Acid lime (Citrus aurantifolia Swingle) is an important commercial fruit crop, cultivated from terai to high hill landscapes of Nepal. However, production and productivity is very low due to various reasons including infestations by various diseases and pests, lack of diseases and pests resistant and high yielding varieties. In this context, determination of genetic variation at molecular level is fundamental to citrus breeders for the development of elite cultivars with desirable traits. In the present study, Random Amplified Polymorphic DNA (RAPD) marker technique has been employed to assess genetic diversity in 60 acid lime landraces representing different agro-ecological zones of eastern Nepal. Nine selected arbitrary primers generated 79 RAPD fragments of which 75 were polymorphic (94.94%). Phenogram was constructed by NTSYS-PC ver. 2.21i using UPGMA cluster analysis based on Jaccard's similarity coefficient to deduce overall genetic diversity and relationships of the acidlime genotypes under study. Sixty acid lime landraces formed seven clusters and similarity value ranged from 38% to 98% with an average of 72%. Genetic variation at different agro-ecological zones was assessed using Popgene ver. 1.32 and found 47% to 69.6% polymorphism. Shannon's index and Nei's gene diversity showed highest level of acid lime diversity in Terai zone (PPB, 69.62%; H, 0.213; I, 0.325) followed by mid-hill zone (PPB, 67.09%; H, 0.208; I, 0.317). The results obtained will be useful to citrus breeders for elite cultivar development. The RAPD-PCR technique is found to be the rapid and effective tool for genetic diversity assessment in acid lime landraces of Nepal.

Acid lime (Citrus aurantifolia (Christm.) Swingle) is an important fruit crop, which has high commercial value and is cultivated in 60 out of the 77 districts representing all geographical landscapes of Nepal. A lack of improved high-yielding varieties, infestation with various diseases, and pests, as well as poor management practices might have contributed to its extremely reduced productivity, which necessitates a reliable understanding of genetic diversity in existing cultivars. Hereby, we aim to characterize the genetic diversity of acid lime cultivars cultivated at three different agro-ecological gradients of eastern Nepal, employing PCR-based inter-simple sequence repeat (ISSR) markers. Altogether, 21 polymorphic ISSR markers were used to assess the genetic diversity in 60 acid lime cultivars sampled from different geographical locations. Analysis of binary data matrix was performed on the basis of bands obtained, and principal coordinate analysis and phenogram construction were performed using different computer algorithms. ISSR profiling yielded 234 amplicons, of which 87.18% were polymorphic. The number of amplified fragments ranged from 7–18, with amplicon size ranging from ca. 250–3200 bp. The Numerical Taxonomy and Multivariate System (NTSYS)-based cluster analysis using the unweighted pair group method of arithmetic averages (UPGMA) algorithm and Dice similarity coefficient separated 60 cultivars into two major and three minor clusters. Genetic diversity analysis using Popgene ver. 1.32 revealed the highest percentage of polymorphic bands (PPB), Nei’s genetic diversity (H), and Shannon’s information index (I) for the Terai zone (PPB = 69.66%; H = 0.215; I = 0.325), and the lowest of all three for the high hill zone (PPB = 55.13%; H = 0.173; I = 0.262). Thus, our data indicate that the ISSR marker has been successfully employed for evaluating the genetic diversity of Nepalese acid lime cultivars and has furnished valuable information on intrinsic genetic diversity and the relationship between cultivars that might be useful in acid lime breeding and conservation programs in Nepal.

Pesticides have wide spread use and their toxic residues have been reported in various environmental matrices. Thus, the determination of pesticide residues in food commodities such as vegetables, cereals, fruits, and other environmental components like soil, water has become increasingly essential requirement for consumers, producers and authorities for food quality control. Therefore, monitoring of residue level and exposure assessment of organophosphate group of pesticides namely dichlorovos and methyl parathion was carried out in 30 winter and summer vegetables supplied in Kathmandu valley. It was found that the MRL value of dichlorovos exceeded in three types of vegetables whereas in case of methyl parathion the MRL value exceeded in two types of vegetables only. The estimated average daily intake (EADI) of dichlorovos exceeded the acceptable daily intake (ADI) in 18 vegetables, whereas, regarding methyl parathion the EADI exceeded the ADI in 10 types of vegetables. Nepal Journal of Science and Technology Vol. 13, No. 2 (2012) 45-50 DOI: http://dx.doi.org/10.3126/njst.v13i2.7713

Visualizing the present biodiversity status of Nepal, an integrated approach comprising of biodiversity documentation, bio-prospecting, sustainable utilization and conservation has become an urgent need. Realizing the importance of medicinal plants altogether 45 plants were collected from Khumbu region with the altitudinal variation of 2582m to 4470m. Among the collected plant species 12 have been characterized as highly valuable. The important biochemical constituents and morphology of these plants have been described in this paper.

Random amplified polymorphic DNA (RAPD) is a simple and reliable method to detect DNA polymorphism and has been used extensively for genetic diversity studies. In the present investigation the RAPD reaction and cycling conditions were optimized for generating RAPD fingerprints of ten Nepalese strains of Bacillus thuringiensis Berliner (Bt) isolated from an altitudinal range of 70 meter above sea level (masl) to 5050 masl. To determine the optimum conditions, different concentrations of MgCl2, template DNA, Taq DNA polymerase, primer, dNTPs as well as different cycling programs were analyzed. Reproducible amplification patterns were obtained using 0.4 μM of primer, 2.5 mM of MgCl2, 125 ng of template DNA, 0.2mM of dNTPs and 1U Taq DNA polymerase in 25 μl of the reaction volume. Cycling programs were also optimized. Out of 100 arbitrary primers screened, amplification performed with 24 primers generated the best RAPD fingerprints. The optimized RAPD-PCR conditions and the selected primers are suitable for further work on genetic diversity analysis of Nepalese isolates of Bt. Key words: DNA fingerprint; primer; Taq DNA polymerase; template DNA DOI: 10.3126/njst.v9i0.3171 Nepal Journal of Science and Technology 9 (2008) 91-97

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Of the 30 species (including five varieties) of the genus Swertia in Nepal, nine have been reported to possess medicinal properties. Among these, S. chirayita is the most valuable species, with high demand in domestic and international markets. Nepal's S. chirayita and related species are being recklessly exploited for commercial purposes. Two problems that have emerged with this lucrative market are (a) adulteration and fraudulent labeling of S. chirayita, and (b) depletion of S. chirayita and allied species from their natural habitats. To address the problem of adulteration and conservation, we studied molecular genetic diversity in S. chirayita populations and developed a molecular diagnostic tool for the purposes of authentication. We studied intra-specific genetic diversity in S. chirayita using Polymerase Chain Reaction (PCR)-based Random Amplified Polymorphic DNA (RAPD) technique. As a preliminary step, we identified optimal RAPD-PCR reaction and cycling conditions by varying PCR reaction parameters such as concentration of template DNA, MgCl 2 , dNTPs, primer, Taq DNA polymerase and RAPD-PCR programs. The optimized PCR reaction and cycling conditions were then used in subsequent RAPD profiling experiments for the study of genetic diversity within S. chirayita populations from various geographical locations. Genetic diversity characterization of S. chirayita populations at the molecular level would furnish information with significant applications in the conservation and sustainable utilization of S. chirayita and its allied species in Nepal.

Owing to the high demand, Swertia chirayita populations in the wild are being depleted beyond its regeneration capacity. S. chirayita is one of the most valuable medicinal plants of Nepal in trade. Present Molecular investigation was undertaken to understand the level of genetic diversity in five S. chirayita populations of Nepal using Polymerase Chain Reaction (PCR)-based Random amplified polymorphic DNA (RAPD) technique. Thirty four accessions of S. chirayita along with six outlier accessions were analyzed using 26 arbitrary primers. Of the total 285 amplified bands scored for S. chirayita, 263 bands (92.28%) were polymorphic. Two major clusters were revealed in the phenogram generated from cluster analysis using NTSYS-PC software (version 2.21i) for the geographic populations under study. Principal Coordinate Analysis further substantiated the results of the phenograms. Swertia chirayita populations from Sankhuwasabha and Terathum were found to be genetically closest (68%, similar) whilst Nagarjun and Terathum were found to be most distant (33%, similar).The high genetic polymorphism reflected in S. chirayita populations indicates the good survival potentiality and adaptability in changing environmental scenario. The results thus produced might be helpful to plant breeders for elite cultivar development. The RAPD-PCR technique is found to be the rapid and effective tool for genetic diversity assessment in S. chirayita populations and generated insights for the formulation of conservation strategy of this vulnerable species together with its phytochemical distinctiveness.