A BC5 recombinant population (RVPM7D) of 90 lines, originally produced by Worland et al. (1988), was used in the current study to define the relationship between yield-, GPC- and Pch1-mediated eyespot resistance. This population was developed by crossing the eyespot susceptible line Hobbit ‘sib’ (HS) and the Pch1 containing substitution line Hobbit ‘sib’-VPM7D (HS/VPM7D). HS/VPM7D is an intergenotypic single chromosome substitution line where chromosome 7D of Hobbit ‘sib’ was replaced by chromosome 7Dv of Ae. ventricosa Tausch (2n = 4x = 28, genomes DvDvMvMv) (Doussinault et al. 1983; Maia 1967). This population was previously used by Chapman et al. (2008) to map Pch1 to the distal end of chromosome 7DV.
A subset of 34 lines of the HSxHS/VPM7D population representing all the recombination haplotypes identified in the population was used for yield and GPC assessment across eight field trials conducted across four consecutive years (one trial in 2014, three in 2015, three in 2016 and one in 2017). These trials were carried out in the following locations of the United Kingdom:
John Innes Centre—Church Farm, Bawburgh, Norfolk (52° 37′ 46.4″ N 1° 10′ 29.4″ E) in 2014, coded as 2014_JIC;
RAGT Seeds Ltd, at Stapleford (52° 14′ 27.5″ N 0.16° 28′ 3″ E) and Great Shelford (52° 15′ 84.9″ N 0.14° 47′ 7.0E), Cambridgeshire, in 2015 (coded as 2015_RAGT_SF and 2015_RAGT_WH, respectively); at Elmdon, Essex (52° 07′ 09.3″ N 0.1° 05′ 98.4″ E and coded as 2016_RAGT_BT) and Ickleton, Cambridgeshire (52° 08′ 63.8″ N 0.13° 54′ 5.3″ E and coded as 2016_RAGT_SD);
Limagrain UK Ltd, at Burnt Fen (52° 12′ 16.9″ N 0° 53′ 04.2″ E), Littleport, Ely CB7 4SU in 2015 (codes as 2015_Limagrain) and at Lower Barn (52° 11′ 56.3″ N 0° 51′ 11.1″ E), Gedding, Bury Saint Edmunds IP30 0QD in 2016 (coded as 2016_Limagrain);
Morley St. Botolph Wymondham, Norfolk in 2017, coded as 2017_JIC.
For 2014_JIC and 2017_JIC, all entries were planted as three replications in randomized complete block designs. The plots were 4 × 1.5 m corresponding to 6 m2.
2015_Limagrain and 2016_Limagrain trials were randomized using an Alpha design with six replications and six sub-blocks of seven varieties/replicate. Plot size was 5.4 m2 with plot length of 6 m and plot width of 1.55 m.
The RAGT trials were conducted in a randomized complete block design with 2 blocks, each block containing 1 replicate. A plot size of 7.2 m2 (6 m × 1.2 m) was used.
All trials were run using standard agronomic packages of fertilisers, pesticides and growth regulators.
Aiming at high-quality genomic DNA, DNA extraction of the parental lines was performed using the CTAB method (Nicholson et al. 1996). Instead a, 96-well extraction protocol adapted from Pallotta et al. (2003) was used for the DNA extraction of the populations lines.
Both HS and HS/VPM7D were genotyped by Axiom® wheat HD Genotyping Array (Winfield et al. 2016). After analysing all the SNPs mapping to the 7D chromosome, a set of SNPs polymorphic between the two parental lines located across the full chromosome were selected. KASP primers were designed on these SNPs using PolyMarker (http://www.polymarker.info/) (Ramirez-Gonzalez et al. 2015). Thermodynamic properties of designed primers were verified after adding the standard FAM or HEX compatible tails (LGC ltd). 7D-specific KASP markers were initially tested against HS and HS/VPM7D, and those that were polymorphic were then assayed across the RVPM7D population.
Nine SSRs, namely Xbarc97, Xgdm67, Xgdm86, Xgdm150, Xgwm37, Xgwm428, Xwmc14, Xwmc221 and Xwmc273 and two biochemical markers, RC3 and Amy-D2, were also included in the analysis and carried over from Chapman et al. (2008).
For the KASP assay, 2 µl (5 ng/µl) of the extracted DNA was added to 0.056 µl of primer mix (12 µl each of specific primer, 30 µl of the common primer and 46 µl deionized water) and 2 µl of KASP master mix (LGC). The PCR amplification included an initial denaturation step of 94 °C for 15 min followed by 10 cycles of touchdown PCR (annealing 62 °C to 56.6 °C, decreasing 0.6 °C per cycle), then 25 cycles of 94 °C for 10 s and 60 °C for 1 min. After amplification, plates were read into the Tecan Safire plate reader and genotyped using the Klustercaller™ software (version 188.8.131.52, LGC).
PCR reactions were prepared in a 6.25 µl final volume containing 2.5 µl DNA (10 ng/µl), 3.125 µl Taq Mastermix (Qiagen) and 0.625 µl of the relevant primer pair (2 µM). A common PCR programme was used throughout consisting of a denaturing step of 95 °C for 5 min; followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min, with a final elongation step of 72 °C for 7 min. Where required, PCR products were then purified using QIAquick Gel Extraction Kit (Qiagen), sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (following the manufacturer’s instructions) and aligned in VectorNTI® (ThermoScientific).
Map construction and QTL analysis
The genetic map of chromosome 7D was generated in JoinMap© (version 3.0) (Stam 1993) using default parameters. Mapping data were combined with phenotypic data from the field for QTL analysis. Predicted mean scores were calculated for each line using a general linear model (GLM) in Genstat v.19 (Copyright 2009 Lawes Agricultural Trust, Rothamsted Experimental Station, UK). The QTL analysis was carried out using the predicted mean score data from each phenotype trial individually as well as using a data set in which the data from all trials were combined.
The identification of QTLs was done using Single Trait Linkage Analysis of Genstat v.19 (Copyright 2009 Lawes Agricultural Trust, Rothamsted Experimental Station, UK) in three different steps: (1) initial genome-wide scan by simple interval mapping (SIM) to obtain candidate QTL positions; (2) one or more rounds of composite interval mapping (CIM), in the presence of cofactors, which are potential QTL positions detected at the previous step; (3) fit the final QTL model. Default threshold based on the estimation of the effective number of tests (Li and Ji 2005) was selected for the QTL analysis.
Thousand grain weight
TGW, grain length and grain width were performed using the Marvin grain analyser (GTA Sensorik GmbH, Neubrandenburg, Germany) using grain from the field grown BC5 plants.
JIC and RAGT field plots were harvested with Zurn 150 plot combine harvesters, which have on board weighing systems. For the JIC trials, the yield figure is the total from 6 m2. After machine threshing, the grain was weighed and the yields were corrected to a moisture content of 14%. The plot weights for RAGT trials were adjusted to 14% moisture content and calculated as tonnes/hectare (t/ha).
2015_Limagrain trial was harvested with a Wintersteiger Delta combine harvester, which has on board weighing system. 2016_Limagrain, instead, was harvested using a Wintersteiger Classic combine harvester. So, the whole plot was bagged on the side of the combine and then the grain weighed in the barn.
Grain protein content
GPC was assessed using near-infrared (NIR) spectroscopy with the method previously published by Chia et al. (2017). A FOSS 6500 wavelength scanning near-infrared microscope incorporating ISIscan™ Routine Analysis Software was used to measure protein content, moisture content and grain hardness for each sample according to the manufacturer’s instructions. Each sample of ~ 5 g of grain was run in duplicate using a ring cup. The sample spectra were compared with calibration set spectra taken from samples with known protein content, moisture and hardness compositions.
Analysis of variance was performed on yield, grain protein content and TGW scores to assess the variation attributable to line, blocks and interactions between line and blocks, using a general linear model (GLM) in Genstat v.19 (Copyright 2009 Lawes Agricultural Trust, Rothamsted Experimental Station, UK). Predicted mean scores were calculated for each line using the GLM for use in the QTL analysis.