Interaction between row‐type genes in barley controls meristem determinacy and reveals novel routes to improved grain

Summary Hordeum species develop a central spikelet flanked by two lateral spikelets at each inflorescence node. In ‘two‐rowed’ spikes, the central spikelet alone is fertile and sets grain, while in ‘six‐rowed’ spikes, lateral spikelets can also produce grain. Induced loss‐of‐function alleles of any of five Six‐rowed spike (VRS) genes (VRS1‐5) cause complete to intermediate gains of lateral spikelet fertility. Current six‐row cultivars contain natural defective vrs1 and vrs5 alleles. Little information is known about VRS mechanism(s). We used comparative developmental, expression and genetic analyses on single and double vrs mutants to learn more about how VRS genes control development and assess their agronomic potential. We show that all VRS genes repress fertility at carpel and awn emergence in developing lateral spikelets. VRS4, VRS3 and VRS5 work through VRS1 to suppress fertility, probably by inducing VRS1 expression. Pairing vrs3, vrs4 or vrs5 alleles increased lateral spikelet fertility, despite the presence of a functional VRS1 allele. The vrs3 allele caused loss of spikelet identity and determinacy, improved grain homogeneity and increased tillering in a vrs4 background, while with vrs5, decreased tiller number and increased grain weight. Interactions amongst VRS genes control spikelet infertility, determinacy and outgrowth, and novel routes to improving six‐row grain.


Fig. S5 Combined grain area from Bowman, single vrs mutants and double vrs mutants.
Violin plots show grain area distribution from all grain including those from central spikelets, lateral spikelets, and (when present) additional spikelets. Grain harvested from the main culm and tallest tiller per individual. n = 10 individuals.

Fig. S6 Tiller outgrowth rate over time in Bowman and single vrs mutants and double vrs mutants.
Graphs show duration and rate of tiller production in Bowman, single and double vrs mutants. Slope indicated by red lines showing rate of tiller production and goodness of fit indicated by R 2 correlation. Black circle symbols indicate window of active tiller outgrowth; dag, days after germination.

Fig. S7 Early tillering phase across Bowman and single vrs mutants (a)
Tiller number was recorded at five days after germination (dag) and repeated at two to three day intervals until 23 dag (n=10 individuals/ genotype). Plants were grown in 7 cm pots under long day glasshouse conditions. Tiller number included tillers with visible spikes as well as 'vegetative tillers' without emerged spikes. Dots represent means (SD). (b) Apex stage was determined at each time point in plants grown along side. VM, vegetative meristem; DR, double ridge; TSM, triple spikelet meristem; GP, glume primordium; LP, lemma primordium; SP, stamen primordium; AP, awn primordium; WA, white anther; GrA, green anther. Methods S1 Supplemental methods for genotyping and qPCR (1) KASP Genotyping. Each reaction contained 1 µl of 20 ng genomic DNA, 3 µl H2O, 4 µl 2x KASP master mix (LGC Genomics) and 0.11 µl allele-specific primers (LGC Genomics, listed below). Reactions were performed according to the following steps: 2 min at 20°C; 15 min at 94°C; 10 touch-down cycles of 20 sec at 94°C, 1 min at 62°C, with -0.7°C per cycle; 32 cycles of 20 s at 94°C, 1 min at 55°C, 2 min at 20°C. All assays were run on an ABI 3700 Step-One Plus Real Time PCR machine (Applied Biosystems).
(2) VRS1 Genotyping. PCR amplicons (primers listed below) were purified with ExoSAP-IT™ PCR Product Cleanup Reagent (ThermoFisher Scientific) and sequenced using the BigDye Terminator version 3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems). Samples were sequenced on an ABI3730 and trimmed sequences were analyzed with Sequencher 5.2.3 software (GeneCodes).
(3) Quantitative RT-PCR (qRT-PCR). The qRT-PCR for each gene was run in three (technical and biological) replicates and expression measured using TaqMan technology (Roche). Relative expression levels were calculated using two stable expressed internal reference genes, HvACTIN2 and PROTEIN PHOSPHATASE 2 (HvPP2A). Sequences of gene-specific oligonucleotides (listed below) and probe numbers were designed using Roche Universal Probe Library Design Center website.