Genetic manipulation of stay- green traits

for crop improvement

Shantanu DasRegn.no. : 11-AMJ-65

Dept. of Plant breeding and Genetics

Contents

Introduction.

Strategy for development of genotype with

stay green traits.

Physiological basis of stay-green traits.

Importance of stay green traits.

Genetic manipulation of stay-green traits.

Case study on stay-green QTL introgression.

Ideotype of stay-green genotype.

The relationship between stay-green traits and grain yield.

Conclusion.

What is stay-green ?

The extended foliar greenness during grain filling under post anthesis drought.

Stay green is one such trait, and genotypes possessing this trait maintain more photosynthetically active leaves

than genotypes not possessing this trait .

Genotype with stay green traits is called stay green genotype.

Where, When and Why stay green traits is required?

It’s required specially in a drought environmental condition.

To keep greenness of leaves alive for longer period of time, specially during the grain filling stage.

To maintain or increase higher grain yield.

Stay-green results when the plant’s normal process of senescence is disrupted.

Strategy for development of genotype with stay green traits.

Keeping alive for longer is the fundamental strategy for increase

Crop yield

Market value

Physiological basis of stay green traits

Components of stay green traits.

Key components determining stay green:

Total plant leaf area (TPLA): (+ve) correlated with green leaf area at maturity (GLAM) .

Duration of leaf senescence: (+ve) correlated with GLAM.

Rate of leaf senescence (LS): (-ve) correlated with GLAM

NB: GLAM is an indicator of stay-green.

Classes of stay green

There is a five way of stay green on the basis of time and duration of occurrence of senescence.

Type A stay greens (delayed initiation of yellowing)

Type B stay greens (initiate senescence on schedule, but comparatively slow)

Type C stay greens (arise due to specific defects in chlorophyll

degradation pathway.)

Type D stay greens (cell are dead)

Type E stay greens ( chlorophyll content is remain same but

enzyme activity is reduce)

Five ways to stay‐green.

Importance of chlorophyll in stay green traits.

Chlorophyll pigment is responsible for greenness of the leaves.

Chlorophyll pigment involved in photosynthesis.

The result of photosynthesis is biomass production.

When there wii be degradation, during the reproductive stage , ultimate biomass production will be less.

Chlorophyllase (CLH) is responsible for degradation of chlorophyll a to chlorophyllide a (Chlide a)

When a mutants disrupt the activity of chlorophyllase (CLH), it leading to stay-green phenotypes.

Chlorophyll degradation initiation

×

Relation of N2 with stay green traits

Chlorophyll pigment is consist of N2, Mg+2 and others constituent.

Stay-green can be viewed as a consequence of the balance between N demand by the grain and N supply during grain filling stage.

While N2 is the constituent of most of the

proteins (Rubisco involved in photosynthesis).)

Chemical structure of chlorophyll

Importance of s

tay-green traits

1. Importance of stay green traits in agronomical crops.

Delayed senescence is useful only when it contributes to increase yield.

N uptake during grain filling is higher in stay-green genotype than senescent hybrids. (Borrell and Hammer, 2000).

In some crop it leads to drought resistance.

It leads to lodging resistance.

2. Importance of stay green traits in horticultural crops.

In horticultural crop it increases market value.

Several approaches have been utilized to manipulate hormone level or responses in transgenic plants to reduce the incidence of postharvest yellowing.

It extends shelf-life and helps in long term transportation.

Genetic manipulation of stay green traits

A. Identification of QTL for stay green.

Requirement for QTL mapping:

1. A suitable mapping population generated from phenotypically contrasting parents.

2. Reliable Phenotypic screening of mapping population.

3. A saturated linkage map based on molecular markers.

4.Appropriate statistical packages to analyze the genotypic information in combination with phenotypic information for QTL detection.

F2 F2:F3

P2

F1

P1x

Phenotypic screening

Screening is going on Trials plot

DonorRecipient

1. and 2. Phenotypic screening of mapping population.

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

3. Overview of ‘marker

genotyping’

Stay-green QTL have been identified – Few to mention

Crop Identified QTL Reference

English blue grass (Festuca Pratensis )

Recessive nuclear allele sid

Thomas(1997)

SorghumStg1, Stg2, Stg3, and Stg4. Wenwei Xu et al.

(2000)

Arabidopsis recessive gene fiw maped on chromosome 4

Nakamura et al. ( 2000)

Stay-green QTL have been identified – Few to mention

Crop Identified QTL Reference

RiceRecessive mutant gene sgr(t) on chromosome 9

Cha et al. (2002)

Rice46 main QTL for six trait are localized in 25 chromosomal region

Jiang et al. (2003)

Wheat (Triticumaestivum L.)

QSg.bhu-1A, QSg.bhu-3B and QSg.bhu-7D

Uttam Kumar et al. (2010)

B. Introgression for stay green QTL

Molecular approaches

F2

P2

F1

P1 x

ResistantSusceptible

MARKER-ASSISTED SELECTION (MAS)

Marker assisted breeding

F2

P2

F1

P1 xResistantSusceptible

Marker assisted breeding

Marker assisted backcrossing

Marker-assisted backcrossing (MAB) There are three levels of selection in which markers may be applied in backcross breeding.

Foreground selection : to screen for the target trait.

Recombinant selection: to minimize linkage drag.

Background selection: to the recovery of the recurrent parent genome.

CASE STUDY

K. NGUGI, W. KIMANI1 and D. KIAMBI1

INTROGRESSION OF STAY-GREEN TRAITS INTO A KENYAN FARMER

PREFERED SORGHUM VARIETY

A MAB breeding programme was conducted to introgress stay green QTL from ICRISAT sorghum line E36-1 well-characterised for stay-green QTL, into the background of the Kenyan farmers’ preferred sorghum variety, Ochuti.

The parental lines E36-1 has 3 stay green QTLs, SBI-01, SBI-07 and SBI-10 located at various chromosomes.

E36-1 donor parental line Ochuti recurrent parental line

Make the backcross and to select the genotypes with Stay-green QTL

Ochuti

X Ochuti Recurrent parent

Collection of leaves at 15 days after sowing

for genotyping

F1 XCollection of leaves

from 96 F1 progenies

for genotyping

BC1F1 Leaves collection from 128 individual

for identifying genotype with the Stay-green QTL

E36-1 Donor parent

Collection of leaves at 15 days after sowing

for genotyping `

Contd.

Seven forground marker were used to identify individuals of F1 generation that had stay green QTLs transferred into Ochuti.

Out of 96 F1 genotype only five genotypes that had at least one stay green QTL introgressed, 3 with two QTL ( SBI-07 and SBI-10) and 2 other F1 genotypes had only one QTL (SBI-10) transferred into Ochuti.

A total of 128 BC1F1 DNA samples together with the two positive controls (the two parents) were genotyped with the five foreground SSR markers.

Ideotype of genotype with stay green traits

Plant should have spread and deep root system.

Genotype with stay green traits should show slow rate of leaf senescence(LS).

Delay onset of leaf senescence(LS).

Genotype should have more total plant leaf area (TPLA).

The relation between the stay green traits and grain yield

Grain yield was correlated positively with green leaf area at maturity and negatively with rate of leaf senescence (from the study of nine genotypes varying in stay-green, Borrell et al.,1999).

GLAM Grain yield ⍺

Rate of leaf senescence ⍺

CONCLUSION

Several stay-green QTL have been identify in Several crops like sorghum, wheat, rice etc.

By hybridization programme, its is enable to transfer identified stay-green QTL to the genotype having good agronomic background.

Incorporation of stay-green trait in a genotype will increase the ultimate grain yield and market value which

will contribute to our national economy.