Engineering Centromeres for

Haploidy Induction in

Grain Legumes

ICRISAT Asia Regional Planning Meeting, Patancheru, India, February 10-12, 2014

Why Double Haploids ??

• Haploids are individuals with a single set of chromosomes which are doubled to produce DH to create instant homozygous lines.

• A quicker method to obtain 100% pure inbred lines; Increase genetic gains per cycle

• Increase efficiency of breeding cycle and developmental costs

• Quantitative Genetics Studies

( Quantitative Inheritance, Mapping QTLs, gene identification etc.)

50% 99.2 % pure

Traditional Inbred Process takes 7-8 generations

Genetic Purity

50% 100 % pure

Genetic Purity

Double-Haploid Inbred Process takes 2 generations

Cons: No uniform protocol, species and genotype dependent, demand technical expertise

Methods of haploid production

In vitro methods Culture of haploid gametophyte cells

• Anther/ microspore/pollen culture

• Ovary/ ovule culture

In vivo methods

Haploid inducing genes and genotypes

Stock 6, ig – Maize ; hap - Barley

Uniparental genome elimination in distant crosses

Bulbosum technique/method - Barley

Legumes have been lagging behind with respect to the

development of doubled haploid protocols

Currently, no crop improvement program uses DHs on a routine

basis for any legume

Source: Berkeley open computing facility

Tail Histone fold domain (HFD)

CENH3

H3

1 2 3 Loop1 Loop2

58% identity to histone H3 HFD

The CENH3 histone-fold domain controls kinetochore localization, but N-

terminal histone tail is required for CENH3 interactions with various

kinetochore components.

CENH3-1 +/- CENH3 +/+

CENH3-1 +/- 68

CENH3 +/+ 38

cenh3-1 -/- 0

cenh3-1 null allele is embryo lethal (Arabidopsis, Tomato, Strawberry)

Isolated by TILLING

Splice acceptor point mutation

CENH3-1 +/-

CENH3-1 +/-

X

Transgenic mediated-Genome elimination

Isolation of cenh3 null allele

Ravi & Chan, 2010

• Lethality can be rescued by

CENH3 variants

Tail Histone fold domain

CENH3

Tailswap

cenh3 -/- + GFP:CENH3

cenh3 -/- + GFP:Tailswap

Complementation by altered CENH3 variants

Ravi & Chan, 2010

• Good localization to centromeres

CENH3 differences between parents induce uniparental genome elimination

Inducer crossed with wild type to induce haploids

Ravi & Chan, 2010

Genome elimination: a promising approach

for haploid induction in recalcitrant legumes?

Strategy

Cross Inducer with desired parent to induce haploids

Isolation of cenh3 knock-outs

Complementation by altered CENH3 variants

Haploid to DH conversion by colchicine treatment

CENH3 protein alignment from Legumes

N terminal tail

C terminal histone fold domain

N terminal tail

C terminal Histone fold domain

CENH3 protein alignment across gene pools

Pigeonpea & Chickpea

N terminal tail

C terminal histone fold domain

C terminal histone fold domain

Creating cenh3 knockouts

(Chickpea/Pigeonpea)

To achieve CENH3 gene knockout

1. Zinc finger nucleases (low rate of success, difficult selection protocols,

expensive)

2. TALENs

3. CRISPER-CAS9

Zinc finger nucleases (ZFNs) ; TALENs (transcription activator-like

effector nucleases); and CRISPRs

• Work by guiding a nuclease to a designated site of

choice and instigate a double-strand break (DSB).

• Cellular DNA repair mechanisms take over and repair

the break

• Proven effective in generating targeted mutations,

homologous recombination, and targeted insertions

and deletions in plants.

• TALENs, are chimeric proteins with a designed DNA recognition domain attached to the non-specific FokI nuclease domain

• This nuclease must dimerize to create a double strand break (DSB)

• Two TALENs (pair) are required to generate a designer nuclease

How TALENs Work?

How CRISPR

system

Work?

1. Should functionally complement cenh3 null mutant

2. Should not have reproductive defects

Identifying Suitable CENH3 variants is the key!

Should be tailored and tested according to crop of

interest

•(GFP:Tailswap/

• GFP:CENH3/

• CENH3 variants from wild relatives?

Complementation is critical

General perturbations in the centromere structure is sufficient to cause

post zygotic incompatibility leading to production of haploids

Cell cycle check points in plants is more relaxed to allow only the

segregation of chromosomes with wild type centromeres

……resulting in haploidy induction using “Transgenic-mediated breeding”

generating Non-GM Double Haploids !!

Inducer Desired genotype

War of the parental genomes

Battle ground : Embryo sac

Maternal genome Paternal genome

CENH3AlteredCENH3

Vs

Survivors

1. Diploid

2. Aneuploid

3. Haploid

60-70% death

Diploids

Aneuploids

Haploids

Mitotic missegregation

1. No tissue culture is needed for haploid induction, thereby removing a major source of genotype dependence.

2. Same inducer produces maternal and paternal haploids.

3. Crossing haploid inducer as the female shifts paternal chromosomes into maternal cytoplasm, which could potentially accelerate the production of cytoplasmic male sterile lines for making hybrid seed.

Potential advantages

ICRISAT

Pooja Bhatnagar- Mathur

KK Sharma

D. Srinivas Reddy

P. Sudhakar

PM Gaur

Trushar Shah

Research Team

IISER

Ravi Maruthachalam

Late Simon Chan UC Davis

Ann Brit UC Davis

Deborah Delmer UC Davis

Jim Dunwell University of Reading

ICRISAT Management

Acknowledgements

Thank you