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diff --git a/assays/AssessmentOfMorphologicalAndGrowth-relatedTraits/protocols/AssessmentOfMorphologicalAndGrowth-relatedTraitsProtocol.md b/assays/AssessmentOfMorphologicalAndGrowth-relatedTraits/protocols/AssessmentOfMorphologicalAndGrowth-relatedTraitsProtocol.md
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@@ -0,0 +1,31 @@
+## 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)**
+$$MSHI=\frac{SKWP}{TSWM}$$
\ No newline at end of file
diff --git a/assays/AssessmentOfPhotosynthesis-relatedParameters/README.md b/assays/AssessmentOfPhotosynthesis-relatedParameters/README.md
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@@ -0,0 +1,5 @@
+## 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).
\ No newline at end of file
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diff --git a/assays/StatisticalAnalysis/protocols/ClimateChamberExperiment.md b/assays/StatisticalAnalysis/protocols/ClimateChamberExperiment.md
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@@ -0,0 +1,9 @@
+## Climate chamber experiment
+
+The adjusted entry means of carbon assimilation-related parameters from the climate chamber experiment were calculated based on the following model:
+ 
+**(7)**
+$$y_{(A)ijklmr} = \mu + G_i + ZS_j + D_k + (D:TW)_{kl} + S_m + (G:S)_{im} + \epsilon_{(A)ijklmr}$$
+where $y_{(A)ijklmr}$ was the carbon assimilation-related parameter, $(D:TW)_{kl}$ the effect of the *l*th time window in the *k*th date of measurement, and $\epsilon_{(A)ijklmr}$ the random error. To estimate adjusted entry means for carbon assimilation-related parameters of six barley inbreds, $G_i, ZS_j, S_m$ and $(G:S)_{im}$ were treated as fixed effects, as well as $D_k$ and $(D:TW)_{kl}$ as random effects.
+
+The relationship between photosynthesis-related parameters and morphological or growth-related parameters of the inbreds was evaluated by Pearson’s correlation coefficient between adjusted entry means.
\ No newline at end of file
diff --git a/assays/StatisticalAnalysis/protocols/EstimationOfHeritability.md b/assays/StatisticalAnalysis/protocols/EstimationOfHeritability.md
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+++ b/assays/StatisticalAnalysis/protocols/EstimationOfHeritability.md
@@ -0,0 +1,15 @@
+## Estimation of heritability
+
+Broad-sense heritability $(⁠H^2)$ was estimated for both field and climate chamber experiments based on the following method:
+
+**(8)**
+$$H^2 = \frac{\sigma_G^2}{\sigma_G^2 + \frac{\bar{\upsilon}_\delta^BLUE}{2}}$$
+where $\sigma_G^2$ was the genotypic variance calculated based on the above models with a random effect for $G_i$ and $\bar{\upsilon}_\delta^BLUE$ was the mean variance of the difference of two genotypic means (Holland et al., 2003; Piepho and Möhring, 2007).
+
+To avoid the effect of the varying number of replicates, the $H^2$ of photosynthesis-related parameters was estimated for each developmental stage based on the following equation:
+ 
+**(9)**
+$$H^2 = \frac{\sigma_G^2}{\sigma_G^2 + \frac{\sigma_{G:E}^2}{5} + \frac{\sigma_e^2}{5}}$$
+where $\sigma_{G:E}^2$ was the variance of the interaction of barley inbreds and environments, and $\sigma_e^2$ was the residual variance.
+
+We used the statistical software R to perform all statistical analyses.
\ No newline at end of file
diff --git a/assays/StatisticalAnalysis/protocols/FieldExperiment.md b/assays/StatisticalAnalysis/protocols/FieldExperiment.md
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+++ b/assays/StatisticalAnalysis/protocols/FieldExperiment.md
@@ -0,0 +1,20 @@
+## Field experiment
+
+Due to the strong dependence of photosynthesis on light intensity (Ogren, 1993), we considered three light intensity clusters when analysing field measurements: LL, ML, and HL conditions. These light intensity clusters were identified by K-means clustering of PAR and LEF. In addition, we also compared three main developmental phases of barley, i.e. slow expansion phase (SEP) (ZS<30), rapid expansion phase (REP) (30≤ZS<60), and anthesis and senescence phase (ASP) (ZS≥60). These two factors, light intensity (L) and developmental phase (S), each with three levels, were considered when analysing the MultispeQ parameters from the field experiments based on the following linear model with the quantitative covariates light intensity (PAR) and developmental stage (ZS):
+
+**(5)**
+$$y_{(p)ijklmnopqr} = \mu + G_i + E_j + (G:E)_{ij} + ZS_k + L_l + S_m + (G:L)_{il} + (G:S)_{im} + M_n + D_o + PAR_{ijklmnor} + T_{ijklmnor} + (E:R)_{jp} + (E:R:B)_{jpq} + \epsilon_{(p)ijklmnopqr}$$
+
+where $y_{(p)ijklmnopqr}$ was the observed MultispeQ parameter across all light conditions and all developmental stages, μ the general mean, $G_i$ the effect of the *i*th inbred, $E_j$ the effect of the *j*th environment, $(G:E)_{ij}$ the interaction between the *i*th inbred and the *j*th environment, $ZS_k$ the effect of the *k*th Zadoks score of barley development, $L_l$ the effect of the *l*th light intensity cluster, $S_m$ the effect of the *m*th barely developmental phase, $(G:L)_{il}$ the interaction between the *i*th inbred and the *l*th light intensity cluster, $(G:S)_{im}$ the interaction between the *i*th inbred and the *m*th barley developmental phase, $M_n$ the effect of the *n*th MultispeQ device, $D_o$ the effect of measurement date, $(E:R)_{jp}$ the effect of the *p*th replicate nested within the *j*th environment, $(E:R:B)_{jpq}$ the effect of the *q*th block nested within the *p*th replicate in the *j*th environment, $PAR_{ijklmnor}$ the light intensity of each measurement, $T_{ijklmnor}$ the ambient temperature of each measurement, and $\epsilon_{(p)ijklmnopqr}$ the random error.
+
+To estimate adjusted entry means for MultispeQ parameters of all inbreds, $G_i, E_j, (G:E)_{ij}, ZS_k, L_l, S_m, (G:L)_{il}, and (G:S)_{im}$ were treated as fixed effects, and $M_n, D_o, (E:R)_{jp}, (E:R:B)_{jpq}$ as random effects, $PAR_{ijklmnor}$ and $T_{ijklmnor}$ were covariates. Furthermore, we calculated adjusted entry means for all inbreds for each light intensity cluster as well as each developmental phase.
+
+In addition, to evaluate the effect of each fixed factor and covariate, analysis of variance (ANOVA) was conducted.
+
+To assess the heritability of each photosynthesis-related parameter at each developmental stage, which was considerably shorter than the above-mentioned three developmental phases, data were separated into eight stages from Zadoks principal growth stages. The adjusted entry means were calculated based on the following model:
+
+**(6)**
+$$y_{(pd)ijlnopqr} = \mu + G_i + E_j + M_n + D_o + PAR_{ijklmnor} + T_{ijklmnor} + (E:R)_{jp} + (E:R:B)_{jpq} + \epsilon_{(p)ijklmnopqr}$$
+where, $y_{(pd)ijlnopqr}$ was the photosynthesis-related parameter for each developmental stage across all other factors. Due to convergence problems, the interaction between $G_i and E_j$ was removed from this model.
+
+To assess the similarities among the barley genotypes with respect to their photosynthesis parameters, we performed hierarchical clustering by Ward’s minimum variance theory (Ward, 1963) using the adjusted entry means of PSII parameters and SPAD at three different developmental phases. Furthermore, principal component analysis (PCA) was conducted by using the adjusted entry means calculated for each inbred in each of the developmental phases described before. In addition, a PCA was conducted by using the environmental factors (temperature and precipitation) of the inbreds at the country of origin. The relationship between photosynthesis-related parameters and morphological or growth-related parameters of the inbreds was evaluated by Pearson’s correlation coefficient among adjusted entry means.
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@@ -0,0 +1,3 @@
+## Climate chamber experimental conditions and design
+
+Based on the results of the field experiments, six representative barley inbreds (HOR1842, IG128216, IG31424, ItuNative, K10877, and W23829/803911) were selected for a climate chamber experiment. The experimental design was a randomized complete block design with three replicates/blocks. A total of 32 plants were planted for each inbred in total. The gas exchange measurements were performed 21, 24, 26, 31, 36, 39, 46, 51, 57, 74, 87, 88, 100, and 102 days after sowing (DAS). At each measurement day, three measurements were taken from three different plants of an inbred, where always one plant from each of the blocks was studied. In addition, the Zadoks score for each evaluated plant was rated. In addition, we harvested three plants per inbred (i.e. one per block) and then measured dry weight of aboveground biomass per plant 26, 36, 46, 57, 74, 102, 113, and 142 DAS. The growth conditions in the climate chamber were as follows: 14 h/10 h light/dark photoperiod, 18 °C/16 °C temperature, and 55% relative humidity. The maximal light intensity measured at 15 cm from the light panel was 750 μmol m−2 s−1.
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+## Field Experimental Design
+
+Twenty-three spring barley (*Hordeum vulgare*) inbred lines (inbreds) were selected from a worldwide collection of 224 barley landraces and cultivars based on their genetic and phenotypic diversity (Weisweiler et al., 2019). These 23 barley inbreds are the parents of the double round-robin population (Casale et al., 2022). All 23 inbreds were grown at three different locations [Bonn (50°37ʹ31.82″ N 6°59ʹ18.508″ E), Cologne (50°57ʹ34.345″ N 6°51ʹ36.407″ E), and Düsseldorf (51°10ʹ42.599″ N 6°48ʹ8.268″ E)] in Germany in 2021. In Bonn, the experimental design was an α-design with two complete replications, where the plants were sown in 10 m2 plots on 31 March 2021. In Düsseldorf, the experimental design was an α-design with three complete replications. The experimental units were single rows with 33 kernels per row, and six rows together were considered as one plot (replicate). The plants in Düsseldorf were sown on 31 March 2021. In Cologne, two distinct trials were performed, which are named in the following as ‘mini-big plot’ and ‘big plot’ trials. Within each of these two trials, 23 inbreds were grown as replicated checks in an augmented design. In the mini-big plot trial, each plot had a size of 2.25 m2. The entire mini-big plot trial had 320 plots, in which the 23 inbreds were grown as replicated checks twice. The big plot trial, in which each plot had a size of 10 m2, comprised also 320 plots and the 23 inbreds were also grown as checks but only one time. Fertilization and crop protection followed local practices. Air temperature and precipitation were recorded during the field experiments at all three locations (Supplementary Fig. S1). In Bonn, the field site has chernozem-para-brown soil, and the field sites, in Cologne and Düsseldorf, have para-brown soil (Bundesanstalt für Geowissenschaften und Rohstoffe).
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@@ -0,0 +1,11 @@
+<img src=./SupplementaryFigureS1.png width=60%>
+
+**Supplementary Fig. S1**: Air temperature and precipitation recorded during the field
+experiments in Bonn, Cologne, Düsseldorf. Dashed, solid, and dotted represent the
+maximum, average, and minimum temperature of one day, respectively. Green, purple
+and orange color represent three location Bonn, Cologne and Düsseldorf, respectively. The
+temperature data of Düsseldorf from 2021/04/24 to 2021/04/25 was missing. 
+
+## Figure Source
+
+https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jxb/75/16/10.1093_jxb_erae198/3/erae198_suppl_supplementary_figures_s1-s7.pdf?Expires=1734506953&Signature=GYSCYBYfRHkM41-kJRdWE4i4nrdF~9zwYjFWz3Wb-OizhbqKvIOp94dhTVr~cQkZtjMTZCiUq9dZswP0UQz6uua9Gp67LhRa7DxwkiTHmibaVmWE8yjh3XEwwax8B7VBPss~M4YcQuzatLXyAj01TK0AEIvXAxIyvMRINrLIOaTk9KKmkyMqbpUQXHAzGVrOuBaqW-vIsbpymNvnvIo2KyJGOVw2El9MEhdQAXjRPh94dbJcvD9U-h5-tqUpMffYyWtK6NFExYLdYKeMu-WmhzP-eyLCmAYlIDB2KnMH6aruSgtecGP1iL2pPgt~CUBdlHpURLS2AjX4el3G~2Jf4w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA
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