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 _publication/Gao[[:space:]]et[[:space:]]al._2024[[:space:]]-[[:space:]]Exploring[[:space:]]natural[[:space:]]genetic[[:space:]]variation[[:space:]]in[[:space:]]photosynthe.pdf filter=lfs diff=lfs merge=lfs -text
 _publication/erae198_suppl_supplementary_figures_s1-s7.pdf filter=lfs diff=lfs merge=lfs -text
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assays/AssessmentOfMorphologicalAndGrowth-relatedTraits/protocols/AssessmentOfMorphologicalAndGrowth-relatedTraitsProtocol.md

+## Assessment of morphological and growth-related traits
+
+To determine the relative growth rate (RGR) of the 23 barley inbreds, aboveground biomass data were collected in the field experiment in Düsseldorf at six different time points during the vegetative period: at 62, 69, 76, 83, 97, and 125 DAS. Plants of one row (initially 33 kernels were sown) per plot were harvested for the 23 genotypes with three replicate plots. Wild animals visited the trails, and therefore the number of damaged plants for each row was recorded.
+
+The dry weight per row per plot was used to estimate the dry mass per plant (DMP), which was needed for the assessment of growth curve parameters, using the following equation:
+
+**(1)**
+$$DMP=\frac{DM}{(TNP-NDP)+0.8\times NDP}$$
+
+where TNP was the total number of plants, NDP the number of damaged plants, 0.8 was the completeness of the damaged plants based on the observation during the harvest. DMP calculated as described above, was corrected separately for each time point for replicate and block effects. The corrected values were then used for further analyses.
+
+In the climate chamber experiment, the total aboveground DMP was measured by weighing at eight different time points (26, 36, 46, 57, 74, 102, 113, and 142 DAS) except for the two inbreds IG31424 and HOR1842, for which only the initial and the final DMP were determined at 26 and 142 DAS. Three replicates per genotype were collected for each time point.
+
+To assess the relationship between DMP and time, logistic (Verhulst, 1838), power-low (Paine et al., 2012), and quadratic regression (Lithourgidis and Dordas, 2010) models were fitted. The quadratic regression model was used:
+
+**(2)**
+$$y_r=a+bt-ct^2$$
+
+where *a* represents the initial biomass, *b* and *c* the growth rate parameters. This model had a high coefficient of determination (⁠$R^2$⁠) and the highest heritability across all 23 barley inbreds. Thus, the quadratic regression was used for estimation of RGR. $RGR_a$, $RGR_b$, $RGR_c$ represent the parameters in quadratic regression *a*, *b*, and *c*, respectively.
+
+Morphological parameters were collected in multi-year and multi-environment field experiments that took place in the years 2017–2021 at Düsseldorf, Cologne, Mechernich, and Quedlinburg (Shrestha et al., 2022; Wu et al., 2022). Not all locations were used in all years to assess all parameters. Flag leaf length (FL, cm) and width (FW, cm), plant height (PH, cm), flowering time (FT), awn length (AL, cm), spike length (EL, cm), and spikelet number in one row of the spike (SR), seed length (SL, mm), seed width (SW, mm), seed area (SA, mm2), and thousand grain weight (TGW, g), grain weight (GW, kg per 10 m2), and net straw weight (NSW, kg per 10 m2) were measured as morphological parameters. FL, FW, AL, EL were measured by ruler, SL, SW, and SA were measured by MARViN seed analyser (MARViNTECH GmbH, Germany), and TGW was measured by MARViN and a balance.
+
+The same set of morphological parameters were also measured in the climate chamber experiment. FL and FW were collected at 74 and 102 DAS with three replicates, and spike-related traits (AL, EL, SR, SL, SW SA, and TGW) were collected at 142 DAS with three replicates. Additionally, the total stem (without spike) weight per plant (SWP, g), total spike weight per plant (SKWP, g), total stem weight of main stem (TSWM, g), and spike weight of main stem (SKWM, g) were also collected in the climate chamber experiment. Harvest index (HI) was calculated using the following equation:
+
+**(3)**
+$$HI=\frac{SKWP}{DMP}$$
+
+In addition, harvest index of main stem (MSHI) was calculated using the following equation:
+
+**(4)**
+$$MSHI=\frac{SKWP}{TSWM}$$
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assays/AssessmentOfPhotosynthesis-relatedParameters/protocols/AssessmentOfPhotosynthesis-relatedParametersProtocol.md

+## Assessment of photosynthesis-related parameters
+
+In the field experiments, the top fully expanded leaves of three representative plants from each plot were measured from seedling stage (ZS13; Zadoks et al., 1974) to dough development (ZS87) using a MultispeQ V2 device (Kuhlgert et al., 2016). We used the measurement protocol ‘Photosynthesis RIDES’, by which the intensity of actinic light was automatically set to the ambient light intensity measured by the built-in light sensor. The following parameters were used for the further analyses: linear electron flow (LEF), the fraction of open PSII centers (qL), the quantum yield of PSII (Phi2), the maximum efficiency of PSII in light-adapted state (Fvʹ/Fmʹ), the total NPQ (NPQt), the quantum yield of NPQ (PhiNPQ), the quantum yield of non-regulated dissipation processes (PhiNO), and relative chlorophyll content (SPAD). In addition, the MultispeQ also recorded environmental parameters, such as the intensity of photosynthetically active radiation (PAR), ambient temperature, ambient humidity, and ambient pressure.
+
+In the climate chamber experiment, gas exchange parameters were measured multiple times from tillering stage (ZS21) to dough development (ZS89) alongside the MultispeQ measurements. The measurements were made on the top fully expanded leaves on the main stem. Three different light intensities (PAR=400, 800, 1500 μmol m−2 s−1) were used as simulated low-light (LL), medium-light (ML) and high-light (HL) conditions for the MultispeQ measurements. Leaf-level gas exchange measurements were performed by LI-6800 (LI-COR Biosciences Inc., Lincoln, NE, USA). Three replicates per genotype were measured from 1 h after the onset of the light period. The settings inside the LI-6800 chamber were as follows: PAR was kept at 1500 μmol m−2 s−1 (as in the simulated HL) with 50% blue and 50% red light, 400 μmol m−2 s−1 air flow rate, 10 000 rpm fan speed, 55% relative humidity, and 18 °C air temperature. The proportion of blue and red light was chosen to mimic the HL conditions in the field. The humidity and air temperature in the LI-6800 chamber were chosen to mimic the growth condition in the climate chamber and, thus, reduce the time needed for stabilization prior to the measurements. The CO2 concentration inside the LI-6800 chamber was 400 ppm during pre-acclimation, which lasted between 10 and 15 min. After the pre-acclimation, photosynthetic CO2 response (A–Ci) curves were measured according to the dynamic assimilation technique (Saathoff and Welles, 2021). CO2 ramps were started from 1605 to 5 ppm with ramping rates of 200 ppm. The A–Ci curves were then analysed using the ‘plantecophys’ package (Duursma, 2015) in R version 4.0.3 to estimate the maximum rate of carboxylation (Vc,max), the maximum rate of electron transport (Jmax), and triose phosphate utilization (TPU).
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