Materials and methods

2.2.1 Plant material and cultivation

The populations used in these experiments are listed in Table 2.1. All populations were developed from a cross between Espada or Young, elite Australian bread wheat cultivars carrying Rht-D1b or Rht-B1b and the bread wheat Rht18 donor line HI25M (courtesy of Greg

Rebetzke and Allan Rattey CSIRO), which was derived from a cross between Icaro

(tetraploid, Rht18) and Halberd (hexaploid). Genotyping information for populations is given in Table 2.2. In 2012, F5 families (Expt 1) with each line derived from a single F2 plant from the biparental cross HI25M (Rht18) × Espada were sown in rows at Black Mountain,

Canberra (Latitude: -35º 16", Longitude: 149º 6" E) and 13 sequential harvests of main stems from five plants were taken from terminal spikelet (TS) to maturity. Main stem was classified as the tallest stem from a plant. In 2013 two populations were sown, F6 families (Expt 2) were sown in plots at Ginninderra Experiment Station (GES), Canberra (Elevation 600m, Latitude: -35º 12", Longitude: 149º 4" E). Quadrats were harvested at physiological maturity and plot harvests were made with a machine harvester (Dominator, CLAAS). BC2F4 families with each line derived from a single BC2F2 plant (Expt 3) were sown in rows at Black Mountain and harvested at 15 sampling times. The main stems from five plants were harvested from TS to maturity. In 2014 three populations were sown, BC2F5 (Expt 4) at GES in plots and five tillers were harvested at three sampling times (10 days before anthesis, 7 days after anthesis and 28 days after anthesis). BC2F4 families (Expt 5) from HI25M × Espada and BC2F4 (Expt 6) from HI25M × Young (Rht-B1b) were sown in rows at GES and five plants per row were harvested at 14 days after anthesis. There were four genotypes in every experiment (Table 2.2) and there were five lines per genotype. Each line was chosen after genotyping (see Section 2.2.2). Lines in all experiments have two replicates and lines in Expt 4, Expt 5 and Expt 6 were randomised. Plants grown at Black Mountain were irrigated while GES was rainfed.

Table 2.1 Populations deployed in growth and yield studies with sowing dates

ID Population Parent 1 Parent 2/

Recurrent

Sowing pattern

Spike and stem

growth Grain yield

Expt 1 F5 HI25M Espada Row 18th Sep 2012

Expt 2 F6 HI25M Espada Plot 24th May 2013

Expt 3 BC2F4 HI25M Espada Row 15th May 2013

Expt 4 BC2F5 HI25M Espada Plot 19th June 2014 19th June 2014

Expt 5 BC2F4 HI25M Espada Row 20th _{June 2014}

Expt 6 BC2F4 HI25M Young Row 20th June 2014

Experiments at Black Mountain were sown in single rows (12 meters long) spaced by 30 cm with Granulock15 (N, P, S of 14.3, 12, 10.5) at a rate of 110 kg/ha and top dressed with 80 kg/ha urea at booting stage. Expt 1 and 3 at Black Mountain were irrigated when the soil was dry. Plots at GES were sown using an Agrowdrill (Agrowplow) with 50 g seed per plot with a 15 cm between-row spacing, and 2-5 cm within row spacing. Plots were 6 m long and 10 rows wide and the quadrats were 1.2 m long and 0.3 m wide. Fertiliser was applied at sowing (Granulock 15) at 110kg/ha. The soil type in Expt 2 and Expt 4 at GES were grey brown clay (Alluvial flats) and shallow red podzolic soil respectively. The row tests Ext 5 and 6 in GES were arranged in the same way as those in Black Mountain but cultivated in the same way as plots. Plots were not irrigated and the temperature and rainfall data were extracted from GES Automatic Weather Station and listed in Appendix Table 2.1. Weather data from GES were also applicable to Black Mountain, except that temperature used to calculate thermal time in Expt 1 and Expt 3 was from a temperature logger placed at the site.

Expt 4, 5 and 6 sown in plots and rows at GES were severely damaged by birds at physiological maturity. Plot harvests were not made and only limited data could be collected.

2.2.2 Genotyping

Families from HI25M crossed with Espada or Young were genotyped with markers for Rht- D1b or Rht-B1b designed byEllis et al. (2002). Rht18 is tightly linked with SSR marker WMS4603 (Spielmeyer et al. unpublished), and lines with Rht18 carried the 239 bp allele in contrast with 220 bp in lines lacking Rht18. All lines used in phenotypic experiments were fixed according to the Table 2.2.

Table 2.2 Pedigree information in populations with four genotypic classes developed from HI25M and Espada (Rht-D1b) or Young (Rht-B1b)

Genotype Rht-1 Rht18 Rht18 Rht-D1a/B1a Rht18 Rht-D1b/B1b Rht-D1b/B1b - Tall Rht-D1a/B1a - Double dwarf Rht-D1b/B1b Rht18 2.2.3 Morphological measurements

Plant height was measured from the soil surface to the tip of the spike. Plant height and internode length were measured using a ruler and recorded in millimetres. The whole stem (free from leaf sheath) was dissected into different internodes and named in order from top to bottom as: Peduncle, P-1, P-2 and P-3 as shown in Figure 2.1.

Flowering time was determined using the Zadok’s scale in all lines in Expt 1 and 3 when around 50% or more of ears were flowering (anthers visible) and 7 days past first flowering in Expt 2 and 4.

Grain weight, grain number, biomass and yield were measured after threshing (Wheat Head Thresher, Model: WHTA010002 220v, Precision Machine Co., Inc.), and grain number was calculated using a seed counter (Contador, PFEUFFER GmbH). Biomass was determined

for the dried plants, and harvest index was calculated as the ratio of grain weight to biomass. Yield in the plots was recorded as grain weight during final machine harvest.

2.2.4 Experiment design and data analysis

Randomisation was arranged at the line level across four genotypes in the experiments conducted at Black Mountain and GES. Statistical analysis was performed for the effect of genotype using ANOVA in Genstat (V16th Edition) and the l.s.d. was provided. In Expt 2, where no genotypic difference was observed, contrast comparison model in ANOVA in Genstat was also used to look at chosen sets of comparisons individually.

Figure 2.1 Stem was dissected into 4 sections recorded as peduncle, P-1, P-2 and P-3+ (includes the lower internodes) in 4 genotypic classes: Rht18, Rht-D1b, Tall and Double dwarf.