• To study differentially expressed genes under different salinity regimes.
• To validate key differentially expressed genes

Treatments

Factor A : Salinity levels

T1: Normal water EC < 2.0 dS/m

T2: Sea water with EC:  40  dS/m

Factor B : Stage of sampling

T1: Before imposition of saline water

T2: After 12 h of imposition of saline water

T3: After 5 days of imposition of saline water

Location & agro climatic sub region

:

Gujarat (Coastal belt of South Gujarat)

Name of Research scheme and B.H.

BH: 12099

Year and Season

:

2024

Organisms

:

Oryza coarctata (Roxb.)

Statistical Design

-

Experimental detail

:

• Sampling site: Oryza coarctata (Roxb.) (From Purna estuary and surroundings).
• Molecular work :
  • • Transcriptome profiling will be carried out from RNA samples under various salinity regimes.

Treatments

Factor A : Salinity levels

T1: Normal water EC < 2.0 dS/m

T2: Sea water with EC:  40  dS/m

Factor B : Stage of sampling

T1: Before imposition of saline water

T2: After 12 h of imposition of saline water

T3: After 5 days of imposition of saline water

Methodology

1. Plant material

• Oryza coarctata (Roxb) Tateoka plants will be collected from coastal region of south Gujarat, India.
• The plants will be brought to the laboratory along with soil and water. The plants will be washed carefully to remove soil from the leaves and roots. Then same will be subjected to different treatments. The plant samples in three biological replicates after 12 h of the treatment will be collected for RNA extraction.
• Growing media: the selected plants will be grown on soil less culture media (Perlite, Soilrite, etc. (as per availability)
• After establishment of the seedlings, the plants will be supplied with saline water as per treatment throughout growing period.

RNA isolation and sequencing

1. Total RNA will be isolated from each tissue sample (control, salt treated) using TRI reagent (Sigma Life Science, USA). The quantity and quality of RNA samples will be checked using Nanodrop (Thermo Fisher Scientific) and Agilent Bioanalyzer .
2. Transcriptome assembly

De novo transcriptome assembly will be performed using various tools, including Velvet (v1.2.01), Oases (v0.2.04), ABySS (v1.2.6), Trinity (vr2012-05-18) and CLC Genomics Workbench (v4.7.2),GSEA, Squish meta tool (as per availability).

1. Functional annotation

BLASTX searches against rice proteome, UniRef90, UniRef100 and NCBI non-redundant nucleotide databases, will be  performed to assign a putative function to each Coarctata transcript.

1. Read mapping and gene expression analysis

To estimate the gene expression, all the high-quality reads from each condition will be aligned on the transcriptome assembly using the RNA-Seq Analysis utility of CLC Genomics Workbench (as per availability).

1. Gene ontology and pathway enrichment analysis

The best Arabidopsis hit corresponding to each coarctata transcript will be identified using BLAST search to study gene ontology (GO) enrichment. GO enrichment of different sets of differentially expressed genes will be performed using the BiNGO tool

1. Real-time PCR analysis

Real-time PCR analysis will be used  for validation of the identified genes.

Observation to be recorded

:

• Mineral composition of leaves and Roots
• Na:K ratio  (Shoot)
• Na:K ratio  (Root)
• Biomass (g/pot)
• Plant height (cm)
• Chlorophyll content (SPAD value)
• Gene expression fold change will be recorded.

Expected Outcome

:

The information related to potential genes related to salt tolerance will be generated from the present investigation. The understanding of extreme salt tolerance trait as wel as related gene network would be useful to check the possibilities of introgression/transfer of key salt tolerance genes in cultivated rice in order to develop a salt tolerance variety for farming community of the Gujarat.