Citrus, one of the major genes of Rutaceae family and most economically important fruit tree and widely cultivated throughout the country. The Citrus have high nutritional value and medicinal value. The three varieties obtained from various districts in Kerala were used in this study. The phylogenetics is the study of the evolutionary relationships among different species which have common ancestor. These relationships are shown in the form of phylogenetic trees composed of branches which indicate the descendents and nodes which represent the most recent common ancestors.
In order to assess the phylogenetic relationships among the 6 samples belongs to the 3 Citrus species (Citrus aurantifolia, Citrus maxima, Citrus limon) a chloroplast gene rbcL and matK was successfully amplified and sequenced. For this study DNA was extracted by using CTAB method. This extracted DNA was analysed by spectrophotometry method for checking purity of DNA.The samples were gel electrophoresed by 1% agarose gel electrophoresis at 80 volts. After electrophoresis the gel is examined in gel documentation system. The DNA band was observed under UV light looking florescent Orangish red colour. The extracted DNA was amplified by PCR method and PCR sample was applied for electrophoresis for checking DNA bands. PCR sample were purified sent for sequencing. Sequences obtained were subjected to editing and alignment using ClustalW programme of Bio Edit and phylogenetic analysis was performed using MEGA software.
High similarity observed between selected verities. The locally collected gene sequence-based phylogeny presented here provides support for the early studies of speciation within the Rutaceae. An understanding of the main phylogenetic relationships between Citrus species will help to fine-tune the taxonomy of Rutaceae.
Table of Contents
Studies on genetic relationships among locally cultivated Citrus varieties in Kerala employing matK and rbcL gene using PCR technique and RFLP markers
Abstract
1. Introduction
1.1 Objectives
1.2 Scope of the study
1.3 Taxonomical classification
2. Review of literature
2.1 Uses and importance
2.2 Molecular markers
3. Hypothesis
4. Materials and Methods
4.1 Study area
4.2 Sample collection
4.3 Description of the species
4.3.1 Citrus aurantifolia/lime:
4.3.2 Citrus maxima/pomelo:
4.3.3 Citrus limon/lemon:
4.3.4 Species identification and morphological characterization
4.4 Isolation of DNA
4.5 Quantification of DNA
4.6 PCR primers
4.7 PCR amplification
4.8 Data sequencing
4.9 Sequence alignment
4.10 Phylogenetic and molecular evolutionary analysis
4.11 Statistical analysis
5. Results and discussion
5.1 Blast search for rbcL gene of Citrus species
5.2 Blast search for matK gene of Citrus species
5.3 Sequence alignment
5.4 Construction of phylogentic tree
5.3 Phylogenetic tree using matk gene
5.4 Calculation of the genetic distance
6. Conclusions
Research Objectives and Focus Areas
The primary objective of this study is to determine the phylogenetic relationships among locally cultivated Citrus varieties in Kerala (Citrus aurantifolia, Citrus maxima, and Citrus limon) using PCR-based amplification and sequencing of the matK and rbcL genes to better understand their evolutionary lineages and taxonomical placement.
- Application of DNA-based genetic markers to evaluate morphological diversity in Citrus.
- Use of chloroplast genes (matK and rbcL) for accurate phylogenetic reconstruction.
- Characterization of genetic distances between specific Citrus species through BLAST analysis and tree construction.
- Integration of molecular systematic data to support taxonomical classification within the Rutaceae family.
Excerpt from the Book
1. Introduction
The genus Citrus, which includes mandarin, orange, lemon, grapefruit and lime, has high economic and nutritional value. This genus belongs to the subfamily Aurantioideae, which is one of the 7 subfamilies of the family Rutaceae. Therefore, phylogenetic study of both the genus Citrus and of the subfamily Aurantioideae is important (Penjor et al., 2013). The Aurantioideae consists of 2 tribes with 33 genera (Swingle et al., 1967). These 2 tribes are each composed of 3 subtribes: the tribe Clauseneae, which includes Micromelinae, Clauseninae, and Merrillinae; and the tribe Citreae, which includes Triphasiinae, Citrinae, and Balsamocitrinae.
None of the Clauseneae species develop axillary spines, and the odd-pinnate leaves have alternately attached leaflets. The fruits are usually small and carry semi-dry or juicy berries, except in Merrilli (Swingle et al., 1967). In contrast, nearly all the species develop axillary spines in the Citreae. The simple leaves are easily distinguished from those of the tribe Clauseneae (Swingle et al., 1967). The genus Citrus belongs to the “true citrus fruit trees.” The characteristics of Citrus species include asexual reproduction, high mutation frequency, and cross compatibility between species. Because of these characteristics, there is great morphological and ecological diversity among Citrus species Citrus species can be classified into 3 clusters: a citron cluster, a pummelo cluster, and a mandarin cluster. The samples used in this study belong to pummelo cluster (Penjor et al., 2013).
Chapter Summary
1. Introduction: Provides an overview of the taxonomy, economic importance, and biological characteristics of the Citrus genus within the Rutaceae family.
2. Review of literature: Summarizes existing research on Citrus utilization, medicinal benefits, and the application of various molecular markers for plant systematics.
3. Hypothesis: Outlines the premise that morphological variations in Kerala Citrus varieties correlate with genetic diversity, testable via matK and rbcL genes.
4. Materials and Methods: Details the field study area, sample collection procedures, DNA isolation, PCR amplification, and the bioinformatics tools used for sequencing and phylogenetic analysis.
5. Results and discussion: Presents the findings from the BLAST searches, sequence alignments, and the resulting phylogenetic trees and genetic distance calculations.
6. Conclusions: Discusses the significance of phylogenetic nomenclature and the utility of molecular phylogenetics in understanding evolutionary relationships within Rutaceae.
Keywords
Anti-oxidant, matK, Citrus, PCR, RFLP, Underutilized fruit, Rutaceae, Phylogenetic tree, DNA extraction, rbcL, Genetic distance, Molecular markers, Speciation, Taxonomy, Sequence alignment
Frequently Asked Questions
What is the core focus of this research?
The research investigates the genetic relationships among three locally cultivated Citrus species (Citrus aurantifolia, Citrus maxima, and Citrus limon) in Kerala, India.
Which molecular markers are utilized?
The study utilizes two chloroplast genes, rbcL (Ribulose-1,5-bisphosphate carboxylase oxygenase) and matK (Maturase K), for molecular identification and phylogenetic analysis.
What is the primary objective?
The primary goal is to use DNA-based markers to assess the phylogenetic relatedness of these specific varieties and to understand the morphological and genetic variations among them.
What methodology is applied?
The study employs DNA extraction via the CTAB method, followed by PCR amplification of the selected genes, sequence editing using BioEdit, and phylogenetic tree construction using MEGA software.
What does the main body cover?
The main body covers the taxonomic background, literature review on molecular markers, detailed experimental protocols (DNA isolation, PCR, sequencing), and the interpretation of the resulting phylogenetic trees.
Which keywords define this work?
Key terms include Citrus, PCR, matK, rbcL, phylogenetic tree, and genetic distance.
How does the matK gene compare to rbcL in this study?
The study notes that the matK gene has a relatively faster mutation rate than the rbcL gene, making it a valuable tool for studying the phylogeny of different Citrus genera.
What were the findings regarding genetic distance?
The research calculated pairwise genetic distances and observed high similarity between samples within the respective species groups, providing insight into the taxonomy of these Citrus varieties.
How were the samples identified?
Samples were identified using a combination of baseline surveys, traditional knowledge from local farmers, and subsequent morphological characterization alongside molecular DNA analysis.
- Quote paper
- Dr. Prem Jose Vazhacharickal (Author), Sajeshkumar N. K. (Author), Jiby John Mathew (Author), Jaina James (Author), 2016, Studies on genetic relationships among locally cultivated Citrus varieties in Kerala employing matK and rbcL gene using PCR technique and RFLP markers, Munich, GRIN Verlag, https://www.grin.com/document/370769
