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Genetic Diversity of Potato Cultivars Assessed by Rapd Marker

M A Haque
Departmentof Biotechnology, BSMRAU, Gazipur

N Mustafy
Departmentof Biotechnology, BSMRAU, Gazipur

D R Gupta
Departmentof Biotechnology, BSMRAU, Gazipur

A H Molla
Dept.of EnvironmentalScience, BSMRAU, Gazipur

M H S Rahman
Scientific Officer
BINA, Mymensingh

M M Islam
Scientific Officer
BARI, Gazipur, Bangladesh

Abstract/ Executive Summary:

An experiment on Bangladeshi eight potato cultivars was done through RAPD marker at the Biotechnology laboratory of the Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur-1706, Bangladesh. This experiment was conducted to know the genetic diversity of the potato germplasm for potato improvement program. Genomic DNA was extricated from youthful leaves and PCR response was performed. The PCR intensified DNA profile was pictured on agarose gel, recoloring with ethidium bromide. All nine primers were polymorphic and among them OPB 01, OPB 04, OPD 01, OPD 03, OPD 04 and OPE 20 produced the highest number of allels (6), followed by the primers OPA 03, OPA 04 and OPC 03 five alleles. Nei's hereditary separation uncovered that low hereditary separation was (0.5556) among the variety Espirit and Courage and most elevated hereditary separation (1.00) was found in Raja assortment with all different varieties aside from Kufri bahar (0.8889). The results showed that, low-and high-level hereditary separation exists between the varieties where Raja had most exceptional hereditary separation from every single other variety which can be utilized for further potato reproducing program.

Keywords: DNA Extraction, Molecular Marker, PCR, Allele and Hereditary.

Location: Plant molecular laboratory, Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Potato

Objectives:

The aim of the study was to characterize eight different cultivars of potato through RAPD markers

Methodology:

The experiment was directed at the plant molecular laboratory, Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh. Sample collection Eight cultivars of potato viz. Cardinal, Diamant, Espirit, Raja, Courage, Lady rosetta, Asterix and Kufribahar were collected from Tuber Crops Research Centre (TCRC), Bangladesh Agricultural Research Institute (BARI), Gazipur and were used as experimental materials. In vitro grown micro plants from sprouts were used as the source of plant material. A sum of 40 individuals (five from every cultivar) were taken arbitrarily to complete the examination DNA extraction Approximately 20 mg fresh young tender leaf were taken into microfuge tube and 300 μl tissue lysis buffer and 10 μl of RNAse solution were added and homogenized by homogenizer. Centrifugation was carried out of the sample to remove the available solids to it. After that 300 μl of nuclease free water was added and eachplant lysate were transferred from the extraction tube to plant DNA extractor machine Maxwell 16 (origin: Promega, USA) to extract DNA. Primer selection and PCR amplification Nine RAPD primers(Table 1) were selected to know the molecular polymorphism among the potato varieties. PCR reactions were performed using Promega PCR master mix kit. Master mixture is a premixed, ready-to-use solution containing Taq DNA polymerase, dNTPs, MgCl₂ and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR. Gel electrophoresisFor gel electrophoresis 0.5g of agarose powder was poured into microwavable flask along with 50mL of 1xTBE buffer. Microwave was done for 1-3 min until the agarose was completely dissolved and there was a nice rolling boil. Agarose solution was cooled down for 5 minute and ethidium bromide (EtBr) was added to a final concentration of approximately 5 μl with 50 ml agarose gel (usually about 2-3μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows to visualize the DNA under ultraviolet (UV) light. The agarose was poured into a gel tray with the well comb in place. Newly poured gel was kept at room temperature for 20-30 minutes until it was completely solidified. After solidification of gel, the loading buffer was added to each sample and also with the ladders. Once solidified, the agarose gel was placed into the gel box (electrophoresis unit) and the gel box was filled with 1xTBEbufferuntil the gel was covered. Molecular weight ladder was loaded into the first and last lane of the gel and samples were loaded into the other lane ofthe gel. The gel was run at 100V until the dye line was approximately 75-80% of the way down the gel. After the electrodes were disconnected from the power source, the gel was removed from the gel box carefully. Documentation of the DNA samples After electrophoresis, the gel was taken out carefully from the gel chamber and was placed on high performance ultra-violatelight box (UV trans-illuminator) of gel documentation for checking the DNA was observed as band and photographed by a gel cam polaroid camera.RAPD data scoring and analysis RAPD markers are dominant marker with an assumption that each band represented the phenotype at a single allelic locus (Williams et al., 1990). Two molecular weight markers, 1kb and 100 bp DNA ladder were used to estimate the size of the amplified products by comparing the distance traveled by each fragments with known sized fragments of molecular weight markers. All of the distinct bands or fragments (RAPD markers) were given identification numbers according to their position on gel and scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer.The scores obtained from all primers in the RAPD analysis were then pooled to create a single data matrix. The data matrix was used to estimate Polymorphic loci, Nei’s (1972) gene diversity, Genetic distance and creating a UPGMA (UnweightPair Group Method of Arithmetic Means) dendrogram among the germplasm using a computer program.(NPcys-PC).Nei’s genetic distance was computed from frequencies of polymorphic markers to estimate genetic relationship amongthe studied eight potato genotypes using the Unweighted Pair Group Method of Arithmetic Means (UPGMA) (Sneath and Sokal, 1973).

Source of Information: Eco-friendly Agril. J. 12(11):78-84, 2019 (November)

Article Link (Online Journal): www.efaj-international.com

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The aforesaid results indicate Raja variety may be utilized for further potato breeding program to improve the present varieties of Bangladesh and the primer OPB 01 and OPD 01 may be used for the identification of potato genotypes in Bangladesh.

Knowledge Management: Journal