Molecular Identification of Aspergillus flavus Using RAPD Markers

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.7(2)pp47-54(2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.


Aspergillus flavus RAPD
The aim of this study is to isolate and identify A. flavus and study the genetic diversity among these isolates by using RAPD.Eleven collected samples were characterized depending on its morphological state, then DNA was extracted from them.RAPD markers are randomly banding with sites of genome more than, markers, where the primer UBC 809achieved discriminative power (19.1) and 43 bands, while6 achieved discriminative power (17.1) with 32 bands.There were more efficiency in specific binding, then RAPD primers have great binding to produce unique band, when 9 primers from 10 primers, 9 produced (5) unique bands, while RAPD markers showed low ability to produce unique bands, 3primers from 9 primers were produced as unique bands.The dendrogram of RAPD was reverted than isolates number 5 and 7 which had the genetic diversity 0.33361, while the isolates number 5 and 6 had the lowest genetic similarity 0.98521 in contrast with markers which showed isolates number1 and 2 genetic diversity 0.97826 while the isolates number 5 and 7 had the lowest genetic similarity 0.10253.
Random Amplified Polymorphic DNA (RAPD) analysis can be performed as a method for study genetic diversity with large number of different strains of microorganisms.It is inexpensive and requires less amount of DNA [Bornet et al., 2001].Moreover, RAPD analysis is technically being commonly used as an indicator for determination the genetic diversity, while Inter-simple sequence repeat-technique analysis based on variation found in the regions between microsatellites has been used in genetic fingerprinting gene tagging and detection of clonal variation [McPherson, 2001].This technique which involved amplification of DNA segment present in between two identical microsatellite repeats regions by addition the oriental in opposite direction with suitable distances.This method has been reported to produce more complex markers patterns than the RAPD markers.In addition, this method is more reproducible than RAPD method because primers are designed to anneal temperature to microsatellite sequences which are longer than RAPD primers, allowing higher annealing temperature to be used.It also because of multi locus finger printing profile obtained, it has been found to be an efficient, low cost, simple operation, and high stability [Zietkiewics et al., 1994].The aim of the studyis the detection of the unique bands and polymorphism between isolates and comparative study between RAPD and markers for genetic diversity between different A. flavus isolates.

Aspergillus flavus Isolates
A total of 11 A. flavus isolates were isolated from Zea Mays grains and were examined according to their microscopic features, and were sub cultured on sabouraud Dextrose Agar for using in DNA extraction.As shown in Figure 1(a) and (b).

Genomic DNA Extraction
The DNA was extracted by smallscale method commercial kit (Bionner-Korea).DNA Purity was measured depending on optical density by spectrophotometer.DNA quality was visualized by agars gel electrophoresis with ethidium bromide and visualized under UV light [Sambrook et al., 2001]

Molecular Analysis RAPD Assay
Three of RAPD primers were used in this study, the primers was synthesd by (Bioneer-Korea) in lyophilized form and dissolved in sterile distilled water to get  Amplification of genomic DNA was performed with the following master amplification reaction (Table 2).RAPD-PCR premix (final reaction volume = 20 µI).No. of cycles = 40 cycles between initial denaturation and final extension, the following table shows the RAPD program (Table 3).Followed by a hold at 4°C [Hatti et al., 2010], each PCR amplification reaction was repeated twice to ensure reproducibility of the products analyzed by electrophoresis in a 1.5% agars gels with 0.5µl stained ethidium bromide at 7vt/cm for 3hours.
Each PCR amplification reaction was repeated twice to ensure reproducibility of the products analyzed by electrophoresis in a 1.5% agars gels with stained ethidium bromide 0.5µl at 5vt/cm for 2hour.

Data Analysis Estimation of Molecular Weight
Computer software Photo-Capture M.W. program was used to determine molecular weight based on comparing the RAPD-PCR and -PCR products depending on molecular weight of bands and number bands of a 2000bp DNA ladder Bioneer (which consist of 13 bands from 100 to 2000 bp.).

Estimation of Polymorphism, Efficiency, and Discriminatory Power
Data generated for molecular weight RAPD and markers result bands were a score for each bands on the molecular size (1 for present, 0 for absence) the commercial soft word [Bibi et al., 2010].Only major bands consistently amplified were scored.Polymorphism of each primer was calculated based on the following formula: Polymorphism % = (Np / Nt) × 100 Where Np = the number of polymorphic bands of random primer and Nt = the total number of bands of the same primer.Efficiency and discriminatory power of each primer were calculated according to the formula below: • Efficiency=number of polymorphic bands to each primer / total number of bands to the same primer.• Discriminating power= number of polymorphic band to each primer / total number of polymorphic band to all primer X100 %.Primer efficiency ranged between (0-1).Discrimination power of each primer

RESULTS AND DISCUSSION RAPD-PCR Analysis
Tables 4, 5, 6 &7 and Figs. 2, 3, 4 & 5 summarize all information obtained from RAPD assay, and based on RAPD assay, the data developed from the PCR analysis demonstrated that some primers generate several bands, while other generates only a few bands.A total of three RAPD primers were used for studying the genetic differences between eleven A. flavus isolates, amplified 341 bands,126 bands were polymorphic, with average of (3-43) polymorphic bands, that OPD-20 produce 3 polymorphic bands only, were OPE_16 can be produce 43 polymorphic bands with average range size (100-2000)bp.(Fig. 2).Some isolates could be distinguished from all other isolates with selection of these primers, for instance OPE_16 primers can produce higher discrimination power 19.1 bands only, while OPL-05 gave 2 unique bands patterns.
Table 4:The polymorphic, monomorphic and unique bands with their molecular weight for primer OPE_16.
Table 5:The polymorphic, monomorphic and unique bands with their molecular weight .
Table 6:The polymorphic, monomorphic and unique bands with their molecular weight for primer OPc_16.
Table 7: Distinct characteristics of , primers including in the study: primers name, total number of bands, number of polymorphic bands, number of unique bands, percentage of polymorphism, primer efficiency and discrimination value.
Cluster analysis illustrated genetic relationship among seven of A. flavus isolates showing two major clusters (Figure5 and Table 6), the first cluster contained two main groups, first group, 5 and 7 isolated in one sub group cluster with low genetic distance 0.3336.These were introduced from environmental sources and isolated number 1 formed separated line due to different in isolate source, while isolate number 2 and 6 formed another sub clusters with genetic distance 0.48652.These isolates were introduced from environmental and clinical sources.Second group contained isolate number 3 only, during clusters analysis showing the levels of genetic relatedness also dendrogram indicates difference between isolates based on source of the isolates.Present result showed multiple differences in isolates of A. flavus which came from two factors including genetic factor and environment factor, also the results indicate that the clinical isolate has greater genetic variability than the environment isolates during gene distance and dendrogram.Genetic difference may come from clinical ones, on the other hand the clinical isolates of patients constitute one group, according to genetic characteristic, with the environmental isolates.Genetic difference observed in this study come from adept fungi to grow and isolates that infected patients to reactive and generally more variability in relation to the original strain [Latge,2010].Genetic diversity may be attributed to mutation or recombination that occurs in fungal cell into resistance to anti-mycotic treatment or under environmental stress [Tramutoli,2005].Environmental and clinical isolates of A. flavus may be different in genotype consisted of gene involved in transport, regulation of transcription, and metabolism of molecular with 1-3 carbon and paroxysm all proteins [Gercia et al., 2011;Hynes et al., 2006].
In this study, each of genetic distance based on RAPD markers doesn't show geographic profiling between isolates.It has been reported that the dendrogram generated by markers is better with genealogy and the pedigree of the markers than RAPD results.On the another hand, it has been found that the data on RAPD genetic distance have more relationship with the geographic distribution in comparative with markers data that based on number of chromosomes.Markers are highly polymorphic and are useful in studies on genetic diversity [K. S. Wu et al.,1994].Numbers of analysis studies used both markers and RAPD technique and found that markers produce more information with fewer numberof primer than the number of RAPD primers.During this study it was found that a number of polymorphic bands were still higher [Lanham and Brennan, 2000;Nagoaka, 1979].Less primers means less time, less DNA, less supplied, and less samples.RAPD markers don't have the specific target comparing to markers.In fact, markers are known to be more sensitive than RAPD markers.In this study, it was obvious that the dendrogram based on RAPD markers was not in accordance with the dendrogram based on markers, thus, both dendrogram are in agreement with the groups of geographic origin, but RAPD markers greatly agree with these groups than markers.The differences in clustering pattern of genotypes using RAPD and markers also may be attributed to markers sampling error and the level of polymorphic detected [M.E. Ferandez et al., 2012].

CONCLUSION
Markers produced high rate from polymorphism depending on polymorphic rate.The technique can produce high level from unique bands a comparative with another markers that are less efficient in dendrogram results.

Fig. 1
Fig. 1(b): Top view of A. flavus under on SDA at 25-27°C after 7 days of

Fig. 5 :
Fig. 5: Dendrogram illustrated genetic fingerprint and relationship between A. flavus isolates developed from RAPD data.

Table 1 :
The names and sequences of the primers used in this study.

Table 3 :
The RAPD program.

Table 8 :
values of genetic distance between A. flavus Isolates calculated according toNei and Lei,  1979.