This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University Abstract Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.

1. Loss of pollen-specific phospholipase NOT LIKE DAD
triggers gynogenesis in maize
Professor
Department of Genetics
Sohag University
abdelsabour.khaled@agr.sohag.edu.eg
18.12.2017
Prof. Dr. Abdelsabour Kahled
2017/2018
Published in EMBO Journal

2. Loss of pollen-specific phospholipase NOT LIKE
DAD triggers gynogenesis in maize
Reproduction et Développement
des Plantes
ENS de Lyon
Sohag University
Faculty of Agriculture
Department of Genetics
Laurine M Gilles, Abdelsabour Khaled, Jean-Baptiste Laffaire, Sandrine Chaignon1, Ghislaine Gendrot,Laurine M Gilles, Abdelsabour Khaled, Jean-Baptiste Laffaire, Sandrine Chaignon1, Ghislaine Gendrot,
Jérôme Laplaige, Hélène Bergès, Genséric Beydon, Vincent Bayle, Pierre Barre, Jordi Comadran, Jean-Pierre
Martinant, Peter M Rogowsky & Thomas Widiez.
Prof. Dr. Abdelsabour G. A. Khaled
Professor of genetics
Faculty of Agriculture
Sohag University
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3. Induction of maternal haploid
in vivo gynogenesis: in vivo induction of maternal haploid
1n 1n 1n 1n
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genome)
Sexual reproduction
1n
gynogenesis
F1

4. Creation of Doubled Haploid (DH) plants:
Creation of DH plants
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5. Double fertilization and gynogenesis:
Gynogenesis in maize
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6. Doubling haploid technology
• Advantage of Doubling Haploid (DH) technology in plant breeding:
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7. In maize: Spontaneous gynogenesis is very rare (0.1%).
Inducer lines: Stock 6 (common ancestor).
QTL: 8 paternal QTLs were uncovered in inducer lines (Barret et
al, 2008; Prigge et al, 2012) and 2 maternal QTLs in receiving
females (Wu et al, 2014).
QTL region
females (Wu et al, 2014).
One parental QTL located on chromosome 1, called ggi1
(Barret et al, 2008) and qhir1 (Prigge et al, 2012).
ggi1/qhir1 locus: 243 kb on ch#1 (Dong et al, 2013), (66%).
qhir8 locus: 789 kb on ch#9 (Liu et al, 2015), (20%).
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8. Challenges
The challenging to elucidate this mechanisms for two reasons:
The partial nature of the induction makes it difficult to predict
for a given pollen tube/embryo sac interaction.
Comparative studies: heterogeneity of the genetic background:Comparative studies: heterogeneity of the genetic background:
No near-isogenic material is available due to the polygenic
nature of the induction capacity.
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9. Inducer and wild-type lines
• Inducer lines characters:
• All steps up to the development of the trinucleate pollen are normal,
• No biologically relevant differences in the number of nuclei, neither in mature pollen
nor in germinated pollen tubes (Mahendru & Sarkar, 2000).
• The Differences between inducer and wt lines:
• During pollen tube germination since pollen from non-inducer lines germinates faster
than pollen from inducer lines (Xu et al, 2013).
• Cytological study: detected remnant sperm cell nuclei in the embryo sac after
pollination with inducer pollen (Swapna & Sarkar, 2012).
• Molecular studies: revealed remnants of paternal genetic material in haploid embryos
(Zhao et al, 2013).
• General decrease in kernel number and an increase in the number of defective kernels
(Xu et al, 2013).
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10. Objectives:
• Identify the genetics behind maize haploid inducers,
• Understand molecular/cellular mechanisms,
• Development of breeding tools.
How to make kids that don't look like their dad?
• Development of breeding tools.
This work presents:
1- the positional cloning of the ggi1/qhir1 QTL,
2- Provides evidence that a mutation in the NLD is the key factor triggering
gynogenesis in maize.
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11. Maize lines and haploid induction
• Maize (Zea mays) plants cultivated in field or grown in a S2 greenhouse ( Lyon
and Saint Martin de Hinx, France).
• Highly recombinant inbred lines (F2i3S2) derived from a DH99 X PK6 cross.
• The public inbred lines of the diversity panel were a gift from A. Charcosset
(Camus-Kulandaivelu et al, 2006).
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• Score of haploid induction (100 kernel):
• ♀ F564 × DH7 (glossy1) × ♂ tested lines,
• ♀ PDH3 × PDH8 (liguleless) × ♂ tested lines (Melchinger et al, 2013).

12. Genotyping and BAC library screen
• Genotyping:
• DNA and RNA (pollen) were extracted using a CTAB.
• RNA extraction from others tissues was extracted with TRI reagent.
• Genotyped in 1,536-well microplate (He et al, 2014).
• KASP markers:
SNP (A/G) INDEL (-/TTTGTTTTGCA)
CNV (T/C) PAV (C/-)
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CNV (T/C) PAV (C/-)
• BAC library screen:
• The BAC libraries Zma-B-PK6 (HindIII) and Zma-B-PK6e (EcoRI) /
pIndigoBAC5 vector (Chalhoub et al, 2004).
• Selected BAC clones were sequenced with a PacBio RS II.
• Sequence assembled with the SMRT Analysis software (ver 2.3.0).
• GenBank accession number: KX852318.

13. • Transgenic plants:
• Resistance cassette (Actin promoter, Bar gene and Nos-ter).
• GFP cassette (CsVMV promoter, GFP gene, and Nos-ter).
• Maize transformation:
•Agrobacterium-mediated transformation of maize inbred line A188.
Maize and Arabidopsis transformation
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•Agrobacterium-mediated transformation of maize inbred line A188.
• Number of T-DNA insertions was evaluated by qPCR.
• Integrity of the transgene: PCR with primers situated next to the left border.
• Arabidopsis transformation:
•Arabidopsis ecotype Col-0 was transformed with the floral-dip method.

14. • Creation of 96 highly recombinant inbred lines of DH99 x PK6.
• Based on haploid induction tests and mapping with 10 markers in the
umc1144/bnlg1811 interval.
• Two inducer lines with the shortest PK6 genome region were backcrossed to DH99.
• Haploid induction tests:
• ♀ glossy1 x ♂ tested lines.
Fine mapping of the ggi1/qhir1 locus
• ♀ glossy1 x ♂ tested lines.
• ♀ liguleless x ♂ tested lines.
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ggi1/qhir1 locus

15. • Backcrossed plants Selfed plants 10,275 plantlets were screened with 3 markers.
• 531 recombinant plants were obtained.
• The phenotypic characterization of the new recombinants exploited the strong
correlation between haploid induction rate and segregation distortion (Barret et al,
2008; Prigge et al, 2012; Xu et al, 2013).
Fine mapping of the ggi1/qhir1 locus
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16. • Genotyping of 48 recombinants for segregation distortion.
• The strongest segregation distortion was observed with G471240_1 marker.
• Among 48 families, 28 contained homozygous allele.
• 28 used as pollen parents in test cross.
Fine mapping of the ggi1/qhir1 locus
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17. •The results indicated that the
locus responsible for haploid
induction was located between
markers GRMZM2G471240_13
(71-19-29) and 155K10_55 (71-
39-162 and 71-29-69) in a 166-
kb interval on the B73 genome.
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18. Structure of the confidence interval in the PK6 genome
• BAC library was created from PK6 gDNA.
• Alignment of the sequences:
• Replacement of a 156-kb of B73 by a sequence of 70 kb in PK6.
• Absence of genes G2320 and G2313 in the PK6 sequence.
• Transposable elements (TE) in the PK6 genome existed in the proximity of the ggi1/qhir1.
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19. Structure of the confidence interval in the PK6 genome
• An initio analysis:
• 13 genes in the 80-kb of genotype PK6.
• 10 genes carried annotations related to retrotransposon families.
• 2 genes, ESH_18 and ESH_19, had more than 10,000 and 182 copies in the B73.
• One gene ESH_8 (2G471240) annotated as patatin-like phospholipase.
• Phospholipases (PLs) are enzymes that cleave phospholipids, the major• Phospholipases (PLs) are enzymes that cleave phospholipids, the major
component of all cellular membranes.
•In comparison with B73 and DH99, the PK6 allele carried a 4-bp insertion in
exon 4 corresponding to marker 2G471240_14.
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4bp insertion
TE TE TE TE TE TE TE TE TE TE TE TE TE TE TE TE TE TE TEPK6

20. • Reference maize sequence (B73)
• Survey of GRMZM2G471240_14:
• Survey: 92 lines and 12 haploid inducer lines.
• The 4-bp insertion haploid induction:
• 4 Lines (EM1201, Nys302, Mo1W and Tx303) possessing the same haplotype
as PK6, but differing only by the 4-bp insertion were not able to induce haploids.
TE TE TE
• Reference maize sequence (B73)
• PK6 inducer line
TE TE TE TE TE TE TE TE TE TE TE TE
4bp insertion
• EM1201
% haploid induction
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21. • This candidate gene was named NOT LIKE DAD (NLD) because
the PK6 allele induces maternal embryos without paternal
contribution.
• Enzyme hydrolyzes phospholipids:
Identification of “NOT LIKE DAD” gene = NLD
• Broad roles: metabolism, stress, development, signaling
(Scherer et al. 2008).
• Earlier aberrations in pollen development were observed in the
plc2 mutant of Arabidopsis (Li et al, 2015).
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22. NLD codes for a patatin-like phospholipase
• Analysis of the deduced amino acid sequence of NLD:
• The deduced NLD amino acid sequence contained a patatin/phospholipase domain.
• The 4-bp insertion in NLD exon 4 found was also found in cDNA.
• Frameshift (4-bp) that replaces the last 49 aa of the wild-type protein by an
unrelated aa sequence of 20 aa followed by a STOP codon.
NLD-WT
NLD-PK6
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23. Alignment of aa sequences of NLD
histidine
tyrosine
glutamine
glutamic acid
stop
20 aa
49 aa
lysine
asparagine
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24. • Phylogenetic analysis
of maize and
Arabidopsis patatin-like
PLA placed NLD
among class II patatin-
like PLA
Phylogenetic analysis of maize and Arabidopsis patatin‐like PLA proteins
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0.2

25. NOT LIKE DAD expression
• qRT–PCR: Expression of NLD was
restricted to the mature stage of the
tassel.
• Comparison DH99 and PK6: showed
slight but significant increase of NLD
expression in mature tassel.
Sekhon et al., 2011, The Plant Journal 66: 553-563 &Sekhon et al., 2011, The Plant Journal 66: 553-563 &
Downs et al., 2013, Plant Physiology 161: 1830-1843.
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26. Expression of NLD
• Distinguish between anther and pollen expression:
• (pNLD::GUS): 2.6-kb pNLD from genotype B73.
• GUS staining was found in pollen grains only.
• NLD promoter activity : Specifying an expression of NLD in mature pollen.
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27. Expression of pNLD in female wt
• ♀ wild type line x ♂ pNLD::GUS.
• After pollination :
• Wild-type silk at 17 HAP, ovules at 17 , 48 and 72 HAP.
• These results suggest that the NLD promoter is active during the entire
fertilization process.fertilization process.
(17 HAP) 17 HAP 48HAP 72HAP
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28. Wild-type NLD abolishes the haploid induction
• Create a near-isogenic inducer line (A188NLD–PK6)/4 generations of backcrosses.
• The haploid induction rate dropped from 3.59% (BC0) to 0.50% (BC3).
• Confirming the need of other favorable loci for enhanced haploid induction.
• A188 was a non-inducer line (0 haploids out of 4,487 seedlings tested).
• NLDDH99-OE crossed with A188NLD–PK6. Zero haploid induction.
• The presence of wild-type NLD abolished the A188NLD–PK6 inducer capacity.
• Conclusion that the PK6 allele of NLD was responsible for maternal haploid
induction at the ggi1/qhir1 locus.
WT and NLD‐over‐expressing line
NLDDH99-OE line 9/1 A188
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29. • Truncation of NLD protein leads to loss of plasma membrane localization:
• pUbi-(NLD-DH99)::CITRINE in Arabidopsis.
• pUbi-(NLD-PK6)::CITRINE signal was absent in the plasma membrane.
• This result suggested that the truncated NLD-PK6 was mis-localized and its
absence in the plasma membrane was the cause for haploid induction.
pNLD expression in Arabidopsis roots
absence in the plasma membrane was the cause for haploid induction.
pUbi NLD-PK6 CITRINEpUbi NLD-DH99 CITRINE
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30. • pUbi-(NLD-DH99)::CITRINE
• pUbi-(NLD-PK6)::CITRINE
• NLD::citrine wt signal appears to the plasma membrane of the sperm cells.
pNLD expression in maize pollen
I
pUbi NLD-PK6 CITRINEpUbi NLD-DH99 CITRINE
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pUbi NLD-DH99 CITRINE

31. Gilles et al., EMBO J 2017
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Journal cover

32. 31The EMBO Journal, 2017, 36 (6): (707 – 717)

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34. Thank to:
- Abdelsabour Khaled
- Sandrine Chaignon
- Ghislaine Gendrot
- Jerome Laplaigne
- Vincent Bayle
- Peter Rogowsky
- Jean-pierre Martinant
- Jordi Comadran
- Jean Baptiste Laffaire
SeedDev Team
Laurine GILLES
CNRGC (Toulouse)
Hélène Bergès
Genséric Beydon
GDEC
(Clermont Ferrand)
Pierre Barret
Abdelsabour Khaled
33