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Variations of Genotypes of Radish at Molecular Level Using Isozyme Analysis for the Identification of Self-incompatible Lines

N. A. Ivy
Associate Professor
Genetics and Plant Breeding, BSMRAU, Gazipur-1706, Bangladesh.

M. S. Biswas
Department of Horticulture, BSMRAU, Gazipur-1706, Bangladesh.

G. Rasul
Genetics and Plant Breeding, BSMRAU, Gazipur-1706, Bangladesh.

T. Hossain
Crop Botany, BSMRAU, Gazipur-1706, Bangladesh.

M. A. K. Mian
Genetics and Plant Breeding, BSMRAU, Gazipur-1706, Bangladesh.

Abstract/ Executive Summary:

The experiment was conducted to characterize 78 radish genotypes using esterase and peroxidase isoenzyme. The results of the experiment revealed a wide range of diversity among the genotypes based on their esterase and peroxidase isoenzyme banding patterns. Five bands at different Rf values varying from 0.06 to 0.56 were observed in peroxidase enzyme system and formed 10 zymotypes. Zymotype P₁ was the most frequent which includes 26.92% of the total genotypes. Lowest frequency of 2.56% was observed in the zymotype P₁₀. Band 3 and 4 consist of same frequency (23.31%) of the genotypes at Rf values 0.20-0.30 to 0.30-0.50. It was also found that there was no common band for all the genotypes. Thus the genotypes have wide range of genetic variation. On the other hand thirteen electrophoretic zymotypes (P₁-P₁₃) were observed in Esterase enzyme system formed by 13 bands at different Rf values varying from 0.04 to 0.93. It appeared that electrophoretic zymotype P₆ was the most frequent (25.64%) and zymotype P₁₃ was least frequent (2.56%). The zymotype P₁ had maximum number of bands. From the distribution of esterase bands among the radish genotypes, it was observed that band 3 had the most frequent of 18.75% followed by band 2 and 4 had the same frequency 16.67% among all the genotypes. A dendogram based on the two polymorphic enzyme activities all the genotypes were classified into eight major clusters designated as I, II, III, IV, V, VI, VII and VIII. The highest number of 21 genotypes was found under the cluster number I that represented 27% of the total genotypes followed by cluster VI contained 17 genotypes. The lowest number (4) of genotypes was found under cluster VIl followed by cluster III contained 5 genotypes. The result indicated that zymotype of higher frequency are the representative of less variation. The lower frequency of the germplasm in different zymotypes indicated higher variation among the genotypes.

Keywords: Radish Esterase, Peroxidase isoenzyme, Characterization

Location: Department of Genetics and Breeding, BSMRAU

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Radish

Objectives:

To know the banding pattern among the parents and hybrids and also the inheritance pattern of parental bands by all the hybrids indicated the authenticity of the hybrids of radish.

Methodology:

Young leaf material were taken from plant sample into a small eppendorf tube with a small amount of sea sand as a crushing agent. Exactly 0.70 µl extraction buffer solutions were added and crushed the sample using a glass rod. The sample was dissolved by vortex until become homogenization. Dissolved samples were centrifuged at 15000 rpm for 4°C for 3 minutes. Samples were kept in a refrigerator until it used. Gels of different concentrations were prepared using the stock solution. The stock solutions were stored in dark and cold condition. Freshly prepared ammonium per sulphate (APS) solution was used. The glass plates (one was notched and the other was plain) and the gaskets with the clips were assembled first to make plate sandwich. A comb was inserted into the sandwich. A level of marking was made on the glass plate, 4-5 mm lower than the teeth of the comb, by using marker pen. The comb was then removed. The solution of separation gel except APS and tetramethylene diamine (TEMED) were gently mixed in a 50 ml conical flask. After words APS and TEMED were added and mixed very carefully, Immediately after adding JAPS and TEMED, the separation gel solution was poured with its notched glass plate upside. Small amount of water or ethanol overlaid to a level of 2-3 mm over the gel solution carefully. Then the solution was allowed for polymerization about 30-60 minutes until a clear line between the gel and water is seen. After polymerization of the separating gel the overlaid water was discarded by using tissue paper. The solution for stacking gel were mixed as separating gel and introduced to fill the upper portion of the glass plates. Immediately a comb was inserted through the stacking gel solution carefully, so that the comb did not trap any air bubbles. About 20-30 minutes was allowed for polymerization. The comb was removed by pushing it up and washed the wells with a small amount of electrode buffer. After removing the clips and gaskets, the glass plate with gel was attached to the electrode assembly. About 430 ml of the electrode buffer was poured in the electrophoresis chamber slowly and gently to avoid trapping of air bubbles at the bottom of the gel. Electrophoresis was performed by connecting the electrode assembly with the power supply for 3-4 hours depending on the concentration of gels at 20 mA constant current per gel. The electrophoresis was stopped at the time when tracking marker stain reaches 4-5 mm to the gel bottom. After removing the gel sandwich from the electrode assembly, the gel was separated from the glass plate gently with a steel spatula keeping under slow running tap water. The gel was then put into the staining solution. The isozyme, which was stained to observe the variation in banding pattern are esterase and peroxidase. Solution A was prepared by mixing 40 ml methanol and 200 mg of 0-phynyl-diamine in 50 ml of 1M Na-acctate buffer. To the mixture 50 ml methanol was added. Solution A was prepared by mixing 100 mg fast violet B salt in 1 ml buffer. Solution B (substrate buffer) was prepared by adding 100 mg 1-naphthyl acetate and 100 mg 2-naphthyl acetate in 10 ml acetone and then 100 ml water was added to it. Solution B was added to solution A, the gel was incubated until bands appeared. For each enzyme similarity coefficient (S,y) values of electrophoretic pattern were subjected to cluster analysis employing the unweighted pair group method using arithmetic averages (UPGMA). For cluster analysis of overall isozyme electrophoretic patterns, the value I was put for the presence of the electrophoretic pattern and value 0 was used against the absence of the pattern for each genotype. The original data was transformed to Z-scores prior to cluster analysis.

Source of Information: Global Journal of Biotechnology & Biochemistry 5 (1): 19-26, 2010; ISSN: 2078-466X.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The result indicated that zymotype of higher frequency are the representative of less variation. The lower frequency of the germ plasm in different zymotypes indicated higher variation among the genotypes. A dendogram based on the two polymorphic enzyme activities all the genotypes were classified into eight major clusters designated as I, II, III, IV, V, VI, VII and VIII. The highest number of 21 genotypes was found under the cluster number I that represented 27% of the total genotypes followed by cluster VI contained 17 genotypes. The lowest number (4) of genotypes was found under cluster Vll followed by cluster III contained 5 genotypes.

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