This presentation describes about approaches to engineer apomixis, an alternative of sexual pathway, in major food crops for fixation of hybrid vigor through clonal seeds.
Pteris : features, anatomy, morphology and lifecycle
synthetic apomixis by gene editing
1. KIRAN K.M
PGS20AGR8449
Department of genetics and plant breeding
MASTERS SEMINAR II
UNIVERSITY OF AGRICULTURAL SCINECE, DHARWAD
Synthetic Apomixis by genome editing:
An Approach to fix hybrid vigour
2. This [hybrid rice] technology is not for farmers who are still struggling at the level of 2 or 3 tons per
hectare. – S.S. Virmani, IRRI, 1998
Is benefit of “hybrid technology” reach up-to bottom hands ?
5. Our way through…
How to capture MiMe technology ? : Mine out the genetic control
Introduction : A general look into apomixis mechanism
Where should tap to induce apomeiosis ?
How to hold hybrid heterozygosity over generations though seeds ?
: Idea of Clonal fix strategy
What are approaches to achieve parthenogenic development of
MiMe gametes
What are the hurdles ahead to overcome?
Conclusion
7. An apospory-specific genomic region is conserved
between Buffelgrass (Cenchrus ciliaris L.) and Pennisetum
squamulatum Fresen.
Buffelgrass (Cenchrus ciliaris L.)
Apomixis is transmitted by a large non-recombining
chromosomal region named the apospory-specific
genomic region (ASGR)contains multiple copies of
PsASGR-BABY BOOM-like (PsASGR-BBML;
Pennisetum squamulatum
Taraxacum officinale
DIP locus : Diplospory
PAR locus : Parethenogensis
In apomictic plant, apomixis is
restricted to female meiosis only
8. Boechera retrofracta
APOLLO (apomixis-linked locus) :
o Have apoalleles’ and ‘sexalleles (heterozygous) in apomictic
Boechera spp.
o sexual genotypes : Homozygous for sex alleles
UPGRADE2 (unreduced pollen grain development)
Three independent loci,
• LOA : to control Apospory,
• LOP : To control parthenogenesis
• AutE : To control Autonomous endosperm
:Sexual and apomictic seed formation in
Hieracium requires the plant polycomb-group
gene FERTILIZATION INDEPENDENT ENDOSPERM
Hieracium (HAWK WEED)
9. 1. Apomixis is a Consequence of Developmental Asynchronies
2. Apomixis is a Mutation-Based Phenomenon
3. Apomixis is an Ancient Switch, Polyphenic to Sex, and
Epigentically Regulated
The Molecular Basis of Apomixis
Apomixis depends on alteration of different species-specific genes controlling
and/ or co-ordinating developmental steps within reproductive modules to
produce genetically balanced clonal progeny
10. Non- Coding
RNA
Transposable
elements
Pseudogenization
Hyper- hypo
methylation
Cell-specific
expression of genes
• MITE transposon insertion in the promoter region of the candidate gene
(CitRWP) controlling sporophytic apomixis in Citrus.
• Apomictic dandelion (Taraxacum officinale) and hawkweed (Hieracium
piloselloides) carry MITE transposons in the upstream region of
parthenogenesis gene (PAR)
Gene regulation at natural apomixis-associated loci
11. Transcriptome comparison of apomictic Boechera, Hieracium and
Hypericum with related sexual lines revealed changes in siRNA
synthesis and RdDM related gene expression
Epigenetic control over apomixis
The analysis of Arabidopsis mutant ovule epigenetically controlled by
sRNA-mediated silencing pathway involving ARGONAUTE 9 (AGO9)
protein showed transition from sexual to apomictic-like phenotype.
The regulation of MEDEA (MEA) gene involved in the control of fertilization-
independent development of endosperm is associated with the epigenetic
regulatory polycomb repressive complex 2 (PRC2), which silencing of gene
expression via trimethylation of histone H3 at lysine 27 (H3K27me3
(Schmidt A, 2013)
12. 1)Mimicking Gametophytic apomixis
2) Mimicking Sporophytic apomixis
1. Producing unreduced non recombinant gametes (either by skipping
meiosis or by blocking the reductional division of meiosis),
2. Develop an embryo parthenogenetically
3. Promoting endosperm development to complete the formation of a
seed.
1. Inducing an ectopic embryo within the ovule
2. Arresting or delaying egg cell progression or zygote development in
the fertilized meiotic female gametophyte
Approach to engineer Synthetic apomixis
15. Mutation that eliminates recombination and pairing (Atspo11-1)
Mutation that modifies chromatid segregation (Atrec8)
16. A genotype in which meiosis is totally replaced by mitosis without affecting
subsequent sexual processes. : MiMe for ‘‘mitosis instead of meiosis
MiMe Technology- Turning meiosis into mitosis
The three genes conferring the MiMe genotype are strongly conserved
among plants, suggesting that apomeiosis may be engineered in any plant
species
17. Conclusion
1. First, spo11-1 abolishes meiotic recombination
2. Second, the mutation of REC8 causes the separation of sister
chromatids at first meiotic division, instead of the distribution of
homologous chromosomes
3. osd1 causes the skipping of the second meiotic division
4. Doubling of ploidy of MiMe genotype after at each generation when
self-fertilized
Dugout other genes involved in the meiosis process – MiMe generation
Check feasibility in other crops: Commercial crops
Need to unravel genetic mechanism of parthenogenic development of
MiMe gametes and endosperm development
Steps ahead
18. Gene Gene product/function Reference
dmc1 Involved in meiotic recombination. Mutants of DMC1 exhibit
defective in meiotic double strand break formation
Couteau et
al., 1999
ahp2 involved in bivalent formation and homologous
chromosomes segregation
Schommer et
al., 2003
scc3 SCC3 protein is essential for the maintenance of the
centromere cohesion
Chelysheva et
al., 2005
asy1 ASY1 plays an essential role in homologous chromosome
synapsis.
Caryl et al.,
2000
tdm1 TDM1 is essential for meiotic termination after meiosis II. Cifuentes et
al., 2016
TAM/CYC
A1;2
Type-A cyclin required for the transition of meiosis I to
meiosis II
D’Erfurth
et al., 2010
Genes and their related functions involved in Apomeiosis
19. Gene Species Gene product/function Reference
DYAD/SWI1
(SWITCH 1)
Arabidopsis Regulator of meiotic chromosome
organization & sister chromatid
cohesion
Ravi et al., 2008;
Mercier R., 2001
AM1
AtSPO11-1
Maize
Arabidopsis
SWI1 ortholog
Topoisomerase-like transesterase
Pawlowski et al.,
2009 ,Grelon, 2001
AtSPO11-2
AtREC8
Arabidopsis
Arabidopsis
AtSPO11-1 paralog
Arabidopsis Cohesin necessary for
centromere cohesion and
kinetochore orientation
Stacey et al., 2006
Chelysheva et al.,
2005
OsREC8
OsOSD1
Rice i)A key component of the meiotic
cohesion complex
ii) Cause omission of M-II division
Mieulet et al.,
2016
PAIR1 Rice Essential protein for the initiation of
meiotic recombination
Nonomura et al.,
2004
22. Identified of Ososd1 (Oryza sativa OSD mutation) mutant in rice from
Oryza Tag Line insertion line insertion library
Generated MiMe in rice
Turning rice meiosis into mitosis
Mieulet et al., 2016 Cell research
Does MiMe technology could generated in Rice ?
Ososd1 mutation + Meiotic recombination +
suppression mutation
Chromatid segregation modifying
recombination /Sister chromatid
separation mutation
pair1-4 Osrec 8-3
23.
24. The single mutants pair1, Osrec8 and
the double mutants pair1 Osrec8 and
pair1 Ososd1 plants were fully sterile.
In contrast, the pair1 Osrec8 Ososd1
triple mutant showed higher fertility,
similar to Ososd1 single mutant
like in Arabidopsis, the combination
of pair1, rec8 and osd1 mutations
turns meiosis into mitosis in rice
known as OsMiMe genotype
28. Does MiMe technology alone enough to hold heterotic potential indefinitely ?
?
cv
cv
1) Uniparental
genome Genome
elimination
2) Induced
parthenogenesis
29. Genes involved in haploid induction
Gene Description crop
cenh3 Alteration of centromere-specific histone CENH3 can
induce genome elimination and haploid formation
Arabidopsis
MTL1 MTL1 encodes a pollen-specific phospholipase which is
involved in fertilization. Mutation of MTL1 gene can
induce haploid formation
Maize
DMP
DOMAIN
MEMBRANE
PROTEIN
Another gene expressed specifically in pollen,(Zhong
et al., 2019).
Loss-of-function mutations in Arabidopsis DMP8 and
DMP9 trigger haploid induction (Zhong et al., 2020)
Maize,
Eudicots ,
monocots
30. Genes involved in parthenogenesis
Gene Description crop
PAR The dominant PAR allele of dandelion is specially
expressed in egg cells and can trigger
embryogenesis without fertilization
Taraxacum
msi1 MSI1 gene functions in chromatin assembly.
Mutants of MSI1 can produce parthenogenetic
embryos
Arabidopsis
ASGR-BBML Expressed in unfertilized egg cells of apomictic
Pennisetum squamulatum and activation of its
expression in sexual pearl millet can also trigger
parthenogenesis
Pennisetum,
Cenchrus
31. Their HI-inducing modification involved adding an N-terminal GFP tag to a fusion
of the histone 3.3 tail with the CENH3 histone fold domain
Dissimilar centromere
behave differentially during
cell division
Centromere histone-3 (CENH-3) mediated haploid induction
32. Efficient elimination line for diploid gametes
GEM Line : Genome Elimination caused by a Mix of
CENH3 variants
Arabidopsis ; MiMe plants were a mixture of Col-0
from Atspo11-1-3/Atrec8-3 and No-0 from osd1-1 (S1).
cenh3-1 GFP-tailswap cenh3-1 GFPCENH3
cenh3-1 GFPCENH3
cenh3-1 GFP-tailswap
Marimuthu et al., 2010
MiMe coupled with genome elimination
33. Analysis of crosses between GEM and MiMe or dyad.
• Up to 34% of the progeny were clones of their parent, demonstrating the
conversion of clonal female or male gametes into seed
• Although not fully penetrant, this demonstrates clonal propagation
through seed in a manner akin to the outcome of apomixis.
34. Genotype analysis of MiMe x GEM and GEM x MiMe
offspring..
The maternal diploids (diploid eliminants) retained the heterozygosity of
the mother plant at all tested loci
Lower seed set low and the trait was not fully penetrant (24–42% diploids)
35. CRISPR/Cas9-mediated deletions/ manipulating a single centromere protein,
the centromere-specific histone CENH3 in CENH3 lead to haploid induction on
outcrossing
The CRISPR/Cas9-mediated deletions in the a N helix terminal can induce
haploids opens a new avenue for the generation of haploid-inducing lines.
[ lower HI rate -0.065–0.86% in maize]
Single amino acid substitutions in the histone fold domain of CENH3 lead to
haploid induction-[frequency as high as 44%] (Karimi-Ashtiyani et al., 2015;
Kuppu et al., 2015).
Requirement of separated crossing with F1 hybrid plant each
time is not entertained
36. • Genes responsible of haploid induction in maize stock 6 have been
identified as a defect in Matrilineal, a spermspecific phospholipase gene-
GRMZM2G471240 (MATRILINEAL) 4-bp insertion (CGAG) in the
induction lines compared to the B73 genome
• The mutation of the MATRILINEAL (MTL) gene (also known as NOT LIKE
DAD and PHOSPHOLIPASE A1), which encodes a spermspecific
phospholipase, triggers haploid induction in maize
• DOMAIN MEMBRANE PROTEIN (DMP), another gene expressed
specifically in pollen, can lead to independent haploid induction inmaize
(Zhong et al., 2019).
Haploid Induction by genetic disruption of MTL or DMP -
option for paternal genome elimination
37. 2019
• Combine fixation of heterozygosity and haploid induction by simultaneous editing of
all four genes (REC8, PAIR1, OSD1 and MTL) in hybrid rice and obtained plants that
could propagate clonally through seeds
‘Chunyou84’ (CY84)
Inter-subspecific hybrid rice
Chunjiang 16A (16A)
Japonica male-sterile line
Paternal C84
Indica-japonica
intermediate-type line
38. The triple mutant of REC8, PAIR1 and OSD1
meiosis genes- MiMe (Mitosis instead of
Meiosis
Editing the MATRILINEAL (MTL) gene (involved
in fertilization)- Induce paternal genome
elimination
39. 1) To test the feasibility of MiMe technology in hybrid
rice varieties
CRISPR–Cas9 vector
targeting OSD1, PAIR1 and REC8
Simultaneously edited the REC8, PAIR1 and OSD1 genes in the hybrid CY84 using
multiplex CRISPR–Cas9 system9
I. Observed chromosome segregation pattern
II. Analysis of ploidy of spores – FISH analyses
using a 5 S rDNA-specific probe, which
identifies chromosome 11 of rice
III. Ploidy analysis flow cytometry
IV.ten insertion-deletion (indel) markers
analysis
Hybrid CY84
40. The chromosomes of CY84 and MiMe were probed
by digoxigenin-16-dUTPlabeled 5 S rDNA (red
signal, indicated with a white arrow) in spores,
showing one signal in wild-type CY84 and two
signals in MiMe. The DNA is stained with 4′,6-
diamidino-2-phenylindole (DAPI, blue signal)
41. Panicles of wild-type CY84 and MiMe.
The fertility of MiMe was as high as
that of wild-type CY84
Panicles and grain shape of CY84 and the progeny
of MiMe.
The progeny of MiMe displayed reduced fertility,
increased glume size and elongated awn length.
43. 2) Generation of a haploid inducer line by editing the
MTL gene in hybrid rice variety CY84
Normal vegetative growth, but the seed-setting rate was
reduced to 11.5%
44. • Twelve indel markers (one per chromosome) that were polymorphic between the two
parents were used to determine the genotype of the progeny of the mtl plants.
• 11 plants from 248 mtl progeny appeared to be homozygous for all markers-
HI rate= 4.4%
Plants homozygous at all markers in the progeny siblings of mtl were identified as
haploid or DH.
45. Flow cytometry showed that nine of these plants were indeed haploid, whereas two were
diploid, presumably resulting from spontaneous doubling of haploid embryos.
46. Whole-genome sequencing of the
haploid, DH and RID plants.
Twelve blocks represent 12
chromosomes. The SNPs of C84
allele are in red, the SNPs of 16 A
allele are in blue, and the coexistence
of both alleles is in yellow
• Simultaneous editing of REC8, PAIR1 and OSD1 genes did not have
obvious adverse effects on the growth and reproduction of the hybrid.
• The MTL gene used to induce paternal genome elimination had
• Negative effects on hybrid fertility
• Not fully penetrant
47. 3) Simultaneous editing of all four genes (REC8,
PAIR1, OSD1 and MTL) in hybrid rice
• Combined fixation of heterozygosity and haploid induction
• Fix plants displayed a reduced fertility(4.5%) due of the MTL mutation
• The Fix plant was able to produce clonal seeds with the same ploidy
and heterozygous genotype
48. The clonal Fix (Diploid progeny of Fix) displayed a low seed-setting rate
that was similar to that of parent Fix plant
Improvements in fertility, such as by modifying the MTL gene or looking
for different haploid-inducing genes, will be required technology or clonal
fix technology to be commercialized for rice.
52. Ectopic Expression of BBM Induces Somatic Embryo Formation in Arabidopsis and Brassica.
Boutilier et al., 2002
The BABY BOOM (BBM)
AINTEGUMENTA-LIKE (AIL) AP2/ERF
domain transcription factor is a major
regulator of plant cell totipotency, as it
induces asexual embryo formation
when ectopically expressed
BBM genes from Arabidopsis thaliana and
Brassica napus can ectopically induce somatic
embryos; however, a role for these genes in
the initiation of zygotic embryos has not been
established.
53. Pennisetum squamulatum
The large non-recombining ASGR transmitting
apomixis contains multiple copies of PsASGR-BABY
BOOM-like (PsASGR-BBML; Gualtieri et al., 2006
ASGR An APETELA 2 (AP2)-domain-containing
gene and member of the BBML clade of the
AINTEGUMENTA-LIKE (AIL) gene family
PsASGR-BBML express in egg cells before fertilization
and induces parthenogenesis
BBML gene expression under the control of its own promoter in sexual pearl
millet yielded haploid embryos (Conner et al., 2015).
PsASGR-BBML can induce parthenogenesis under the control of either its own
promoter or an Arabidopsis egg-cell-specific promoter [DOWNREGULATED IN dif1
45 (DD45), At2g21740], which drives gene expression in egg cells in monocot
crops, such as maize, rice (Joann A. Conner, 2017) and in sorghum
Induced Parthenogenesis of MiMe gamete
54. 1 Department of Plant Biology, University of California, Davis, CA, USA.
2 Innovative Genomics Institute, Berkeley, CA, USA. 3Department of Genetics, Development and Cell Biology, Iowa State
University, Ames, IA, USA.
Nature, 2018
• Rice cultivar Kitaake (O. sativa L. subsp. japonica)
• Halfstrength Murashige and Skoog’s (MS) medium containing 1% sucrose
and 0.3% phytagel in a growth chamber for 12 days, under a 16 h light:8 h
dark cycle at 28 °C and 80% relative humidity.
I. To make the ectopic expression of BBM1 in the rice egg prior
fertilization to induce parthenogenesis
II. Combining egg cell expressed rice BBM1 with CRISPR/Cas generated
MiMe knockouts
55. Does parent of origin have effect on embryogenesis ?
• Pointed multiple BBM-like genes in rice, at least three—BBM1, BBM2
and BBM3 (Os11g19060, Os02g40070 and Os01g67410, respectively)
• CRISPR–Cas9 system to generate bbm1 bbm3 and bbm2 bbm3 double
mutant (NO triple mutant obtained)
• BBM1/bbm1 bbm2/bbm2 bbm3/bbm3 plants were recovered and
selfed
• viability of the bbm1 bbm2 bbm3 triple-mutant seeds was severely
affected (2 out of 191 viable compared with the expected 48 out of 191
• Performed reciprocal crosses of BBM1/bbm1 bbm2/bbm2 bbm3/bbm3
to BBM1/BBM1 bbm2/bbm2 bbm3/bbm3 plants
56. BBM1/BBM1 bbm2/bbm2 bbm3/bbm3 bbm1/BBM1 bbm2/bbm2 bbm3/bbm3
Female
Male
X
BBM1/BBM1 bbm2/bbm2 bbm3/bbm3
Male
bbm1/BBM1 bbm2/bbm2 bbm3/bbm3
Female
X
Result of maternal transmission of bbm1 allele
Result of paternal transmission of bbm1 allele
57. Paternal expression of BBM1 in zygotes
Expression of BBM1 fused to a GFP reporter was detected by antibody staining.
GFP expression is observed only when BBM1–GFP is transmitted by the male parent
58. Male-genome-derived expression of BBM1—acting redundantly with other
BBM genes—triggers the embryonic program in the fertilized egg cell.
Subsequent activation of expression of the female BBM1 allele by the male
BBM1 results in biallelic expression, with both parental alleles eventually
contributing to embryo patterning and organ morphogenesis
The embryo arrest and abortion due to triple knock down of the genes
BBM1, BBM2, and BBM3 were fully rescued by the male transmitted BBM1
59. Parthenogenesis induction by expression of BBM1 in
the egg cell.
T-DNA region of the binary vector
The expression of BBM1 in the egg cell initiated parthenogenesis in emasculated
flowers , but the seeds aborted in the absence of endosperm .
Agrobacterium mediated transfer of the
construct into rice egg cell
(BBM1- ee transgenic plant)
Cloning BBM1 downstream to Arabidopsis DD45
promoter and upstream of the nopaline synthase
terminator in pCAMBIA1300.
60. Development of parthenogenetic embryos (red arrowhead) by egg-cell-specific expression of
BBM1 in carpels of an emasculated BBM1-ee plant at nine days after emasculation.
In the absence of fertilization, endosperm development is not observed (black arrow).
In fertilized control wild-type (4 days after pollination (DAP)) carpels, the development
of both embryo and endosperm is observed
61. 3n
2n
n + n
n+n
n
3n
n
Non viable/ non
endospermous
Viable Diploid
Viable haploid-
Parthenogenic
Transgenic plant
62. Analysis of Self-pollinated T1 progeny from BBM1-ee transgenic plants
Self-fertilization produce viable
seeds(Endosperm development )containing
parthenogenetically derived haploid
embryos.
• Haploids Small size compared with their
diploid siblings, as well as by their sterile
flowers owing to defective meiosis
• The haploid induction frequency was 5–10%
(T1 plants) and reached around 29% in
homozygous T2 line 8C—this frequency was
maintained through multiple generations
A BBM1-ee parthenogenetic
haploid panicle showing no anthesis
Check for haploid viable seeds (Endo-spermous)
63. Pollen viability in haploids was assessed
by Alexander staining.
BBM1-ee haploid anther with
non-viable pollen
wild-type anther with viable
pollen
64. The expression of BBM1 in the egg cell can initiated parthenogenesis
in emasculated flowers haploid inducer homozygous T2 line 8C
(around 29% )
Inference
The genome editing to substitute mitosis for meiosis (MiMe)
should combined with the expression of BBM1 in the egg cell for
asexual propagation via seeds to retain genome-wide parental
heterozygosity
The next step forward ?
65. Material and Method
Schematic of the CRISPR–Cas9 plasmid construct used for genome editing of the three
MiMe rice genes.
pCAMBIA2300 MiMe CRISPR–Cas9 was transformed in embryogenic calli
derived from pDD45::BBM1#8c haploid inducer lines
Rice cultivar : Kitaake (O. sativa L. subsp. japonica)
•Three candidate genes (OSD1, Os02g37850; PAIR1, Os03g01590 and REC8,
Os05g50410) for creating MiMe mutations in rice were selected and sgRNAs
sequences 5′-GCGCTCGCCGACCCCTCGGG-3′, 5′-GGTGAG GAGGTTGTCGTCGA-
3′ and 5′-GTGTGGCGATCGTGTACGAG-3′, respectively, for CRISPR–Cas9-based
knockout
Schematic of genome integrated pDD45::BBM1 in the BBM1-ee plant
66. Haploid BBM1-ee plant + MiMe Haploid embryo FERTILE
n n n
MMC of Haploid
BBM1-ee plant
NO recombination and
reduction division
(MiMe)
Parthenogenic
development of egg
cell
egg cell
• The three rice MiMe genes were subject to genome editing by CRISPR–
Cas9 in haploid and diploid plants carrying the BBM1-ee transgene.
• Unlike BBM1-ee haploids, the MiMe + BBM1-ee haploids were fertile with
normal anther development, suggesting that meiosis was successfully
replaced by mitosis
Self pollination
Diploid
(Double haploid)
Haploid
(S-Apo ;Synthetic-
Apomictic)
<few plants only>
Embryo/ Haploid
progeny
T0
T1
67. • Study found haploid progeny from two MiMe + BBM1-ee (Synthetic-
Apomictic) haploid mother plants at frequencies of 26% and 15%, due
to parthenogenesis
• These haploid induction frequencies were maintained for the next two
generations.
• These results show that haploid S-Apo plants can be propagated
asexually through seeds.
Diploid + Tetraploid
T0 – Transgenic plant/
MiMe Haploid
T1
Diploid
(Double haploid)
Haploid
(S-Apo ;Synthetic-
Apomictic)
Asexual
reproduction
T2
Sexual
reproduction
68. T0 – Transgenic plant/
Diploid MiMe
MiMe Tetraploid S-Apo Diploid
(S-Apo ;Synthetic-
Apomictic)
Diploid BBM1-ee plant + MiMe Diploid embryo
Obtained diploids at frequencies of 11% and 29% from the progeny of two
diploid S-Apo (that is, MiMe + BBM1-ee) T0 transformants and rest tetraploids
T1
69.
70.
71. MiMe mutations and confirmation of clonal
progeny from S-Apo plants.
a, Sequence chromatograms at mutation
sites of MiMe genes in wild-type, T0 diploid
S-Apo mother plant and two diploid
progeny from each of T1, T2 and T3
generations of S-Apo line 1 (n = 7). Red
arrows point to mutation sites. PAIR1 and
REC8 are biallelic whereas OSD1 is
homozygous
72. Asexual propagation without genetic segregation can be engineered in a
sexually reproducing plant, and illustrates the feasibility of clonal
propagation of hybrids through seeds in rice
The maternal : paternal genome ratio of 2:1 is maintained in the endosperm in both
the pathways, ensuring normal seed development.
Conclusion
73.
74. Mimicking sporophyte apomixis
Ectopic embryo induction
within the ovule
Arresting or delaying egg cell progression
or zygote development in the fertilized
meiotic female gametophyte
The complete arrest of zygote development will impose arrestment of the
endosperm and seed failure due to embryo-endosperm signaling and
communication
Gene Description crop
RWP CitRWP gene is co-segregated with citrus nucellar embryo and
preferentially expressed in nucellar embryo initiation cells.
Citrus
AGL11 MADS-box transcription factor, expressed at the apomictic
nucellar embryo stage .
Zanthoxylum
bungeanum
BBM Transcription factor Brassica
75. Genetic analysis in citrus revealed, somatic embryogenesis
is likely regulated by the gene CiRKD1, which encoding an
RWP-RK domain-containing transcription factor
CitRKD1 at the polyembryonic locus comprised multiple alleles that were
divided into two types, polyembryonic alleles with a MITE insertion in the
upstream region and monoembryonic alleles without it.
Additionally, a C2H2 zinc-finger domain-containing transcription factor gene
(CitZFP), which is homologous to the dandelion parthenogenesis gene (PAR),
is specifically expressed in apomictic cells.
Xia Wang et al., 2017
This indicates further study on the function and regulation of CitRWP and CitZFP genes
in citrus are necessary for utilization of sporophytic apomixis in apomixis breeding
76. • Autonomous endosperm in sexuals is complex process rely on genome
balance, epigenetic gene regulation, parent-of-origin effects founded upon
the contribution of the male gamete, and regulatory pathways underlying
embryo-endosperm developments
• Maternally expressed polycomb repressive complex 2 (PRC2), which catalyzes
histone H3 lysine 27 methylation (H3K27me3) to repress gene expression,
involved in the control of endosperm development
• Mutations in fertilization independent endosperm (FIE) and other
Polycomb group genes leading to Autonomous endosperm development in
Arabidopsis
• Orthologs reported in both rice (OsFIE1 and OsFIE2) and maize (ZmFIE1 and
ZmFIE2) produce distinct phenotypes.
Autonomous endosperm development
77. Genes involved in endosperm development
Gene Description crop
ORC3 Defective ORC3 mutants exhibit normal female
gametophyte but the development of embryo and
endosperm is abolished.
Paspalum
FIE The expression of FIE is negatively correlated with
parthenogenesis capacity. Mutant of FIE allows
endosperm development without fertilization.
Malus,
Arabidopsis
fis FIS controls seed development after double
fertilization. In the fis mutants, partial
development of seed can occur without pollination
Arabidopsis
78. Autonomous endosperm
development
Using Cas9 as a recruiting platform for demethylase would allow activation of
repressed female genes, for instance to erase genomic imprinting and trigger
autonomous endosperm
79. Tonosaki K, 2020
This study, demonstrate that mutation of the rice (Oryza sativa) gene
EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component
of PRC2, causes an autonomous endosperm phenotype involving proliferation of
the central cell nuclei with separate cytoplasmic domains, even in the absence of
fertilization
• A CRISPR/Cas9 guide RNA sequence targeting 10th exon of OsEMF2a that encodes a
zinc-finger domain
• OsU6pro:sgRNA:polyT binary vector: pZH_OsU3gYSA_MMCas9 harboring SpCas9 and
hygromycin phosphotransferase gene (HPT) expression constructs using AscI and PacI site
80. Multiple nuclear cytoplasmic domains were
present in the enlarged cavity of the embryo
sac in emf2a-3/ + plants at 4 DAE
Accumulation of starch grains and protein
storage vacuoles
(Double stained with iodine and safranin)
82. Maternal OsEMF2a is involved in seed development after fertilization
Reciprocal Crosses Normal
seeds
Shriveled
seeds
Wild-type X emf2a-3/+ 97.73% 2.27%
emf2a-3/+ X Wild-type 62.50% 37.50%
Wild-type X emf2a-5/+ 100.00% 0.00%
emf2a-5/+ X Wild-type 64.29% 35.71%
Does OsEMF2a gene involved in other functions ?
emf2a / + selfing
OsEMF2a controls the timing of cellularization :
1. Delayed development
2. Coenocytic endosperm showed unusual vacuolated structures and centripetal
proliferation without cellularization, especially at the embryo periphery
83. OsEMF2a targets MADS-box transcription factor genes
• Transcriptome and H3K27me3 ChIP-seq analyses using
endosperm from the emf2a mutant identified downstream
targets of PRC2.
• These included 4100 transcription factor genes such as type-I
MADS-box genes(OsMADS77 and OsMADS89;), which are likely
required for endosperm development.
EMBRYONIC FLOWER2a-containing PRC2 controls endosperm developmental
programs both before and after fertilization.
Identifying key regulators that repress nuclear divisions in the central cell and the
developmental transitions in endosperm will require more detailed investigations
further to achieve full benefit
Inference
84. Current hurdles to exploit synthetic apomixis
• The MiMe combined with CENH3 system depends on hybrid
pollination , which restricted the commercial production of clonal
seed.
• In both MiMe combined with MTL1 system and MiMe combined with
BBM1 system, the clonal seeds exhibit relatively low fertility,
possibly due to the low frequency of parthenogenesis,
• The MiMe combined with BBM1 system requires self-pollination
to initiate endosperm development and thus sexual seeds are also
produced together with the clonal seeds.
• MiMe + BBM1 include transgene BBM promoter, and it leak
through pollen into environment
• Lower expressivity penetrance and fertility of clonal fix seeds
86. Modify different
genes to mimic
apomixis- like steps
Optimizing fittest
combination of
mutants
Maximum trait
expressivity
Modulate gene
expression
87. Asexual propagation through synthetic apomixis should extend to most
cereal crops.
The efficiency of clonal propagation inculding, frequency of parthenogenesis,
which could potentially be improved in the future with different promoters
Minimizing endosperm imprinting effects specifically on endosperm
development and starch mobilization
Expand data collection for genomic dissection of apomixis loci to unfurl its
secret: thus to maximize fertility
clear regulatory network of each component of apomixis, cell-to-cell
signalling will provide strong foundation for engineering of apomixis in crops
Produce a fertile highly expressive apomict, with no off-target effects of
gene editing.
Future direction
88. CONCLUSION
Knowledge on how genes are interconnected
in different metabolic pathways will be
important for editing different reproductive
genes without disrupting the process that lead
to a mature seed
![This [hybrid rice] technology is not for farmers who are still struggling at the level of 2 or 3 tons per
hectare. – S.S. Virmani, IRRI, 1998
Is benefit of “hybrid technology” reach up-to bottom hands ?](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)