DOI: 10.5061/dryad.np5hqbzrd
title: Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner: Additional data (images, phylogenetic alignments and trees)

description: Flower development is an important determinant of grain yield in crops. In
 wheat, natural variation for the size of spikelet and floral organs is
 particularly evident in Triticum polonicum, a tetraploid subspecies of
 wheat with long glumes, lemmas, and grains. Using map-based cloning, we
 identified VRT2, a MADS-box transcription factor belonging to the SVP
 family, as the gene underlying the P1 locus. The causal P1 mutation is a
 sequence substitution in intron-1 that results in both increased and
 ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic
 variation studies, we propose that the intron-1 mutation in VRT-A2 is the
 unique T. polonicum species defining polymorphism, which was later
 introduced into hexaploid wheat via natural hybridizations. Near-isogenic
 lines differing for the T. polonicum long-glume (P1) locus revealed a
 gradient effect of P1 across florets. Transgenic lines of hexaploid wheat
 carrying the T. polonicum VRT-A2 allele show that expression levels of
 VRT-A2 are highly correlated with spike, glume, grain, and floral organ
 length. These results highlight how changes in expression profiles,
 through variation in cis-regulation, can impact on agronomic traits in a
 dosage-dependent manner in polyploid crops. Here, we deposit additional
 data that did not fit into the manscurpt itself. These are images of whole
 spikes and of spikelets of Triticum polonicum, Triticum petropavlovskyi,
 Triticum aestivum 'Arrancada' landrace group, the hexaploid and
 tetraploid near-isogenic lines described in the manuscript, as well as
 imags from the CSxPol F2 population. We also upload a phylogenetic tree,
 including the protein sequences and where they were sourced from, as well
 as a description of the methods used.
description: <strong>Images of whole wheat
 spikes:</strong> Individual wheat
 spikes (<em>Triticum polonicum</em>, <em>Triticum
 petropavlovskyi</em>, <em>Triticum aestivum</em>
 'Arrancada' landrace group) were harvested and placed on black
 cloth to image them. A scale bar and the pot label were included in every
 image to measure dimensions and correctly identify each
 accession/genotype.  
 <strong>Images of wheat
 spikelets:</strong> After imaging the
 whole wheat spikes as described above (<em>Triticum
 polonicum</em>, <em>Triticum petropavlovskyi</em>,
 <em>Triticum aestivum</em> 'Arrancada' landrace
 group), one or two spikelets from the centre of each spike were cut off
 and placed on black cloth for imaging. A scale bar and the pot label were
 included in every image to measure dimensions and correctly identify each
 accession/genotype. A cropped version of these images have been used for
 Figure 3B and C in the manuscript.  
 <strong>Alignment of StMADS11-like
 proteins:</strong> 1) Protein sequences
 were downloaded from public databases: -
 AmIncomposita|Q2UZM5_ANTMA: UniProt
 https://www.uniprot.org/uniprot/Q2UZM5 -
 AtSVP|AT2G22540: Ensembl Plants
 http://plants.ensembl.org/Arabidopsis_thaliana/Gene/Summary?g=AT2G22540;r=2:9579647-9583901;t=AT2G22540.1;db=core - AtAGL24|AT4G24540: Ensembl Plants http://plants.ensembl.org/Arabidopsis_thaliana/Gene/Summary?g=AT4G24540;r=4:12670897-12674106;t=AT4G24540.1;db=core - StMADS16|PGSC0003DMT400024247: Ensembl Plants http://plants.ensembl.org/Solanum_tuberosum/Gene/Summary?g=PGSC0003DMG400009363;r=4:64959661-64967188;t=PGSC0003DMT400024246;db=core - StMADS11|PGSC0003DMT400083739: Ensembl Plants http://plants.ensembl.org/Solanum_tuberosum/Gene/Summary?g=PGSC0003DMG400033570;r=1:82400439-82408299;t=PGSC0003DMT400083739;db=core - ZMM26|GRMZM2G046885: Ensembl Plants http://plants.ensembl.org/Zea_mays/Gene/Summary?g=GRMZM2G046885;r=5:215341117-215349179;t=GRMZM2G046885_T01;db=otherfeatures - ZMM19|GRMZM2G370777: Ensembl Plants http://plants.ensembl.org/Zea_mays/Gene/Summary?g=GRMZM2G370777;r=4:181856997-181864042;t=GRMZM2G370777_T01;db=otherfeatures - BdMAD44|Bradi3g58220: Phytozome 12.1 https://phytozome.jgi.doe.gov/pz/portal.html#!gene?search=1&detail=1&method=5010&searchText=transcriptid:32815705<br> <br> - HvBM10|HORVU6Hr1G077300: Ensembl Plants http://plants.ensembl.org/Hordeum_vulgare/Gene/Summary?g=HORVU6Hr1G077300;r=chr6H:529572891-529579621;t=HORVU6Hr1G077300.1;db=core - OsMADS22|Os02g0761000: Ensembl Plants http://plants.ensembl.org/Oryza_sativa/Gene/Summary?g=Os02g0761000;r=2:32038935-32044840;t=Os02t0761000-01;db=core - TaSVP-A1|TraesCS6A02G313800: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS6A02G313800;r=6A:550637111-550643896;t=TraesCS6A02G313800.1;db=core - TaSVP-B1|TraesCS6B02G343900: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS6B02G343900;r=6B:605795238-605801747;t=TraesCS6B02G343900.1;db=core - TaSVP-D1|TraesCS6D02G293200: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS6D02G293200;r=6D:403969791-403976317;t=TraesCS6D02G293200.1;db=core - ZMM21|GRMZM5G814279: Ensembl Plants http://plants.ensembl.org/Zea_mays/Gene/Summary?g=GRMZM5G814279;r=9:6696564-6707154;t=GRMZM5G814279_T01;db=otherfeatures - BdAGL33|Bradi1g45812: Phytozome 12.1 https://phytozome.jgi.doe.gov/pz/portal.html#!gene?search=1&detail=1&method=5010&searchText=transcriptid:32793162 - HvVRT-2|HORVU7Hr1G036130: Ensembl Plants http://plants.ensembl.org/Hordeum_vulgare/Gene/Summary?g=HORVU7Hr1G036130;r=chr7H:82946101-82953235;t=HORVU7Hr1G036130.1;db=core - OsMADS55|Os06g0217300: Ensembl Plants http://plants.ensembl.org/Oryza_sativa/Gene/Summary?g=Os06g0217300;r=6:5952599-5963309;t=Os06t0217300-01;db=core - TaVRT-A2|TraesCS7A02G175200: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS7A02G175200;r=7A:128826237-128833021;t=TraesCS7A02G175200.1;db=core - TaVRT-B2|TraesCS7B02G080300: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS7B02G080300;r=7B:90189439-90195307;t=TraesCS7B02G080300.1;db=core - TaVRT-D2|TraesCS7D02G176700: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS7D02G176700;r=7D:128912281-128919539;t=TraesCS7D02G176700.1;db=core - ZMM20|GRMZM2G059102: Ensembl Plants http://plants.ensembl.org/Zea_mays/Gene/Summary?g=Zm00001d027957;r=1:18299864-18321135;t=Zm00001d027957_T001;db=core - BdMAD18|Bradi1g72150: Phytozome 12.1 https://phytozome.jgi.doe.gov/pz/portal.html#!gene?search=1&detail=1&method=5010&searchText=transcriptid:32799834 - HvBM1|HORVU4Hr1G077850: Ensembl Plants http://plants.ensembl.org/Hordeum_vulgare/Gene/Summary?g=HORVU4Hr1G077850;r=chr4H:605649505-605668683;t=HORVU4Hr1G077850.1;db=core - OsMADS47|Os03g0186600: Ensembl Plants http://plants.ensembl.org/Oryza_sativa/Gene/Summary?g=Os03g0186600;r=3:4519405-4525692;t=Os03t0186600-01;db=core - TaSVP-A3|TraesCS4A02G002600: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS4A02G002600;r=4A:2402444-2412218;t=TraesCS4A02G002600.1;db=core - TaSVP-B3|TraesCS4B02G302600: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS4B02G302600;r=4B:589180023-589195403;t=TraesCS4B02G302600.1;db=core - TaSVP-D3|TraesCS4D02G301100: Ensembl Plants http://plants.ensembl.org/Triticum_aestivum/Gene/Summary?g=TraesCS4D02G301100;r=4D:469304325-469319944;t=TraesCS4D02G301100.1;db=core The FASTA file with all protein sequences used is deposited here as well (StMADS11-like_MADS-box_proteins.fa).   2) The protein alignment was made using MUSCLE integrated into the MEGA X software with default settings: Gap Open penalty: -2.9 Gap Extension Penalty: 0 Hydrophobicity Multiplier: 1.2 Clustering Method (Iteration 1,2): UPGMA Clustering Method (Other iterations): UPGMA Min Diag Length (lambda): 24 The alignment file is deposited here in FASTA format (StMADS11-like_MADS-box_proteins-alignment.fas).   3) A phylogenetic tree was made from the alignment file using MEGA X software with the following settings: Statistical method: Neighbor-joining Test of Phylogeny: Boostrap-method No. of Bootstrap Replications: 1000 bootstraps Model/Method: Poisson model Rates among sites: Uniform rates Pattern among lineages: Same (Homogenous) Gaps/Missing Data Treatment: Pairwise Deletion The phylogenetic tree is deposited here in Newick format (StMADS11-like_MADS-box_proteins-alignment_newick-tree.txt). The tree was also saved as a tif file, and the three grass SVP groups shaded with different colours in Adobe Illustrator 2021; this image was used for Supplementary Figure 8 in the manuscript.