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Comparative Karyotype and Rapd Analysis for Characterizing Three Varieties of Lycopersicon Esculentum Mill

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

ELHAM ISHRAT
Department of Botany, University of Dhaka, Dhaka

MD YAHIA ZAMAN
Department of Botany, University of Dhaka, Dhaka

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

Abstract/ Executive Summary:

Three tomato varieties (Lycopersicon esculentum Mill.) viz. BARI tomato-11, BARI tomato-2 and BARI tomato-3 released from Bangladesh Agriculture Research Institute (BARI) were characterized by karyotype analysis and RAPD markers. BARI tomato-11 and BARI tomato-2 were found to possess 2n = 24 metacentric chromosomes. On the other hand, 2n = 25 metacentric chromosomes were observed in BARI tomato-3 revealing a trisomic variety. The karyotype of the three varieties was very symmetric and each of the varieties showed specific and unique RAPD fingerprinting. Therefore, based on karyotype and RAPD analysis the three tomato varieties could be characterized.

Keywords: Karyotype, RAPD, Tomato

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Tomato

Objectives:

In this study, three tomato varieties released from BARI were considered to - (a) confirm diploid chromosome number of the varieties, (b) elucidate aneuploidy or other chromosomal aberration and (c) characterize the varieties based on karyotypic analysis and RAPD fingerprinting

Methodology:

Three varieties of tomato namely, (i) BARI tomato-11 (Jhumka), (ii) BARI tomato-02 (Ratan) and (iii) BARI tomato-03 were used in this study. These varieties were collected from BARI and maintained in the Botanical Garden, Department of Botany, University of Dhaka, Bangladesh. Healthy roots of the grown plants from the field were collected and pretreated with 0.002 M 8-hydroxyquinoline for 30 min at 18°C followed by 15 min fixation in 45% acetic acid at 4°C. These were then hydrolysed in a mixture of 1 N HCl and 45% acetic acid (2 : 1) at 60°C for 8 s. The root tips were stained and squashed in 1% aceto-orcein. Tender 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 PCR reaction mixture for 25 μl contained template DNA (25 ng) 2 μl, de-ionized distilled water 18.8 μl, Taq buffer A 10× (Tris with 15 mM MgCl₂) 2.5 μl, primer (10 μM) 1.0 μl, dNTPs (2.5 mM) 0.5 μl and Taq DNA polymerase (5U/μl) 0.2 μl. PCR amplification was done in an oilfree thermal cycler (Biometra UNOII, Germany) for 46 cycles after initial denature at 94⁰C for 5 min, denature at 94⁰C for 1 min, annealing at 36⁰C for 30 s, extension at 72⁰C for 3 min and final extension at 720C for 5 min. Six primers of Operon Technologies, USA viz. Batch-7736-030 (GAA ACG GGT G), Batch-7736-031 (GTT GCG ATC C), Batch-7736-032 (TGC CGA GCT G), Batch-7736-033 (GGG TAA CGC C), Batch-7736-034 (TCA CGT CCA C) and Batch- 7736- 036 (CCC GCC TTC C) series were used. The PCR products were analyzed after gel electrophoresis. The photographs were critically examined 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 three tomato varieties using UPGMA (Sneath and Sokal 1973).

Source of Information: Bangladesh J. Bot. 41(2): 149-154, 2012 (December)

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Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results showed that BARI tomato-3 is quite different from the rest two varieties of tomato in respect to - (i) 2n = 25 chromosome instead of 2n = 24, revealing a trisomic nature. (ii) lowest total length of 2n chromosome complement (14.25 μm) and range of individual chromosome length (0.75-0.41μm), (iii) no RAPD fragment was found in primer batch 7736-031, 7736-034 and 7736-036, (iv) showed characteristic specific bands in primer 7736-030 and 7736-034 and (v) the tree dendrogram showed that this variety was placed in different cluster far away from other two varieties at linkage distance 17.5. With the help of above parameters, BARI tomato-3 could be easily characterized. This variety is a primary trisomic. Trisomic produces unbalanced gametes due to irregular distribution of chromosomes at anaphase and generally resulting in 50% sterile gametes. This lowers the yield of BARI tomato-3. Therefore, BARI authority must characterize tomato varieties before releasing those to the farmers.

Knowledge Management: Journal