Milestones and outputs (including HRD activities) - For the Consortium

 

Moisture stress, salinity, heat, cold and submergence tolerance Microbes

 

Time schedule of activities and activity milestones

 

S.No.

Objective/Activity

Activity

Milestones

Expected Output

Responsibilitiy

 

1

2

3

 

 

1.

Selection of different extreme enviornments for survey and collection of soil, water and sediment samples.

The sites proposed to be surveyed are with regards to extreme conditions of

a)   Salinity/ Sodicity/ Acidity

 

Survey and collection of soil samples from extreme enviornments.

Yes

 

 

Samples from different sites will be available for isolation of microbes.

NBAIM. Mau,

IARI, New Delhi,

CIFRI, Barrackpur,

CMFRI, Cochin,

CIFT, Cochin

NRCG, Junagarh

 

b) Moisture

Survey and collection of soil samples from extreme enviornments.

Yes

 

 

Samples from different sites will be available for isolation of microbes.

NBAIM. Mau,

IARI, New Delhi,

NRCG, Junagarh

 

 

b)   Temperature

(Low and High)

Survey and collection of soil samples from extreme enviornments.

Yes

 

 

Samples from different sites will be available for isolation of microbes.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

NRCG, Junagarh

 

 

c)    Temperature

(Low and High)

Survey and collection of soil samples from extreme enviornments.

Yes

 

 

Samples from different sites will be available for isolation of microbes.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

NRCG, Junagarh

 

 

d) Submergence (Anoxia)

Survey and collection of soil samples from extreme enviornments.

Yes

 

 

Samples from different sites will be available for isolation of microbes.

CRRI, Cuttuck,

CIFT, Cochin,

CMFRI, Cochin,

 

2.

Analysis of diversity of predominant genera from various extreme enviornments

Isolation of microbes from samples collected from extreme environments of the country

Yes

Yes

 

Development of database for microbes isolated from extreme environments.

Generation of baseline information in the country.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

3.

Sequencing of rDNA genes for identification of selected isolates/strains.

Generation of sequence database of native isolates and identification of isolates.

 

Yes

 

Submission of DNA sequences in GenBank of native isolates.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CIFT, Cochin,

CRRI, Cuttuck,

NRCG, Junagarh

 

4.

Specific primers for known genes will be used in PCR to identify and isolate candidate genes from microbes.

PCR amplification of relevant genes using specific primers

Yes

 

 

Allelic variants from indigenous isolates will be identified.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

5.

Construction of genomic library in suitable vector and screening of clones for identification of novel genes.

Genomic library construction.

 

Yes

Yes

Identification of novel genes for abiotic stress tolerance.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

6.

Transposon mutagenesis of the seleted strains for screening and isolation of the mutants of interest.

Development of mutants using Transposons.

 

Yes

Yes

Mutants of negative for relevant trait Will be developed.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

7.

Identification of the genes disrupted by transposon insertion in the mutants by cloning and DNA sequence analysis of transposon flanking sequences.

DNA sequences and identification of new genes.

 

Yes

Yes

Validation of novel genes for abiotic stress tolerance.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

8.

Metagenomic library construction and sequence based screening for novel genes for candidate traits.

Isolation DNA from soil and construction of metagenomic library.

Yes

Yes

Yes

Metagenomic library construction for unculturables.

NBAIM. Mau,

IARI, New Delhi,

CRRI, Cuttuck,

 

9.

Bioinformatics analysis of the identifies genes and the encoded proteins and putative biochemical pathways responsible for the stress tolerance.

Identification of the genes and genes sequences.

 

Yes

Yes

Novel genes from indigenous isolates identified.

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

CIFRI, Barrackpur,

CMFRI, Cochin,

CRRI, Cuttuck,

CIFT, Cochin,

NRCG, Junagarh

 

 

 

Trait-wise Distribution of work elements among participating Institutes - microbes

 

Trait

Species

Location/Resources

Activity

Institute

 

Salt tolerance, dessiccation, high temperature

 

Fungi

Both aquatic and soil

Fungal isolation and identification, gene identification by Molecular & Metagenomic approach

NBAIM. Mau,

NRCG, Junagarh

 

Heat & cold tolerance, Desiccation & Acid soil

Soil Bacteria

Thar desert, Jaisalmerin Rajasthan, Arid ecoregion, Leh Ladakh, RohtangPass, Manikaran, Rhishikesh and acid soils from NE regions.

 

Isolation & Identification of microbes, variable tolerance study, Gene identification by Molecular & Metagenomic approach.

Microbiology Lab, IARI, New Delhi

Salt tolerance

Aquatic bacteria, Phytoplankton (Blue-green algae) and Zooplankton

 

Esturine mangrove ecosystems

(Sunderbans in west Bengal, Mahanadi-Bhitarkanika and Chilka lake in Orissa, Godavari-Vizag-vijaywada in Andhra Pradesh)

Microbe isolation & identification, salt tolerance study, ,Gene identification by Molecular & Metagenomic & Bioinformatic analysis

(metagenomic component to the carried out in collaboration with CRRI, Cuttack)

CIFRI, Barrackpur,

CRRI, Cuttack

High Temperature Tolerance

Aquatic Bacteria

Orissa and west Bengal

Microbe identification, heat tolerance study, Gene identification by Molecular & Metagenomic & Bioinformatic analysis

 

CIFA, Bhubaneswar

 

Salt Tolerance and Anoxia/ Submergence

Marine Microbes

Marine environments of Kerala and Lakshwadeep coast and Kutch region in Gujarat

Microbe identification, identification, salt tolerance study, transposon mutagenesis and Molecular & Metagenomic & Bioinformatic analysis

 

CIFT, Cochin

 

Salt Tolerance and Anoxia/ Submergence

Marine Microbes

Marine environments of Tamilnadu,Kerala and Andhra Pradesh

 

Algal identification, identification, salt tolerance study, transposon mutagenesis and Molecular & Metagenomic & Bioinformatic analysis

 

CMFRI, Cochin

Anoxia/ Submergence

Soil and Aquatic Microbes

Orissa, West Bengal

Microbe identification,

Gene identification and Bioinformatic analysis.

Metagenomic study of samples collected by CIFRI, Barrackpur

 

CRRI, Cuttack

Validation of genes for abiotic stress tolerance

 

 

Aiotic stress tolerance gene validation

NRCPB, New Delhi

 

 

Moisture stress, salinity/sodicity, acidity, heat/cold and submergence tolerance - Rice

 

Time schedule of activities and activity milestones

 

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibilitiy

 

1

2

3

 

 

1.

Identification of core collection of rice that would represent total diversity

Analysis of the available passport data as well as agro-ecological, morphological and molecular data on the whole germplasm collection of rice to constitute a core collection

 

Yes

 

 

A core collection of about 6000 rice accessions including landraces and wild relatives

NBPGR

2.

Validation of cire and Identification of mini-core collection of rice

Phenotypic and Molecular markers-based analysis of core collection of rice and constitute a mini-core collection

 

Yes

Yes

 

A mini-core collection of about 1000 accessions representing and diversity

NRCPB, NBPGR, DRR, CRRI. IARI, IGKVV, CAU-B & RCNEHR

 

3.

Generation of phenotypic information for the mini-core collection

Phenotypic evaluation of the mini-core collection in multiple environments for target traits (stress tolerance,yield,cooking and nutritional quality

 

 

Yes

Yes

Phenotypically characterized mini-core set of rice

NBAIM. Mau,

IARI, New Delhi,

CIFA, Bhubaneswar

NRCG, Junagarh

 

4.

Identification of candidate genes through differential expression analysis for selected traits.

To use rice DNA chips in gene expression analysis for identifying possible candidate genes for trait such as abiotic stress tolerance.

 

Yes

Yes

 

Information on candidate genes related to target traits.

DUSC, CAU-B, DRR

5.

Selection of SNPs and SSRs for whole genome and candidate gene based genotyping.

Study of the whole genome SSR and SNP information as well as genic region of the rice gemone, standalone assays for high throughput genotyping.

Yes

 

 

Selected set of SSR and SNP markers (Total about 4000) covering the whole genome spaced at an physical interval of about 100kb and standardized genotyping methods.

 

NRCPB, DUSC, CAU-B

6.

Generation of SSR and SNP database for mini-core collection.

SSR and SNP genotyping

Of the mini-core collection and understanding the diversity pattern and haplotype structure.

 

 

Yes

Yes

 

NRCPB, NBPGR, DRR, CRRI, IGKVV, IARI, CAU-B, & RCNEHR

7.

Allele mining for the selected/known candidate genes.

Association the phenotypic information of the mini-core collection with the known candidate gene based genotypic information

 

 

Yes

Yes

Novel alleles of genes for the target traits.

NRCPB, NBPGR, DRR, IIT-K, IARI, CAU-B & RCNEHR

8.

Association mapping and identification of new genes/QTLs for the target traits.

Associating the phenotypic information on the mini-core collection with the whole genome genotypic information

 

 

Yes

Yes

Novel genes/QTLs for the chosen trait.

NRCPB, NBPGR & IASRI

9.

Validation of marker-trait associations in biparental mapping populations.

Use of the available mapping population segregating for abiotic stress tolerance for validation of linkages.

 

 

 

Yes

Validated markers for QTLs for abiotic stress tolerance

NRCPB, DRR, IGKVV, CRRI, IARI

10.

Marker associated Backcross transfer of useful genes/QTLs

Generation of backcross derived lines with new gene combinations.

 

Yes

Yes

New genotypes for use in Rice improvements/function analysis of traits.

DRR, IGKVV, CRRI, IARI

11.

 

Validation of candidate gene in transgenic of model systems.

Genetic transformation of Arabidopsis/Rice using identified candidates/transcription factors.

 

 

Yes

Yes

Functionally validated genes.

DUSC, VPKAS, IIT-K, CAU-B, RCNEHR

12,

Cloning and characterization of orthologs of new/candidate genes from bajra.

Pcr CLONING, Sequencing and sequence analysis of new genes for stress analysis from pearl millet.

 

 

Yes

Novel orthologs of rice from pearl millet, which is highly stress tolerance.

NRCPB, IIT-K

 

*Responsibility:

 

NRCPB, New Delhi: T. Mohapatra, N.K. Singh, K.C. Bansal and K. Gaikwad

NBPGR, New Delhi: K.V. Bhat, I.S. Bisht, R.K. Singh and Amit Singh

DRR, Hyderabad: V. Rai, N. Sarla, T. Ram, D. Subrahmanyam and L. V. Subbarao

CRRI, Cuttack: G.J.N. Rao, J.N. Reddy, R.K. Sarkar, P. Swain, P. Krishnan, H.N. Subudhi, S Marndi, and J. Meher

IARI, New Delhi: A.K. Singh, C. Viswanathan

IASRI, New Delhi: A.R. Rao, S.B. Lal and Sudeep

DUSC, Delhi: J.P Khurana and S. Kapur

IGKVV, Raipur: G. Chandel, S Verulkar

CAU, Barapani: W. Tyagi and M. Rai

Res Comp NEHR: A. Pattanayak, S Gupta, P. K. Ghosh

IIT, Kharagpur: S. K. Sen, A. Basu, M.K. Maiti and B. S. Das

VPKAS, Almora: P.K. Agrawal, K. Babu, J.P. Aditya and A. Kumar

 

Work allocation & sharing: Rice across traits

 

Name & Designation

Institute/ Organization

Work Allocation

T. Mohapatra, PS

NRCPB, New Delhi

Coordination of whole activities in rice,

selection of markers, Genotyping of core

and mini-core collections, expression

analysis, phenotyping for abiotic stress

tolerance, association analysis to discover

new genes and alleles

 

N. K. Singh, PS

NRCPB, New Delhi

Expression analysis, genotyping and phenotypic of the mini-core collection

K. Gaikwad, SS

NRCPB, New Delhi

Genotyping and phenotyping of the mini-core collection, allele mining using the candidate genes

V. Rai, SS

DRR, Hyderabad

Genotyping of the core and mini-core using SSR markers, expression analysis to identify candidate gene for drought tolerance and candidate gene based allele mining

 

 

N. Sarla, PS

DRR, Hyderabad

Genotyping of the core using SSR markers

T. Ram, PS

DRR, Hyderabad

Generation of core and mini-core, phenotyping of the mini-core

L. V. Subbaro, PS

DRR, Hyderabad

Generation of core and mini-core, phenotyping of the mini-core

D. Subrahmanyam, SS

DRR, Hyderabad

Physiological analysis of core and mini-core

G.J.N. Roa

CRRI, Cuttack

Generation of core and mini-core, SSR genotyping, QTL mapping, allele mining

J. N. Reddy

CRRI, Cuttack

Generation of core and mini-core, phenotyping for submergence tolerance, SSR genotyping, QTL mapping, AS

R. K. Sarkar

CRRI, Cuttack

Phenotyping for salinity and submergence tolerance, MAS

P. Swain

CRRI, Cuttack

Phenotyping for drought tolerance, MAS

P. Krishnan

CRRI, Cuttack

Phenotyping for high temperature tolerance, MAS

J. Mehar

CRRI, Cuttack

Phenotyping for high temperature tolerance, MAS

H. N. Subudi

CRRI, Cuttack

Generation of core and mini-core, phenotypic of the mini-core collection

S. Marandi

CRRI, Cuttack

Phenotyping for Salinity tolerance

A. K. Singh

IARI, New Delhi

Phenotyping and genotyping of the core and mini-core using SSR markers, candidate gene based allele mining for BLB resistance, MAS for BLB and BPH resistance.

 

C. Viswanathan

IARI, New Delhi

Phenotyping of the mini-core for drought tolerance

K.V. Bhatt

NBPGR, New Delhi

Generation of core and mini-core collection, and genotyping using SSR markers, allele mining using known/candidate genes

I.S. Bisht

NBPGR, New Delhi

Generation of of core and mini-core collection, and genotyping using SSR markers, allele mining using known/candidate genes

R.K. Singh

NBPGR, New Delhi

Generation of of core and mini-core collection, allele mining, and genotyping using SSR markers

A. Singh

NBPGR, New Delhi

Allele mining, and genotyping using SSR markers

J. P. Khurana

DU, South Campus

Expression analysis using microarrays and functional validation

S. Kapur

DU, South Campus

Expression analysis using microarrays and functional validation

G. Chandel

IGKVV, Raipur

Phenotyping for drought tolerance, SSr genotyping of core and mini-core, validation for association in biparental populations

S. Verulkar

IGKVV, Raipur

Phenotyping for drought tolerance, SSr genotyping of core and mini-core, validation for association in biparental populations

P.K. Agarwal

VPKAS, Almora

Phenotyping for drought and cold tolerance, validation for gene function in transgenics

K. Babu

VPKAS, Almora

Phenotyping for drought and cold tolerance, validation for gene function in transgenics

J. P. Aditya

VPKAS, Almora

Phenotyping for drought and cold tolerance

A. Kumar

VPKAS, Almora

Phenotyping for drought and cold tolerance

W. tyagi

CAU, Barapani

Phenotyping for cold and acidity tolerance, allele mining for cold tolerance, functional validation in trangenics

M. Rai

CAU, Barapani

Phenotyping for cold and acidity tolerance, allele mining for cold tolerance, functional validation in trangenics

A. Pattanayak

ICAR Res Compl for NEHR, BArapani

Phenotyping for cold and acidity tolerance, allele mining for cold tolerance, functional validation in trangenics

S. Gupta

ICAR Res Compl for NEHR, BArapani

Phenotyping for cold and acidity tolerance

P. K. Ghosh

ICAR Res Compl for NEHR, BArapani

Phenotyping for cold and acidity tolerance

S. K. Sen and associates

IIT, Kharagpur

Identification of candidate and their functional validation in transgenics

A. R. Rao and associates

IASRI, New Delhi

Statistical and computational genomics support

 

Moisture stress tolerance: Maize

Time schedule of activities and activity milestones

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibilitiy

 

Y1

Y2

Y#

 

 

14.

Collection of relevant germplasm for the target trait, including targeted exploration in the country

Assembling a core panel of atleast 300 genotypes for drought stress tolerance

Yes

 

 

Avaliability of relevant indigenous and exotic germplasm for bioprospecting and allele mining for drought stress tolerance

BMP & FH (in collaboration with other partners)

15.

Phenotyping of ~300 genotypes under drought stress at multiple locations (New Delhi; Uchani; Hawalbagh)

Intensive evaluation of atleast 100 genotypes each year under control and stress condition at three locations

 

Yes

Yes

Yes

Identification of sources of tolerance to drought stress suitable to different agro-ecologies

BMP; FH; RP; DP; PKA; AK

16.

Intensive molecular characterization of 96 selected genotypes using genomic and genic SSRs and SNP markers

SSR and SNP genotyping of selected genotypes (complete by Y2)

Yes

Yes

 

Profiles and database of genomic and genic SSRs and SNPs on phenotypically contrasting genotypes

 

BMP; FH; PKA; BKB

17.

Allele mining for 10 selected candidate genes associated with drought stress tolerance

Primer design, sequence analysis and/or SNP analysis of selected genotypes for 10 candidate genes

 

 

Yes

Yes

Analysis of candidate gene variation in selected contrasting genotypes for drought stress tolerance

BMP; FH

18.

SNP and haplotype pattern mining based on sequence analysis of candidate genes

Computataional analysis of sequence data for SNP and haplotyp pattern mining in candidate genes

 

 

 

Yes

Detection of a series of SNPs and haplotypes in candidate genes

BMP; FH

19.

Association genetic analysis for identification of marker-trait associations

Statistical analysis of genotypic and phenotypic datasets for identification of functionally relevant SNP/haplotype in candidate genes

 

 

 

Yes

Identification of favourable alleles )SSRs & SNPs_ and haplotypes for potential use in molecular brreding

BMP; FH

 

IARI, New Delhi: BMP: BM praanna; FH: Firoz Hossain

VPKAS, Almora PKA: PK Aggarwal; BKB: B Kalyan Babu; AK: Arun Kumar

CCSHAU, Uchani RP: Rishi Pal; DP: Dharam Pal

 

Excess water Stress Tolerance: Maize

Time schedule of activities and activity milestones

 

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibilitiy

 

Y1

Y2

Y3

 

 

1.

Collection of relevant germplasm for the target trait, including targeted exploration in the country

Assembling a core panel of atleast 300 genotypes for drought stress tolerance

Yes

 

 

Avaliability of relevant indigenous and exotic germplasm for bioprospecting and allele mining for drought stress tolerance

BMP & FH (in collaboration with other partners)

2.

Phenotyping of ~300 genotypes under excess water stress at four locatons (Pantnagar; Barapani; New Delhi; Uchani)

Intensive evaluation of atleast 100 genotypes each year under control and stress condition at three locations

 

Yes

Yes

Yes

Identification of sources of tolerance to excess water stress in North Eastern India

FH; MZKW; SSV; NKS; AP; SG; PKG; RP; DP

3.

Intensive molecular characterization of 96 selected genotypes using genomic and genic SSRs and SNP markers

SSR and SNP genotyping of selected genotypes (complete by Y2)

Yes

Yes

 

Profiles and database of genomic and genic SSRs and SNPs on phenotypically contrasting genotypes

 

BMP; FH; NKP

4.

Transcriptome profiling for excess water stress tolerance and identification of fiffrentially expressed genes

Microarray analysis for excess water stress using selected highly contrasting genotypes

 

Yes

Yes

Identification of significant differentially expressed genes under excess water stress

BMP; FH

5.

Allele mining for 6 selected candidate genes associated with excess water stress tolerance

Primer design, sequence analysis and/or SNP analysis of selected genotypes for 6 candidate genes

 

 

Yes

Yes

Analysis of candidate gene variation in selected contrasting genotypes for excess water stress tolerance

BMP; FH

6.

SNP and haplotype pattern mining based on sequence analysis of candidate genes

Computational analysis of sequence data for SNP and haplotype pattern mining in candidate genes

 

 

Yes

Detection of series of SNPs and haplotypes in candidate genes

BMP; FH

7.

Association genetic analysis for identification of marker-trait associations

Statistical analysis of genotypic and phenotypic datasets for identification of functionally relevant SNP/haplotype in candidate genes

 

 

 

Yes

Identification of favourable alleles )SSRs & SNPs_ and haplotypes for potential use in molecular brreding

BMP

 

IARI, New Delhi: BMP: BM Pranna; FH: Firoz Hossian

GBPUAT; Pantnagar: MZKW: MZK Warsi; SSV: SS Verma; NKS: NK Singh

ICAR-NEH, Barapani: AP: A Pattnayak; SG: S Gupta; PKG: PK Ghosh

CCSHAU, Ucani: RP: Rishi Pal; DP: Dharam Pal

 

Moistue Stress: Sorghum

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibilitiy

1.

Scrrning of available sorghum genetic stocks for drought/heat tolerance

Phenotypic trait such as stay green, Root mass, length and density, yield components, total biomass will be analysed under stress and controlled conditions

 

Yes

 

 

A core set of Sorghum defined for carryuing out allele mining

NRCS

2.

Allele mining in selected set from the core set of sorghum

A Pcr based method will be followed for allele mining using candidate genes primers

 

 

Yes

 

Cloned and sequenced alleles available

NRCS

3.

Search for polymorphism and molecular marker development

In silico analysis of sequences and identification of SNPs will be done. Molecular makers will be designed

 

Yes

 

Useful alleles identified and molecular markers developed

NRCS

4.

Genotyping in remaining core set

Using the SNP markers larger set will be genotyped

 

 

Yes

Core set genotyped and alleles identified

NRCS

5.

Validation of genotypes of sorghum for drought tolerance and susceptibility

Selected genotypes will be given stress treatment and will be characterized based on phenotypic markers

 

Yes

 

True drought tolerant and susceptibility genotypes identified

MPKV

6.

Construction of cDNA library (subtracted cDNA)

Isolation of total RNA from tolerant and susceptibe genotypes after stress and mRNA isolation and RT-PCR

 

Yes

 

cDNA library (subtracted cDNA) ready

MPKV

7.

Sequencing of cDNA library

`cDNAs will be sequenced by outsourcing

 

Yes

 

Sequences available

MPKV

8

Expression analysis of identified clones

Expression will be checked in Yeast.bacterial expression system

 

 

Yes

Validation of identified genes

MPKV

 

Work allocation & sharing: Sorghum

Name & Designation

Institute/ Organization

Work Allocation

Monica Dalal, SS

NRCS, Hyderabad

Phenotyping of available genetic stock for drought stress tolerance, allele mining. Molecular marker development genotyping and cloning and expression analysis of genes

 

R. MAdhusudhan, SS

NRCS, Hyderabad

Phenotyping of available genetic stock for drought stress tolerance, allele mining. Molecular marker development genotyping and cloning and expression analysis of genes

 

H. S. Talwar, SS

NRCS, Hyderabad

Phenotyping of available genetic stock for drought stress tolerance, allele mining. Molecular marker development genotyping and cloning and expression analysis of genes

 

Ashok Jadhav, Professor

MPKV, Rahuri

Validation of genotypes for drought tolerance and susceptibility cDNA (subtractive), screening and analysis of new genes

 

A.A. Kale

MPKV, Rahuri

Validation of genotypes for drought tolerance and susceptibility cDNA (subtractive), screening and analysis of new genes

 

V. P. Chimote

MPKV, Rahuri

Validation of genotypes for drought tolerance and susceptibility cDNA (subtractive), screening and analysis of new genes

 

 

Moisture Stress Tolerance: Cucumis sp.

 

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

1.

Analysis of ecogeographical data and identification of core collection in cucumber, muskmelon, C. callosus

Core collection of cucumber (250) and muskmelons(150)

 

X

 

 

Designation of cores in cucumber and melons

NBPGR

2.

Multi-location characterization of core collection for tolerance to drought.

Characterization of core collections

X

X

 

Diversity in core

NBPGR, IIHR, IIVR, RAU

 

3.

Identification characterization of SSR primers and construction of saturated linkage maps in cucumber. Muskmelons

Markers (1000) for genotyping

 

X

X

Polymorphic SSR markers

NBPGR, IIHR, RAU

4.

Phenotyping of the core collection under standard controlled condiation in four location

Characterization of core for target traits

 

X

X

Accession with specific traits

NBPGR, IIHR, IIVR, RAU

5.

Genotypic of the core collection using AFLP, SSR and SNPs for candidate genes

AFLP, SSr and SNP profiles of core accessions (~450 markers)

X

X

X

Molecular profiles of core accessions

NBPGR, IIHR, IIVR, RAU

6.

Association analysis to identify genes/markers for target traits

Identification of accessions and marker tags

 

X

X

Donor accessions for target traits

NBPGR, IIHR, RAU

7.

Fine mapping of selected genes for validation & use in markler assisted transfer of the trait

Tagging of target genes

 

X

X

Marker tags for MAS

NBPGR, IIHR, RAU

8.

Identification of putative genes through differential expression analysis for selected traits

To use differential display and subtractive hybridization for identifying putative genes for moisture stress tolerance

 

X

X

Information on candidate genes related to moisture tolerance

NBPGR, IIHR, IIVR, RAU

9.

Cross validation of results in Cucumis Arabidopsis among the patterns for specific traits identified

Identification nof genes and allele for tarfet traits

 

X

X

Allelic variation for deployment

NBPGR, IIHR, RAU

 

NBPGR: KVB- KV Bhat, MKR- MK Rana;

RAU, Bikaner: RAS- RAmavtar Sharma

IIVR, Varanasi: SP-Sudhakar Pandey, ML-M. Lokanath

IIHR: MP-M. Pitchaimuthu, ASK- Ashokan, VR- V. Ravindram, RS- Ravishanker

 

Moisture Stress Tolerance: Lathyrus sp.

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

1.

Analysis of ecogeographical data and identification of core collection in Lathyrus

Core collection of Lathyrus (300 accessions)

 

X

 

 

Designation of cores in Lathyrus

NBPGR

2.

Multi-location evaluation of core collection for tolerance to drought

Characterization of core collections

X

X

 

Diversity in core

NBPGR, IIT (KGP)

3.

Identification and characterization of SSR primers and construcation of saturated linkage in maps in Lathyrus

Markers for genotyping (500 SSRs)

 

X

X

Polymorphic SSR markers

NBPGR

4.

Phenotyping of the core collection under standard and controlled conditions in three locations

Characterization of core for target traits

 

X

X

Accessions with specific traits

NBPGR, IIT (KGP)

5.

Genotyping of core collection using AFLP, SSR and SNPs for candidate genes

AFLP, SSR and SNP haplotypes of core collection

X

X

X

Molecular profiles of core accessions

NBPGR

6.

Assocaition analysis to identify genes for target traits

Identification of accessions and marker tags

 

X

X

Donor accessions for target traits

NBPGR

7

Fine mapping of selected genes for use in marker assisted transfer of the traits

Tagging of target genes

 

X

X

Markers tags for mAS

NBPGR

8.

Cross validation of results in Arabidopsis system

Identification of genes and alleles for target traits

 

X

X

Allelic variations for deployment

NBPGR, IIT (KGP)

 

IIT, KGP: SKS- S. K. Sen, AB- Asitava Basu, MKM- MK Maity, BSD- BS Das

NBPGR: KVB- K. V. Bhat, MKR- MK Rana

 

Moisture Stress tolerance: Ziziphus sp.

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

1.

Collection, screeing and identification of reference collection in Ziziphus

Reference collection of Ziziphus

 

X

 

 

Designation of cores in Ziziphus

CIAH

2.

Phenotypic of the reference collection using lab. Based techniques

Characterization of reference for target traits

 

X

X

Accession with specific traits

CIAH

3.

Isolation and cloning of candidate genes through subtractive analysis

Putative genes for tolerance to drought

 

X

X

Information on candidate genes related to drought tolerance

CIAH, NRCPB

4.

Expression profiling and identification of putative genes for drought tolerance

Confirmation of genes identified

 

X

X

Identification of putative genes for drought tolerance

NRCPB

5.

Validation of genes in Arabidopsis

Confirmation of genes for tolerance

 

X

X

Novel genes for plant transformation

NRCPB

 

CIAH: PNS-PN Sivalingam, DS-Dhurinder Singh

NRCPB: JCP-JC Padaria, KNK-Kanika, PKA-PK Ananda Kumar

 

Heat Tolerance: Vigna sp.

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

1.

Analysis of ecogeographical data and identification of core collection in moth bean including wild species

Core collection of moth bean (225 accessions)

 

Yes

 

 

Designation of cores in moth bean

NBPGR

2.

Screening and classification of core collection for the tolerance

Characterization of core collection

Yes

Yes

 

Diversity in core

NBPGR, RAU

3.

Isolation and cloning of candidate genes for heat tolerance based on homology

Identification & cloning of genes for heat tolerance

 

Yes

 

A set of cloned genes involved in heat tolerance mechanism from moth bean

NBPGR, RAU

4.

Validation of isolated genes through transformation in moth bean/ Arabidopsis

Functional validation

 

Yes

Yes

A system to study genes further through expression analysis

RAU

5.

Identification of candidate genes through differential expression profiling

Information on heat stress induced genes

 

Yes

Yes

Information on heat stress induced genes

NBPGR, RAU

6.

Allele mining for the selected genes

Identification of elite germplasm lines

 

Yes

Yes

Novel alleles of genes for the target traits

NBPGR, RAU

 

RAU, Bikaner : RAS-Ramavtar Sharma

NBPGR : KVB_KV Bhat, MKR-MK Rana

 

Heat and Cold Stress tolerance: Camel and Goat

Milestones and outputs (including HRD activities)

S.No.

Activity

1

2

3

Expected Output

Responsibility

1.

To identify population structure and breed specific haplotypes-under extremes of temperature conditions.Identification of various in camels and goats for various candidate genes associated with thermoregulatory mechanisms in mammals in different geographical locations having extremes of temperature variations.

Yes

 

 

Allele associated with heat and cold stress in camels and goats

 

 

 

 

 

 

NBAGR

2.

Transcriptome analysis of goats and camels inhabiting the extreme climatic regions of the country

 

Yes

 

 

3.

Extimating of relative frequency of the SNPs between extreme temperature conditions

 

Yes

 

 

4.

Association of the SNP/haplotypes with the adaptive traits

 

Yes

Yes

 

5.

Analysis of expression of genes associated with thermoregulatory mechanism in camel and goat.

Approach to Research:

Comparative analysis of apparently normal goat and camel blood lymphocytes at normal conditions and under heat stress at various temperatures and duration of exposure

Yes

Yes

 

Quantification of mRNA profile for heat and cold stress

In camels and goats under hot arid and temperate regions.

CIRF, NDRI

Comparison of hot arid region goats with thermoneutral conditions.

IVRI, NDRI

Comparison of temperate region goats with thermoneutral conditions.

SKUAST

Comparison of temperate region goats and camel at thermoneutral conditions and stress

NRCC

Comparison of hot arid region camels under thermoneutral and stress conditions

6.

In vitro differential gene expression related to heat/cold temperatures

 

Yes

 

 

7.

Establishment of relationship between the identified differential heat/cold (Thermoregulatory) gene in different genetic groups.

 

Yes

Yes

 

 

Anoxia: Catfish

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

9.

Identification and characterization of genes for Anoxia resistance in Clarius batrachus and Channa striatus

       Collection of Clarius batrachus from different locations

       Construction of cDNA libraries by Suppression Subtractive Hybridization (SSH) technique

       Full length normalized cDNA library construction of different tissues

 

Yes

 

 

 

Yes

 

 

 

Yes

 

 

 

 

Yes

 

 

 

 

 

 

 

 

 

Yes

       Availability of base material with abiotic stress (anoxia)

NBFGR, CIFA

10.

Full length normalized cDNA library construction of different tissues.

The Normalized cDNAs

 

Illumina GA-sequencing

 

Gene annotation

 

Cloning

Yes

 

Yes

 

 

 

 

 

Yes

 

Yes

 

Yes

 

 

 

 

 

Yes

 

Yes

Genome resource for abiotic stress

 

Anoxia transcriptome characterization

NBFGR, CIFA

11.

Quantitaive Real-Time PCR for differentially expressed genes.

 

RACE

Designing of RT primers

 

Differential expression

 

Full length cDNA construction

 

 

Yes

 

Yes

 

Yes

Genome Resorce

Expression profile

Sequence information

NBFGR, CIFA

 

CIFA: Dr. H.K. Burman, Dr. K. Kumar, Dr. C. Debaraj

NBFGR: Dr. Vindhya Mohindra, Dr. Ravindra Kumar

 

Salt tolerance: Shrimp and Scrampy

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

9.

Identification and characterization of genes for salinity stress in giant freshwater prawn.

M. Rosenbergii/ M. malcolmsoni-CIFA, Bhubneswar and P. Mondon (CIBA)

       Collection of larval populations of M. Rosenbergii/ M. malcolmsoni and P. Mondon under varied salinity conditions

       Collection & preservation of different tissue samples from brackish water during season and from freshwater

       Construction of cDNA libraries by SSH techniques from wild and acclimatized samples.

       Construction of cDNA libraries by normalized cDNA from wild & acclimatized samples

       Conversion of normalized cDNAs to Illumina GA-sequencing ready libraries followed by Illumina GA(Solexa) sequencing

       Assembling sequences for cutom bioinformatics.

       Cloning normalized cDNA into pGEMT-easy vector.

       Sequencing of full length cDNA by RACE

       Estimation of transcript levels by Real-Time PCR

       Report writng

 

Yes

 

 

 

 

 

Yes

 

 

 

 

 

 

Yes

 

 

 

 

 

Yes

 

 

 

 

 

 

 

 

 

 

 

 

Yes

 

 

 

 

 

Yes

 

 

 

Yes

 

 

Yes

 

 

 

 

 

 

 

Yes

 

 

 

 

 

 

Yes

 

 

 

 

 

Yes

 

 

 

Yes

 

 

Yes

 

 

 

Yes

 

       Availability of base material with abiotic stress (anoxia)

NBFGR, CIFA

2.

Fuvtional characterization of novel genes identified in P. mondon-CIFE, Mumbai

Sequencing og 5 and 3 ends of the 3 genes over-expressed in P.mondon acclimatized to 35 ppt and 1 gene over-expressed in specimens acclimatized to 0 ppt. using RACE

 

Estimating transcripts levels at different salinities in P.mondon gill and muscle tissue using Real time PCR and valiadating relation with adaption to salinity stress

 

Cloning of cDNA sequences in a constitute His-tag expression vector and over-express in a eukaryotic cell lines

 

Purification of the His-tagged protein using Ni coloumn and raise antibodies in rabbit.

 

Sub-cellular localization of the novel proteins involved in salt homeostasis in the gill and muscle tissues of marine tiger shrimp using antibodies

 

Tracing the change in expression levels of these proteins in different salinities using immuno-histochemical techniques and hypothesizing their roles in salt homeostasis

 

Report writing

Yes

 

 

 

 

 

 

 

Yes

 

 

 

 

 

 

Yes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Yes

 

 

 

 

 

Yes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Yes

 

 

 

 

 

Yes

 

 

 

 

 

Yes

Complete cDNA sequences pf 3 P. mondon genes over-expressed during saline stress and 1 over-expressed in freshwater

 

Validation of the importance of these sequences for saline adaptation

 

 

 

 

 

 

Purified proteins that are important for saline adaptation

 

Sub-cellular location of the above proteins

 

 

Validation of the role of these proteins in saline adaptation

 

Final report

CIFE, Mumbai

 

CIFA: Dr. H. K. Barman, Dr. P. Jayashanker

CIBA: Dr. M.S. Shekhar, Dr. G. Gopalkrishna, Dr. C. Gopal, Dr. K. Vinaya Kumar, Dr. S. Kannappan

CIFE: Dr. Aparna Chaudhari, Dr. Gayatri Tripathi, Mr. Gireesh Babu P.

 

Cold Tolerance: Trout

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

1.

Collection of fish samples from different high altitude colder environments for the study of cold tolerant. The sites proposed to be surveyed with regards to extreme cold conditions in J&K, Himachal Pradesh and Uttarakhand upland regions,

Survey and collection of fish samples from extreme environments,

 

Yes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Samples collected from different sites.

DCFR

2.

Isolation of DNA/RNA samples collected following standard techniques and optimized protocols.

Isolation of DNA/RNA

Yes

 

 

 

Development of standardized protocols.

DCFR

3.

Molecular identification and sequence variation between the exotic and indigenous trout sp. Of collected samples for cold tolerant genes

Identification of known genes through public domain.

Yes

 

 

 

Allele mining

DCFR

4.

Specific primers for known genes will be used in PCR to identify and isolate candidate genes from fishes

PCR amplification of relevant genes using specific primers

 

Yes

 

Allele variants from indigenous fish species will be identified

DCFR

5.

Construction of genomic library in suitable vector and screening of clones for identification of novel genes.

Genomic library construction

 

Yes

 

Identification of novel genes for cold tolerance

DCFR

6.

Cloning and sequencing of genes in vectors

Genomic library construction

 

 

Yes

Development of cDNA library

DCFR

7.

Bioinformatic analysis of the identified genes

Submission in GenBAnk

 

 

Yes

Bioinformation available of cold tolerant genes in indigenous fish species

DCFR

 

DCFR: Dr. A. Barat, Senior Scientist and Dr. G.K. Sivaraman, Scientist

 

Statistical and Computational Genomics

Time Schedule of activity and activity milestones

S.No.

Objective/Activity

Activity

Milestones

Milestones and when to be attained

Expected Output

Responsibility

 

1

2

3

 

1.

Statistical and Computational analysis of genomic data generated from plants, animals and fish genetic resources

Identification, procurement and installation of statistical and computational software;

Statistical analysis of generated molecular and phenotypic data;

Undergoing national and international trainings

 

 

Yes

 

 

 

 

Yes

Yes

Establishment of centralized facility for statistical and computational analysis or genomic data

Establishing association between genomic and marker data

ARR, VKB, SBL,SD

2.

Development of algorithms for supporting need based genomic analysis

New user friendly analytical tools for gene discovery

 

Yes

Yes

Efficient gene prospecting in genetic resources

ARR, VKB, SBL, SD

3.

Training scientific personnel in the field of statistical genomics and Bioinformatics

Providing training to 2 batches of 20 participants each year.

Yes

Yes

Yes

Upgrading skills and building expertise among the scientists working in genomics and Bioinformatics

ARR, VKB, SBL, SD