DOI: 10.5281/zenodo.16970720
title: Strain, procedures and tools for reproducible genetic transformation and genome editing of Spirodela polyrhiza (L.) Schleid.

description: The dataset contains all the source data and original raw files for the manuscript: 

 

Strain, procedures and tools for reproducible genetic transformation and genome editing of Spirodela polyrhiza (L.) Schleid.

 

Verónica Barragán-Borrero1, Amanda de Santana Lopes2, Enrico Diniz Rodrigues Batista2, Martin Höfer2, Rana Elias1, Abhisek Chakraborty2, Arturo Ponce-Mañe1, Clotilde Descombes1,3, Laura Diezma-Navas1, Lydia Petraki1,4, Meret Huber2**, Shuqing Xu2**and Arturo Marí-Ordóñez1**.

 

1 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

2 Institute of Organismic and Molecular Evolution (IomE), Johannes Gutenberg University, Mainz Biozentrum I, Hanns-Dieter-Hüsch-Weg 15 D-55128, Mainz. Germany.

3 Current address: Institute of Plant Sciences Paris‐Saclay (IPS2), Centre National de la Recherche Scientifique, Université Paris‐Saclay, Gif Sur Yvette 91190, France.

4 Current address: Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.

 

** The authors responsible for correspondence and distribution of materials integral to the findings presented in this article are:

Arturo Marí-Ordóñez (arturo.mari-ordonez@gmi.oeaw.ac.at)

Shuqing Xu (shuqing.xu@uni-mainz.de)

Meret Huber (meret.huber@uni-mainz.de)

 

Original images, protein and RNA blots, qPCR data, Sanger sequencing files, and any other type of source data, are sorted by figure and figure panel. NGS data has been deposited to NCBI, accession numbers of datasets used in each figure are listed accordingly in this document. 

 

Contribution of each author to the source data presented here is shown at the end of the document. 

 

The content of each file is:

 

FIGURES:

 

FIGURE 1:

-        1B: Table with callus induction rate raw measurements of 4 S. polyrhiza clones under four tested protocols.

-        1C: Original pictures of S. polyrhiza callus induction.

-        1D: Table with callus induction raw rate measurements of S. polyrhiza clones.

-        1E: Original callus induction pictures of several S. polyrhiza clones.

-        1F-G: Callus growth rates raw measurement table and original pictures of S. polyrhiza SP162 and 9509 clones during regeneration.

 

FIGURE 2:

-        2B: Original pictures of S. polyrhiza callus induction at day 0.

-        2C: Original pictures of S. polyrhiza callus induction after 4 weeks.

-        2D: Original pictures of S. polyrhiza dark compact calli derived from root and pocket after 4 weeks and a callus after 10 weeks.

-        2E: Original pictures of S. polyrhiza pale green calli after 12 and 16 weeks.

-        2F: Raw calli growth rates measurement table and original side by side picture of S. polyrhiza SP162 dark and pale callus.

-        2I: Original picture of SP162 callus after 4 weeks of regeneration.

-        2J: Original pictures of SP162 callus acclimatation in magenta and dishes and close view of regenerating calli.

-        2K: Original pictures of SP162 regenerating frond.

 

FIGURE 3:

-        3A: Table with raw survival rate data of SP162 calli under different selection media.

-        3B: Images of SP162 calli under different antibiotic selection strength at start time, one and two weeks including replicates.

 

FIGURE 4:

-        4B: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 2-, 7- and 14-days post transformation under white and UV light.

-        4C: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 3- and 4-weeks post transformation under white and UV light.

-        4D: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 5- and 6-weeks post transformation under white and UV light.

-        4E: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 10-weeks post transformation under white and UV light.

-        4F: Raw Southern blot autoradiogram and EtBr gel staining prior to membrane transfer for the detection of pZmUBQ::eGFPT-DNA integration events in SP162 calli.

 

FIGURE 5:

-        5A: Original pictures of SP162 callus transformed with pZmUBQ::eGFP after 4-weeks of regeneration under white and UV light.

-        5B: Original pictures of SP162 callus transformed with pZmUBQ::eGFP after 6-weeks of regeneration under white and UV light.

-        5C: Original pictures of SP162 regenerated fronds transformed with pZmUBQ::eGFP after 12-weeks of regeneration under white and UV light.

-        5D: Raw Southern blot autoradiogram and EtBr gel staining prior to membrane transfer for the detection of pZmUBQ::eGFPT-DNA integration events in SP162 regenerated lines.

-        5E: Original image and raw biomolecular scanner files of SP162 pZmUBQ::eGFP regenerated fronds.

-        5F: Raw qPCR data for eGFP expression in SP162 pZmUBQ::eGFP regenerated lines.

-        5G: Western blot and Coomassie staining raw image files for the detection of GFP from SP162 pZmUBQ::eGFP regenerated lines.

-        5H: Original pictures of SP162 callus and regenerated fronds transformed with pZmUBQ::RUBY.

-        5I: Original pictures of SP162 regenerated fronds transformed with pZmUBQ::DsRed2 under white and UV light.

 

FIGURE 6:

-        6B: SpZMET gDNA, intron-exon annotated gDNA, protein, and protein domains annotated sequences in fasta(.fas) and genebank (.gbk) formats.

-        6C: Original pictures of non-transgenic SP162 callus and transformed with pZmUBQ::eGFP or pZmUBQ::zCas9i:P2A:eGFP under white and UV light.

-        6D: Western blot and Coomassie staining raw image files for the detection of Cas9 and GFP from SP162 pZmUBQ::eGFPand pZmUBQ::zCas9i:P2A:eGFP calli.

-        6E: Original image and raw biomolecular scanner files of SP162 pZmUBQ::eGFP regenerated lines.

-        6F: Western blot and Coomassie staining raw image files for the detection of Cas9 and GFP from SP162 pZmUBQ::zCas9i:P2A:eGFP regenerated fronds.

-        6G: Original image of EtBr staining of agarose gel electrophoresis of SpZMET locus PCR in SP162 and pZmUBQ::zCas9i:P2A:eGFP regenerated fronds.

-        6H/J: See Figure S8 source files.

 

FIGURE 7:

-        7A: Original raw biomolecular scanner files of SP162 infiltrated with pZmUBQ::eGFP. Also used for Supplemental Figure S12.

-        7B: Original pictures of SP162 infiltrated without or with pZmUBQ::eGFP Agrobacterium 21 d.p.i.

-        7C: Original scanner images of bacteria titration plates and table with estimated cfu/frond extracted from individual fronds 21dpi.

-        7D: Raw qPCR data and statiscal analysis report for eGFP expression in SP162 after infiltration of pZmUBQ::eGFP.

-        7E: Western blot and Coomassie staining raw image files for the detection of GFP protein from SP162 infiltrated with pZmUBQ::eGFP.

-        7H: Original pictures of SP162 infiltrated with pZmUBQ::eGFP or pUBQ10::sXVE::eGFP under white and UV light with or without estradiol.

-        7H: Northern blots (autoradiography) raw image files for the detection of GFP- derived, SpTAS3 and snoRNA U6 + miR159controls in SP162 infiltrated with pZmUBQ::eGFP or pZmUBQ::FPPFhp.

 

SUPPLEMENTAL FIGURE S1:

-        S1A: Original pictures of S. polyrhiza SP162 fronds grown in a beaker in SH-media and N-media at different times (weeks) of turion induction.

-        S1B: Original pictures of S. polyrhiza SP162 developing turions in fronds grown in N-media and released turions.

-        S1C: Original pictures of a turion induction culture in N-media before turion harvest.

-        S1D: Original pictures of turions in storage.

-        S1G: Original pictures of germinating turions.

-        S1E-F: Table with turion germination rates after different times of stratification. 

 

SUPPLEMENTAL FIGURE S2:

-        Original pictures of S. polyrhiza SP162 fronds after 1, 2, 3 or 4 weeks in callus induction media.

 

SUPPLEMENTAL FIGURE S3:

-        S3A: Original pictures of S. polyrhiza SP162 calli after 1 week in frond regeneration media containing TDZ or Zeatin.

-        S3B: Original pictures of S. polyrhiza SP162 calli after 2 weeks in frond regeneration media containing TDZ or Zeatin.

 

SUPPLEMENTAL FIGURE S4:

-        S4A: Original pictures of S. polyrhiza SP162 calli after 6 weeks of regeneration initiated with TDZ or Zeatin.

-        S4B: Original pictures of S. polyrhiza SP162 calli after 12 weeks of regeneration initiated with TDZ or Zeatin.

 

SUPPLEMENTAL FIGURE S5:

-        For source data see Figure 3B

 

SUPPLEMENTAL FIGURE S6:

-        S6A: Original pictures of S. polyrhiza SP162 regenerated non transgenic fronds and expressing GFP or RUBY at the Mari-Ordonez Lab.

-        S6B: Original pictures of S. polyrhiza SP162 calli and regenerated transgenic fronds expressing dsRED or GFP selected with Hygromycin or Spectomycin at the Hubert Lab.

  

SUPPLEMENTAL FIGURE S8:

-        S8A-E: Raw sanger sequencing trace data ab1 files for analysis of CRISPR/Cas9 editing results in Figure S6 and Figures 6H and 6J.

 

SUPPLEMENTAL FIGURE S9:

-        S9A: Original pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing GFP or RUBY.

-        S9B: Source images, frond length measurement tables and statistical analysis results of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing GFP or RUBY (the images correspond to day 8 of the growth curve perform in Figure S8).

 

SUPPLEMENTAL FIGURE S10:

-        S10A: Original and processed pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated lines expressing GFP or RUBY for Ilastik pixel and object classification of live and senescent frond tissue areas during growth time-course. Ilastik pixel classification probabilities and object classification output images for each picture are also contained in the folder. 

-        S10B-D: Data summary tables of total, relative and senescent areas in pixel and cm2 obtained from Ilastik analysis.

-        S10F: Summary report of curve fitting for relative surface area fold-change (Figure S8C) data.

 

SUPPLEMENTAL FIGURE S11:

-        S11A: Original pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET.

-        S11B: Source images, frond length measurement tables and statistical analysis results of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET (the images correspond to day 7 of the growth curve perform in Figure S9C-F).

-        S11C: Original and processed pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated lines expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET for Ilastik pixel and object classification of live and senescent frond tissue areas during growth time-course. Ilastik pixel classification probabilities and object classification output images for each picture are also contained in the folder. 

-        S11D-F: Data summary tables of total, relative and senescent areas in pixel and cm2 obtained from Ilastik analysis.

 

SUPPLEMENTAL FIGURE S12:

-        S12A: FACS report and raw data files for 1C estimation of regenerated fronds.

-        S12B: Table with estimation of the genome size (1C) of regenerated fronds based on FACS data.

 

SUPPLEMENTAL FIGURE S13:

-        See Figure 7A source data.

 

SUPPLEMENTAL FIGURE S14:

-        Original images of transiently expressed GFP at 3, 6 and 10 dpi.

 

SUPPLEMENTAL FIGURE S15:

-        S15A: Original images of estradiol-inducible GFP in calli.

-        S15B: Original images of estradiol-inducible GFP in regenerated fronds.

 

SUPPLEMENTAL FIGURE S16:

-        S16D: Arabidopsis and Spirodela miR390 sequences.

-        S16E: SpTAS3 sequence in fasta format between both miR390 target sites.

 

 

NGS DATASETS:

 

All the NGS data generated for this study can be found under the bio-project number PRJNA1308930. The genome assembly and annotation have been deposited on Figshare: https://figshare.com/articles/dataset/Genome_assembly_and_annotation_of_i_Spirodela_polyrhiza_i_strain_SP162_ID_5676_/29942036. 

The data was used to generate the following figure panels:

-        Supplemental Figure S7.

 

Additionally, publicly available small RNA sequencing data from SRA BioProject ID PRJNA1164696 was used to generate Supplementary Figure S16E.

 

 

AUTHOR DATA AND FIGURE CONTRIBUTION:

 






FIGURE



PANEL



DATA/ANALYSIS CONTRIBUTED BY





1



1B



MHO





1C



MHO, RE





1D



MHO, RE





1E



MHO





1F



MHO, RE





1G



VBB, RE





2



2B



RE





2C



RE





2D



RE, VBB





2E



RE, VBB





2F



RE, VBB





2I



VBB





2J



VBB





2K



VBB





3



3A



ERB





3B



ASL





4



4B



VBB





4C



VBB





4D



VBB





4E



VBB





4F



VBB





5



5A



VBB





5B



VBB





5C



VBB





5D



VBB





5E



VBB, AMO





5F



VBB





5G



VBB





5H



VBB





5I



ERB





6



6A



VBB, CD





6B



CD, AMO





6C



CD, VBB





6D



VBB





6E



VBB, AMO





6F



VBB





6G



VBB





6H



VBB, AMO





6J



VBB, AMO





7



7A



APM





7B



APM





7C



APM, AMO





7D



APM





7E



APM





7F



ERB





7H



APM





S1



S1A



LP, LD





S1B



LP, LD





S1C



LP, LD





S1D



LP, LD





S1E



LP, LD





S1E



LP, LD





S1F



LP, LD





S2



S2



RE





S3



S3A



VBB





S3B



VBB





S4



S4A



VBB





S4B



VBB





S5



S5



ASL





S6



S6A



VBB, AMO





S6B



ERB, ASL





S7



S7



AC, SX





S8



S8A-D



VBB, AMO





S9



S9A



VBB, AMO





S9B



VBB, AMO





S10



S10A



VBB, AMO





S10B



AMO





S10C



AMO





S10D



AMO





S10F



AMO





S11



S11A



VBB, AMO





S11B



AMO





S11C



VBB, AMO





S11D



AMO





S11E



AMO





S11F



AMO





S12



S12A



RE





S12C



RE





S13



S13



APM





S14



S14



ASL





S15



S15



ASL





S16



S16



AMO