1. Genome elimination in plants can be induced by crossing species with differing centromere structures or CENH3 proteins, as these differences may cause chromosomes to improperly segregate and be lost. 2. Experiments in Arabidopsis thaliana found that modifying the N-terminal tail domain of CENH3 caused uniparental genome elimination of the wild type parent. 3. Similar results were found in maize, where RNAi silencing of CENH3 or expressing mutated versions induced haploid production at rates up to 3.6% of offspring. Modifying centromere-associated proteins like CENH3 provides a method for haploid induction in plant breeding.

1. Welcome

2. Haploid induction by centromere
mediated genome elimination

3. Importance of haploidy in plant breeding
• Haploids are valuable tools
• Haploids when doubled produce
– True breeding lines (purelines or inbreds)
– Perfect homozygotes
– Accelerate plant breeding
– Useful in mapping of traits
• Homozygosity is achieved in one generation eliminating the need for several generations of self-
pollination
• Especially in biennial crops and in crops with a long juvenile period
• For self incompatible species, dioecious species and species that suffer from inbreeding depression
due to self-pollination, haploidy may be the only way to develop inbred lines

4. Methods of haploid generation
In vitro haploid generation
• In vitro anther culture in Datura
by Guha and Maheshwari, 1964,
1966
• Published protocols for in vitro
haploid culture in ~ 250 plant
species belonging to almost all
families of the plant kingdom
(Maluszynski et al., 2003)
• High genotype dependency
within species
• Recalcitrance of some
important agricultural species
• Expensive
In vivo haploid induction
• Wide hybridization
– Hordeum vulgare X H. bulbosum/
Zea mays
– Triticum aestivum X Zea mays/
H. bulbosum/ Sorghum bicolour/
Pennisetum glaucum
– Triticale/Secale cereale/Avena sativa
X Zea mays
– Solanum tuberosum X S. phureja
• Haploidy inducing genes
– Haploid inducer Stock 6 in maize
– W23 Indeterminate gametophyte (ig)
gene in maize
– Haploid initiator gene (hap) in barley
• Pollination using irradiated pollen
• Polyembryony

5. Haploid InducerCrop
X
Gametes
2n=8 2n=8
n=4
n=4
Haploid

6. Uniparental genome eliminationis a widespread outcome of distant
geneticcrosses
• Described in diverse taxa of plant species, fishes and amphibians
• First reported in tobacco (Clausen and Mann, 1924)
• Production of viable haploid progeny by uniparental genome elimination reported from distant
hybridization crosses mostly in plants belonging to Solanaceae and Poaceae
• Uniparental genome elimination can also occur in intraspecific crosses (Maize Stock 6 strain & hap
mutant in barley)
• Chromosomes of one parent attach poorly to spindle microtubules, remain as laggards on the mitotic
spindle, mis-segregated and lost during mitosis
• As centromeres are indispensible for accurate chromosome segregation, parental differences in
centromere activity might be the reason for preferential genome elimination

7. Centromere Biology
 Centromeres are usually associated with
 DNA elements
 Centromere-specificrepeats
 Centromere-specifictransposons
 Kinetochore proteins
 Histones
 Centromere-specifichistoneH3 variant
 CENP‐Aassociatedproteins

8. Components of centromere
• Monomeric size of the centromeric satellite repeats are 150–180 bp
– In Arabidopsis thaliana centromeres : 178-bp
– In Zea mays: ~156 bp (CentC)
– In Oryza sativa: 155-bp (CentO)
– In Homo sapiens: ~171-bp (alpha satellite DNA)
• Tandem arrays that can stretch for many thousands of nucleotides
• Interspersed with centromeric retrotransposons
– In Zea mays: CRM
– In Oryza sativa: CRR
• These DNA sequences are predominately found in active centromeres, but recent
findings have demonstrated that these features are not essential to centromere
localization
• In the majority of eukaryotes, centromere positioning appears to be an epigenetic, rather
than sequence-based, phenomenon (Liu et al., 2015)
• Centromeres are epigenetically marked by association with a centromere-specific
histone H3 variant (CENH3)

9. CenH3
• Epigenetic “mark” of the centromere (Allshire and Karpen, 2008)
• Nucleosomes comprise of an octamer of the core histones H2A, H2B, H3, and H4 that can
wrap 147 nucleotides of chromatin
• CENH3 replaces canonical histone
H3 within active centromeres
• But not every H3 is replaced
• In plants CENH3 is deposited into
centromeres almost exclusively during
the G2 phase of the cell cycle

10. Structure of rice Cen8
FISH mapping of the CentO repeat
(green) on rice pachytene
chromosomes
Chr. 8 with the smallest CentO array (~65 kb)
Characterization of the ~750-kb CENH3-
binding domain of Cen8 by mapping of 454
sequence reads derived from ChIP against
rice CENH3
Mapping of trimethylated H3 Lys 36
(H3K36me3), a euchromatic histone
modification mark, within Cen8. Black bars
represent relative enrichment of
H3K36me3 across Cen8.
A diagrammed core domain of rice Cen8, consisting
of interspersed blocks of CENH3 nucleosomes (red
circles) and H3 nucleosomes (blue circles).

11. STRUCTURE OF CENH3
1. C-terminalHistoneFoldDomain
2. N-terminaltail
Comparison between CenH3 and Canonical Histones (H3) (Malik et al., 2009)

12. • Satellite repeats often evolve rapidly, so it can be species-specific or it can be
present in a group of closely related species.
• Rapid evolution of centromeres adds an evolutionary argument that favors
their involvement in uniparental genome elimination.
• Centromere differences may be the reason behind infertility or lower fitness in
interspecies hybrids
• This would create reproductive isolation ultimately leading to speciation.
Rapid evolution of centromeres

13. Hordeum vulgare
Hordeum bulbosum
Temp < 18⁰C
2 Environments
6-13-d-old ovaries
B
In situ Hybridization
Immunostaining
A
Temp > 18⁰C

14. Anaphase chromosome segregation behavior of normally segregating (A)
and lagging (B) H. bulbosum chromosomes in an unstable H. vulgare × H.
bulbosum hybrid embryo. Chromosomes of H. bulbosum (green) were
identified by genomic in situ hybridization using labeled genomic DNA of
H. bulbosum. Chromosomes of H. vulgare are shown in blue.
< 18 ⁰C > 18 ⁰C

15. Anaphase chromosomes of an unstable (A) and stable (B) H. vulgare × H.
bulbosum hybrid embryo after immunostaining with anti-grass CENH3 and
anti–α-tubulin. The centromeres of lagging chromosomes (arrowheads) are
CENH3-negative

16. Interphase nucleus of an unstable H. vulgare × H. bulbosum hybrid embryo (A) after
immunostaining with anti-grass CENH3 (B), genomic in situ hybridization with H.
bulbosum DNA (C, red), and in situ hybridization with the Hordeum centromere-
specific probe BAC7 (D). GISH, genomic in situ hybridization. (E) Only approximately 7
of the 14 more or less equally sized centromeric FISH signal clusters are overlapping
with the position of strong CENH3 signals. Hence, interphase centromeres of H.
bulbosum carry less CENH3 protein. BAC7-positive centromeres without CENH3-
signals are shown (arrows).

17. Characterization of micronuclei of unstable H. vulgare × H. bulbosum hybrid
embryos. Micronuclei are H. bulbosum-positive after genomic in situ
hybridization (A) CENH3-negative (B) and RNA polymerase II-negative (C)
after immunostaining but enriched in H3K9me2-specific heterochromatin-
specific markers (D). Arrowheads indicate micronuclei.

19. • Genome elimination in Arabidopsis thaliana was discovered serendipitously through
experiments that aimed to study structure-function relationships within CENH3 (Ravi and
Chan, 2010 ; Ravi et al., 2010 )
• In order to dissect domains required for CENH3 targeting and function, several chimeras
that combined regions of conventional H3 and CENH3 with GFP (Ravi et al., 2010 ).
• One such chimera, termed “GFP-tailswap” contained GFP tagged to the N-terminal “tail”
domain of H3.3 tail fused to the C-terminal histone-fold domain of CENH3.
• Another chimera “GFP-CENH3” had GFP tagged to the N-terminal “tail” of CENH3.

20. • cenh3-1, an embryo-lethal null mutant in A. thaliana allows to completely replace
native CENH3 with modified variants
• cenh3-1 is a G-to-A transition at 161st nucleotide isolated by the TILLING
procedure
Ravi et al., 2010
Plants were transformed by the Agrobacterium floral dip method
1. GFP-CENH3 plants (cenh3-1 mutant plants rescued by GFP-
CENH3)

23. Percentage of haploids induced

24. Haploid Arabidopsis thaliana produced by crossing plants expressing altered CENH3 to wild
type. a, GFP–CENH3 and GFP–tailswap transgenes used in this study. Tail, N-terminal tail domain;
HFD, C-terminal histone fold domain. b, c, Chromosome spreads from mitotic telophase in
diploid and haploid A. thaliana, respectively. d, e, Chromosome spreads from late diplotene in
diploid and haploid A. thaliana, respectively. In d and e, chromosomes 2 and 4 are joined at their
nucleolar organizer regions independent of homologue pairing (arrows). f, Haploids (right) have
narrower rosette leaves than diploids. g, Haploids (right) have smaller flowers than diploids.
Diploid Haploid

25. Haploid Arabidopsis thaliana yield spontaneous diploid progeny. a–l, Meiosis in diploid (a–d)
and haploid (e–l) A. thaliana. Meiosis II cells show a central organelle band (arrows), indicating
that they have completed meiosis I. Panels e–h show unbalanced reductional segregation (3-2)
in haploid meiosis. Panels i–l show non-reductional segregation (5-0) in haploid meiosis,
forming haploid dyads (l).m, Spontaneous chromosome doubling in somatic cells of haploid A.
thaliana plants produces fertile diploid branches on otherwise sterile haploid plants.

26. Mechanismof uniparental genome elimination

27. Effect of various modifications of transgenic CENH3 variants in an
Arabidopsis thaliana cenh3 null mutant
(Ravi et al., 2010; Maheshwari et al., 2015)

28. Variation in CENH3, specifically in the N-terminal
tail causes genome elimination
Maheshwari et al., 2015

29. • CENH3 could be silenced by RNAi
• Create a cenh3 mutation
 site-specific mutagenesis technology
 Genome editing
 Zinc finger nucleases
 TALENs
 CRISPR/Cas9

30. Experimental overview of the four CENH3 complementation strategies tested
Kelliher et al., 2016

31. Summary of haploid induction rate (HIR) data following outcrosses
with CENH3-altered transgenic maize lines
A004A:3

32. (A) Photograph of the ear crossed by pollen from individual *A004A:3, which exhibited a 3.6%
haploid induction rate (3 haploids found out of 84 embryos). (B) Putative diploid plant
(*A004A:3-104) and haploid plant (*A004A:3-104) which was male and female sterile. They
were also shorter and had thinner leaves. (C,D) Adult leaf samples were tested to confirm ploidy
status.
CENH3-based haploid induction in maize

33. Point mutations in CENH3 histone fold domain induce haploids
Kuppu et al., 2015
Karimi-Ashtiyani et al., 2015

34. Multiple sequence alignment of CENH3 Histone Fold Domain (HFD) of Arabidopsis
thaliana, Brassica rapa, Solanum lycopersicum, Zea mays, Saccharomyces cerevisiae and
Homo sapiens.
Kuppu et al., 2015

35. Schematic representation of transgenic CENH3 point mutant
transformation and crossing

36. Haploid plantsproducedby genome elimination in crosses of CENH3point mutants by Ler
gl1-1

37.  .
 .
 .
 .
Haploid induction and seed abortion frequency of transgenic lines
Kuppu et al., 2015

38. TILLING population (3000 plants)
4 Point mutations in HFD of CENH3
A86V, R176K, W178
A86V homozygotecenh3-1/cenh3-1
A86V
Kuppu et al., 2015
Haploid induction and seed abortion frequency of TILLING lines

39. Schematic comparison of transgenic two-step vs. non-transgenic one step haploid inducers

40. Regulatory issues
Molecular point of view----Non-transgenic
GMO Interpretation of law
Based on the product
Based on the process

41. Conclusions
• True breeding lines indispensible for development and
production of crop varieties
• Haploid production techniques are limited to few crop species
and/or varieties
• Centromere localization is determined by the presence of
nucleosomes carrying CENH3 rather than by the underlying
DNA sequence
• Manipulation of CEN3 can lead to haploid induction
• Domain swapping with addition of fluorescent tag to the NTT
• Complementing with CENH3 from different species
• Point mutations in the highly conserved C terminal HFD
• Since CENH3 is universal in all plants this technology can be
translated to the majority of crop species

42. Practical Implications
• Shown to be useful in trait mapping (Seymour et al., 2012), reverse breeding (Wijnker et al., 2012), and
a variety of other applications (Ravi et al., 2014) in Arabidopsis
• In spite of the great success in model plant Arabidopsis, CENH3- mediated genome elimination needs
to be tested in other crop species–its application to maize (Kelliher etal., 2016) is very encouraging
• The possible delay in implementing this technology may be due to the lack of CENH3 knockouts in
other species
• The recent development of efficient CRISPR-CAS9 based gene targeting conveniently addresses this
issue
• The point mutants of CENH3 that can produce uniparental haploids without involvement of
transgenics can be utilized
– Could be identified in existing TILLING populations of crop species OR
– Could be induced in a single step by CRISPR-Cas9 mediated changes