Munankarmi, Nabin Narayan2026-04-272026-04-272013https://hdl.handle.net/20.500.14540/26426Citrus is one of the most important fruit crops widely cultivated in tropical and subtropical regions of Nepal. Citrus crops cover about 30% of the total area under fruit cultivation. Its cultivation must be promoted to increase yield to fulfill the national and international demand. A large amount of genetic variation exists within and among Citrus spp. as a result of long history of cultivation, frequent bud mutation, interspecific and intergeneric hybridization, apomixes and human selection. As morphological traits are inadequate in differentiation of closely related cultivars and biochemical markers doesn’t generally distinguish between cultivars differentiated by mutations, DNA based molecular markers adds a new and reliable dimension to identification, phylogeny construction and genetic diversity assessment. Genetic diversity assessment based on molecular marker tools is essential for the selection of parental genotypes for breeding of elite cultivars having desirable qualitative (disease resistant, drought tolerant, off season production etc.) and quantitative (Total Soluble Solid, Tritable Acidity, Vitamin C content, juice percent etc.) traits via Marker Assisted Selection (MAS) and Marker Assisted Introgression in variety development programs. Present investigation consists of two parts viz. diversity assessment of acid lime (C. aurantifolia Swingle) landraces of different agro-ecological zone of eastern Nepal using ISSR (Inter-Simple Sequence Repeat) markers and diversity assessment of Citrus spp. of Nepal using SSR (Simple Sequence Repeat) markers. Sixty accessions of acid lime landraces from the different agro-ecological zone of eastern Nepal were analyzed using 21 ISSR markers. Out of 234 scored amplified bands for acid lime accessions, 204 were found to be polymorphic (87.18%). Binary data matrix was created based on presence and absence of ISSR bands. Cluster analysis using ISSR (qualitative) data was performed in the NTSYS-PC (Numerical Taxonomical System) ver. 2.21i statistical package for studying genetic diversity and relationship among various acid lime accessions. Similarity matrices and phenograms were generated by using Simple Matching, Jaccard’s and Dice coefficient of similarity and these were compared based on correlation coefficient and consensus indices values. Dice coefficient of similarity in constructing phenogram using UPGMA (Unweighted Pair Group Method of Arithmetic Averages) module was found to be the best for deducing genetic relationship among acid lime accessions. It revealed two major clusters and three minor clusters showing overlapping of acid lime accessions from different agro-ecological zones. The acid lime landraces from High-hill were found to be genetically closest (86.02%, similar) while from Terai were genetically most distant (79.33%, similar). Principal Co-ordinate Analysis (PCO) computed using MVSP (Multi-Variate Statistical Package) ver. 3.21 substantiated the results of phenogram. AMOVA (Analysis of Molecular Variance) analysis performed using GenAlEx (Genetic Analysis in Excel) ver. 6.5 showed high variations within agro-ecological zones (86%) and low among agro-ecological zone (14%). Similarly, 45 citrus accessions belonging to different species from Paripatle, Dhankuta and Kathmandu Valley were analyzed using 12 SSR markers. Genetic diversity and relationship among the Citrus spp. was studied in the similar manner as that of acid lime using NTSYS-PC ver. 2.21i statistical package. In this study, Jaccard’s coefficient of similarity in constructing phenogram using UPGMA module was found to be the best for deducing genetic relationship among Citrus spp. accessions under study. It revealed four clusters with kumquat (Fortunella margarita and F. hindsii) and rootstock citrus (Poncirus trifoliata and Old Citranze) out of major clusters. Principal Co-ordinate Analysis (PCO) computed using MVSP ver. 3.21 substantiated the results of phenogram. In present investigation, 60 putative alleles were detected at 12 SSR loci in 45 samples varying from 2 (TAA 27) to 8 (TAA 52) with an average of 5 per locus. Loci TAA 41 (6 alleles, PI value 0.075 and PIC value 0.802) and TAA 52 (8 alleles, PI value 0.089 and PIC value 0.785) were found to be the most informative loci for the investigation of diversity of Citrus spp. in this investigation on the basis of number of alleles detected, PI values and PIC values. The Shannon’s Information Index, Allele frequency, Observed and Expected Heterozygosity and Fixation Index were computed using GenAlEx ver. 6.5 and their values showed high level of genetic variation among the Citrus spp. The result provides genetic information about intraspeicfic diversity and help in identification of similarity group that could be useful for the selection of parental plants to be used in future breeding programs. The results will be helpful for the plant breeders in elite cultivar development purposes. It also provides information about interspecific, intraspecific and even intergeneric genetic similarity and distances of Citrus at molecular level using SSR markers and paves the way to tackle the long standing problem of Citrus germplasm evaluation, classification and identification in Nepal. Keywords: Citrus spp., acid lime, ISSR, SSR, genetic diversity, molecular markeren-USGenetic diversityAssessmentThesis