significant associations were with wmc505a for severity and index at Ottawa, and with gwm493 for FDK at Ottawa and Charlottetown (Table 7). In 2008, the gwm493 marker showed the highest (P > 0.001) and most consistent associations with severity, index, and FDK at the U of M (Carman) and with DON in Charlottetown (Table 8). In the Alsen/Helios population, Xgwm533 at the 3B locus was predominately the marker associated with FHB resistance over all environments. The Alsen variant of Xgwm533 repeatedly was significantly associated with resistance including incidence, index, and FDK at Carman, severity and index at Ottawa in 2007, and FDK at Charlottetown in 2008 (Tables 9  and 10).
Alsen also contributed the STS3B-66 resistant allele that significantly reduced severity and index in both seasons, but in different environments (Tables 9 and 10). For the Infinity/ND3085 population, the ND3085 variant for Xgwm493 was trait-related to incidence, severity, index, and FDK in Charlottetown in 2007, and in 2008 to FDK and DON. As with the ND744 variant for Xgwm493, the ND3085 variant revealed a consistent and significant association with resistance for the different FHB measurements. In 2008, ND3085 contributed a strong resistance effect marked by Xwmc505a, which affected FDK and DON content in Ottawa and disease incidence, severity, and index in Charlottetown (Table 6).

3BSc genomic region
Markers at the 3BSc locus showed an effect on FHB traits in both the Infinity/ND744 and Infinity/ND3085 populations. While in Infinity/ND744 resistance was contributed by the resistant donor ND744, in Infinity/ ND3085 the susceptible parent Infinity contributed FHB resistance at the 3BSc locus. ND744 alleles associated with the Xgwm285, Xgwm566, and Xwmc653 markers functioned to significantly reduce FHB symptoms in the different locations and during both years (Tables 7 and 8).
The Infinity variant for the Xwmc418, Xgwm285, Xwmc471, and Xwmc653 markers in Infinity/ND3085 contributed reduced FHB incidence, severity, index, and FDK at the 3BSc locus in 2007, while in 2008, the Infinity variant was effective in reducing disease symptoms only for Xwmc418 and Xwmc653 markers at variable levels of significance. No effect on DON accumulation of Infinity alleles at the 3BSc locus was detected when the population was grown in the different environments and during both years (Tables 5 and 6). Note: Infinity is listed twice because each population was grown as a separate test with its own parental controls. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively.

5AS genomic region
The 5AS locus marker Xgwm304a of the FHB resistance QTL was effective in all three populations, while Xgwm293 was only effective in the Infinity/ND744 and Alsen/Helios populations. The ND744 and Alsen alleles associated with the 5AS markers Xgwm304a and Xgwm293 functioned strongly in the U of M Carman and Ottawa environments to reduce FHB symptoms, FDK, and DON accumulation. Inconsistent associations were observed between these markers and the different FHB measurements at Charlottetown and AAFC Carman in the Infinity/ND744 population (Tables 7 and 8). In the Alsen/Helios population, Helios alleles marked by both Xgwm304a and Xgwm293 contributed weak resistance to disease incidence, severity, and index when the population was grown at Charlottetown (Tables 9 and 10). In Infinity/ND3085, the ND3085 variant for Xgwm304a showed a measurable effect only at U of M Carman and Ottawa, not Charlottetown or AAFC Carman (Tables 5  and 6).

4DL genomic region
The reported SSR marker Xwmc48 for FHB resistance on the 4DL QTL showed a weak and inconsistent contribution of resistance in the Infinity/ND3085 and Infinity/ ND744 populations. The ND3085 variant of this marker contributed a resistance allele that reduced incidence at Carman in 2007 and severity at Charlottetown in 2008. Similar to ND3085, the ND744 allele marked by Xwmc48 showed associations with resistance to disease severity and index at Carman in 2007 and resistance to disease severity, index, and FDK at U of M Carman in 2008. Note: SD, standard deviation.

2DL genomic region
The Infinity/ND744 and Alsen/Helios populations did not show any effect of the 2DL locus. Infinity in the Infinity/ND3085 population contributed resistance alleles at the 2DL locus, with significant associations observed between the marker Xgwm349 and resistance at all locations in 2007 and only at Charlottetown in 2008. Note: Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. Note: Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively.

6BS genomic region
The Xwmc397 marker showed associations with FHB resistance in the Infinity/ND3085 and Alsen/Helios populations and not in the Infinity/ND744 population, while Xgwm608 showed associations only in the Infinity/ ND744 population. The 6BS locus contributed FHB resistance inconsistently in the different populations. The Infinity variant for Xwmc397 in the Infinity/ND3085 population contributed the resistance at Ottawa and U of M Carman in 2007 and at Charlottetown and U of M Carman in 2008. With the Infinity/ND744 population, it was the resistance donor ND744 that contributed weak resistance alleles and only when the population was grown in Ottawa. In the Helios/Alsen population, both parents at the same marker showed association with FHB resistance; the Alsen variant for Xwmc397 contributed resistance to incidence at Charlottetown and the Helios variant for the same marker contributed resistance to severity and index at U of M Carman. Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error.
Markers associated with resistance loci on 3BS and 5AS revealed resistance effects in Alsen, ND3085, and ND744. ND3085 further possessed a resistance allele in chromosome 4DL. ND744 contributed to resistance also with the 3BSc, 4DL, and 6BS loci. Unexpectedly, Infinity contributed to resistance with chromosomes 2D, 3BSc, and 6BS in the Infinity/ND3085 population, and Helios with chromosome 5AS in Alsen/Helios population.

Discussion
The results over years and across locations confirmed FHB response based on prior knowledge of the more resistant parents Alsen, ND3085, and ND744, the more moderately resistant Helios, and the more susceptible Infinity.
The variation in response of the Fhb1 locus over environments confirmed an effect of environment on the Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error.  Buerstmayr et al. (2003), Cuthbert et al. (2006), andMcCartney et al. (2004) have shown that Xgwm493 and Xgwm533 are associated with Fhb1 and our results further confirm the value of the markers. While the consistent and strong association of Xgwm493 and Xgwm533 with resistance indicated the value of these markers, the inconsistent results for Xwmc505a with Infinity/ND744 and Infinity/ND3085 populations suggested this marker may not be as closely associated as Xgwm493 and Xgwm533. Similarly, the weak performance of STS3B-66 in the Alsen/Helios population would indicate a less desirable marker because of a weaker association with resistance. The lack of consistent associations between these markers and resistance is likely related to the distance of the marker from the resistance gene. The 3B region is more complex than a single resistance factor. Schweiger et al. (2016) indicated that the Fhb1 region spans 860 kb with high confidence genes unique to Fhb1 being in a very compact cluster within the region. McCartney et al. (2004) referred two different resistance loci on chromosome 3BS, one more distal and one more proximal to the centromere. The markers we are referring to for Fhb1 on 3BS are the more distal group. The markers Xgwm285, Xgwm566, and Xwmc653, which affirmed strong and consistent associations to resistance in the Infinity/ND744 population, are more proximal to the centromere and we refer to them as being at the 3BSc locus. The results demonstrated that ND744 carried the resistance at the 3BSc locus and because ND744 is derived from Sumai 3, the resistance allele was likely transferred from Sumai 3. Although the Infinity/ND3085 population also segregated for resistance at the 3BSc locus, the resistance was identified from Infinity for the markers Xwmc418, Xgwm285, Xwmc471, and Xwmc653, and was therefore not derived from Sumai 3. The fact that Infinity contributed resistance alleles but does not have Sumai 3 in its pedigree indicates the allele or alleles, depending on whether more than one gene exists in the interval, are not unique to Sumai 3. Mayer et al. (2014) explained the complexity of the QTL regions. Indeed, each QTL region may contain several genes and the dissection and understanding of the functional characterization of each gene localized in the QTL regions is a very challenging task as some genes can be effective in one background but not another. Although Infinity does not have Sumai 3 in its pedigree, it is possible for the two lines to share common alleles from a common parent in the ancestry of each line.
Because the Alsen/Helios population showed no effect at the 3BSc locus, either Alsen does not possess the resistance allele at this locus or the resistance was not segregating. ND744, ND3085, and Alsen are not as resistant to FHB as Sumai 3, therefore the three derivatives most likely did not acquire all the resistance loci that Sumai 3 has to offer. Therefore, the possibility exists that Alsen did not acquire the 3BSc locus. Alternatively, Helios has some resistance to FHB and, like with the 3BS locus, may have shared a common ancestral parent with Alsen and Sumai 3 somewhere in its ancestry and received a common resistance allele. This is an example of the importance of understanding the alleles possessed by parents before undertaking marker-assisted breeding. Selection using the Sumai 3 3BSc markers in the Alsen/ Helios population would have been a waste of effort and resources because of no segregation at the locus.
The results for the 5AS QTL markers suggested the transfer of the FHB resistance alleles of Sumai 3 through Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error. ND744, ND3085, and Alsen. Although Infinity has Frontana in its pedigree, which is known to possess an FHB resistance gene on 5AS (Steiner et al. 2004), apparently Infinity did not acquire the allele. The allele at 5AS was very effective in both the Infinity/ND744 and Alsen/ Helios populations because highly significant associations were demonstrated with several FHB measurements across multiple environments. The results are consistent with previous research in which the Xgwm293 and Xgwm304a markers were reported to flank the Qfhs.ifa-5A QTL in CM82036, which is a Sumai 3 derived line (Buerstmayr et al. 2002). Surprisingly, the Helios variant for Xgwm293 and Xgwm304a was associated with Type I resistance in Charlottetown in two years, suggesting that Helios possessed an allele at the 5AS locus that functions under certain environmental conditions. Although 5AS Sumai 3 resistance was apparently transferred to Alsen because in the majority of environments FHB resistance was attributed to the Alsen molecular variants of the markers, apparently other alleles may Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error.
dominate, in this case from Helios, in certain environments. This is an example of a locus expressing genetic trade-off between environments (Mitchell-Olds 2013).
Our results also showed that the 6BS locus produced an effect on FHB within the Infinity/ND744, Infinity/ ND3085, and Alsen/Helios populations. The results from Ottawa of Xwmc397 showed the transfer of Sumai 3 resistance to ND744 was possible, with resistance at this locus attributed to ND744. Our results are consistent with Cuthbert et al. (2007), who fine-mapped the locus, which included the Xwmc397 marker, and named the gene Fhb2. Surprisingly, the Infinity molecular variant for the Xgwm397 marker was associated with weak resistance to severity, index, and FDK in the Infinity/ ND3085 population. The resistant parent ND3085 did not contribute the resistance, suggesting that ND3085 may not have inherited the resistance allele from Sumai 3 at this locus. The fact that the effect was consistent over environments for the Infinity/ND3085 population indicates the effect is not simply a statistical aberration. Given Infinity is common in the Infinity/ ND3085 and Infinity/ND744 populations, these results are not easy to explain. One possibility is the presence of an allelic series at the 6BS locus with more than one allele conditioning resistance. The Infinity allele shows weaker expression in the test environments, whereas Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error. the ND744 allele is more strongly expressed in more environments.
The 6B locus in the Alsen/Helios population showed a scenario similar to 5AS in the same population where molecular variants of both parents were associated with resistance with the Xgwm397 marker. The Alsen allele functioning at Charlottetown and the Helios allele functioning at the U of M Carman location suggests the alleles from Alsen and Helios show environmentdependent differential expression. This would be another example of a QTL genetic trade-off between environments (Mitchell-Olds 2013).
Given that ND744 and ND3085 carried FHB resistance alleles marked by Xwmc48 at the 4DL locus, this indicates the resistance could be transferred from Sumai 3. The relationship between Xwmc48 and previously published FHB resistance QTL on chromosome 4DL was explored through comparative mapping. Xwmc48 has been reported on chromosome 4DL in the SSR consensus map by Somers et al. (2004). The potential transfer of the 4DL locus from Sumai 3 to Alsen could not be determined. The lack of effect of the 4DL locus in the Alsen/Helios population indicates no segregation, which could be because the two parents both possess the same resistance allele or because both do not possess the resistance allele.
The effectiveness of the 2DL FHB resistance QTL in the Infinity/ND3085 population with Infinity contributing the resistance allele indicates that the resistance allele from Sumai 3 was not transferred to ND3085. The lack of effect of the 2DL locus in Alsen/Helios and Infinity/ ND744 populations suggests either both parents in each cross possess the resistance allele or both do not possess the resistance allele. Given that Infinity in the Infinity/ ND3085 cross contributed a resistance allele, it is likely that the lack of segregation in the Infinity/ND744 population is due to ND744 also possessing the resistance allele. Therefore, it is possible that ND744 derived resistance at this locus from Sumai 3. With Helios having an intermediate reaction to FHB, it is possible the lack of segregation at the 2DL locus with Alsen is due to the presence of resistant alleles. The Xgwm349 marker is assigned to the long arm of chromosome 2D (Röder et al. 1998) and mapped 2 cM from Xgwm539 ). Handa et al. (2008)  Note: The number of alleles varies from a total of 80 due to the failure of some lines to amplify for some markers. Asterisks (*, **, and ***) indicate significance at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. SE, standard error. the resistance allele was contributed from Wuhan. The resistance from Sumai 3 on 2DL is not straightforward, and our results provide little clarification. More testing is needed to fully understand the contribution of resistance on 2DL from Sumai 3.
While our study targeted six genomic regions for Sumai 3 FHB resistance, the results affirmed the research of Buerstmayr et al. (2002Buerstmayr et al. ( , 2003, Cuthbert et al. (2006), and Xue et al. (2011) that the 3BS distal and 5AS genomic regions were among the most effective and stable. The results are consistent with Fhb1 and Qfhs.ifa-5A resistance alleles in ND744, ND3085, and Alsen being transferred from Sumai 3. These results validate the use of markers for Sumai 3 FHB resistance QTL transferred to breeding populations from ND744, ND3085, and Alsen. With respect to the other Sumai 3 loci, ND744 acquired the 3BSc, 4DL, and 6BS resistance, ND3085 the 4DL, and Alsen the 6BS resistance.
In addition to validating which Sumai 3 loci were transferred to breeding populations through intermediary parents, the project revealed some other interesting outcomes. Infinity, the common parent in the two populations, contributed resistance QTL in three of the six tested loci in the Infinity/ND3085 population, but showed no effect at the 2DL locus and was inferior at the 3BSc and 6BS loci in the Infinity/ND744 population. These results demonstrate that lines with little resistance can contribute resistance alleles in crosses. The results also suggest that an allelic series with a ranked effect may also be at play. Seda (2017) reported that, in many cases, alleles for FHB resistance are contributed by susceptible parents. Sometimes these are due to passive resistance (associated with flower structure, heading date, or height), but in other cases they are QTL conferring active resistance from the susceptible parent. The wide ranges in disease scores across the populations further support the role of transgressive segregation for FHB resistance.
Only three of the six loci segregated in the Alsen/ Helios population, which may be due to Helios carrying some resistance loci. It is not inconceivable for the lack of segregation at the 2DL, 3BSc, and 4DL loci to be a result of Helios having resistance alleles at one or more of these loci. This cross stresses the importance of resistance locus validation in breeding material where two lines produce similar FHB resistance phenotypes based on resistance from different loci.
In breeding, the assumption is that most markers associated with QTL from preliminary mapping studies can be directly used for MAS for enhancing FHB resistance breeding efficiency. However, the effect of certain alleles may vary among studies because of different genetic backgrounds, environments, and sampling variation (Holland 2007;Pumphrey et al. 2007;Salameh et al. 2011). Anderson et al. (2007) also suggested pleiotropic effects and linkage drag of the different reported FHB QTL to be examined to avoid undesirable consequences on agronomic or quality traits to make MAS successful. McCartney et al. (2007) evaluated the effects of FHB resistance QTL alleles from several sources like Nyuubai, Sumai 3, and Wuhan 1 on FHB resistance, and agronomic traits in elite Canadian spring wheat backgrounds. They showed that FHB resistance tended to be improved when more resistance QTL were incorporated. However, they found that the Sumai 3 5AS resistance allele was negatively associated with grain protein content and that the Wuhan 1 resistance allele was associated with increased plant height.
We found that some markers were polymorphic in one cross, but not another. Most of the markers we used were originally identified using specific biparental mapping populations, and polymorphism at the marker site may vary with genetic background (Garvin et al. 2015). A related challenge of identifying polymorphic markers in an interval occurs with determining if the proximity of the marker is such that it still has sufficient association with the trait to be useful in changing gene frequencies in breeding. For example, our study showed that some markers, such as Xgwm566 or STS3B-66, were weakly and inconsistently associated with resistance in the same 3BSc chromosomal region as other markers, such as Xgwm493 or Xgwm533, which were strongly associated with and consistently affected FHB resistance in the 3BS region. The genetic distance between markers and the resistance genes will affect the strength of association. Bernardo (2013) stated the importance of understanding the genetic distance between a linked marker and the gene of interest, to determine the likely recombination rate with which to evaluate the usefulness of the marker to a breeding program.
A dominant feature of the genetics of resistance to FHB is the inconsistent effect of certain alleles across environments. The priority for breeding is the utilization of QTL that are expressed consistently in different genetic backgrounds, across environments, and which produce larger effects and therefore have a lower genotype × environment interaction. By evaluating the three populations in different locations over 2 yr, we were able to determine the four markers, Xgwm493 and Xgwm533 on 3BS and Xgwm293 and Xgwm304a on 5AS, were associated with the strongest effect on FHB resistance and were the most stable in the different environments and in different genetic backgrounds. Our results are consistent with previous reports on the effectiveness and power of these markers to detect Fhb1 and Qfhs.ifa-5A. Markers associated with the 2DL, 4DL, and 6BS loci are less desirable because they were weakly associated with FHB resistance and the resistance was inconsistently expressed over years and across locations.
Wheat resistance to FHB is a complex trait and the evaluation of FHB resistance is time-consuming and labor-intensive. Therefore, marker-assisted selection can be a valuable contribution to improving FHB resistance, but breeders need to know which resistance loci survive intergenerational transfer from the source of resistance for which markers were developed. We demonstrated a strategy of testing the effectiveness of intergenerational transfer of FHB resistance loci from a resistance source, Sumai 3, to breeding populations, which assists the breeder in choosing markers for effective loci. The results determined the transfer of Sumai 3 resistance alleles for Fhb1 and Qfhs.ifa-5A through intermediary parents ND3085, ND744, and Alsen, allowing for effective marker-assisted selection of the resistance at these loci for reduced FHB incidence, severity, FDK, and DON. The effectiveness of transfer of 2DL, 3BSc, 4DL, and 6BS Sumai 3 resistance was less consistent because of lack of segregation and genetic trade-offs among environments, requiring more testing in specific crosses involving ND3085, ND744, and Alsen. With the advent of next-generation sequencing technology, a high quantity of single nucleotide polymorphism (SNP) markers associated with FHB resistance have been identified. Testing these SNP markers with these populations could clarify the transferability of Sumai 3 resistance alleles.