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Molecular Characterization of Onion (allium Cepa) Using Rapd Markers

MANIRUZZAMAN
Scientific Officer
Biotechnology Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur 1701,

M. E. HAQUE
Scientific Officer
Biotechnology Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur

M. M. HAQUE
Senior Scientific Officer
Spices Research Centre, BARI

M. A. SAYEM
Agriculture Extension Officer
DAE

M. AL- AMIN
Principal Scientific Officer
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

A polymerase chain reaction (PCR) based approach, namely random amplified polymorphic DNA (RAPD) analysis was applied to l0 varieties of onion (Allium cepa) in order to assess the degree of polymorphism within the genes and to investigate if this approach was suitable for genetic studies of onion. For this study, ten cultivars of onion were evaluated for variability using a set of 15 random l0-mer primers. The polymorphisms in PCR amplification products were subjected to the unweighed pair group method for arithmetic averages (UPGMA) and plotted in a phenogram. The dendogram constructed from the similarity data showed that all the cultivars analyzed were related. Among them, 12 of the primers revealed scorable (168 bands) polymorphisms between cultivars of A. cepa and the rest did not show polymorphism in their genetic level. In this study, it was found that Bermis and India-2 were more dissimilar and on the other hand, Faridpuri and Bhati were the most similar in their genetic level.

Keywords: RAPD, Onion, Genetic diversity, Polymorphism

Location: Bangladesh Agricultural Research Institute, (BARI), Gazipur

Start Date: 01-01-2007

End Date: 30-06-2008

Program Area: Variety and Species

Commodity: Onion

Objectives:

In this study, selected nine varieties and one line (OF-5) were taken to investigate the genetic relation among the varieties

Methodology:

The study was conducted from January 2007 to June 2008 at Biotechnology Division, Bangladesh Agricultural Research Institute, (BARI), Gazipur. A total of 9 varieties and one line of Allium spp., where some were BARI released, two of them were Indian and one of them was Bermis viz., BARI Peas-I, BARI Peas-2, BARI Peas-3, India-I, India-2, Bermis, White Onion, OF-5, Faridpuri, and Bhati.Total DNA was isolated by CTAB method with slight modifications according to MaaB and Klass (1995). After treatment with 10 μg/ml RNase A for half hour at 3 7°C, the DNA was purified with propanol. The purified DNA was dissolved and stored in TE buffer, and the concentration was determined fluorometrically Amplification was carried out using 15 arbitrary 10 bp primers (OPA-4, OPA-9, OPA- 15, OPA-16, OPAB-4, OPAB-18, OPG-13, OPB-6, OPC-5, OPC-7, OPC-9, OPD-1, OPD-3, OPE-17 and RE-01), obtained from Biobasic, Canada. The primers were chosen after initial screening of more than 25 different primers, based on the production of distinct and reproducible bands in PCR reactionsTwenty μl of the isolated DNA were used for PCR (approximately 100 ng) amplification. The amplification conditions were optimized according to Friessian et al. (1997). Full of the reaction mixtures was separated on 1.5% agarose gel in lx TAE buffer followed by staining with ethidium bromide and were documented using UV protected gel documentation system. The DNA profiles were scored manually, directly from photographs of the gels, by assigning a value of 1 for band presence and 0 for band absence. The scores of band presence or absence were then used to calculate a pair-wise genetic distance matrix using different coefficients. Finally, a phenogram based on UPGMA cluster analysis of the genetic distance matrix was prepared with help of the NTSYS-pc program (Applied Biostatistic Inc. New York, 1993). Similarity of RAPD patterns was determined by the calculation of F- values.

Source of Information: Bangladesh J. Agril. Res. 35(2) : 313-322, June 2010 ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

From the genetic similarity index, the pair-wise genetic similarity coefficients indicated that the variety Faridpuri and Bhati are very much closest (83.87%) to each other followed by Bhati and BARI Peas-2 (81.25%), White and Faridpuri (80%).The results suggested that their genotypes are more similar to each other. So, in crop improvement programme, these genetically similar parents could not be chosen in the crossing programme to creat genetic variability. On the other hand, the lowest similarity (34.29%) was found in Bermis and India-2 as well as in Bermis and Faridpuri (40%), Bhati and Bermis (41.18%), White and BARI Peas-1 (41.18%), and Bermis and India-I (41.18%), White and Bermis (42.42%), BARI Peas-3 and Bermis (43.75%) and BARI Peas-1 and India-2 (44.44%). It means that Bermis and India-2 are the most dissimilar in their genetic level. So, these genetically dissimilar parents should be chosen in crop improvement programme for creating genetic variability.A dendrogram based on UPGMA cluster analysis of the RAPD data showed three clearly distinct groups of the 10 varieties. BARI Peas-1, OF-5 and Bermis are on the same cluster, but BARI Peas-1 and OF-5 are closer to each other. Again BARI Peas-2, Faridpuri, and Bhati are on the same cluster, but Faridpuri and Bhati are closer to each other. On the other hand, BARI Peas-3, India-2, India-1, and white are on the same cluster group. But BARI Peas-3 and India-2 are more closer to each other. It was also same in India-1 and White onion.The two dimensional graphical view of Principal Coordinate Analysis (PCA) showed the spatial distribution of the varieties along the two principal axes. The varieties viz., Bermis, BARI Peas-I, India-2, OF-5 and BARI Peas-2 were found placing far away from the centriod of the cluster and the rest of the varieties were placed more or less around the centriod. The results indicated that the varieties placed far away from the centriod were more genetically diverse, while the varieties placed near around the centriod possessed more or less similar genetic background. However, centriod may be defined as the vector representing the middle point of the cluster which contained at least one number for each variable. The connecting line between the each variety and the centriod represented eigen vectors for the respective varieties.

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