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Genetic Diversity Analysis in Cashew (anacardium Occidentale L.) Germplasm Using Rapd Marker

T. Bhadra
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

A.Z.M. Obaidullah
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Sabiha Sultana
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

M. Ahmed
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

M.M. Islam
Agrotechnology Discipline, Khulna University, Khulna 9208, Bangladesh

Abstract/ Executive Summary:

Anacardium occidentale L., commonly known as cashew nut, belongs to the family Anacardiaceae. It is regarded as a high valued fruit nut crop world-wide. Potential of this economically important nut is under-utilized in Bangladesh in spite of having all favorable agro-climatic conditions. The objective of the present investigation was to characterize six cashew accessions using Random Amplified Polymorphic DNA (RAPD) markers. Four random primers viz. OPE-02, OPE-18, OPK-03 and OPB-15 were used to amplify DNA segments. A total of 33 reproducible bands were obtained, out of which 11 were monomorphic and 22 were polymorphic. On average 74.12% polymorphism was observed. . Primers OPB-15 and OPK-03 yielded 100% polymorphism and OPE 02 and OPE 18 produced 33.33% and 63.16 % polymorphism, respectively. Cluster analysis revealed two main distinct groups, first group included GP-1 and the second consisted of five genotypes viz. GP-2, GP-3, GP-4, GP-5 and GP-6 The major cluster- II was further subdivided into two minor clusters i.e. minor cluster- III and IV. Minor cluster- III contained only one genotype GP-4. Minor cluster- IV consists of four rest genotypes. The genetic distance between the groups was found low and varied from 0.002 to 0.0308. Maximum genetic distance was observed between GP-1 and GP-2 cashew germplasm and minimum between GP-5 and GP-6. The low genetic distance which is unusual for this out crossing long-lived tree species, indicates the probability of having common ancestry among the germplasm or may be due to the use of a narrow range of populations for the investigation.

Keywords: Anacardium occidentale, DNA, RAPD, Genetic relationship, Cashew

Location: Germplasm Bank of Agrotechnology Discipline of Khulna University.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cashewnut

Objectives:

The present investigation, the diversity and genetic relationship of six cashew germplasm accessions were analyzed by using RAPD markers.

Methodology:

Plant materials- Six germplasm accessions of cashew plant were taken for the study. Those were GP-1, GP-2, GP-3, GP-4, GP- 5 and GP-6. The plant materials were collected from Germplasm Bank of Agrotechnology Discipline of Khulna University. DNA extraction- The young leaves from the established plants were used for DNA extraction. The leaf samples were wrapped with aluminum foil paper and freeze-dried at -86ºC in ultra low freezer for lyophilizing the sample. The leaves were finely ground in liquid nitrogen and approximately 200 mg of powdered leaf was taken in sterile 1.5 mL eppendorf tube. Norgenbiotek DNA isolation kit was used for DNA extraction. The purified DNA of leaves was quantified by using Spectrophotometer (Multiskan GO Microplate, Thermo Fisher Scientific, Germany) at 260 nm wave length. The final concentration of the template DNA was adjusted at 30 ng μL^-1 for PCR and stored at -20°C. denaturation at 94 °C for 30 seconds, annealing at 37°C for 1 minute and initial extension at 72°C for 1 minute and repeat for 40 cycles, finally at 72°C for 5 minutes and hold at 4°C. Agarose Gel Electrophoresis for separation of PCR amplicons- The amplified products were resolved by electrophoresis on 1% agarose gels stained with ethidium bromide (0.5 µg mL^-1) run at 60 volts in Tris Acetate EDTA buffer (1.0 × TAE) for one hour. Resulted amplicon profiles of all the isolates across the primers were visualized under UV Tech Gel documentation System with computer program (Biometra gel documentation, A Biodoc Analyze 2.2 version, Germany). A molecular weight marker 1 kbp (Direct load, Sigma Aldrich, USA) was used. Data analysis- The DNA profiles were scored visually from gel images. Clear and reproducible amplified bands were chosen in the analyses. The presence of a band was designated as (1) and absence as (0). The data obtained by scoring the RAPD profiles were subjected to the calculation of similarity matrix using Jaccard’s coefficients (Zahra et al., 2012). The similarity values were used for cluster analyses. Sequential agglomerative hierarchical nonoverlapping (SAHN) clustering was applied using unweighted pair group method with arithmetic averages (UPGMA) method. Dendrograms were plotted using Restdist (version 3.69) computer programs by Joseph Felsenstein of the University of Washington, USA. Polymorphism percentage was calculated as the proportion of polymorphic bands over the total number of scored bands of respective primer.

Source of Information: J Bangladesh Agril Univ 17(4): 461–465, 2019

Article Link (Online Journal): DOI: https://doi.org/10.3329/jbau.v17i4.44606

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

A total of 33 reproducible bands and 74.12% polymorphism were observed. Primers OPB-15 and OPK-03 yielded 100% polymorphism respectively. Cluster analysis revealed two main distinct groups, first group included GP-1 and the second consisted of five genotypes viz. GP-2, GP-3, GP-4, GP-5and GP-6 The major cluster- II was further subdivided into two minor clusters i.e. minor cluster- III and IV. The genetic distance between the groups was found low and varied from 0.002 to 0.0308. Maximum genetic distance was observed between GP-1 and GP-2 cashew germplasm and minimum between GP-5 and GP-6. In spite of having some limitations such as use of limited number of germplasm, the result of this study could be used as a guideline for future diversity assessment and genetic analysis of cashew in Bangladesh. The analysis of genetic relationships in cashew using RAPD banding revealed that the method is appropriate enough to evaluate the genetic relationships in cashew nut tree and thus the RAPD markers can generate wide array of polymorphism for varietal identification, study of genetic diversity and genetic conservation, and could be successfully used in cashew breeding program.

Knowledge Management: Journal