Genetic diversity assessment of citrus SPP. of Nepal using PCR-based molecular marker techniques
Abstract
Citrus 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 marker