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Genetic Diversity in Pointed Gourd (trichosanthes Dioica Roxb) Revealed by Random Amplified Polymorphic DNA (rapd) Markers

A.S.M.M.R. Khan
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute Gazipur-1701 Bangladesh

M.S. Islam
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute Gazipur-1701 Bangladesh

M.H. Rashid
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute Gazipur-1701 Bangladesh

A.K.M.M. Alam
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute Gazipur-1701 Bangladesh

M.G. Rabbani

Abstract/ Executive Summary:

Genetic variation in 64 pointed gourd accessions was investigated using the Randomly Amplified Polymorphic DNA (RAPD). Out of 45 random primers screened five were selected, which gave 38 clear and bright fragments, out of which 30 (79.5%) fragments were considered polymorphic. The proportion of polymorphic loci across all loci was 96%. The number of bands per primer was five to eleven. The highest genetic distance 0.6419 was observed between the accession PG035 and PG051, PG035 and PG056 and PG035 and PG021. While the lowest genetic distance 0.00 was observed between the accessions PG042 and P043 and between PG042 and PG044. The UPGMA dendogram constructed based on RAPD analysis in 64 pointed gourd accessions were found to be grouped in twelve major clusters. Cluster VIII is a broad one which includes 21 accessions and only a single accession formed in cluster VII (PG021). RAPD analysis showed promise as an effective tool in estimating genetic polymorphism in different accessions of pointed gourd.

Keywords: Genetic variation, Pointed gourd accessions, Amplified Polymorphic DNA (RAPD)

Location: Regional Agricultural Research station, BARI, Ishurdi, Pabna

Start Date: 00-00-2005

End Date: 00-00-2005

Program Area: Variety and Species

Commodity: Pointed gourd

Objectives:

The present investigation was carried out for analyzing the amount of genetic variation in pointed gourd accessions and classifying them to assist in selection of parent genotypes in a breeding program. They can facilitate rapid screening of large numbers of genotypes for polymorphic loci.

Methodology:

Sixty four pointed gourd accessions were used in this study. All the accessions were obtained from Regional Agricultural Research station, BARI, Ishurdi, Pabna during the month of November 2005. The accessions were different in their origin and their phenotypic character. In order to carryout RAPD analysis DNA was extracted from young growing leaves from each accession using following phenol: chloroform: isoamyl alcohol purification and ethanol precipitation method. Finally, the DNA samples were stored at -20^oC. DNA concentrations were determined at 260 nm spectrophotometrically and the quality verified by electrophoresis on a 1.4% garose gel. Initially, 45 decamer primers from three kits (20 from kit A, 20 from kit B and 5 from kit C) of random sequence (Operon Technologies, Inc., Alameda, California, USA) were screened on a sub sample of three randomly chosen pointed gourd accession to test their suitability for amplifying pointed gourd. Primers were evaluated on the basis of intensity or resolution of RAPD bands, repeatability of markers consistency within individuals and potential to differentiate accessions (polymorphism). A final subset of five primers exhibiting better quality banding patterns were tested three times on sub samples to be certain that the bands obtained were not mere artifacts of the RAPD method but rather true amplified products; they were then selected for analyses of the entire sample set of 64 accessions. The amplification condition was based on Williams et al. with some modification. PCR reactions were performed on each DNA sample in a 10 μL reaction mix containing 1 μL of 10X ampli Taq polymerase buffer, 2 μL of 10μM primer, 1 μL of 250 μM dNTPs (Gene, Banglar, India), I unit of Ampli Taq polymerase (Gene, Banglar, India), 1.5 mM MgCl₂ and 50 ng genomic DNA made up to 10 μL with sterile deionized water. DNA amplification was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf). The reaction mix was preheated at 94^oC for 3 min followed by 45 cycles of one min denaturation at 94^oC, one min annealing at 34^oC and elongation or extension at 72^oC for two minutes. After the last cycle, a final cycle of seven minutes at 72^oC was added to allow complete amplified fragments. The amplified product from each sample was separated electrophoretically on 1.4% agarose gel (Fisher Biotech, USA) containing ethidium bromide in TBE buffer at 120 V for 1½ hrs. Molecular weight DNA markers (pUC18/ Sau 3A I-pUC18/Taq I Digest and 100 bp DNA Ladder) were electrophoresed alongside the RAPD reactions. DNA bands were observed on UV-transilluminator and photographed by a Gel Cam Polaroid camera (Type 667). The RAPD markers were scored visually on the basis of their presence (1) or absence (0), separately for each accession and each primer. For more accuracy, band scoring was performed by two independent persons. Bands not identified by the two readers were considered as non scorable. The scores obtained using all primers in the RAPD analysis were then pooled for constructing a single data matrix. This was used for estimating polymorphic loci, and gene diversity genetic distance (D) and constructing an unweighted Pair group Method of arithmetic mean (UPGMA) dendogram among accessions using POPGENE (version 1.31) computer program. Estimation of gene frequencies of RAPD loci was based on the assumption of a two allele system. Of the two alleles, only one is capable of amplification of a RAPD band by primer annealing at an unknown genomic position (locus). The other is the ‘null’ allele incapable of amplification, mainly because of loss of the primer annealing site by mutation. The two allele assumption was in most cases acceptable, because co dominant loci showing band shifts are few. In this system only a null homozygote was detectable as negative for the RAPD band of interest. Under the assumption of Hardy-Weinberg equilibrium the null allele frequency (q) may be (N/n) ½, where N and n are the number of band negative individuals observed and the sample size, respectively. The frequency of the other allele (P) is 1-q. The assumption of the two allele system enables us to calculate the Nei’s genetic distance from the RAPD pattern.

Source of Information: Thai Journal of Agricultural Science 2009, 42(2): 61-69

Article Link (Online Journal): www.thaiagj.org

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The RAPD analysis discovered sufficient genetic variations among the pointed gourd accessions. No information on genetic structure of the pointed gourd was available in Bangladesh. This is the first attempt to study genetic structure of pointed gourd. Only 30 polymorphic markers were generated which was possibly not sufficient to cover pointed gourd genome particularly the regions influencing the expression of quantitative traits. Hence, sufficient and more efficient RAPD primers showing maximum number of polymorphic bands or other available markers systems could be utilized for analysis of pointed gourd. The result of the present study indicated that RAPD can be, used for identification of the available pointed gourd gremplasm. It can also be used to study relationship of pointed gourd germplasm among each other for further improvement through cross breeding.

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