Genetic and molecular analysis of two new loci controlling flowering - - PowerPoint PPT Presentation

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Apr 14, 2023 591 likes •760 views

Genetic and molecular analysis of two new loci controlling flowering in garden pea, Pisum sativum . A. S. M. Mainul Hasan PhD candidate School of Biological Sciences University of Tasmania General biology of flowering Plant organ producing

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Genetic and molecular analysis of two new loci controlling flowering in garden pea, Pisum sativum.

A. S. M. Mainul Hasan

PhD candidate School of Biological Sciences University of Tasmania

SLIDE 2

❖ Plant organ producing flowers is known as inflorescence. ❖ Shoot/Vegetative apical meristem (SAM) transforms into an inflorescence meristem at the reproductive stage. ❖ Timing and synchronization with season is important. ❖ Plants require exposure to specific photoperiods/day length. ❖ Other important environmental factors – ➢ Vernalization (exposure to cold) ➢ Ambient temperature ❖ Endogenous cues- ➢ Circadian clock ➢ Developmental age ➢ Gibberellin ➢ Sugar, Hormones

http://slideplayer.com/slide/7009388/

General biology of flowering

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Flowering time mechanism in the model system, Arabidopsis thaliana

❖ Mobile signal called florigen later discovered as a protein, FLOWERING LOCUS T (FT). ❖ Integrates environmental factors. ❖ Expressed in leaves and moves to shoot apex ❖ Forms a complex with FD to induce floral meristem identity genes. ❖ Regulated by: Daylength – CONSTANS (CO) Vernalization – FLC

Arabidopsis thaliana

Amb.temp. Vernalization Photoperiod FLC Autonomous Circadian clock Gibberellin Shoot apex Leaf AP1 FT FD LFY CO FT Age

Flowering time mechanism in important crop plants

❖ Knowledge developed in the past decade. ➢ Cereals: rice, maize, sorghum, barley, wheat ➢ Legumes: barrel medic, soybean, pea ➢ Nightshades: potato, tomato ❖ The process is somewhat evolutionary conserved. ❖ However, diverged mechanism have been evolved in these plants. ? ? ? ? ? ?

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❖ One of the most agronomically important legume, member of Fabaceae family. ❖ Domesticated in fertile crescent/middle east. ❖ Good source of carbohydrate, proteins and vitamins. ❖ Self-pollinating, annual plant species. ❖ Vernalization responsive, long day plant. ❖ Model species for flowering time studies in legumes.

Source: http://www.slideshare.net/ipga/brian-clancey-presentation Source: http://www.pulseaus.com.au/growing-pulses/bmp/field-pea Source: http://mapsofworld10.xyz/global-map/

Pea (Pisum sativum)

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Strategy: Gene discovery by mutant analysis

Courtesy: Jim Weller

LUX ELF4 ELF3b PHYA GIGANTEA FTa1

Genes that promote flowering Genes that repress flowering

Courtesy: Jim Weller

Late - Flowering mutants Early - Flowering mutants

late4

A. thaliana:

Current knowledge on flowering time in pea, Pisum sativum

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❖ Two novel loci promoting flowering (LATE3 and LATE4). ❖ LATE3: 3 recessive mutant alleles (late3-1, late3-2, late3-3) ❖ LATE4: 2 recessive mutant alleles (late4-1, late4-2) ❖ Extremely late flowering

16 NFI 40 NFI 39 NFI

Long days ≥ 16 hours *Leaf area of single leaflet at node 10 *Diameter of stem between node 9 and 10 * After growth of 12 days

Relevant background information of the project

W T la te 3 -1 la te 3 -2 la te 3 -3 la te 4 -1 la te 4 -2 5 1 0 1 5 L e a f a re a (m m 2 ) W T l a t e 3

l a t e 3

l a t e 4

1 0 2 0 3 0 4 0 5 0 N o d e o f flo w e r in itia tio n (N F I) W T la te 3 -1 la te 3 -2 la te 3 -3 la te 4 -1 la te 4 -2 1 2 3 4 5 S te m d ia m e te r (m m ) W T la te 3 -2 la te 4 -1 1 0 2 0 3 0 4 0 5 0 R o o t d ry w e ig h t (m g )

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1. Develop genetic map.
2. Identify/evaluate candidate genes.

How do they function to regulate flowering? Overall goal: Identify the LATE3 and LATE4 genes and understand their function

3. Explore genetic interactions with other flowering loci (especially early mutants).
4. Explore regulatory interactions with other known flowering genes.

What are the LATE3 and LATE4 genes?

Goal and strategies

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cv. Terese x late3-1

PsLGIII

CDK8 BTB1

0.5 cM

TPR1 SPS1 NIP2 UNI FRI AAP2 RKP1 BTB1

0.25 Mb

TPR1 SPS1 NIP2 UNI FRI RKP1 CDK8 AAP2

Mt Chr. 3 PsLGV

AAR3 ICE1 MCO1 CYCC1 PAP1 ATPB1

3cM

COLa ABI3 WRI11

late4-1 x cv. Terese

ICE1 ATPB1 MCO1

2.0 Mb

COLa ABI3 WRI11 AAR3 CYCC1 PAP1

Mt Chr. 7

Cyclin dependent kinase 8 (CDK8), Cyclin C (CYCC1) which are components of the eukaryotic mediator complex are positional candidates for Late3 and Late4 respectively

1. What are the LATE3 and LATE4 genes?

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Eukaryotic mediator complex ❖ Eukaryotic mediator complex is involved in transcriptional regulation and deeply conserved among eukaryotes. It has 4 components: head, tail, middle and CDK8 module. ❖ CDK8 and CYCC1 are components of the CDK/CDK8 module along with MED12 and MED13. ❖ Head, tail and middle module (core mediator) promote transcription of genes along with general transcription factors by initiating RNA polymerase II activity. ❖ CDK8 module acts in maintaining optimum transcription of genes by blocking re-initiation through binding with core mediator which is crucial for various biological processes in eukaryotes.

Ref: Yang et. al., 2016 Ref: Davis et. al., 2012

1. What are the LATE3 and LATE4 genes?

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Multiple independent functionally significant mutations in candidate genes confirms identity

1. What are the LATE3 and LATE4 genes?

late3-1: Mutation at 5’UTR causing potential alternative start codon late3-2: Mutation at donor splice site causing skipping of entire exon 7 late3-3: Mutation at acceptor slice site causing retention of 7 bp of intron 4 late4-1: Mutation at 5th position of intron 5 causing generation of 5 different splice variants late4-2: Mutation at exon 4 causing introduction of premature stop codon and generation of 4 different splice variants PsCDK8 PsCYCC1

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PsCDK8 and PsCYCC1 act in the same regulatory pathway

LD >= 16h *Leaf area of single leaflet at node 10

2. How do LATE3 and LATE4 function to regulate flowering?

W T la te 3 -1 la te 4 -2 la te 3 -1 la te 4 -2 1 0 2 0 3 0 4 0 5 0 N o d e o f flo w er in itia tio n (N F I)

W T la te 3 -1 la te 4 -2 la te 3 -1 la te 4 -2 5 1 0 1 5 L e a f a re a (m m 2 )

LD >= 16h

* Yeast two hybrid assay, mated diploid yeast cells grown on selective medium for 7 days at 30°C

PsCDK8 and PsCYCC1 proteins physically interact

2. Strong positive interaction control

10. PsCDK8 bait +

PsCYCC1 prey

11. PsCYCC1 bait +

PsCDK8 prey * Yeast two hybrid assay, mated diploid yeast cells grown on selective medium for 4 days at 30°C

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PsCDK8 and PsCYCC1 promote expression of key pea flowering genes

2. How do LATE3 and LATE4 function to regulate flowering?

2 0 4 0 6 0 8 0 1 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 D a y s a fte r s o w in g % T FIIa tra n script P IM in ap ex 2 0 4 0 6 0 8 0 1 0 0 2 4 6 8 D a y s a fte r s o w in g % T FIIa tra n script F T c in a p e x 2 0 4 0 6 0 8 0 1 0 0 1 0 2 0 3 0 4 0 5 0 D a y s a fte r s o w in g % T FIIa tra n script V E G 2 in a p e x 2 0 4 0 6 0 8 0 1 0 0 1 0 2 0 3 0 D a y s a fte r s o w in g % T FIIa tra n script V E G 1 in a p e x 2 0 4 0 6 0 8 0 1 0 0 5 1 0 1 5 D a y s a fte r s o w in g % T FIIa tra n script F T a 1 in le a f 2 0 4 0 6 0 8 0 1 0 0 2 0 4 0 6 0 8 0 1 0 0 D a y s a fte r s o w in g % T FIIa tra n script F T b 2 in le a f

W ild ty p e late 3-1 late 4-1

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PsCDK8,PsCYCC1 LF(PsTF1c) LF is epistatic to PsCDK8 and PsCYCC1

PsCDK8 and PsCYCC1 suppress expression of important pea flowering repressor gene