A F₉-derived recombinant inbred line (RIL) population consisting of 125 lines from the cross Kenyon x 86ISMN 2137 was tested in this study. Each RIL was generated by single seed descent from a unique F2 individual. Kenyon is a Canada Western Red Spring (CWRS) variety with the pedigree Neepawa^*5/Buck Manantial (Hughes and Hucl, 1991). Neepawa was the most widely grown CWRS variety in the 1970s to mid-1980s in western Canada (McCallum and DePauw, 2008), and a prominent in the pedigree of many current CWRS wheat varieties. Buck Manantial was the source of the leaf rust resistance gene Lr16 in Kenyon. 86ISMN 2137 is a spring wheat line with resistance to tan spot (Singh and Hughes, 2005), Septoria nodorum blotch (Feng et al., 2004), and Septoria tritici blotch (McCartney, unpublished data), however, its pedigree is unknown.

The RIL population, parents, and checks were tested in six environments over 2012 and 2013 (Carman MB 2012, Winnipeg MB 2012, Carman MB 2013, Charlottetown PEI 2013, Ottawa ON 2013, and Winnipeg MB 2013). The check lines were 5602 HR, 93 FHB 37, AC Barrie, AC Cora, AC Morse, AC Vista, CDC Teal, ND 2710, Neepawa, Roblin, Shaw, and Snowbird. Entries were randomized in an alpha lattice with 12 incomplete blocks of 12 plots each, with 3 replicates per environment. The experimental unit was a 1 m row.

The date of 50% anthesis was recorded for each plot in all locations, except Charlottetown, PEI. On this date, anthesis had begun for 50% of the main tillers in that plot. Carman and Winnipeg tests were spray inoculated twice [on the recorded anthesis date and 2–3 days later (confirmed for Carman and Winnipeg)]. The inoculum consisted of the isolates M7-07-1 (3-ADON), M9-07-1 (3-ADON), M1-07-2 (15-ADON) and M3-07-2 (15-ADON) for the Carman nurseries and the Winnipeg 2012 nursery. In Winnipeg 2013, the isolates used were M9-07-01 (3-ADON) and M1-07-02 (15-ADON). The inoculum concentration was adjusted to 50,000 macroconidia per L and applied at a rate of 50 ml per row with a backpack sprayer. During the period the population was being inoculated, a misting system irrigated each day for 10 min every hour over a 12 h period (6:00 pm–6:00 am) in Carman. This misting system kept the plots wet overnight to promote F. graminearum infection. In Winnipeg, plots were irrigated with a sprinkler head irrigation system for 1 h following inoculation. The goal of this irrigation was to maintain wet soil to result in a humid micro-environment in the nursery, rather than to directly apply water to the canopy over the entire night.

In PEI, five F. graminearum isolates were used to produce macroconidia for inoculation. Inoculum was prepared to 50,000 macroconidia per ml and inoculated on plots three times per week over the course of flowering in the trial. Inoculum was applied with a standard pesticide sprayer delivering 200 L/ha water. The field was misted for 2 min bursts at a rate of 650–700 L/ha; misting was done every half hour from hour 7:00–10:30, at 15 min intervals thru to 19:00, half hour intervals to 21:00 and then on an hourly basis until 7:00. Misting nozzles were Naan Dan 501/2 (yellow) (Southern Drip Irrigation Ltd, Lethbridge, Alberta) spaced 10 feet apart in rows which ran down the center of the plots to ensure complete and overlapping coverage.

The Ottawa FHB nursery was inoculated by spreading infected corn and barley kernels on the soil surface as described by Xue et al. (2006). The corn and barley was inoculated with a mixture of three F. graminearum isolates (DAOM178148, DAOM232369, and DAOM212678; Canadian Collection of Fungal Cultures, AAFC, Ottawa, Canada), colonized, and then dried down. Inoculated corn is spread between the rows at a rate of 80 grams/meter² at about 6 weeks after planting. Plots were irrigated twice daily for 30 min with irrigation sprinklers to promote conditions favorable for infection by F. graminearum.

Plant height, anthesis date, FHB incidence, and FHB severity data were collected. Plant height was measured from the soil surface to the top of main tiller spikes (excluding awns if present). FHB incidence was the percentage of spikes with visual FHB symptoms. FHB severity was the percentage of the spike with visual FHB symptoms, when only considering diseased spikes. FHB incidence and severity data were converted into FHB visual rating index (VRI). VRI = (FHB incidence ^* FHB severity)/100.

Least squares means were calculated for anthesis date, plant height, and VRI with JMP Genomics 6.0 (SAS Institute Inc.) using a mixed model. Wheat lines were considered fixed effects, and environment, rep, and incomplete block were considered random effects.

Ninety-seven RILs were genotyped with a combination of microsatellite, diversity array technology (DArT) (Akbari et al., 2006), and single nucleotide polymorphism (SNP) markers. Genomic DNA was extracted from freeze-dried leaf tissue with the DNeasy 96 Plant Kit (Qiagen, Toronto, Canada). DNA was quantified with PicoGreen stain (Molecular Probes, Inc., Eugene, Oregon, USA). SNP markers were genotyped on the RIL population and parents using the Illumina Infinium 9K wheat SNP beadchip (Illumina, San Diego, CA) (Cavanagh et al., 2013). The raw data were analyzed with GenomeStudio V2011.1 software (Illumina, San Diego, CA). The genotype calls from GenomeStudio were converted into allele scores for linkage mapping in Excel. Markers with greater than 10% missing data or strong segregation distortion were excluded from mapping. Microsatellite markers were tested as previously described (McCartney et al., 2004).

The linkage map was developed with MapDisto version 1.7.7 (Lorieux, 2012). Linkage groups were initially formed with stringent LOD and recombination fraction thresholds and gradually relaxed to a minimum LOD score of 4 and a maximum recombination fraction of 0.20 cM. Loci were ordered using a combination of the “AutoMap,” “Order sequence,” and “Compare all order” functions. The “Branch and Bound II” and “Seriation II” ordering methods were used in combination with the sum of adjacent recombination fractions (SARF) and count of crossover events (COUNT) as fitting criteria. For each linkage group, the shortest linkage map was selected from the linkage map solutions generated using these different mapping algorithms and criteria. The Kosambi mapping function was used to calculate map distances (cM) from recombination fractions.

QTL analysis was conducted with QTL IciMapping version 4.0.6.0 (Li et al., 2008) using interval mapping (IM) and inclusive composite interval mapping (ICIM). For linkage bins with more than one marker, a single marker was selected with the least missing data to represent the linkage bin for QTL analysis. Analysis for additive effect QTL was conducted with 0.1 cM steps and the 5% LOD significance threshold was calculated with 10,000 permutations. The LOD threshold for declaring QTL was 3.22 for additive effect QTL analysis for both IM and ICIM based upon this permutation analysis. Additive effect QTL were reported when the LOD score exceeded 3 in two or more environments, or one or more environments plus the pooled dataset, based upon IM or ICIM. For the reported QTL, QTL statistics were reported for environments in which the LOD score exceeded 2.

Analysis for epistatic QTL was conducted with 2.0 cM steps and a default LOD significance threshold of 5.0. Determining a 5% LOD significance threshold by permutation analysis was not possible for epistatic QTL analysis because of the computational power required. Linkage maps and QTL scans were illustrated with MapChart v. 2.2 (Voorrips, 2002). For anthesis date and plant height, epistatic QTL were reported when the LOD exceeded 3.5 in four or more environments, or three environments plus the pooled dataset, based upon IM or ICIM. For FHB resistance, epistatic QTL were reported when the LOD exceeded 3.5 in seven or more combinations of environment (individual environments or pooled dataset) by traits (VRI, FHB incidence, FHB severity). The more stringent criteria was applied to FHB resistance because the three FHB resistance traits were pooled for considering QTL. For the reported QTL, QTL statistics were reported for environments in which the LOD score exceeded 3.5.

To confirm the presence of a reciprocal translocation, immature spikes were harvested from F₁ plants. Spikes were fixed (9 95% ethanol: 6 chloroform: 1 glacial acetic acid) at −20°C for 24 h and stored in 70% ethanol at −20°C, changing the ethanol once a day for 3–4 days. Anthers were macerated in acetocarmine to liberate pollen mother cells (PMCs) and stain the chromatin. PMC preparations were warmed on a hot plate and gently squashed to spread the chromosomes. Cells in metaphase I of meiosis were analyzed under a compound microscope to confirm the presence of quadrivalents.

Trait data histograms for pooled datasets of the Kenyon/86ISMN 2137 RIL population are reported in Figure 1. Histograms for anthesis date, plant height, VRI, FHB incidence, and FHB severity from each FHB nursery are presented in Supplementary Figures S1–S5, respectively. All traits were approximately normally distributed. The earliest and latest RILs flowered within 8.3 days of each other (Figure 1, Supplementary Table S1). Kenyon and 86ISMN 2137 had very similar flowering dates, but some transgressive segregation was present in the population. Plant height varied widely with the shortest and tallest RILs differing by 36.5 cm (Figure 1, Supplementary Table S1). There was some transgressive segregation for plant height, but most RILs had means within the means of the parents.

The resistant checks 93FHB37 and ND 2710 had the lowest FHB VRI, incidence, and severity in the field FHB nurseries (Table 1, Supplementary Table S1). AC Vista, Roblin, and CDC Teal were the most susceptible to FHB based upon the VRI, incidence, and severity data, which was consistent with previous experience with these lines. The varieties 5602HR, AC Barrie, Neepawa, Snowbird, and AC Cora had intermediate FHB data. AC Morse, the sole durum wheat check, had intermediate to moderate susceptibility based upon VRI data, which is also typical. However, it should be noted that AC Morse would typically have very high Fusarium-damaged kernels (FDK) and deoxynivalenol (DON) accumulation relative to bread wheats with similar VRI scores. Cumulatively, these findings were all consistent with past experience with these wheat lines in FHB nurseries.

The wheat line 86ISMN 2137 was amongst the most susceptible to FHB based on VRI in these field nurseries (Table 1). Kenyon was more resistant to FHB than 86ISMN 2137 on average, and had a VRI score less than 86ISMN 2137 in four of the six field tests. Kenyon also had a higher VRI score than Neepawa in every field test, which was unexpected given its pedigree (Neepawa^*5/Buck Manantial). The mean of Kenyon/86ISMN 2137 RIL population was similar to its parents, but there were individual RILs which were more resistant and susceptible to FHB. This result suggested transgressive segregation for FHB resistance in this population.

Correlation analysis revealed a consistent negative correlation between FHB VRI and plant height (r = −0.55) (Supplementary Table S2). Anthesis date and VRI were not highly correlated, and the correlation was not consistent (Supplementary Table S2). For instance, FHB VRI and anthesis date were negatively correlated in Winnipeg 2012, Ottawa 2013, and Winnipeg 2013, but positively correlated in Carman 2013 and completely uncorrelated in Carman 2012.

The Kenyon/86ISMN 2137 linkage map was 2647 cM in length and consisted of 25 linkage groups and 3081 loci. Most of the wheat genome was covered by 22 of the linkage groups, with chromosome 1A consisting of two relatively large linkage groups of 82 and 29 cM (Supplementary Table S3). The three remaining linkage groups were small (7, 15, 13 cM) and were assigned to 1D (as linkage group 1D.1), 3D (as linkage group 3D.2), and 5D (as linkage group 5D.1) chromosomes based upon comparison to published maps (Cavanagh et al., 2013).

A problem was identified in the 5B and 7B linkage maps. Under the linkage mapping conditions described above in the Materials and Methods, markers on chromosomes 5B and 7B formed a single linkage group. A reciprocal translocation was suspected in one of the parents. This was investigated by examining chromosome pairing during metaphase I in pollen mother cells. Cytology revealed the presence of quadrivalents during this phase of meiosis (Figure 2), which was also consistent with a translocation in one of parents. Markers on chromosomes 5B and 7B were subsequently separated with the maximum recombination fraction of 0.02 and a minimum LOD score of 5. This strategy successfully separated the markers for the two chromosomes, except for a single linkage group that represented the translocation breakpoint. The 5B and 7B markers, that were successfully separated based upon the stringent linkage group criteria above, were then mapped as outlined in the Materials and Methods. This resulted in four linkage groups that represented chromosomes 5B and 7B (i.e., a linkage group on either side of the translocation breakpoint for each chromosome). The markers in the translocation breakpoint linkage group were sorted into their appropriate chromosomes based upon the chromosome that these markers were previously mapped in other populations (Somers et al., 2004; Cavanagh et al., 2013). The linkage maps for chromosomes 5B and 7B were then re-calculated and a single linkage group was developed for each chromosome. The markers involved in the translocation breakpoint are indicated in Supplementary Table S3. These results were consistent with a reciprocal translocation in one of the parents of the mapping population, such that the 5B and 7B markers were genetically linked near the translocation breakpoint.

Three additive effect QTL for anthesis date were identified (Table 2). The QTL were located on chromosomes 2D, 4A, and 5B, and were named QAnth.crc-2D, QAnth.crc-4A, and QAnth.crc-5B. Kenyon alleles increased days to anthesis for QAnth.crc-2D and QAnth.crc-5B, and decreased days to anthesis for QAnth.crc-4A. QAnth.crc-2D and QAnth.crc-5B exceeded the LOD threshold more frequently than QAnth.crc-4A.

Analysis for epistatic QTL identified additional QTL for anthesis date. Three digenic epistatic interactions were identified (Supplementary Table S4). The most consistent identified epistatic interaction was between loci on chromosomes 5B and 5D. The r² value and estimated additive^*additive effect of the interaction was generally higher for IM than ICIM. Other epistatic interactions were identified between loci on chromosomes 3B and 3D, and 3D and 7D. None of the additive effect QTL for anthesis date were detected as an epistatic QTL, and vice-versa.

Four additive effect QTL for plant height were identified (Table 2). These QTL were located on chromosome 1B, 2D, 3D (linkage group 3D.1), and 7B, and were named QHt.crc-1B, QHt.crc-2D, QHt.crc-3D, and QHt.crc-7B. The Kenyon allele increased plant height for each of these QTL, which is consistent with Kenyon being 15 cm taller than 86ISMN 2137 in field tests. The QHt.crc-1B, QHt.crc-2D, and QHt.crc-3D were detected by IM (exceed LOD 3.22 in at least one environment), while all four height QTL were detected by ICIM. One epistatic QTL interaction was detected between loci on chromosomes 2B and 6B. This interaction was consistently identified by IM, but half of the time by ICIM.

The minor 7B height QTL was located within the translocation breakpoint. The peak of this QTL (38.0 cM) was located 1.9 cM from the most likely position of the translocation breakpoint (36.1 cM). Given this, it is strange that a height QTL was not detected on chromosome 5B at the translocation breakpoint (33.2 cM). Examination of the allele scores between markers at position 33.2 cM on chromosome 5B and position 38.0 cM on chromosome 7B revealed five allele score differences for these locations amongst the RILs. The relatively weak effect of this QTL and the number of allele score differences between these locations was apparently sufficient to prevent detection of the height QTL at the translocation breakpoint on chromosome 5B. Given these results, we recommend caution regarding the accuracy of the location of this QTL (i.e., the QTL could be located at the translocation breakpoint on chromosome 7B, or possibly 5B).

Nine additive effect QTL for FHB resistance were identified by QTL analysis using IM and ICIM with the additive effect module of QTL IciMapping (Table 2). These FHB resistance QTL were located on chromosomes 2D, 4A, 4D, 5B, and 7D, and were named QFhb.crc-2D.1 (chromosome 2D at 8.5 cM), QFhb.crc-2D.2 (chromosome 2D at 20.7 cM), QFhb.crc-2D.3 (chromosome 2D at 37.5 cM), QFhb.crc-2D.4 (chromosome 2D at 98.2 cM), QFhb.crc-4A.1 (chromosome 4A at 51.1 cM), QFhb.crc-4A.2 (chromosome 4A at 124.4 cM), QFhb.crc-4D, QFhb.crc-5B, and QFhb.crc-7D. Kenyon alleles decreased FHB symptoms for QFhb.crc-2D.1, QFhb.crc-2D.2, QFhb.crc-2D.3, QFhb.crc-2D.4, QFhb.crc-5B, and QFhb.crc-7D, and increased VRI for QFhb.crc-4A.1, QFhb.crc-4A.2, and QFhb.crc-4D. QFhb.crc-2D.3 was the most consistently detected. QFhb.crc-4D and QFhb.crc-5B were the least significant FHB resistance QTL. The other FHB resistance QTL were reasonably consistent and detected by both IM and ICIM.

Additional QTL for FHB resistance were identified by digenic epistasis QTL analysis. Four digenic epistatic interactions were identified between loci on chromosomes 1A and 4B, 1B (near QHt.crc-1B) and 7B (QHt.crc-7B), 2B and 6B, and 2B and 6D (Supplementary Table S4). All four interactions were detected in approximately the same number of environments. None of the FHB resistance QTL detected by digenic epistasis loci were identified by additive effect QTL analysis. Similar to anthesis date, the r² value and estimated additive^*additive effect of the interactions was generally higher for IM than ICIM.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.