© by PSP Volume 22 – No 12. 2013 Fresenius Environmental Bulletin 3567 ASSESSING INTRA-SPECIFIC VARIATION WITHIN BACILLUS SPP. STRAINS USING DNA FINGERPRINTING Ömür Baysal* Muğla Sıtkı Koçman University, Faculty of Life Sciences, Department of Molecular Biology and Genetic, P.B. 48000 Kötekli Muğla, Turkey ABSTRACT Bacillus subtilis strains are known to be prominent biologics playing different roles both in biological degra- dation of organic compounds and bio-control of plant dis- eases. In the present study, a schematic diagram was im- proved using efficient ISSR markers, selected in previous screening studies and suggested in view of their cost and labor-effective roles in discrimination of Bacillus spp. strains. To assess their efficiency and use in diversification of Bacillus spp. strains, whole findings were compared with 16S rDNA analysis. The results by selected Inter- Simple Sequence Repeats (ISSR) markers showed conven- ient, highly reproducible results to be a tool for revealing genetic relationships in Bacillus spp. strains, with regard to 16S rDNA. Even ISSRs have no informative knowledge based on gene sequence. Quick studies on structure and dynamics of microbial communities in ecosystems are of importance. Therefore, ISSR fingerprinting is suggested to be an efficient tool in intraspecific discrimination of Bacillus subtilis strains in further studies. KEYWORDS: Molecular markers, intra-specific variation, 16S rDNA, ISSR, Bacillus spp. 1. INTRODUCTION Studies on the structure and dynamics of microbial communities are relevant in microbial ecology to develop bioremediation strategies. For identification of a microbial agent, polymerase chain reaction (PCR) has been success- fully used in different microbiological studies. Particularly, the gene commonly amplified in such cases codes for 16S rDNA, which is a highly conserved region both within a species and among species of the same genus, and is essen- tial for the survival of living organisms [1]. Therefore, 16S rDNA analysis is also considered to be a standard method for the identification of bacteria at family, genus and spe- cies levels [2]. However, further discrimination of individ- ual strains within a given species is needed in order to understand the speciation and host preference occurrence * Corresponding author depending on pathogenic behavior. Consequently, a num- ber of fingerprinting methods have been developed in recent years to discriminate between strains belonging to a given species, such as random amplification of polymor- phic DNA [3] and amplified fragment length polymor- phism (AFLP) analysis [4]. However, these methods are not cost-effective. Inter-Simple Sequence Repeats (ISSR) are dominant markers and detect polymorphisms in mi- crosatellite and inter-microsatellite loci, and do not re- quire prior information of DNA sequences [5]. In contrast to other molecular markers, the target sequences for ISSR primers help in revealing higher numbers of polymorphic fragments than RAPD (Random Amplified Polymorphic DNA) markers. On the other hand, there is also no strict correlation between function of Bacillus isolates and their genotypic and phenotypic variations [6]. A relatively recent study indicated that the function of a large number of genes in B. subtilis remains unknown [7]. Most of the available evi- dence concerning genotypic variation among different Ba- cillus isolates has been acknowledged with assessment of phenotypic variation and cell wall chemistry [8] but not sufficiently informative on genus-based diversification. In the present study, a schematic list using ISSR mark- ers was developed for easy discrimination of Bacillus strains to examine the species variability and to differenti- ate closely related strains at the subspecies level, which was also evaluated with the results obtained by 16S rDNA analysis confirmations. 2. MATERIAL AND METHODS 2.1 Isolation, selection and identification of bacteria In this work, 12 samples were isolated from green- house soils collected during the Methyl Bromide Phase Out Project of the Ministry of Agriculture in Turkey, within 2003-2010. The bacterial suspensions were stored in tubes containing King B medium at 4 ºC, and in flasks con- taining TSB (tryptone soy broth) plus glycerol (20%, v/v) at -20 ºC until use. Whole isolated bacteria were distinguished from other B. subtilis colonies according to bacteriology notes [9], including some tests. Afterwards, identification of each strain was confirmed with 16S rDNA sequences