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Molecular Diversity Analysis in Potato (solanum Tuberosum L.) Through Rapd Markers

M.E. Hoque
Associate Professor
Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh

H. Huq
Associate Professor
Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh

N.J. Moon
M.S. Student
Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh

Abstract/ Executive Summary:

Random Amplified Polymorphic DNA (RAPD) markers were used to study the molecular diversity of 12 popular potato varieties in Bangladesh. DNA was extracted from tender leaf sample for PCR amplification. The PCR amplified DNA profile was visualized on 2% agarose gel, staining with ethidium bromide. Eight RAPD primers were used to evaluate the genetic diversity of potato varieties. Some total of 36 DNA fragments were amplified and out of them 24 were polymorphic. Those primers generated 61.53% of polymorphic DNA band. The primer OPX 04 produced highest (9) number of DNA band and out of 9 amplicon 6 were polymorphic. Lowest number of amplification was observed in the primer OPA-17 and it was only 3. The highest Nei’s genetic distance (0.9701) was noticed between the variety Granola and Provinto. The highest (0.8205) number of genetic identity/similarity was observed between the varieties Cardinal and Diamant. The unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei’s genetic distance revealed that the 12 varieties followed into two main clusters. The present finding showed that there was high level of genetic diversity among the varieties which can be used for parental selection in potato breeding program.

Keywords: Molecular diversity, RAPD, Potato.

Location: Department of Biotechnology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka.

Start Date: 00-12-2012

End Date: 00-03-2013

Program Area: Variety and Species

Commodity: Potato

Objectives:

1)To molecular diversity analysis of some released potato varieties through RAPD markers;

2) To identify the divergence genotypes for potato improvement program.

Methodology:

The experiments were carried out at th e Department of Biotechnology, Sher-e- Bangla Agricultural University, Sher-e-B angla Nagar, Dhaka. Twelve popular released potato varieties were collected from Tuber Crops Research Centre (TCRC), Bangladesh Agricultural Research Institute (BARI), Gazipur and used as experimental materials. The potato varieties were Cardinal, Diamant, Granola, Asterix, Sagita, Courrage, Lady Rosetta, Felsina, Multa, Provinto, Petronige and TPS-1. Good quality, disease free, healthy tuber (breeder stock) were sown in plastic pots and kept in nets house. All management practices were done for raising quality seedlings from those materials. Fresh leaves were collected at 3-4 leaf stage of plant for isolation of DNA. Total genomic DNA from each variety was isolated by CTAB method with slight modification according to Frey et al., (2004). The extracted DNA was purified by propanol and treated with 10μg/ml RNase A for 20-25 min at 37^oC to remove the RNA. The purified DNA was dissolved in TE buffer and quantification of DNA was done through electrophoresis on 1% agarose gel staining by ethidium bromide. The sample DNA was stored at - 20^oC freezer for further use. Seventeen RAPD primers were selected on the basis of previous works to evaluate the molecular polymorphism among the potato varieties. PCR reaction was performed using BIONEER KIT (korea). The PCR reaction having 20.0 μl mixture containing 3.0 μl sterile deionized water, l0X PCR buffer 4.0 μl, enzyme dilution buffer 4.0 μl, 20 mM MgCl₂, 3.0 μl dNTPs (10mM) 1.0 μl top DNA polymerase 0.5 μl, primer 2.5 μl and sample DNA (approx. 40-50 ng) 2.0 μl. The reaction mixture was subjected to the following thermal profile for amplification in a thermocycler: 5 min at 95^oC for initial denaturation, followed by 33 cycle of 1.10 min denaturation at 94^oC, 1.0 min at annealing and 1.30 min at 72^oC for extension. A final extension step was done at 72^oC for 7 min. Electrophoresis was done to visualize the PCR amplified product. It was carried out on 2.0% agarose gel and amplified fragments were visualized by staining with ethidium bromide. The amplified bands were scored as present (1) and absent (0) for each primer. The score of bands were pooled to create a single data matrix. These were used to estimate polymorphic loci. Genetic distance and identity were calculated based on Nei’s (1972). Phylogenetic tree and dendrogram were established based on an unpaired group method of arithmetic means (UPGMA) using the software POPGENE (Version 1.31) (Yeh et al., 1999).

Source of Information: SAARC JOURNAL OF AGRICULTURE, Vol. 11, Issue 2, 2013, pp. 95-102, ISSN 1682-8348, SAARC Agricultural Information Centre.

Article Link (Online Journal): http://www.banglajol.info/index.php/SJA/article/view/18405/12915

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Information on the genetic diversity allows to assist the parent selection and paving the way to genetic gains. The results of present study revealed the existence of high level of genetic diversity among the studied 12 popular widely grown potato varieties in Bangladesh. These varieties can further be used as parental material for fixation of heterosis in potato improvement program.

Knowledge Management: Journal