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Karyotype and Rapd Analysis in Five Potato Varieties (solanum Tuberosum L.)

RIFFAT ARA ALAM
Department of Botany, University of Dhaka, Dhaka

MD AHASHAN HABIB
Department of Botany, University of Dhaka, Dhaka

SHEIKH SHAMIMUL ALAM
Department of Botany, University of Dhaka, Dhaka

Abstract/ Executive Summary:

Two native and three high yielding potato varieties available in Bangladesh were studied cytogenetically at molecular level to get a preliminary idea about their genomes. These varieties possess 2n = 4x = 48 chromosomes. Each variety has definite centromeric formula. Presence of maximum metacentric chromosomes suggested their karyotypes as symmetric type. The range of individual chromosomal and total length of 2n chromosome complements was also distinct in five varieties. Heteromorphicity regarding centromeric position suggesting the occurrence of structural aberration in respective homologous pairs. Seven primer combinations were used for RAPD finger printing. A few common RAPD bands suggested the sharing of genomic fragments among five varieties. However, each variety showed specific characteristic RAPD bands. Therefore, each variety has characteristic karyotype and RAPD markers.

Keywords: Karyotype, RAPD, Potato

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End Date:

Program Area: Knowledge Management

Commodity: Potato

Objectives:

The aim of the present study was (i) to find out 2n chromosome number of each variety, (ii) to elucidate ploidy level, (iii) to find other karyotype features, (iv) to make a RAPD finger printing and (v) to construct a phytogenetic dendogram on the basis of RAPD finger printing.

Methodology:

Five varieties of potato such as Jaam, Laal Sheel, Diamant, Cardinal and Granola were studied in this experiment. Jaam and Lal Sheel varieties were collected from Mymensingh and Bogra, respectively. Whereas, Diamant, Cardinal and Granola were collected from Bangladesh Agricultural Research Institute (BARI). All these varieties were germinated and maintained in the Botanical garden, Department of Botany, University of Dhaka, Bangladesh. Healthy roots were collected and pretreated with 0.002 M 8-hydroxyquinoline for 30 min at 18ºC followed by 15 min fixation in 45% acetic acid at 4ºC. These were then hydrolyzed in a mixture of 1N HCl and 45% acetic acid (2 : 1) at 60ºC for 20 sec. The root tips were stained and squashed in 1% aceto-orcein for 2 h at room temperature and squashed. Leaves were harvested and total genomic DNA was extracted by using modified CTAB method (Doyle and Doyle 1987). DNA concentration was quantified through spectrophotometer (Analylikjena, Specord 50, Germany). The A260/280 readings for DNA samples were 1.6 - 1.8. The amplified products were separated electrophoretically on 1% agarose gel. The gel was prepared using 1.0 g agarose powder containing ethidium bromide 8 μl and 100 ml 1× TAE buffer. Agarose gel electrophoresis was conducted in 1× TAE buffer at 50 volts and 100 mA for 1.5 h. DNA ladder 1kb was electrophoresed alongside the RAPD reactions as marker. DNA bands were observed on UV-transilluminator and photographed by a gel documentation system. The PCR products were analyzed after gel electrophoresis. The photographs were critically discussed on the basis of presence (1) or absence (0), size of bands and overall polymorphism of bands. These were carried out for further investigation. RAPD analysis was then combined to create a single data matrix. This was used for estimating linkage distance (D) and constructing a UPGMA (Unweighted Pair Group Method of Arithmetic Means) Dendrogram among the varieties using computer program “Statistica”. Linkage distances were computed from frequencies of polymorphic markers to estimate genetic relationship between the studied five potato varieties using UPGMA (Lakhanpaul et al. 2000).

Source of Information: Bangladesh J. Bot. 41(1): 105-110, 2012 (June)

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Achievement/Findings:

RAPD is a unique tool for obtaining a clear DNA finger printing of a particular specimen. Each primer combination finds corresponding fragments after PCR reaction. In this study, 7 different primer combinations were tried to get clear polymorphic DNA finger printing in 5 varieties of potato. The five varieties showed some common RAPD bands in different primer combinations revealing the sharing of similar fragments. This also indicates that those fragments had come from common ancestors. In spite of some common bands, each variety had unique polymorphic bands in different primer combination. These bands were not present in other varieties. Therefore, each variety has its own diagnostic RAPD finger printing. On the basis of RAPD finger printing, a cluster analysis was carried out to elucidate the phylogenetic relationship among the five varieties of potato. The cluster analysis showed that the variety Granola was totally different from the rest followed by Jaam at linkage distance 19.0 and Lal Sheel at 16.8. Diamant and Cardinal showed close relationship at linkage distance 7.0. Therefore, it showed that each variety of potato has specific karyotype and RAPD markers.

Knowledge Management: Journal