Towards a functional analysis of the major factors
involved in the reproductive barrier between
Asian and African cultivated species of rice
Andrés GUTIÉRREZ
January 22, 2016

 Model plant for genetics and genomics studies
- Diploid species: 2n = 24, AA
- Genome size small: 390 Mb
- Short growth duration
- Efficient genetic transformation
- Extensive genomic resources:
High-quality reference sequence, dense molecular maps,
mutant libraries
Introduction
Rice (Oryza sativa): One of the most important crops in the world
2
 Oryza sativa complex (AA-genome type)
Source: FAO, 2004

 Many traits of agronomical interest (After Ali et al., 2010)
Introduction
Using AA genome species of rice to discover genes of importance
3
 Cultivated species: O. sativa and O. glaberrima

4
Introduction
Oryza glaberrima: A source for the improvement of Oryza sativa
• Diploid species: 2n = 24, AA
• Genome sequenced
• Resistance or tolerance to abiotic
and biotic stresses
• Traits have been combined with high
yield O. sativa acc.
 Strong reproductive barrier hamper his full utilization
Source: archive.gramene.org

Pre-mating isolation
Prevent the copulation and
fusion of gametes of interspecific
crosses
Post-mating isolation
After mating has occurred
=> prevent fertilization or
formation of zygotes hybrids
Introduction
• Habitat Isolation
• Temporal Isolation
• Behavioral Isolation
• Mechanical Isolation
• Gametic Mortality
Introduction
There are two basic categories of reproductive barriers
5

Pre-zygotic Isolation
Mechanisms
Pollen–pistil interactions
Post-zygotic Isolation
Mechanism
• Gametic incompatibility
• Zygote dies after fertilization
• Hybrid inviability
• Hybrid breakdown
• Hybrid Sterility
Arrest of the development of
young zygotes
Introduction
Post-mating isolation
6
Hybrid Sterility
Inability to form functional gametes in a hybrid due to
disturbances in sex-cell development or in meiosis, caused by
incompatible genetic constitution

Introduction
What is the importance of studying hybrid sterility?
 For understanding biology of reproduction
When, where and how action of genes involved in reproduction take
place
 For studying evolution
Hybrid sterility is one of the mechanisms of reproductive barrier
between species
 For breeding
Sterility decreases yield
Hybrid sterility genes prevent the introgression of useful genes

• This phenomenon is one of the strongest post-zygotic reproductive barriers in
Oryza species
• Use of O. glaberrima in breeding is limited
• Hybrid sterility prevent the introgression of useful genes
Introduction
Hybrid sterility between O. sativa x O. glaberrima
O. sativa
(Asian rice, AA genome)
O. glaberrima
(African rice, AA genome)
F1 hybrid
Totally male sterile
Partially female sterile
8

Introduction
Hybrid sterility between O. sativa x O. glaberrima
 Several loci causing hybrid sterility O. sativa x O. glaberrima => S1 locus exert the strong effect
 Epistatic interactions between these loci and S1
Garavito et al., 2010
9

• Genetics:
- S1 locus is the main factor of hybrid sterility between
O. sativa X O. glaberrima (Sano, 1990)
- S1 is a complex formed by three linked loci S1A, S1 and S1B
• Cytology:
- Abnormalities in gametophytes
Pollen semi-sterility
Normal embryo sac Abnormal embryo sac
Koide et al., 2008
Introduction
Hybrid sterility between O. sativa x O. glaberrima
- The effect of S1 is Universal (Tao, 2010)
10
Garavito et al., 2010
Guyot et al., 2011

• Female factor in a 27.8 kbp region nested in the male factor region
• Both male and female gamete elimination are probably controled
by the same factor(s)
Garavito et al.,2010
Introduction
Fine mapping of the S1 locus
Garavito et al.,2010
11
• Model of gamete elimination

Guyot et al., 2011
Garavito et al.,2010
200 400 600 800
200400600800
Nipponbare(849kpb)
CG14 (813 kpb) S1S1A S1B
O. sativa (Nipponbare)
O. glaberrima (CG14)
ENOD-93 ENOD-93 Ribos_biog F-box ENOD-93
ENOD-93 ENOD-93 Ribos_biog F-box ENOD-93
S1
F-box F-box_dup
F-box
S1A
Introduction
Structural genomics
12
S1B remains to be
elucidated

Introduction
The F-box is a protein motif of approximately 50 amino acids that
functions as a site of protein-protein interaction
Lechner et al. 2006 Current Opinion in Plant Biology. 9:631-638

14
Introduction
F-box proteins are involved in self-incompatibility
and floral organ determination

Introduction
F-box as candidate for S1 & S1A
F-box proteins in rice
F-box at S1 locus
RiceXpro DB
Jain et al., 2007
Classification of 687 F-box proteins based on their domain architecture
15

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
To study the nature of the S1 locus
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1

- O. sativa: Caiapo
- O. glaberrima: MG12
- Introgression line: L229 BC3DH from Caiapo x MG12 (S1
g Introgressed)
CSSLs introgression Lines
S1 region _O. glaberrima
Chr. 6
S1Ag S1
g S1Bg
L229 BC3DH
O. sativa L229 O. glaberrima
17
Materials and Methods
Isolation of the S1 region
Gutierrez et al., 2010

229 line (BC3DH) X O. sativa (Caiapo)
Chr. 6 S1Ag S1
g S1Bg S1As S1
s S1Bs
• Panicle, pollen and embryo sac evaluation
• Cytology observations
• Gene expression analysis
S1Ag
S1As S1Bs
BC4F1
S1
g S1Bg
S1
s
18
Materials and Methods
Genetic stocks

Gutierrez et al., 2015 (submitted)
19
Materials and Methods
Evaluation at different stages of O. sativa, O.glaberrima & L229_F1
Female
gametophyte
development
Male
gametophyte
development

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1
 Panicle, pollen grains and embryo sac fertility evaluation
 Histology analysis
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs

0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
Caiapo MG12 L229-F1
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
O. sativa L229-F1 O. glaberrima
Results
Panicle Fertility
Reduced fertility in the L229-F1
21

L229-F1O. sativa
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
Caiapo MG12 L229-F1
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
Results
Pollen grain fertility
22

Msp
MP
AP
Msp
MP
Results
Male gametophyte development in O. sativa and L229_F1
Pollen grain abortion in the hybrid seems to occur at the early microspore stage
Meiosis
O. sativa
L229_F1
Early Microspore Mitosis Maturation
23

0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
Caiapo MG12 L229-F1
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
O. sativa L229-F1
Results
Embryo sac fertility
43.75% embryo sacs were aborted
in the L229-F1
24

dm
fm
dm
Results
Female gametophyte development in O. sativa and L229_F1
Embryo sac abortion in the hybrid seems to occur after completion of meiosis
MMC Meiosis Maturation
O. sativa
L229_F1
25

Conclusion
 L229_F1 showed around 50% of pollen and embryo sac
sterility and 78% of panicle sterility
 Pollen grain abortion in L229_F1 occur at the early
microspore stage
 Embryo sac abortion in L229_F1 occur after completion of
meiosis
Precise determination of abnormalities

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
Sequence comparison and gene structure
of orthologous F-box alleles

Results
F-box at S1 locus is an FBL (F-box and LRR)
OsFBL-185
Pairwise % Identity
Genomic: 84.0%
CDS: 98.1%

Results
F-box at S1 locus (OsFBL-185)
OsFBL-185 lacks an Arginine at the 15th position of the first exon
F-box domain
LRR domain
29
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima

Results
F-box at S1A locus is an FBL (F-box and LRR)
OsFBL-184
30
Pairwise % Identity
Genomic: 94.4%
CDS: 96.9%

Results
F-box at S1A locus (OsFBL-184)
Two amino acid conversion in the F-box domain and two in the LRR domain
F-box domain
LRR domain
31
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima
O. sativa
O. glaberrima

Conclusion
 There are important structural changes between the
orthologous genes (S1 and S1A)
=> OsFBL-185 lacks an Arginine at the 15th position of the
first exon
=> Two amino acid conversion in the F-box domain and two in
the LRR domain in OsFBL-184

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1
3. To evaluate the temporal and spatial expression of the F-box
candidate genes
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
 qPCR analysis
 in situ hybridization

Highest expression in the hybrid at critical meiosis/mitosis stages
Results
Expression analysis of F-box at S1 locus
O. sativa
O. glaberrima
S1A S1
<
<
<
0
0.2
0.4
0.6
0.8
1
1.2
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm 14 - 17 cm > 18 cm
Relativeexpression
0
0.2
0.4
0.6
0.8
1
1.2
1.4
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm 14 - 17 cm > 18 cm
Relativeexpression
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore
formation
Mitosis I Mitosis II - Maturation
Floral organs
differentiation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Spikelet Palea-Lemma-Glumes Pistil Stamen
Caiapo
BC4F1
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore
formation
Mitosis I Mitosis II - Maturation
Floral organs
differentiation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Spikelet Palea-Lemma-Glumes Pistil Stamen
MG12
BC4F1
34

Specifically expressed in the embryo sac and pollen grains in development
Results
Spatial expression of F-box at S1 locus in female and male
gametophytes
Caiapo L229_BC4F1 MG12
Anti-sense probe
Sense probe
Anti-sense probe Sense probe
Caiapo L229_BC4F1
LNA probe_O. sativa
Caiapo
35
MG12

0
0.5
1
1.5
2
2.5
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm 14 - 17 cm > 18 cm
Relativeexpression
Highest expression in the hybrid at floral organ differentiation and meiosis/mitosis stages
Results
Expression analysis of F-box at S1A locus
S1A S1
O. sativa
O. glaberrima
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm 14 - 17 cm > 18 cm
Relativeexpression
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore
formation
Mitosis I Mitosis II - Maturation
Floral organs
differentiation
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore
formation
Mitosis I Mitosis II - Maturation
Floral organs
differentiation
0
0.5
1
1.5
2
2.5
Spikelet Palea-Lemma-Glumes Pistil Stamen
Caiapo
BC4F1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Spikelet Palea-Lemma-Glumes Pistil Stamen
MG12
BC4F1
36

Conclusion
 The F-box genes of S1 and S1A in the hybrid showed highest
expression at Critical meiosis/mitosis stages
The F-box OsFBL-185 is specifically expressed in the immature
embryo sac and pollen grains

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1
4. To investigate if the F-box plays a role in the development of male
gametophytes
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
Knock-out of the F-box OsFBL-185 using
the CRISPR/Cas9 system

Expression vector pOsUbi-Cas9
Cas9/sgRNA complex
Materials and Methods
Functional validation of OsFBL-185 through CRISPR-Cas9 system
Prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages
sgRNA_S1-1
sgRNA_S1-2 sgRNA_S1-3
Selected sgRNA target sites of OsFBL-185 39

Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
Transformed plants in the T0 generation
 A total of 79 plants were obtained
 Vegetative phenotype similar to WT
 Pollen fertility of 72 lines was observed
WT Transgenic
40
 Pollen fertility of 72 lines was observed

Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
sgRNA_S1-1 sgRNA_S1-2 sgRNA_S1-3
T7 assay
41
 From 72 lines observed:
56 showed a deletion in the OsFBL-185 gene.
From these 56 lines:
22 (39.3%) => partial or complete pollen sterility
34 (60.7%) => pollen fertile

Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
Deletion-Phenotype 1 Deletion-Phenotype 2
Fertile
Semi-sterile Sterile
OsFBL-185 seems to play a significant role in the development of male gametes42
Pollen grain evaluation

4. To investigate if the F-box plays a role in the development of male
gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate
genes
2. To identify patterns of sequence divergence of the F-box candidate
orthologous genes
5. To verify the hypothesis that F-box is actually S1
5. To verify the hypothesis that F-box is actually S1
1. To characterize the sterile phenotype and to precise the cellular stage
where gamete development defect occurs
Functional complementation strategy

Results
Validation of OsFBL-185 through functional complementation
Genetic transformation of Caiapo (O. sativa) with the F-box “S1
g” from O. glaberrima
Over-Express-O. glaberrima-CDS
Over-Express-O. sativa-CDS

T0 generation
Results
Validation of OsFBL-185 through functional complementation
45

O. sativa Over_sat Over_glab
Pollen grain evaluation
Results
Validation of OsFBL-185 through functional complementation
0.00
20.00
40.00
60.00
80.00
100.00
O. sativa Over_sat Over_glab
46
Failure in male gamete development in the O. sativa x O. glaberrima hybrid is
due to allelic interaction between S1
g and S1
s

Conclusion
 OsFBL-185 seems to play a significant role in the
development and viability of male gametes in rice
=> knock-out by CRISPR/Cas9 system
- Mutants with deletions showed a sterile phenotype
 OsFBL-185 F-box is actually S1, the main sterility factor in the
interspecific O. sativa x O. glaberrima hybrid
=> Functional complementation
- Over expression of S1
g in O. sativa show a sterile phenotype
like the natural hybrid

General Conclusion
OsFBL-185 is actually S1, the main sterility factor in the interspecific
O. sativa x O. glaberrima hybrid
48
OsFBL-1850
0.2
0.4
0.6
0.8
1
1.2
1.4
0
0.2
0.4
0.6
0.8
1
1.2
Pollen grain sterility

Pollen abortion in the hybrid is possibly due to the alteration of a
target protein anchored by OsFBL-185
OsFBL-185 may assemble into an active SCF complex which interact with an
unknown target protein => Protein Degradation
49

Perspectives
 Functional analysis: Validation of CRISPR and Complementation results
=> Characterization of T1 plants by co-segregation analysis
 What it is the function of the F-box OsFBL-185?
 Does this protein form an SCF Complex?
 What is the Target Protein?
=> Identification of protein subunits of the SCF complex
=> Bacterial 2 hybrid assays
=> Protein expression and allelic imbalance
 What are OsFBL-185 interactions?
=> Bimolecular fluorescence complementation (BiFC) assay
Elucidation of pathways at the molecular level
in O. sativa and the hybrid 50

Perspectives
 Develop compatible interspecific bridges
Using CRISPR/Cas9 by suppressing the expression of the S1
g allele and/or for large
chromosomal deletions => S1 region
 Identification of factors involved in female gamete development (S1A - S1B loci)
=> RNA-seq and transcriptome profiles of recombinants lines around S1
51

IRD
Mathias Lorieux
Hélène Adam
Laurence Albar
François Sabot
Christine Tranchant
Pierre Larmande
Hélène Pidon
Cecile Monat
Harold Chrestin
Sophie Cheron
Elise Grenon
Anais Roudiere
Myriam Collin
Stéphane Jouannic
Alain Ghesquière
CIRAD
Emmanuel Guiderdoni
Donaldo Meynard
Anne Cecile Meunier
Jérôme Puig
Mumu
Aurore Vernet
Martine Bes
Julie Petit
Acknowledgments
52
CIAT
Silvio James Carabalí
Natalia Franco
Lady Arbelaez
Alex Aguirre
Marco Brito
Victor Lozano
Paul Chavarriaga
Sandra Vidal
Didier Marin

Thank you for your attention