DOI: 10.5061/dryad.ffbg79cth
title: Raw data: Spikelet stop determines the maximum yield potential stage in barley

description: Determining the grain yield potential contributed by grain number is a
 step towards advancing cereal crops' yield. To achieve this aim, it
 is pivotal to recognize the maximum yield potential (MYP) of the crop. In
 barley (Hordeum vulgare L.), the MYP is defined as the maximum spikelet
 primordia number of a spike. Previous barley studies often assumed the awn
 primordium (AP) stage as the MYP stage regardless of genotypes and growth
 conditions. From our spikelet-tracking experiments using the two-rowed
 cultivar Bowman, we found that the MYP stage can be different from the AP
 stage. Importantly, we find that the occurrence of inflorescence meristem
 (IM) deformation and its loss of activity coincided with the MYP stage,
 indicating the end of further spikelet initiation. Thus, we recommend
 validating the barley MYP stage with the IM's shape and propose this
 approach (named Spikelet Stop) for MYP staging. Following this approach,
 we compared the MYP stage and the MYP in 27 two- and six-rowed barley
 accessions grown in the greenhouse and field. Our results reveal that the
 MYP stage can be reached at various developmental stages, which majorly
 depend on the genotype and growth conditions. Furthermore, we found that
 two-rowed barleys’ MYP and the duration reaching the MYP stage may
 determine their yield potential. Based on our findings, we suggest key
 steps for the identification of the MYP in barley that can also be applied
 in a related crop such as wheat.
description: <b>Plant materials
 and growth conditions</b> We conducted one greenhouse and
 one field experiment using a panel of twenty-seven barley accessions
 chosen from a worldwide collection (Alqudah et al., 2014). The field study
 was conducted at the Leibniz Institute of Plant Genetics and Crop Plant
 Research, Gatersleben, Germany (51° 49′ 23″ N, 11° 17′ 13″ E, altitude 112
 m) from March to August 2017 and greenhouse from July to December 2017.
 For both the experiments, we followed similar sowing and pre-treatment
 protocols. Grains were sown on 96 well trays and grown under greenhouse
 conditions (photoperiod, 16h/8h, light/dark; temperature, 20<sup>o
 </sup>C/16<sup>o </sup>C, light/dark) for two weeks.
 Then, the grown plants were vernalized at 4<sup>o </sup>C for
 four weeks and then given hardening in the greenhouse for a week.
 Following the acclimatization, plants were directly transplanted in the
 silty loam soil for the field experiment; however, in the greenhouse
 experiment (photoperiod, 16h/8h, light/dark; temperature, 20<sup>o
 </sup>C/16<sup>o </sup>C, light/dark), plants were
 potted in a 9 cm pot (9 × 9 cm, diameter × height). For the field
 experiment, we followed a single plot per genotype design, in which each
 plot had eight rows with a 15 cm distance between the rows. The rows are
 0.8 m long, wherein we sowed five plants per row. Details of the selected
 barley panel and their field growth conditions were given in tables S1 and
 S2, respectively. The series of experiments on
 cultivar Bowman were performed in the greenhouse and climate chamber. The
 growth conditions and different pot sizes of the experiments are given in
 table S3. In all the greenhouse and climate chamber experiments, plants
 were grown in pots that contain two parts of autoclaved compost, two parts
 of ‘Rotes Substrat’ (Klasmann-Deilmann GmbH, Germany), and one part of
 sand. We followed the standard practices for irrigation, fertilization,
 and control of pests and diseases in these experiments. <b>Methods of phenotyping
 the traits</b> All traits were measured only
 from the main culm of a barley plant due to its higher phenotypic
 stability across growth conditions and its major contribution to the final
 grain yield (Cottrell et al., 1985; Elhani et al., 2007). In all Bowman
 spikelet-tracking experiments, plants were randomly selected, and spikes
 were dissected out almost every alternate day. In the spikelet-tracking
 experiments with the 27 accessions, random plants were dissected every two
 to three days or more, depending on their developmental rate. Dissection
 of spikes was performed according to the methods described in Kirby and
 Appleyard, 1984. Different spike developmental stages were identified by
 following the description provided earlier (Waddington et al., 1983; Kirby
 and Appleyard, 1984). For every stage of a spike, the decimal code
 suggested by Waddington et al., 1983 was given following the letter ‘W’
 (Waddington). A decimal code was assigned to a spike when the specific
 stage was found in a minimum of three or a maximum of four consecutive
 nodes of a spike (Waddington et al., 1983; Kirby and Appleyard, 1984).
 These nodes are always the most developed nodes of a spike (Kirby and
 Appleyard, 1984) and may be found close to the spike base, two to three
 nodes above the spike base, or in the center of a spike. For the potential spikelet number
 (PSN) of a spike, differentiated spikelets and undifferentiated spikelet
 ridges (usually found at the base and tip of a spike) were counted
 (Appleyard et al., 1982). Generally, barley forms three spikelets at every
 rachis node (Bonnett, 1935; Bonnett, 1966; Komatsuda et al., 2007; Koppolu
 et al., 2013), so the number of undifferentiated spikelet ridges were
 multiplied by three. The number of ridges developed on a spike was
 considered as spikelet ridge number (SRN), and the final spikelet number
 (SN) and grain number (GN) of a spike were enumerated after physiological
 maturity. Growing degree days (GDD) or thermal time was calculated from
 the average maximum and minimum air temperature of a day subtracted by the
 base temperature (McMaster and Wilhelm, 1997). We considered 0<sup>o
 </sup>C as the base temperature for barley (Gallagher et al., 1976).
 GDDs and PSN/SRN were taken from three plants, while SN and GN were from
 six plants.    <b>Data
 analysis</b> The data were analyzed using the
 Prism software, version 8.4.2 (GraphPad Software, LLC), and outliers were
 detected by the ‘ROUT’ method (Motulsky and Brown, 2006). Mean value
 comparison of different traits was made with either the multiple Student’s
 t-tests or paired Student’s t-test (parametric). The false discovery rate
 approach of the two-stage linear step-up procedure of Benjamini, Kreier,
 and Yekutieli (Q=5%) (Benjamini et al., 2006) was used to calculate the
 significance of Student’s t-tests. For traits like the Waddington scale
 and Growing Degree Days at MYP, a two-way ANOVA with Tukey’s multiple
 comparison test (alpha=5%) was used to identify the mean values'
 significance. For the spikelet ridge number (SRN), a one-way ANOVA with
 Tukey’s multiple comparison test (alpha=5%) was used to identify the mean
 values' significance. All the replicates of a genotype were analyzed
 individually and without assuming a consistent standard deviation. Linear
 regression was done using the appropriate dependent (Y values) and
 independent (X values) traits. The 95% confidence intervals were
 identified for every linear regression and plotted as confidence bands
 along with the ‘goodness of fit’ line.
description: Please refer to the 'Readme' file/sheet for further
 information on the data.