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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)

Citation

Adamski, Nikolai et al. (2021), 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), Dryad, Dataset, https://doi.org/10.5061/dryad.np5hqbzrd

Abstract

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.

Methods

Images of whole wheat spikes:

Individual wheat spikes (Triticum polonicum, Triticum petropavlovskyi, Triticum aestivum '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.

 

Images of wheat spikelets:

After imaging the whole wheat spikes as described above (Triticum polonicum, Triticum petropavlovskyi, Triticum aestivum '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.

 

Alignment of StMADS11-like proteins:

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

- 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.