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Molecular Characterization of Four Morphological Mutants of Neurospora Crassa

NASRIN AKTER
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

MD. AHASHAN HABIB
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

SALINA PARVIN BEAUTY
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

TAHSINA RAHIM
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

MOHAMMAD NURUL SLAM
mnurul@du.ac.bd
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Four morphological mutants (albino, ropy, conidial band and buff) of Neurospora crassa were characterized based on morphological features and molecular markers. The mycelial color of mutants buff, ropy and conidial band were more or less orange like wild parents . Albino mutant showed colorless mycelium. The germination time of conidia of wild and other mutants ranged from 5 to 7 days while in albino it was 12 days. Esterase and acid phosphatase isozymes analysis of the N. crassa mutants clearly indicated that mutation altered the carotenoid biosynthesis pathway creating the albino mutant due to the effect UV light. Most of the mutants viz; albino, conidial band and buff showed characteristic RAPD banding profile. However, no band was found in wild EmA, Ema and mutant ropy. Highest number of RAPD bands were found in albino. The mutant albino showed very different morphological and molecular features from the rest specimens.

Keywords: Morphological mutant, Molecular characterization, Neurospora crassa

Location: Department of Botany, University of Dhaka

Start Date:

End Date:

Program Area: Variety and Species

Non-commodity: Performance

Objectives:

To characterize the UV induced mutants by PAGE and RAPD markers and to find the correlation between morphological and molecular data.

Methodology:

Two wild ( EmA, Ema) and four induced morphological mutants (albino, ropy, conidial band and buff) of Neurospora crassa were investigated in this study. The wild type strains used in the study were received from Fungal Genetic Stock Centre, Department of Microbiology, University of Kansas Medical School, Kansas, USA. Vogel's minimal medium (VM) was used for the maintenance of cultures. Five-day-old conidia of Ema were irradiated with UV light having wave length of 254 nm. Radiation was given at a distance of 12 cm. Media supplemented with arginine, histidine, leucine, lysine and tryptophan were used for plating and isolating the irradiated conidia. The isolated conidial cultures were incubated at 25°C. Af ter 5 days they were observed and classified according to their morphological and nutritional requirements. Fresh conidia of 4 - 6 days old cultures of selected morphological mutants and Ema were inoculated at the centre of the VM plate for germination. Germination and radial growth of the conidia were observed under the microscope after 1 and 3 hrs intervals, respectively. Linear growth of morphological mutants and Ema were studied through inoculation of mutant and wild type N. crassa at one end of the Race Tube containing VM media solidified with 3% agar. Observations were recorded after 6 hrs interval up to 96 hrs. Mycelia were harvested and total genomic DNA was extracted by using CTAB method. DNA concentration was quantified through spectrophotometer (Analylikjena, Specord 50, Germany). The A260/280 nm readings for DNA samples were 1.6 - 1.8. The PCR reaction mix for 25 µl containing template DNA (25 ng/fll) 2 f1l, de-ionized distilled water 18.8 fll, Tag buffer A lOX (Tris with 15 mM MgCh) 2.5 fll, primer (l0f1M) 1.0 f11, dNTPs (2.5 mM) 0.5 fll, Tag DNA polymerase (5U/µl) 0.2 µl. PCR amplification was done in an oil-free thermal cycler (Biometra UNOII, Germany) for 46 cycles af ter initial denature 94°C for 5 min, denature at 94°C for 1 min, annealing at 36°C for 30 sec, extension at 72°C for 3 min and final extension at 72°C for 5 min. Four random primers were used for RAPD analysis- viz. Primer-10 (GCC ACG GAG A), Primer-11 (CAC GGC TGC G), Primer 12 (GTA TGG GGC T) and Primer-13 (GCG AAC CTC G).

Source of Information: Dhaka Univ. J. Biol. Sci. 23(1): 85-91, 2014 (January)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

With the help of RAPD scoring, a dendogram was made on the basis of genetic distance. The cluster analysis clearly indicated that the albino mutant was totally different from the rest five specimens. On the basis of genetic distance this mutant was separated initially into different clusters. The other five samples however, placed in the same cluster different from that of albino. This differentiation was correlated with the morphological characteristic and isozyme activities. Therefore, a combina tion of classical and molecular data it was possible to characterize different Neurospora crassa mutants.

Knowledge Management: Journal