MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF Citrus GERMPLASM IN SRI LANKA

Abstract

Sri Lanka has a rich variety of Citrus species, ranging from wild-growing limes in village backyards to cultivated hybrid mandarins and oranges in commercial plantations. Despite their economic and cultural importance, the true diversity of Citrus in Sri Lanka has not been fully explored or documented. The main objective of this project is to fill that gap by identifying and characterizing Citrus species across the island using both traditional morphological methods and modern molecular tools. Morphological characterization involved the documentation of traits such as fruit, leaf, tree and floral morphology. Quantitative data were scandalized using z- score normalization, while qualitative data were encoded numerically. Species distances were calculated using Gower distance in the R programming environment. A dendrogram was constructed using the word’s method of hierarchical clustering in IBM SPSS. Based on the morphological characters, five main clusters were identified. Cluster I - Yak dehi, Key lime, Nas naran, and Kaffir lime. Cluster II - Lemon- round, Lemon – long, Cluster III - Sour orange, Sweet orange Cluster IV - Sidaran, Cluster V – Pumelo. Molecular characterization was carried out through DNA barcoding of chloroplast genes. Genomic DNA was extracted using a modified CTAB method, and PCR amplification was performed using rbcL and matK gene markers. PCR products were sequenced at Macrogen, South Korea. The sequences were analyzed using BioEdit software and deposited in GenBank. Phylogenetic trees were constructed using MEGA software to infer genetic relationships among species. The phylogenetic tree based on matK sequences revealed three major clusters: Cluster I - Sour orange, sweet orange, Nas naran, Yak dehi, and Pumelo. Cluster II - Long lemon, Round lemon, Key lime, and Kaffir lime. Cluster III - Sidaran. The phylogenetic tree based on rbcL sequences formed two primary clusters. Cluster I - Sour orange, Sweet orange, Nas naran, Yak dehi, Pumelo, and Sidaran. Cluster II - Long lemon, Round lemon, Key lime, and Kaffir lime. Phylogenetic analysis using both rbcL and matK markers revealed consistent clustering patterns, with the only notable exception being the placement of Sidaran. These findings provide a foundational understanding of the morphological and genetic diversity of Citrus species in Sri Lanka and contribute to future conservation and breeding programs.