SHIVAJI UNIVERSITY, KOLHAPUR - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4336/18/18_synopsis.pdf · The advances in physiology, genetics, and molecular biology ... Sugarcane

Synopsis Ph.D. in Botany, Miss. M. C. Pagariya, VSI, Pune

1

“CANDIDATE GENES AS MOLECULAR MARKERS FOR EVALUATING

AND VALIDATING SUGARCANE GERMPLASM FOR SALINITY STRESS”

SYNOPSIS OF THE PROPOSED WORK

SUBMITTED TO

SHIVAJI UNIVERSITY, KOLHAPUR.

FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

BOTANY

UNDER THE FACULTY OF SCIENCE

BY

MISS. MADHURI CHANDRAKANT PAGARIYA

M.Sc.

UNDER THE GUIDANCE

OF

DR. R. M. DEVARUMATH M.Sc., Ph.D.

MOLECULAR BIOLOGY & GENETIC ENGINEERING DIVISION

VASANTDADA SUGAR INSTITUTE,

MANJARI (BK.), PUNE 412307.

2008


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Ph.D. Synopsis in the faculty of Science

Subject: Botany

1. Research Student : Miss. Madhuri Chandrakant Pagariya

Qualification : M. Sc.

Address : Molecular Biology & Genetic Engineering Div.

Vasantdada Sugar Institute,

Manjari (Bk.), Tal. Haveli, Dist.

Pune- 412 307

Maharashtra, India.

Email – [email protected]

Ph : 020-26902234.

2. Research Guide : Dr. R. M. Devarumath

Qualification : M. Sc., Ph.D.

Address : Molecular Biology & Genetic Engineering Div.


Manjari (Bk.), Tal. Haveli, Dist.

Pune- 412 307

Maharashtra, India.

Email – [email protected]

Ph : 020-26902235.

3. Title of Proposed Thesis: “CANDIDATE GENES AS MOLECULAR

MARKERS FOR EVALUATING AND

VALIDATING SUGARCANE GERMPLASM

FOR SALINITY STRESS”


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4. Introduction

Stress plays a very important role in plant development. Among the biotic and abiotic

stresses, abiotic stress is identified as the most common cause in limiting the growth

of crops and productivity. (Araus et al., 2002). Abiotic stress is one of the major

limitations to food production, reducing average crop yield by more than 50%

(Rodriguez et al., 2006). The advances in physiology, genetics, and molecular biology

have greatly helped in understanding plant responses to various stresses and

manipulate, compared to biotic stresses (Agarwal et al., 2006).

Many genes and transcription factors have been identified to be functionally

associated with abiotic stress and functionally analyzed like DREBs (Dehydration

Responsive Element-Binding proteins), CBFs (C-repeat-binding factor), NACs

(NAM, ATAF, and CUC) are found in plants like rice, Sorghum, wheat (Bhatnagar et

al., 2007; Gao et al., 2007).

Sugarcane (Saccharum spp.) is an important C4 plant. Saccharum along with

Erianthus and Miscanthus constitutes highly complex inter breeding group called as

‘Saccharum complex’. Sugarcane is a perennial crop and grown in tropical and sub

tropical parts of the world, which makes it one of the most productive cultivated

plants (Cordeiro et al., 2006). It is mostly used to produce sugar accounting for almost

two thirds of world production and recently for bio-fuel production.

Sugarcane is a glycophyte confined to tropical and subtropical irrigated regions,

where salinity is an ever-increasing problem (Gandonou et. al., 2008), which

adversely affects the productivity of sugar and cane yield (D’Hont and Glazmann,

2001). The salinity is due to the excess of Na+, Ca

++, Mg

++, SO4

– and Cl

– ions, the

degree of toxicity in decreasing order in sugarcane is SO4–>Na

+>Cl

–>Mg

++. Salinity

causes inhibition of growth, chlorophyll synthesis, PEPCase activity, decreased the uptake of

K+ and Ca

++ ions but stimulated nitrate reductase (Joshi and Naik, 1980).

Molecular markers are one of the important tools having a large number of

applications like localization of a gene for the improvement of plant varieties by

marker assisted selection (MAS) (Ribaut et al., 2006). Abiotic stress associated


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candidate genes have the potential to be used, as molecular markers to study the

association of the particular traits, as their biological function are well understood.

Genetic modification combined with marker-assisted breeding program can

substantially increase plant stress tolerance.

With the advent of suppression subtractive hybridization (SSH) technique, which

provided biologists a powerful tool to develop subtractive cDNA libraries enriched

for rare transcripts (Diatchenko et al., 1996). Towards the goal of identifying and

isolating the ESTs for developing the candidate gene markers for salinity stress, we

aim at using SSH to capture and enrich the rare transcripts expressed in sugarcane as a

consequence of salinity stress.

5. Research problem

In the present investigation an attempt will be made to study differential gene

expression under salinity stress condition in sugarcane to develop candidate gene

markers for using in molecular breeding program.

6. Significance of research work.

The complexity of plants especially sugarcane in response to salinity stress suggests

that a large number of genes are involved in stress response pathways. Identifying

critical genes that contribute to the salinity stress tolerance of sugarcane, ESTs that

have high levels of expression at different time points, involved in salinity stress

pathway based on database analysis.

Identification of the candidate genes and developing a model for molecular marker-

assisted selection to identify the promising sugarcane (Saccharum spp.) cultivars

possessing tolerance to abiotic stress at a very early stage of the breeding programme

and thus help the plant breeders in early selection of desirable genotypes cutting short

the time in variety development.


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7. Brief review of literature

a. Introduction

Sugarcane (Saccharum officinarum L.) is an important glycophyte of tropical and

subtropical regions and is cultivated on 20 million hectares in more than 90 countries

(FAO; http://apps.fao.org). Salt induced stress deters sugarcane and sugar productivity

in many parts of the world and several studies have shown that salinity affects both

germination and plant growth (Chowdhury et al., 2001). The mechanism through

which sugarcane perceives and responds to different stresses in the environment is not

well studied. Identification of salt responsive genes and their role in improved

tolerance of other related stress is very important in sugarcane improvement program.

b. Genomics studies

Functional genomics approach plays an important role in elucidating the mechanism

of abiotic stress tolerance in plants. A variety of genomic tools have been used to

study the molecular mechanism of abiotic stress tolerance in plants like maize, rice

and wheat (Bruce et al., 2002, Fu et al., 2007).

The molecular signals and pathways that govern biotic and abiotic stress responses in

sugarcane are poorly understood. Of which few are ethylene responsive factor (ERF),

abscisic acid-responsive genes accumulation of salt ions and osmolytes which plays

an important role in osmotic adjustment in sugarcane cells under salt stress have been

studied. (Linacero et.al., 2001 ,Trujillo et.al., 2008, Patade et.al., 2008) High level

expression of MIPS, WRKY like transcription factor, 22 kD drought induced protein,

Absicic acid (ABA) inducible gene and HVA22 have been studied by semi

quantitative RT-PCR in sugarcane (Prabu et al., 2008) Molecular breeding and

genetic engineering contributed substantially to our understanding of the complexity

of stress responses. Transcripts encoding for salinity tolerance in rice as well as

mangrove plant, Bruguiera cylindrica roots have been characterized using microarray

and suppression subtractive hybridization (SSH) followed by functional screening of

the unique genes as candidate genes for abiotic stresses. (Kawasaki et al., 2001; Wong

et al., 2007).


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Normal physiology and entire metabolic balance is disturbed due to salinity stress

(Nimbalkar, 1973). Abiotic stress mediated stress tolerance in plants is a complex

quantitative trait regulated by large number of up- and down- regulated genes

(Rodriguez et al., 2006, Simon-Sarkadi et al., 2007).

SSH technology have been used to gain preliminary insights into gene expression

induced by aluminium (Watt 2003), drought stress (Prabu et.al., 2008), cDNA

microarrays and SUCEST datamining have been used to study responses to oxidative

(Kurama et al., 2002,cold (Nogueira et al.,2003, 2005), and drought (Rocha et al.,

2007) in sugarcane.

Multiple signaling pathways regulate the stress responses in plants (Knight and

Knight, 2001) and expression of genes that are induced in response to different stress

(Seki et al.,2001; Chen et al.,2002). The genes coding for regulatory proteins viz.

transcription factors Basic-Leucine Zipper (bZIP), MYB, Dehydration Responsive

Element Binding protein (DREB), protein kinases, Mutagen Activated Protein

Kinases (MAPK), Calmoduline Dependent Protein Kinases (CDPK), Receptor Protein

Kinases, ribosomal proteins, transcription regulator protein kinases and genes

encoding for the proteins that scavenge free radicals (Superoxide Dismutase,

Catalase.) have been extensively studied (Yordanov et al.,2003). Molecular markers

are important tools to identify elite cultivars through adoption of appropriate marker

assisted selection for different traits (Abdel-Tawab et al., 2003; Mutengwa et al.,

2005).

c. Molecular maker studies

Marker assisted breeding methods are becoming increasingly useful in the

development of newer germplasm with improved stress tolerance (Hemamalini et al.,

2000). The primers designed from candidate genes associated with different traits

have been used for mapping disease resistance in wheat (Faris et al., 1999) drought

tolerance in Pinus taeda L.(Santiago et al., 2006) and rice (Vinod et al., 2006).

In order to develop a robust molecular markers system for screening the germplasm

for salinity stress and to understand tolerance mechanism in sugarcane, we propose to


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use the candidate gene approach along with development of cDNA library using SSH

technique. (Pflieger et.al.,2001). The specific primers for the candidate genes and the

ESTs up-regulated in the SSH library during salinity tolerance will be used for

delineating and validating the stress responses in selected tolerant and susceptible

sugarcane varieties/genotypes.

8. Hypothesis

Due to salt stress large no of genes expressed in sugarcane.

9. Objectives:

1. Physiological and biochemical studies of sugarcane at germination and

tillering stage submitted to salt stress and its recovery stage.

2. Construction cDNA-RAPD and SSH libraries for identification of ESTs

associated with salinity tolerance in sugarcane.

3. Identification and validation of salt associated candidate genes using

selected susceptible and tolerant sugarcane genotypes.

10. Methodology / Laboratory (Experimental/ Theoretical) Work

A. Plant Materials:

Moderately salinity tolerant (Co 62175) and susceptible sugarcane varieties (CoC

671, Co 86032 and CoVSI 9805 ) and its wild relatives species (Narenga sp.,

Schelerosthya sp., Erianthus sp.)

B. Physio-Biochemical tests

Following physio-biochemicals tests will be carried out with normal and stress

induced plants to select the sample for SSH. For quantitative estimation of

biochemical constituents standard laboratory manual on Experimental Plant

Physiology and Biochemistry will be referred.


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C. Identification of Candidate gene markers.

I. cDNA-RAPD and SSH Approach:

a. Isolation of total RNA: Extraction of total RNA from salt induced and

control sugarcane plants using the RNeasy Plant Mini Kit following

manufacturer’s instructions.

b. cDNA-RAPD: PCR amplification of quantified second-strand cDNA

(10 ng µl-1

) from both stressed and control plant samples will be

achieved using 10-mer RAPD primers (OPA, AB, G and K, Operon

Technologies, Inc., Alameda, CA) amplifications will be performed in

BIO-RAD icycler programmed.

c. Suppression subtractive hybridization: Suppression subtractive

hybridization will be carried out according to the PCR-select cDNA

subtraction Kit (BD Bioscience, USA).

d. Cloning, Sequencing, data analysis and ESTs identification.

II Bioinformatics Approach:

i. Data mining for candidate gene sequences from different crops like

Sorghum, maize, rice and other grass species.

ii. Sequence analysis and primer designing software’s and using

different bioinformatics tools like ClastalW, MEGA, BioEditR,

BLASTX, BLASTN, and Primer3.

11. Outline of the proposed research work

The work will be carried out in the following phase wise program

Phase I-

Reference work will be carried out

Phase II-

A pot experiment will be executed for salt stress induction.

Morphological, physiological and biochemical studies and

analysis of stress induced and normal plants.

Phase III-

cDNA-RAPD profiling and Suppression Subtractive

Hybridization will be carried out and differentially expressed


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genes library will be construceted, cloned and sequenced.

Sequence data will be analyzed by using different softwares.

Phase IV-

Based on data analysis the candidate gene primers will be

designed and validated on available germplasm. Data will be

compiled with significant findings and writing of Ph.D. thesis

will be undertaken.

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Signature of the student

(Miss. Madhuri Chandrakant Pagariya)

Signature of the Research Guide

(Dr. R. M. Devarumath)

Scientist, MB & GE,


Manjari(Bk.), Pune- 412307

Head of Department/ Institute

.

(Signature and Seal)

Documents

SHIVAJI UNIVERSITY, KOLHAPUR - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4336/18/18_synopsis.pdf · The advances in physiology, genetics, and molecular biology ... Sugarcane