This study was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, Thiruvananthapuram during 2015-2017. The objective of the study was isolation and sequencing of genes related to shattering viz. sh4 and qsh1 in weedy rice biotypes and characterization by expression profiling and phylogenetic analysis. The tBLASTx programme showed that the amplicon of size ~690bp was similar to shattering 4 (sh4) genes of Oryza populations, whereas the amplicon of size ~750bp belonged to qsh1 gene for putative transcription factor qsh1 of rice populations and mRNA of Oryza sativa japonica group.
The result clearly revealed that the gene responsible for shattering viz., sh4 and qsh1 are present in both weedy rice and cultivated variety Uma as evidenced from the PCR amplification of genomic DNA. However, the genes responsible for shattering investigated in the present study viz., sh4 and qsh1 were not expressed in any of the growth stages in cultivated rice variety Uma. The NCBI conserved Domain Search programme, showed that the qsh1 FW2 x RW2 sequence of amplicon at ~750bp (obtained by reaction using weedy rice flag leaf cDNA) belonged to homeobox kn domain. The sequence of the amplicons of qsh1 obtained was the partial fragments of the first ever qsh1 gene for putative transcription factor qsh1 to be isolated from Oryza sativa f. spontanea.
Phylogenetic tree constructed for sh4 FW2 x RW2 sequence, had highest similarity to shattering (sh4) gene of Oryza meridionalis voucher and also had relationship with Oryza rufipogon and Oryza sativa. However, the weedy rice qsh1 FW2 x RW2 query sequence was closely related to qsh1 gene of Oryza rufipogon, together with Oryza sativa indica group and Oryza sativa japonica group. Semi quantitative analysis with sh4 and qsh1 gene primers showed that the sh4 gene was expressed in flag leaf and grains and qsh1 gene was expressed only in the flag leaf. From this study it can be inferred that the gens responsible for shattering in weedy rice biotypes of Kerala viz. sh4 and qsh1 are present both in cultivated rice and weedy rice. However, the genes are expressed only in weedy rice during the flowering stage.
Table of Contents
1 INTRODUCTION
2 REVIEW OF LITERATURE
2.1 Weedy Rice
2.2 Origin of weedy rice
2.3 Weedy rice accessions and cultivated rice
2.4 Seed Shattering in weedy rice
2.5 Genes controlling shattering in weedy rice
2.6 Molecular characterization of seed shattering gene
3 MATERIALS AND METHODS
3.1 PLANT SAMPLE COLLECTION
3.2 IDENTIFICATION OF GENES RELATED TO SHATTERING
3.2.1 Degenerate Primer Designing
3.2.1.1 Primer Analysis
3.2.2 Genomic DNA Isolation
3.2.2.1 SDS method of Genomic DNA Isolation
3.2.2.2 CTAB method of Genomic DNA Isolation from weedy rice seed
3.2.2.3 Qualitative analysis of DNA samples
3.2.2.4 Quantification of DNA
3.2.3 PCR Amplification of Genomic DNA with Degenerate Primers
3.2.4 RNA Isolation
3.2.4.1 CTAB Method for RNA isolation
3.2.4.2 TRIZOL Method for RNA isolation
3.2.5 Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR)
3.2.5.1 Synthesis of cDNA Using Thermo Scientific First Strand cDNA Synthesis Kit
3.2.5.2 PCR Amplification of cDNA with Degenerate Primers
3.2.6 Sequencing of the Amplicons Produced after PCR
3.2.6.1 Gel Elution and Purification Using HipuraTM PCR Product and Gel purification Combo Kit (Himedia)
3.2.6.2 Cloning of PCR products for Sequencing
3.2.6.2.1 Competent Cell Preparation
3.2.6.2.2 Ligation of PCR products
3.2.6.2.3 Transformation of Ligated PCR products into Competent cells
3.2.6.2.4 Plasmid isolation from transformed colonies
4 RESULTS
4.1 IDENTIFICATION OF SHATTERING GENE
4.1.1 Designing of Degenerate Primers
4.1.1.1 Primer Analysis
4.1.2 DNA Isolation
4.1.3 PCR Analysis of Genomic DNA with Degenerate Primers
4.1.5 RNA Isolation
4.1.6 RT-PCR with Degenerate Primers
4.1.7 Sequence Analysis of the Amplicons
4.2 EXPRESSION STUDIES OF THE IDENTIFIED GENE
4.2.1 Semi Quantitative Analysis
5 DISCUSSION
5.1 ISOLATION OF GENES RELATED TO SHATTERING
5.1.1 DNA Extraction and PCR Analysis
5.1.2 RNA Isolation and RT-PCR Analysis
5.1.3 Sequencing and Sequence Analysis
5.2 EXPRESSION STUDIES OF THE IDENTIFIED GENE IN WEEDY RICE
5.2.1 Semi Quantitative Analysis
6 SUMMARY
Research Objectives and Core Topics
The primary objective of this research is to perform a molecular characterization of shattering in weedy rice (Oryza sativa f. spontanea) biotypes found in Kerala. The study focuses on isolating and sequencing specific genes associated with grain shattering and analyzing their expression profiles to better understand the genetic basis of this trait in weedy rice populations.
- Molecular identification and sequencing of sh4 and qsh1 genes in weedy rice.
- Comparative analysis of gene expression in weedy rice and cultivated rice variety 'Uma'.
- Use of phylogenetic analysis to determine the evolutionary relationships of the identified genes.
- Application of RT-PCR techniques to evaluate shattering-related gene expression at different growth stages.
Excerpt from the Book
1. INTRODUCTION
Rice is one of the major staple food crops in the world and is particularly significant in Asia where approximately 90 per cent of the world’s rice is produced and consumed (Ziegler and Barclay, 2008). Improving the productivity of rice has achieved immense importance to feed nearly half of the world’s population (Ratnasekera, 2015). One of the major constraints that obstruct these objectives is the inefficiency in management of problematic weeds such as ‘weedy rice’ in rice fields.
Weedy rice (Oryza sativa f. spontanea) evolved mostly by natural hybridization between wild and cultivated rice is an emerging threat to rice cultivation (Rathore et al., 2013). It is a self-pollinated annual plant that is conspecific to cultivated rice (Xia et al., 2011) and has became a menace infecting rice fields globally and most severely in Asia. In India an infestation level of 50-60 per cent has been reported from other states and yield reduction of 30-60 per cent has been documented in the rice fields of Kerala (Rathore et al., 2016). It is not quite easy to differentiate weedy rice from cultivated ones during vegetative stages and by the time panicle emerges the damage is already done. With diverse biotypes, weedy rice has already infested large rice growing areas across the major rice tracts of Kerala and yield reduction to the tune of 30 to 60 per cent depending on severity of infestation like 3 to 10 mature plants of weedy rice per m2 has been reported by Abraham et al. (2012).
Summary of Chapters
1 INTRODUCTION: Outlines the significance of rice as a global staple and introduces weedy rice as a major threat to agricultural productivity, establishing the need for molecular study.
2 REVIEW OF LITERATURE: Provides an overview of current knowledge regarding weedy rice origin, morphological characteristics, and the genetic basis of seed shattering, including the roles of specific genes like sh4 and qsh1.
3 MATERIALS AND METHODS: Details the experimental procedures including sample collection, DNA/RNA extraction protocols, primer design, PCR/RT-PCR amplification, and sequencing methods.
4 RESULTS: Presents the findings of gene identification through sequence analysis, including BLASTx results and phylogenetic trees, and discusses the expression patterns observed via semi-quantitative PCR.
5 DISCUSSION: Interprets the experimental results in the context of rice domestication and weed management, comparing the identified gene sequences with existing literature and discussing the implications for controlling seed shattering.
6 SUMMARY: Offers a concise recap of the research objectives, the methodologies employed, and the key conclusions drawn from the study of shattering genes in weedy rice.
Keywords
Weedy rice, Oryza sativa f. spontanea, seed shattering, sh4, qsh1, molecular characterization, gene expression, RT-PCR, phylogenetic analysis, rice domestication, Kerala, genomic DNA, transcriptional regulation, crop improvement, agro-ecosystems
Frequently Asked Questions
What is the core focus of this research?
The research is dedicated to the molecular characterization of seed shattering in weedy rice biotypes found in the Kerala region, specifically targeting genes that influence this trait.
Which genes are identified as central to seed shattering?
The study specifically investigates the sh4 and qsh1 genes, which are known to be key quantitative trait loci involved in the seed shattering mechanism in rice.
What is the ultimate goal of this study?
The goal is to isolate and sequence these genes in local weedy rice populations and analyze their expression profiles to contribute to the development of better weed management strategies and crop improvement.
What scientific methods were employed to achieve these results?
The study utilized a combination of molecular techniques, including DNA and RNA extraction, degenerate primer design, PCR and RT-PCR amplification, gene sequencing, and phylogenetic tree construction.
What does the main body of the work cover?
The main body systematically covers the identification of shattering genes via bioinformatics tools, the analysis of their expression across different plant tissues using semi-quantitative methods, and a discussion on their role in rice evolution.
Which keywords define this work?
Key terms include weedy rice, shattering genes (sh4, qsh1), molecular characterization, gene expression, and rice domestication.
How do the researchers differentiate between weedy rice and the cultivated variety 'Uma'?
The study uses comparative molecular profiling, including genomic DNA and cDNA amplification, to observe variations in gene presence and expression between the weedy rice biotypes and the 'Uma' cultivar.
What significance do the results hold for agricultural management?
The findings help elucidate the genetic basis of weedy rice success in the field, providing a scientific foundation for potential future applications like gene silencing to mitigate the impact of weedy rice.
- Citar trabajo
- Shelvy Sree (Autor), 2017, Molecular characterization of shattering in weedy rice biotypes of Kerala, Múnich, GRIN Verlag, https://www.grin.com/document/470265
