Supplementary Figures SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana Linda Zamariola 1, Nico De Storme 1, Katrijn.

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Supplementary Figures SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana Linda Zamariola 1, Nico De Storme 1, Katrijn Vannerum 1, Klaas Vandepoele 2,3, Susan J. Armstrong 4, F. Chris H. Franklin 4, Danny Geelen 1

GENE PROPOSED PROTEIN FUNCTION (TAIR)NASC line At4g35620 Cyclin-B2;2 (CYCB2;2)SALK_067657C At2g16270 Unknown proteinSALK_ At1g18370 HIK (HINKEL); microtubule motorSALK_056766C At5g48310 Similar to unknown protein Arabidopsis thaliana: At4g SALK_134597C At1g76310 Core cell cycle genes;CYCB2;4 (CYCLIN B2;4); cyclin-dependent protein kinase regulatorSALK_073438C At2g44190 Similar to unknown protein Arabidopsis thaliana: At3g SALK_047950C At3g11520 Encodes a B-type mitotic cyclinSALK_026335C At1g18040 Cyclin-dependent kinase D;3SALK_151657C At3g51280 Similar to male sterility MS5 family protein Arabidopsis thaliana: At5g SALK_046426C At5g15510 Similar to unknown protein Arabidopsis thaliana: At3g SALK_096426C At3g55660 Encodes a member of KPP-like gene family, homolog of KPP (kinase partner protein) gene in tomato. SALK_114559C At4g17000 Unknown proteinSALK_131195C At3g14190 Similar to unknown protein Arabidopsis thaliana: At5g SALK_070337; SALK_ At1g77720 Encodes a predicted protein kinase based on sequence similaritySALK_ At4g14330 Similar to AtK1 (Arabidopsis thaliana KINESIN 1): At4g21270SALK_ At5g01910 Unknown proteinSALK_ At1g03780 Homolog of vertebrate TPX2SALK_064340; SAIL_732_CO2 At5g48720 Encodes XRI1 (X-ray induced 1)SALK_056851; SAIL_1162_AO2 At3g23890 Encodes a topoisomerase II that is highly expressed in young seedlingsSALK_ At1g34770 Unknown proteinGK-534A03.01 AT3G44050 Microtubule motor activity, ATP bindingWiscDsLox4G07 Table S1 List of 21 genes obtained from the integrative comparative genomic analysis, their putative protein function, and the T-DNA lines screened for their pollen phenotype, ordered from NASC.

Figure S1 Schematic representation of the intron (black lines) and exon (black rectangulars) structure of the PANS1, PANS2, AtSGO1 and AtSGO2 genes and location of the T-DNA insertions (triangles). The white rectangulars correspond to the 5’ and 3’ UTR regions. Below PANS1 gene are indicated the primers used for gene expression analysis. pans2-2 pans1-1 pans1-2 PANS fwPANS re Atsgo1-2 Atsgo1-1 Atsgo2-1 At3g14190-PANS1 At5g12360-PANS2 At3g10440-AtSGO1 At5g04320-AtSGO2

Bra_ Bra_ Tha_lv m Cru_v m PANS1_AT3G14190 Bra_ Cru_v m Tha_lv m PANS2_AT5G12360 Cpa_evm.model.supercon Stub_PGSC0003DMP Sly_07g Vvi_06G10710 Ptr_09G07440 Phvul_008G Gma_02G44960 Gma_14G03810 Sly_03g Egr_K Osa_RSS1 Zma_ LOC Gra_011G Tca_1EG019923t1 Ccl_ m Csi_orange1.1g039037m Stub_PGSC0003DMP Figure S2 Phylogenetic tree showing PANS1 and PANS2 conservation among Eudicots. The tree was computed with MEGA 4.0 using neighbor joining method (NJ) with the Poisson correction and 500 bootstrap samples. Internal nodes were labeled with boostrap support values. Rice RSS1 and its maize homolog are also shown in the tree. Bra: Brassica rapa; Tha: Thellungiella halophila; Cru: Capsella rubella; Cpa: Carica papaya; Stub: Solanum tuberosum; Sly: Solanum lycopersicum; Vvi: Vitis vinifera; Ptr: Populus trichocarpa; Phvul: Phaseoulus vulgaris; Gma: Glycine max; Csi: Citrus sinensis; Ccl: Citrus clementina; Egr: Eucalyptus grandis; Tca: Theobroma cacao; Gra: Gossipium raimondii ; Osa: Oryza sativa; Zma: Zea mays.

Figure S3 Alignment of PANS1 and PANS2 homologous proteins and identification of three conserved motifs. Colors are reported according to the aminoacid properties, as displayed in the results from the MEME analysis. DEN box D box Unknown motif

Figure S4 Male meiotic spread of a) Col-0 wt and b) pans1-1 at prometaphase II, showing abnormalities in the organelle band configuration of pans1-1. Scale bar = 10 µm. a b

Figure S5 Comparison of Col-0 wt, Atsgo1-2 and Atsgo1-2 Atsgo2-1 phenotypes. Pollen distribution of a) Col-0 wt, b) Atsgo1-2, c) Atsgo1-2 (-/-) Atsgo2-1 (+/-) and d) Atsgo1-2 (-/-) Atsgo2-1 (-/-) analysed with Multisizer 3, Coulter Counter. d) Tetrads analysis of Col-0 wt, Atsgo2-1, Atsgo1-1, Atsgo1-2, Atsgo1-2 (-/-) Atsgo2-1 (-/-). e) Table representing the level of fertility, evaluated counting the number of seeds per silique, and the level of female fertility, evaluated by fertilizing mutant pistils with wt pollen, of Col-0 wt, Atsgo2-1, Atsgo1-1, Atsgo1-2, Atsgo1-2 (-/-) Atsgo2-1 (+/-) and Atsgo1-2 (-/-) Atsgo2-1 (-/-). n.a. = not analyzed. Genotype Number of seeds per silique Number of seeds per silique obtained from crosses with Col-0 wt pollen on mutant pistils Col-0 wt45,9±8,7 (n=36) Atsgo1-1 (-/-) SK ,3±4,4 (n=35)24,8±6,8 (n=11) Atsgo1-2 (-/-) SK255621,6±6,4 (n=47)8,5±6,1 (n=23) Atsgo2-1 (-/-) 48,4±8,2 (n=42)n.a. Atsgo1-2 (-/-) Atsgo2 (+/-) 3,8±1,5 (n=40)3,3±2,1 (n=33) Atsgo1-2 (-/-) Atsgo2 (-/-) 3,1±2,0 (n=40)2,9±2,6 (n=28) e Col-0 wt Atsgo1-2 Atsgo1-2 (-/-) Atsgo2-1 (-/-) a b Atsgo1-2 (-/-) Atsgo2-1 (+/-) cd f

Figure S6 FISH on Col-0 wt (a-c), pans1-1 (d-f) and Atsgo1-2 Atsgo2-1 (g-i) meiocytes. Pachytene (a,a’, d,d’, g,g’), diakinesis (b,b’, e,e’, h,h’), metaphase I (c,c’, f,f’, i,i’). Scale bar = 10 µm. a a’ b b’ cc’ d d’ ee’ ff’ gg’ hh’ ii’ Col-0 wt pans1-1Atsgo1-2 Atsgo2-1