Maize Gametophyte Project - validated Ds-GFP insertions
64 of 83 putative transposable element insertions obtained from the Dooner/Du
Ds-GFP (also called dsg or tdsg) collection at the Maize Genetics Cooperation
Stock Center (acdsinsertions.org)
were verified to have Ds-GFP insertions at the predicted sites by PCR (Table A).
Because these insertions were selected to be located in coding sequence, they
represent putative knockout alleles for the mutated gene. The predicted
insertion site was not found in 19 of 83 Ds-GFP elements, using primers designed
to amplify a Ds insertion at that location (Table B). However, extensive PCR
optimization was not pursued with this material, so although these insertions
are currently designated as 'non-verified', we do not conclude that the
acdsinsertions.org prediction is incorrect.
All lines were also tested for the presence of only a single Ds-GFP insertion by
outcrossing and identifying co-segregating single elements in progeny kernels
based on the fluorescent GFP endosperm phenotype (Fig. 1) and a diagnostic PCR
band. As originally acquired from the Stock Center, a few lines appeared to harbor
more than a single Ds-GFP element, based on outcross segregation patterns. From
these lines, only progeny segregating for a single insertion were tested further.
Several lines also harbored the wx1-m7::Ac element (by PCR), which was used in the
initial mobilization of the Ds-GFP element (Li et al. 2013).
Based on the GFP endosperm phenotype, all 83 single insertions segregate 1:1 when
the insertion line is used as the female in an outcross; however, 9 of the 64
verified and 1 of the 19 non-verified insertion lines are associated with a
male-specific transmission defect. These are likely due to a causal mutation from
the Ds-GFP insertion. Transmission rates through the male, as well as primer
sequences used for PCR genotyping, are available in the tables below, reproducing
select data from Warman et al. (PLoS Genetics 2020) and Warman et al.
(bioRxiv 2020).
Contact: John Fowler
References:
High expression in maize pollen correlates with genetic contributions to
pollen fitness as well as with coordinated transcription from neighboring
transposable elements. Warman C, Panda K, Vejlupkova Z, Hokin S,
Unger-Wallace E, Cole RA, Chettoor AM, Jiang D, Vollbrecht E, Evans MMS,
Slotkin RK, Fowler JE. PLoS Genet. 2020 Apr 1;16(4):e1008462.
https://doi.org/10.1371/journal.pgen.1008462
MaizeGDB reference
A cost-effective maize ear phenotyping platform enables rapid categorization and
quantification of kernels. Warman C, Sullivan CM, Preece J, Buchanan ME,
Vejlupkova Z, Jaiswal P, Fowler JE. bioRxiv 2020
https://www.biorxiv.org/content/10.1101/2020.07.12.199000v1
Gene tagging with engineered Ds elements in maize. Li, Y., G. Segal, Q. Wang,
and H. K. Dooner. 2013 Methods in Molecular Biology: Plant Transposable
Elements, 1057: 83-99. T. Peterson, ed. Springer Science & Business Media, NY.
https://doi.org/10.1007/978-1-62703-568-2_6
Download pdf file
Figure 1. 1:1 segregation of the tdsgR99B02 Ds-GFP element on a maize
ear, resulting from an outcross when the male parent was heterozygous for the
element. The element carries a GFP coding sequence driven by the a-zein promoter
(Li et al. 2013), generating a fluorescent green endosperm phenotype that can be
easily distinguished from wild-type, non-fluorescent seeds (appearing purple due
to the orange filter used for imaging).
Table A. Validated Ds-GFP insertions.
DsGFP
allele |
Male transmission rate (** if
significantly different from 50%) |
V4 Gene |
v3 Gene |
Expression class (based on
associated gene) |
Primer 1 |
Primer 2 |
tdsgR63F09 |
49.4% |
Zm00001d002266 |
GRMZM2G000052 |
seedling_only |
GTTGACGGGATGTAGGAGGTGC |
AGCTGAGAAAAGGCGAACTGGG |
tdsgR80E09 |
49.1% |
Zm00001d004768 |
GRMZM2G111143 |
seedling_only |
TGCCCTGGCAAAGTAGTGCACC |
GCAGCTGCAGTTGTACACAGTACAGAG |
tdsgR83H05 |
49.2% |
Zm00001d005036 |
GRMZM2G007283 |
seedling_only |
ACAGGAAGGGAAGGGGAGGAAG |
ATAGTGGGGAGGAGAAGAGGGC |
tdsgR44E07 |
47.9% |
Zm00001d005798 |
GRMZM2G148333 |
seedling_only |
ATGGGCAAGGCTGTTCAGAGTG |
GGCTGCTCTCGACGACATAAGG |
tdsgR65E02 |
48.0% |
Zm00001d007228 |
GRMZM2G129209 |
seedling_only |
GTCATCCACCATCTCTTCCCGC |
GCAGAGAGATCTAAGGCGCAGG |
tdsgR46C04 |
51.3% |
Zm00001d013295 |
GRMZM2G051403 |
seedling_only |
CTCCACCATGTCCTGACCGAAG |
TAAGGCGCCAACCCAATCTACG |
tdsgR91G06 |
50.8% |
Zm00001d017240 |
GRMZM2G148387 |
seedling_only |
TGGCTGTGACGGTGAGTTGTTC |
TTGAGCTTGCAGTCCAGACGAC |
tdsgR106F04 |
49.4% |
Zm00001d022274 |
AC217975.3_FG001 |
seedling_only |
CAGACAGAACGGGCATCTTCACA |
GGACTCATTCCGGGACATCAGATACT |
tdsgR76E07 |
49.8% |
Zm00001d029047 |
GRMZM2G100288 |
seedling_only |
TCCTCAGGCTCCACTTCTACCC |
TTGTGGCTTCGAGTCGGGATTG |
tdsgR106E07 |
48.6% |
Zm00001d031325 |
GRMZM2G080724 |
seedling_only |
GCCACGCCTCCTCCTCATTATC |
CCACTCTCCAGAAACCACCACG |
tdsgR21D01 |
nd |
Zm00001d034991 |
GRMZM2G004396 |
seedling_only |
TGTACCACCACAGCAATCAGGC |
TCATCAGGGGGAAGCTCGTCAC |
tdsgR59F11 |
nd |
Zm00001d035737 |
GRMZM2G018786 |
seedling_only |
GAGTGCGCTGTGGCGAAGAA |
TGACTTCATCTGCTGCGGCC |
tdsgR53F11 |
48.9% |
Zm00001d035925 |
GRMZM2G127798 |
seedling_only |
TCCAATGTGGACGCACATCGAG |
TGTACGCGTCCAAGATCTGCAG |
tdsgR52B09 |
48.5% |
Zm00001d036283 |
GRMZM2G342243 |
seedling_only |
CTTGACAGAAACGCCAAGACCG |
CAGAGGCACAGGCACAGAACTC |
tdsgR12H07 |
49.4% |
Zm00001d051110 |
GRMZM2G044882 |
seedling_only |
ACCCATGCTTTGCCTTCCCTTC |
AGTTCATGCGGTAGGTGTTGGC |
tdsgR65A10 |
52.3% |
Zm00001d051194 |
GRMZM2G374302 |
seedling_only |
AGGCACAGACCCTACTTCATATCG |
GAGCACGATGATGGGGTTCAGG |
tdsgR82A03 |
33.4% ** |
Zm00001d005781 |
GRMZM2G036832 |
sperm_cell_high |
CGAACTGAGGTGGTCTGCAGAG |
ACTTCCTGTACCATAACCTGCCC |
tdsgR84A12 |
23.1% ** |
Zm00001d005781 |
GRMZM2G036832 |
sperm_cell_high |
CTGAAGTAGCCAGGCATGTCGG |
CATCCACGGTTCAAAACTGCGAC |
tdsgR60D10 |
51.5% |
Zm00001d006218 |
GRMZM2G365613 |
sperm_cell_high |
CACTCATCACTGTACCTGCCCG |
TAAGCACCCATCATCGTCGCTG |
tdsgR87A03 |
50.0% |
Zm00001d012128 |
GRMZM2G100318 |
sperm_cell_high |
CCCTTGCATGCTCTTGTTCCAAG |
TCAGTGCCGGTGATAAGGACTTC |
tdsgR83A02 |
49.2% |
Zm00001d012575 |
AC194405.3_FG021 |
sperm_cell_high |
TGCATGTCCTCACTAATCGCTCC |
CGCATTGTCCAACAACTCTGCC |
tdsgR96B12 |
nd |
Zm00001d015457 |
GRMZM2G417525 |
sperm_cell_high |
TTCGAGATTTTGCAGCGAACGC |
TTCACTGCAACCAGGGCTCATC |
tdsgR35A03 |
51.1% |
Zm00001d021974 |
GRMZM2G172726 |
sperm_cell_high |
TATGTCACCCAAGCGCACCTAG |
TTTGCTCGTTCTCACCGGTCAG |
tdsgR31B01 |
48.0% |
Zm00001d025834 |
GRMZM2G160069 |
sperm_cell_high |
AGGATGTCTGTGCCCCATATGC |
TGCGCCATTTCTTGTTGCTGTC |
tdsgR91F11 |
49.2% |
Zm00001d034788 |
GRMZM2G114899 |
sperm_cell_high |
TGCACTCGTTAACCACCTCACG |
GGTAATTCCCTCCGACAGCAGC |
tdsgR26G07 |
51.0% |
Zm00001d042810 |
GRMZM2G007659 |
sperm_cell_high |
GATCATGCAGCACAACACGGTC |
CTGCTCGGTCTCACAGGTATGC |
tdsgR53C03 |
51.0% |
Zm00001d043076 |
GRMZM2G038252 |
sperm_cell_high |
AAGCCACAATGCAGGTCCCAAG |
TGCCACTTTCCCCATTCCTGTC |
tdsgR106G12 |
49.3% |
Zm00001d044109 |
GRMZM2G099382 |
sperm_cell_high |
GGCGAGAAACTGATGGACTGGG |
TGGGCTGTGACTGAGAAGTTCC |
tdsgR37A04 |
51.3% |
Zm00001d048434 |
GRMZM2G352898 |
sperm_cell_high |
TGCAACGGCAATGCAGTAGTATACC |
CAAGATATTGATACAACGCGCTGCT |
tdsgR81G05 |
51.3% |
Zm00001d002258 |
GRMZM5G876898 |
vegetative_cell_high |
CCCCCTTCAAACACAGCACAAC |
ATCCCGATCTCACCGTCTCCAC |
tdsgR49F11 |
43.9% ** |
Zm00001d003431 |
GRMZM2G012328 |
vegetative_cell_high |
ATAGCGACTCCCAACGAACACG |
TGCTGGATGGTCTTGAACTGGC |
tdsgR83B04 |
48.7% |
Zm00001d003947 |
GRMZM2G142863 |
vegetative_cell_high |
TCGCTCTTGTCTTCCCAGCAAC |
CTCACCGACAGCTTCCTCGAC |
tdsgR52E07 |
47.1% |
Zm00001d007845 |
GRMZM5G827174 |
vegetative_cell_high |
CATGTTCACGTGCAGGTTCTCC |
CTTCGCTCCACGCAAAAGGAAC |
tdsgR34C11 |
48.8% |
Zm00001d012382 |
GRMZM2G045278 |
vegetative_cell_high |
TCGACTGCCTTGCCTTGTGTAC |
CGGTTTGCGTATAGGTTAGCTGC |
tdsgR107C12 |
48.3% |
Zm00001d012382 |
GRMZM2G045278 |
vegetative_cell_high |
ATCTGATGAATCGACGGGCAGC |
GGCCTTAGGACGGGAAATCAGC |
tdsgR67C09 |
44.1% ** |
Zm00001d014731 |
GRMZM2G135570 |
vegetative_cell_high |
CTGTCCATGGCTAACTACGGGC |
TACTTAGGGCGTTTGGCAGAGC |
tdsgR92F08 |
45.1% ** |
Zm00001d014782 |
GRMZM2G153987 |
vegetative_cell_high |
ATTAATCGAGCAGAGCAGGCCG |
GCAGGTTCTCTTGTCCAGGGTG |
tdsgR99B02 |
49.3% |
Zm00001d015242 |
GRMZM2G102912 |
vegetative_cell_high |
CACGCTGATGGAAGAGGAGGTG |
AGGCGAGTGATTTCTCCGATGC |
tdsgR96C12 |
29.5% ** |
Zm00001d015901 |
GRMZM2G082517 |
vegetative_cell_high |
CCTTACCCACCACCACTGCTTC |
CGGTTTGTGTCTTCGAGGAGGG |
tdsgR41F01 |
49.6% |
Zm00001d017840 |
GRMZM2G056252 |
vegetative_cell_high |
CACGGATGCCAACCACACAAAC |
TACGTGTACAACAACCCGGTCG |
tdsgR98H09 |
47.5% |
Zm00001d017958 |
GRMZM5G872068 |
vegetative_cell_high |
AGCACAGGTTACCGCATCAGTG |
ACCCAGTGTACCAAACCCAAGG |
tdsgR33F03 |
43.9% ** |
Zm00001d022250 |
GRMZM2G039583 |
vegetative_cell_high |
GTCTCCTGGTGGTAATCTGCGG |
GAAATGGCCACGGCAGATTGC |
tdsgR31H05 |
50.2% |
Zm00001d025437 |
GRMZM2G136508 |
vegetative_cell_high |
TGCCTCTGTGTCGCAATTCCAG |
AGGAGAAATCAGCACAGCAGCC |
tdsgR23D05 |
51.1% |
Zm00001d026303 |
GRMZM2G126858 |
vegetative_cell_high |
GCGCCCATCCCACCCAAATG |
ACTATTTCCCGAGTGCAGCACC |
tdsgR24D03 |
49.5% |
Zm00001d026445 |
GRMZM2G120136 |
vegetative_cell_high |
AATGGCCAGAGTTCAGCAGGTG |
TTGGTGACTGAATCCTGCTGGC |
tdsgR02D02 |
49.6% |
Zm00001d026490 |
GRMZM2G006894 |
vegetative_cell_high |
AGAGTCCCTCCCGGTTACCAAG |
AAGACCACGCTCGGCATACTTG |
tdsgR35A08 |
48.2% |
Zm00001d027590 |
GRMZM2G172751 |
vegetative_cell_high |
AGCCTCTCCTCGATCCAAGTCC |
GTTGTGCTCGACGAGGTGGATG |
tdsgR72D11 |
49.8% |
Zm00001d027856 |
GRMZM2G035243 |
vegetative_cell_high |
TCGGCAACATACTGAGCTCTGC |
CTGACAATCAGCCGATGTCCAG |
tdsgR04A02 |
43.8% ** |
Zm00001d028437 |
GRMZM2G359879 |
vegetative_cell_high |
CTTCAGCTCGAGGTCACTGCAC |
GGTGTGGTATGAGTTCCTGGCC |
tdsgR27E01 |
48.3% |
Zm00001d028820 |
GRMZM2G016734 |
vegetative_cell_high |
ACCCCAGCTTACACAATCGACC |
TGGTGCAGTTCTGTCGGACAAG |
tdsgR77F09 |
49.2% |
Zm00001d032279 |
GRMZM2G142249 |
vegetative_cell_high |
TGATGCTGCCTTCGCTACGAAC |
TGGCAAGGCTTCTGATTGGAGG |
tdsgR04G10 |
49.6% |
Zm00001d032310 |
GRMZM2G114093 |
vegetative_cell_high |
ACAGCCAGTGTAGAATCATGTTAGC |
TGTCATCTTCAGACGCCAAGCC |
tdsgR01G01 |
48.4% |
Zm00001d032950 |
GRMZM2G124434 |
vegetative_cell_high |
TGAGATCGTGCTGGGCTTTGAG |
TCGTATCGTTTGGACCATGCCC |
tdsgR103E04 |
49.3% |
Zm00001d034799 |
GRMZM5G878153 |
vegetative_cell_high |
GCGTACCCTTCTCGTCCTGCAT |
GGAAACAATTACCCTGCTCGTCCTG |
tdsgR32B05 |
50.3% |
Zm00001d034839 |
GRMZM2G134054 |
vegetative_cell_high |
CCGTTGGTCAGGTACAGGTTGG |
AAATTCCCGCAACTCCCGTACC |
tdsgR45E04 |
50.0% |
Zm00001d034839 |
GRMZM2G134054 |
vegetative_cell_high |
CCGTTGGTCAGGTACAGGTTGG |
GCTCCTCCGTCCGATCCATACG |
tdsgR69C04 |
48.3% |
Zm00001d036330 |
GRMZM2G307402 |
vegetative_cell_high |
TGATCGATCGGTGAAGCAGCAG |
AGGAGGAGGAGGAGGAGGAGAC |
tdsgR101B03 |
50.1% |
Zm00001d037061 |
GRMZM2G012263 |
vegetative_cell_high |
CATCGCCAAGTCCACCGTAGAG |
TCTGCAGGAACCATGGAAGCTG |
tdsgR81E02 |
50.4% |
Zm00001d037061 |
GRMZM2G012263 |
vegetative_cell_high |
CTACAACTTCTCCCAGGACGCC |
GGCAACCGGATGTGCAGATTTG |
tdsgR102H01 |
45.5% ** |
Zm00001d037695 |
GRMZM2G350802 |
vegetative_cell_high |
AGCCCCGTGTAGTTCCCTTTTTC |
TTGCTTGCTAGGCTGGGTTCTC |
tdsgR88B08 |
50.5% |
Zm00001d041514 |
GRMZM2G018372 |
vegetative_cell_high |
TGCCCATCTCCTTGCTCGTTTC |
CAAGGAGACAGCACTGGACTGC |
tdsgR92A10 |
52.4% |
Zm00001d046483 |
GRMZM2G095206 |
vegetative_cell_high |
ACTGTGAAGCCAAACCCTCAGC |
CTGTTCTGCCTTCTCCGTCCG |
tdsgR39B06 |
51.0% |
Zm00001d048384 |
GRMZM2G089699 |
vegetative_cell_high |
CCCACCTCTATCCTTGTGTCTTGG |
TGTCGGCTTGCCATACCATGTC |
tdsgR08A07 |
47.3% |
Zm00001d048785 |
GRMZM5G845021 |
vegetative_cell_high |
GATTCACCTTGACGCACGCAAC |
CTTCCATACCACGCCTACTCGC |
Table B. Unverified Ds-GFP insertions.
DsGFP
allele |
Male transmission rate (** if
significantly different from 50%) |
V4 Gene |
v3 Gene |
Expression class (based on
associated gene) |
Primer 1 |
Primer 2 |
tdsgR48D04 |
nd |
Zm00001d032618 |
GRMZM2G153208 |
seedling_only |
AACGCATTGAGCCATTGACGC |
GAGACAACGCACGTGTGGCAGT |
tdsgR63C12 |
nd |
Zm00001d044212 |
GRMZM2G176903 |
seedling_only |
AAGAGCCGATGTGACAGAGCTG |
GGCCTCTGACAAGCCGATGTAC |
tdsgR98H08 |
nd |
Zm00001d013493 |
GRMZM2G102760 |
seedling_only |
ATGCTTCGAAGGAACTCGCTGG |
ACCGCATCCACACACTCATCAC |
tdsgR21D07 |
nd |
Zm00001d005694 |
GRMZM2G119906 |
seedling_only |
AACCACCGATGACCCCCAAAAG |
TGTCACTTTGTCAGGGCTTCGG |
tdsgR06D07 |
nd |
Zm00001d002570 |
GRMZM2G038851 |
sperm_cell_high |
CTGTACCTCCTCGAGCGTTCTG |
CACAATGGTAAGCGCCTGACTG |
tdsgR89B08 |
37.2% ** |
Zm00001d002824 |
GRMZM2G062554 |
sperm_cell_high |
GCTTGAGAGGGGTTAGAGCTCG |
GGCCTACTTGCGATCACCCATC |
tdsgR105B06 |
nd |
Zm00001d044412 |
GRMZM2G072080 |
sperm_cell_high |
AATGCCTTGCTCACGTATGCTG |
ACGAGGTGCTGTGATATTGCTGG |
tdsgR82B10 |
nd |
Zm00001d034788 |
GRMZM2G114899 |
sperm_cell_high |
TGCACTCGTTAACCACCTCACG |
GGTAATTCCCTCCGACAGCAGC |
tdsgR29A11 |
49.1% |
Zm00001d012674 |
GRMZM2G124365 |
sperm_cell_high |
CCAAGTTTGCATGCGTCGATCC |
ACCCAGCCAAAGAAAGTGACCC |
tdsgR75H08 |
nd |
Zm00001d044290 |
GRMZM2G130375 |
sperm_cell_high |
ATGTCATGCTCGCTCAGGTACC |
GTCTCATCTGCACCCTCACCTG |
tdsgR67H12 |
48.7% |
Zm00001d031678 |
GRMZM2G033828 |
vegetative_cell_high |
TCTTTCTTCTTTGGGCTGGCGC |
AAGTCAGGTCTCCCAAAAGGGC |
tdsgR97C08 |
nd |
Zm00001d009775 |
GRMZM2G050364 |
vegetative_cell_high |
ATGCATTTCAGTGTCTCCCCGC |
GATGTCCGTCTCCATGGTGCC |
tdsgR85A08 |
50.3% |
Zm00001d005053 |
GRMZM2G051491 |
vegetative_cell_high |
CCTCCAGCACCATGTCCGATTAG |
CAGAGAGAGGCCAGACTGATGG |
tdsgR88H09 |
nd |
Zm00001d044192 |
GRMZM2G057733 |
vegetative_cell_high |
GCTGTTCTTAGACGCACGCAAC |
GGACTTGGACGAGCTTAGCGAG |
tdsgR90C03 |
nd |
Zm00001d016444 |
GRMZM2G098278 |
vegetative_cell_high |
GCTTCATCCTCAGCCTCCTTCG |
ATCACTCAACACTCGCACGACC |
tdsgR31B05 |
nd |
Zm00001d014731 |
GRMZM2G135570 |
vegetative_cell_high |
TGTACACACATCTACGCAGGCC |
GACGTCCATCCCTTTCACCACC |
tdsgR02A05 |
47.4% |
Zm00001d042353 |
GRMZM2G140107 |
vegetative_cell_high |
GTCCATCGACGGTGAGAAGAGC |
TGAGTGCCTGTGACCTTGATGC |
tdsgR72F03 |
nd |
Zm00001d037308 |
GRMZM2G168190 |
vegetative_cell_high |
TTGAGTAGTACGAGGCTCCGGG |
AAGGTCGGGTAGAGGGTAGGTG |
tdsgR108A02 |
47.1% |
Zm00001d039693 |
GRMZM2G319167 |
vegetative_cell_high |
GAGAAGCCAACGGAGCCTTAGG |
ACGGCCTCAGAAACTTGACCAC |
Validation of Ds-GFP insertion sites – Methods (from
Warman et al, 2020)
A FASTA file containing 2 kb of genomic sequence surrounding each Ds-GFP
insertion site was used as input to a primer3-based tool to generate a pair
of specific primers to genotype individual plants from each line
(
https://vollbrechtlab.gdcb.iastate.edu/tools/primer-server/).
The primers used for each Ds-GFP line are listed in S6 Table from Warman et al,
and are shown below.
To genotype the plants, two 7 mm discs of leaf tissue were collected from each
plant using a modified paper punch. The samples were collected in 1.2 ml tubes
that fit within a labeled 96 well plate/rack
(
https://vollbrechtlab.gdcb.iastate.edu/tools/tissue-sample-plate-mapper/)
(Phenix Research Products, Candler, NC; M845 and M845BR or equivalent). Genomic
DNA was isolated from the leaf punches with the following modifications.
An additional centrifugation (3,000 g for 10 min.) was added to clear the leaf
extracts prior to loading onto a 96-well glass fiber filter plate (Pall, 8032).
DNA was eluted from filter plates in 125 μL water, and 2 μL was used as template
for PCR. Amplification followed standard PCR conditions using GoTaq Green Master
Mix (Promega) with 4% DMSO (v/v) and amplicons were resolved using agarose
gel electrophoresis. Lines were genotyped using the pair of gene-specific
primers, designed to amplify flanking sequence at the predicted insertion site,
plus one Ds-specific primer (JSR01 GTTCGAAATCGATCGGGATA or JGP3
ACCCGACCGGATCGTATCGG). All lines were also screened by PCR for the presence
of wx1-m7::Ac using primers for wx1 (CACAGCACGTTGCGGATTTC) and Ac
(CCGGATCGTATCGGTTTTCG). Followup PCR to test for co-segregation of GFP
fluorescence with the presence of the insertion used the appropriate set
of three PCR primers (two gene-specific and one Ds-specific) and DNA prepared
either from endosperm or seedling leaves.
Heterozygous lines with PCR-validated Ds-GFP insertion alleles were planted in
the Botany & Plant Pathology Field Lab (Oregon State University, Corvallis, OR).
All insertions were in coding sequence (CDS) sites. Heterozygous Ds-GFP plants
were outcrossed to tester plants (c1 wx1/c1 wx1 or c1/c1 genetic background)
through both the female and the male, with male pollinations made with a heavy
pollen load on extended silks (silks that had been allowed to grow for at least
two days following cutback). Following harvest, resulting ears were imaged
using a custom rotational scanner in the presence of a blue light source and
orange filter for GFP seed illumination. Briefly, videos were captured of
rotating ears, which were then processed to generate flat cylindrical
projections covering the surface of the ear. Seeds were manually counted using
the Cell Counter plugin of the Fiji distribution of ImageJ.
This work was funded by National Science Foundation grants
IOS-1340050
and
MCB-1832186