Plant Material and Population Development

A RIL population was developed from the cross Parshall × Reeder (PR) using single seed decent method. Parshall (PI-613587) is a HRSW cultivar developed from the cross Keene (PI-598224)/ND674, where Keene was developed from the cross (Stoa(Sib)/3/Ias-20*4/H-567.71//Amidon), and the ‘ND674’ (PI- 592759) was developed from the cross (Grandin (PI-531005)*2/ND‘Glupro’ (PI-592759))

(http://wheatpedigree.net/sort/show/55032). Parshall has moderate earliness and good resistance to FHB.

Also, it has good adaptation to ND and surrounding areas, test weight, resistance to lodging and shattering, and end-use quality for domestic and export wheat markets (Underdahl et al. 2008). Reeder (PI-613586) was an experimental line developed from the cross (Ias-20*4/H-567.71//Stoa/3/ND-674) at NDSU in 2000 (http://wheatpedigree.net/sort/show/60730). A total of 110 PR RIL were evaluated in field and GH experiments. Seven checks were evaluated, including the high FHB resistant line [ND2710 (PI-633976)],

two moderately resistant cultivars [Faller (PI-648350) and Alsen (PI-615543)], two moderate susceptible cultivars [Steele-ND (PI-634981) and Barlow (PI-658018)], a susceptible line [‘Vida’ (PI-642366)], and the highly susceptible check (ND2398).

3.3.2. Field Experiment

Multiple phenotypic evaluations were performed for FHB resistance types/traits and HD. Field experiments were conducted for three years (2010-2012) at one location in Minnesota (MN) (Minneapolis, 44°59′N, 93°16′W), in South Dakota (SD) (44°19′05″N, 96°47′00″W), and three locations in North Dakota (ND) [Carrington (47°45′00″N, 99°12′39″W), Prosper (46°96′30″N, 97°01′98″W) and Langdon (48°45′42″N, 98°22′18″W)]. Field experiments were conducted in FHB nurseries using artificial FHB inoculation (Stack et al. 1997). All experiments were conducted in randomized complete block design (RCBD) with four replicates in ND and two replicates in MN and SD. Field experiments were irrigated using overhead misted method (Rudd et al. 2001). Experimental unit consisted of hill/plot 0.3 M long. Each hill/plot contained at least 10 plants. FHB inoculum sprouted in field at flowering time and FHB traits were scored about three weeks after flowering time.

3.3.3. Greenhouse (GH) Experiment

Evaluations in 2011 and 2012 were conducted in RCBD layout with four replicates in eight-inch sized pots. Planting soil was Sunshine Mix #1 (Sun Gro Horticulture, Agawam, MA USA), augmented with 20 g Osmocote® _{slow release fertilizer (Scott’s Company LLC, Marysville, OH USA). Each pot contained}

five plants. At least 5 spikes for each pot were artificially injected using FHB spore-suspension method (Bekele 1995). Briefly, one middle floret, of a single spikelet per spike, was manually injected, using a needle containing about ~4 μl of (100 k/ml) FHB spore suspension. Inoculated spikes were individually covered using plastic misted bags for 72 hours. Severity data was collected about three weeks after injection.

3.3.4. Phenotypic and Genotypic Data Recording

PR population and checks were assessed for all FHB resistance types/traits. Resistance type I, was conferring FHB incidence (INC) and assessed as the percentage of infected spikes per total number of spikes in a plot/hill. INC was assessed only in the field experiments for five environments (combinations of year by location) including MN 2010 (M10), SD 2010 (S10), Carrington 2011 (C11) and Prosper 2011 and 2012 (P11 and 12). Resistance type II, was conferring FHB severity (SEV) and assessed as the percentage of infected spikelets per spike averaged across 10 total randomly chosen spikes per plot/hill in the field, and across five to ten spikes in GH experiments. SEV was recorded for 12 environments, including M10; P10, P11, P12; C11; S10, S11, S12; GH 2011 and 2012 (G11 and G12); and the combined means across field data (FieldSEV); and GH data (GHSEV). Resistance type III, was conferring resistance to the accumulated levels of the toxin deoxynivalenol (DON) and was detected in 10 gm of milled kernels for each genotype using gas chromatography and/or mass spectroscopy (Schwarz et al. 1995). DON was assessed in six total environments; three for ND (P10, P11 and G11), and three for SD (S10, S11, and the combined SDDON environment). DON data in SD was assessed from two replicates, while in ND as a single replicate. Resistance type IV, was conferring the resistance to the high percentage of Fusarium damaged kernel (FDK) and assessed, as the percentage of infected kernels found in 200 kernels randomly chosen from each individual genotype per replicate. FDK was scored in four environments including S10, S11, G11, and G12. Disease index (NDX) was a calculated FHB variable and was the product of multiplying the INC and SEV for each genotype. NDX was assessed in six total environments including the combined data across all environments.

One agronomic trait was evaluated for HD. HD was measured as the number of days from planting until ~50% of the spikes were fully emerged from the boot leaves in each genotype at the growth stage Feekes 10.5. HD was recorded in seven total environments including P10, P11, P12, C11, Langdon 2011 (L11), M10, and the combined data across ND environments.

Genomic DNA was extracted from lyophilized young leaf tissue for each individual RIL, parent and check using Qiagen DNeasy Plant mini kit (Cat# 69106) with minor modifications. For each genotype, 30

µl of DNA (80ng/µl) were sent to Triticarte Pvt. Ltd (Canberra, Australia; http://www.triticate.com.au) for (DArT) analysis (Akbari et al. 2006). Additionally, DNA samples were also for SNP analysis (Wang et al. 2014) at the small grains genotyping lab, USDA, ARS, Plant Science Research Unit, Fargo, ND, US, using the 90 K iSelect wheat SNP chip (Illumina).

3.3.5. Map Construction and QTL Analysis

The scores of all polymorphic DArT and SNP markers were converted according to the parental codes. Linkage maps for each chromosome were constructed using Carthagene Software

http://www.inra.fr/mia/T/Carthagene/.1.2-LKH (De Givry et al. 2005) specifying a RIL genetic model. A

maximum distance of 30 centimorgans (cM) and a minimum logarithm of odds (LOD) threshold of 4 were used to partition markers into linkage groups. Cosegregating markers were merged into single markers. The most likely positions of the markers along the linkage groups were determined using the commands [mrkdouble, mrkmerges, group 0.3 4, mrkselset, build, flips, polish, detail]. The Kosambi mapping function (Kosambi, 1944) was applied to convert recombination rates into map distances in cM. Linkage groups were assigned to chromosomes according to SSR markers and their map information from GrainGenes (http://wheat.pw.usda.gov/ggpages/maps.shtml) and consensus map of DArT markers. Maps were compared to the high-density wheat consensus SSR and AFLP genetic map (Somers et al. 2004) available in GrainGenes. Final maps were compared with the DArT (Huang et al. 2012) and SNP consensus maps using the program Autograph (Derrien et al., 2007; http://autograph.genouest.org/) to confirm accuracy of the marker order. Combined DArT/SNP genotyping revealed 214 polymorphic markers and used to generate the genetic map that plotted 154 markers to 14 different wheat chromosomes.

QTL mapping was carried out by the composite interval mapping (CIM) method (Zeng 1994) using the software QGene 4.0 (Joehanes and Nelson 2008). A scanning interval of 1 cM between markers and putative QTL with a window size of 10 cM was used to detect QTL. The number of marker cofactors for background control was set by forward and revers regression with a maximum of five controlling markers. A QTL was considered significant when one markers was associated at 3 LOD. Permutation test was used at 1000 replications with 30 threads to confirm LOD score threshold for identified QTL in each trait.

Confidence intervals (CI) were obtained using ±2 LOD positions distant from each QTL peak. The proportion of phenotypic variance (PV) explained by a single QTL was determined by the square of the partial correlation coefficient (R2_{). QTL with overlapping CIs were considered as one QTL. QTL with ≥10%}

of PV was considered major, and if repeated across ≥50% of total tested environments, was considered stable. For additive QTL effects, positive and negative signs of the estimates indicate, respectively, the contribution of Parshall and Reeder toward higher trait values. Graphical representation of linkage groups and QTL was carried out using MapChart 2.2 software (Voorrips 2002).