Pakistan Journal of Biological Sciences

Year: 2016 | Volume: 19 | Issue: 4 | Page No.: 143-157
DOI: 10.3923/pjbs.2016.143.157

Characterization of Cladosporium Species by Internal Transcribed Spacer-PCR and Microsatellites-PCR

Mohammed S. Alhussaini, Mohamed Adbo Moslem, Mohammed I. Alghonaim, Abdullah A. Al-Ghanayem, Abdulrahman A.I. AL-Yahya, Hamido M. Hefny and Adbul Moneam Saadabi

Abstract: Background: This investigation compared genetic similarities and diversities within and among Cladosporium species populations using the two PCR-based markers; Internal Transcribed Spacer (ITS)-PCR and microsatellite-PCR. Methodology: Nuclear ribosomal DNA internal transcribed spacers have been used to analyze intraspecific and interspecific relationships in various fungi. In the present study, the internal transcribed spacer (ITS)-PCR and microsatellite-PCR were used to identify the genetic diversities in Cladosporium species. Results: The Internal Transcribed Spacer (ITS) was amplified using polymerase chain reaction combining primers ITS4 and ITS5. The PCR products were digested with three restriction enzymes and separated by agarose gel electrophoresis. Restriction patterns generated by CfoI and Msp I and RsaI were unique for most species assayed. The ITS-PCR fingerprinting methods led to a clear differentiation of the isolates at the species level. Fingerprinting profiles generated readily discriminated between each of the 6 species. Cluster analysis further supported this observation and clusters corresponding to each species were readily identified in the dendrograms. Seven microsatellite primers out of eight primers were unable to generate visible DNA fingerprints. Conclusion: Amplification experiments demonstrated that microsatellite primer, T3B and (GTG)₅ are technically simple tools for assaying genetic variability in Cladosporium spp.

Keywords: RsaI, MspI, ITS-PCR, microsatellite, Cladosporium, DNA fingerprinting and CfoI

INTRODUCTION

Internal Transcribed Spacer (ITS) regions of the ribosomal DNA (rDNA) repeat unit are well-investigated sequences that are present in multiple copies and can be easily isolated by PCR amplification. The ITS regions usually vary between species within a genus. Hence, they have been used in numerous phylogenetic studies of various fungi¹. Analysis of the ITS region is used to distinguish species and higher taxonomic divisions². The DNA fingerprint has been used to study several genera. Restriction Fragment Length Polymorphism (RFLP) of the ITS region is used to distinguish between species within same genera and also species from different genera³.

Microsatellites, also known as Short Tandem Repeats (STRs) and Simple Sequence Repeats (SSRs) are tandem repeats of simple dinucleotide, trinucleotide, tetranucleotide, pentanucleotide or hexanucleotide sequence (two to six bases) that occur abundantly and at random in most eukaryotic genomes⁴. They are typically short and embedded within a unique sequence, thus being ideal for designing flanking primers for in vitro amplification by the Polymerase Chain Reaction (PCR)⁵. Microsatellite markers can be used to detect polymorphism in plants, fungi and animal genomes^6,7. The stability displayed by microsatellites make them perfect markers for use in constructing high-resolution genetic maps to identify susceptibility loci involved in common genetic diseases. They can be used for generating attractive molecular markers that can show codominant inheritance, multi-allelism⁸ and random distribution throughout the genomes⁹.

The aim of this study is to explore the genetic variation between various isolated Cladosporium strains using the Internal Transcribed Spacer-Polymerase Chain Reaction (ITS-PCR) and microsatellites (MP-PCR).

MATERIALS AND METHODS

Fungal isolates: The ITS-PCR and MP-PCR techniques were performed in 23 fungal isolates. Four reference isolates brought from Central bureau; voor Schimmelcultures, Utrecht (The Netherlands) were used as control; Cladosporium cladosporioides (CBS 131.29), C. herbarum (CBS 673.69), C. sphaerospermum (CBS 114326) and C. tennuissimum (CBS 117134). The other 19 fungal isolates, Clad#1 through Clad#19, were four isolates of C. cladosporioides (Fresenius) de Vries, seven isolates of C. sphaerospermum Penzig, five isolates of C. herbarum (Persoon) Link, two isolates of C. macrocarpum Preuss and one isolate of C. chlamydosporis Matsushima. The order of the isolates that resulted in the dendrograms are described in Table 1.

Fungal culture and DNA preparation: Genomic DNA was extracted from 5-7 days old cultures of the fungal isolates, grown in micro-culture tube containing 3 mL of malt extract broth at 25°C. Mycelia were harvested by filtration through two layers of cheesecloth and washed with sterile distilled water. Samples were frozen in liquid nitrogen and were ground to fine powder using mortar and pestle. The DNA was extracted using a modification of the traditional Sodium Dodecyl Sulphate (SDS) extraction procedure reported by Abd-Elsalam et al.¹⁰.

Internal Transcribed Spacer-PCR (ITS-PCR) conditions: The PCR was conducted in a 25 μL reaction volume. Three microliters of DNA template (1 ng quantified using a spectrophotometer) was added to a 5 μL master mix of thermostable DNA polymerase (Jena Bioscience GmbH, Cat. No. PCR-101S), 13 μL of PCR grade water (Jena Bioscience GmbH), 2 μL of 10 pmol of ITS4 primer and 2 μL of 10 pmol of ITS5 primer (GE Healthcare). The thermal cycling parameters were carried out using a thermal cycler (Techne TC-312, Techne, Stone, UK) as follows; initial denaturation at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min, then final extension at 72°C for 10 min. The PCR product was checked on 1.5% agarose containing 0.05 μg mL^–1 ethidium bromide. Photographs were taken after 30 min¹⁰.

ITS-PCR product digestion with restriction enzymes: Internal transcribed spacer fragments were digested using three base cutter restriction enzymes: CfoI, MspI and RsaI. Fifteen microliters of amplified DNA was digested for 2 h at 37°C using a DNA thermal cycler (Techne TC-312, Techne, Stone, UK) as follows; 15 μL of ITS-PCR product, 1 μL of enzyme (1 U), 2.5 μL of buffers provided by the manufacturer and 2.5 μL of PCR grade water (Jena Bioscience GmbH). The resulting DNA fragements were electrophoresed on 1.7% agarose containing 0.05 μg mL^–1 ethidium bromide (Agarose 25). The DNA patterns were visualized and photographed under ultraviolet light (UV)¹¹.

Microsatellite-PCR (MP) conditions: Microsatellite-PCR was conducted in a 25 μL reaction volume. Three microlters of DNA template (1 ng quantified using spectrophotometer) was added to a 7 μL master mix of thermostable DNA polymerase (Jena Bioscience GmbH), 13 μL of PCR grade water (Jena Bioscience GmbH), 2 μL of 20 pmol Microsatellite primer. Microsatellite primers used were; (AGG)₅, (CAG)₅, (GACA)₄, (GTG)₅, (GTGC)₄, (AG)₈C and T3B. Primers were brought from Metabion International AG GmbH. The thermal cycling parameters were as follows; initial denaturation at 94°C for 4 min, followed by 35 cycles consisting of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 2 min and final extension was achieved at 72°C for 7 min. The PCR product was checked on an 1.5% agarose containing 0.05 μg mL^–1 ethidium bromide photographs were taken after 30 min¹⁰.

RESULTS

The current investigation evaluated the potential of DNA fingerprinting molecular marker techniques to discriminate Cladosporium isolates.

Molecular characterization and genetic diversity of Cladosporium species as revealed by Internal Transcribed Spacer (ITS) PCR
Internal Transcribed Spacer (ITS) PCR patterns of 23 Cladosporium spp. isolates digested with CfoI: The ITS-PCR fingerprint patterns were analyzed using PC-windows software; BandMap package. Following conversion, normalization and background subtraction by using mathematical algorithms, levels of genetic similarity between ITS-PCR fingerprints were calculated by Pearson product-moment correlation coefficient. A cluster analysis was performed by using the UPGMA algorithm. The genetic similarity between Cladosporium isolates ranged from 50-68% for inter-specific and 68-100% for intra-specific comparisons. The average of genetic similarity based on ITS-PCR patterns digested with CfoI was approximately 50%. Dendrogram of the ITS-PCR patterns digested with CfoI separated the isolates of Cladosporium spp. into two main clusters. The first main cluster included all Cladosporium cladosporioides isolates at the genetic similarity (GS = 76 %). Isolates 2, 3 and 4 showed genetic similarity (GS = 100%) (Fig. 1).

Internal Transcribed Spacer (ITS) PCR patterns of 23 Cladosporium spp. isolates digested with MspI: The dendrogram constructed with ITS-PCR digested with MspI revealed that all isolates of C. cladosporioides and most of the C. sphaerospermum can be grouped into a major cluster delimited from other Cladosporium spp. This is in good agreement with the classification by morphological characteristics (Fig. 2).

Internal Transcribed Spacer (ITS) PCR patterns of 23 Cladosporium spp. isolates digested with RsaI: The genetic similarity between Cladosporium spp., isolates ranged from 20-42% for inter-specific and 42-100% for intra-specific comparisons.

The application of UPGMA clustering produced two large clusters within the population with a branched-off at genetic similarity of GS = 20%, each consisting of several subclusters (phenons). Dendrogram of the ITS-PCR patterns digested with RsaI separated the isolates of Cladosporium spp., into two main clusters (Fig. 3).

Molecular characterization and genetic diversity of Cladosporium species as revealed by microsatellite-primed PCR (MP-PCR)
Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (AG)₈C primer: The (AG)₈C-primed PCR were normalized and analyzed using the BandMap software. This program calculates the Genetic Similarities (GS) and differences between each MP-PCR patterns, with the Pearson’s product moment correlation coefficient (r) between samples to construct a matrix.

The samples were then clustered using the unweighted pair group method of arithmetic average (UPGMA), which resulted in a dendrogram. The dendrogram showing the genetic relationship among the Cladosporium strains based on the total number of amplified MP-PCR fragments is presented in Fig. 4. The genetic similarity between Cladosporium isolates ranged from 10-14% for inter-specific and 80-100% for intra-specific comparisons. The application of UPGMA clustering produced two large clusters within the population with a branched-off at genetic similarity of GS = 14%, each consisting of several subclusters (phenons).

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (AGG)₅ primer: All 23 isolates of Cladosporium spp., used in this study were typed by PCR amplification with primer (AGG)₅, producing a fingerprinting profile. Under the conditions stipulated, MP-PCR using the proposed primer combinations produced an average of 6 or 18 DNA fragments per isolate. Based on the cluster analysis of the genetic similarities using UPGMA clustering, two main clusters were defined (Fig. 5). Between Cladosporium spp., similarities ranged from 25-28% for inter-specific comparisons. The genetic similarities among all taxa ranged from 28-100% for intra-specific comparisons. The differentiation power of (AGG)₅ primer profiling for identification of Cladosporium spp., isolates was relatively poor.

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (CAG)₅ primer: Figure 6 shows the results of cluster analysis of the MP-PCR profiles. It is noteworthy that this technique yielded 6-13 isolate-specific patterns. The UPGMA analysis of the (CAG)₅ data separated the Cladosporium spp., isolates into two main groups, each of which shared about 20% similarity. Between Cladosporium spp., isolates, similarities ranged from 22-28% for inter-specific and 30-100% for intra-specific comparisons. The two Cladosporium herbarum isolates 14 and 16 share high genetic similarity value (100%). The differentiation power of (CAG)₅ primer profiling for identification of Cladosporium spp., isolates was relatively poor.

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (GACA)₄ primer: Based on genetic similarity values (GS), UPGMA cluster analysis was conducted to graphically display grouping between isolates and species. The clustering resulted in a single tree with no ambiguity (ties) detected in the course of linking closest pairs of isolates. Between Cladosporium spp., isolates, similarities ranged from 40-58% for inter-specific and 58-78% for intra-specific comparisons. Isolates from six Cladosporium species could be separated into two main clusters (Fig. 7). Dendrogram of the (GACA)₄ primer did not provide clear-cut differentiating features between Cladosporium spp., isolates.

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (GTG)₅ primer: The phenogram obtained by cluster analysis of the (GTG)₅-PCR data revealed a moderate level of genetic variability between 23 geographically diverse isolates of Cladosporium spp. between Cladosporium spp., isolates, similarities ranged from 60-80% for inter-specific and 80-100% for intra-specific comparisons. Isolates from six Cladosporium species could be separated into two main clusters (Fig. 8). Some of the Cladosporium spp., isolates that had been identified as a same species showed a tendency to cluster.

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with (GTGC)₄ primer: The (GTGC)₄ banding patterns obtained by agarose gel were concatenated and analyzed as a composite by BandMap software. The normalization setting used were as follows: A resolution of 500 points, a smoothing factor of 3 and background subtraction by the rolling-disk method with intensity setting of 12 and the patterns were clustered by unweighted pair group method of arithmetic average. By UPGMA, all the (GTGC)₄-PCR markers were used to estimate the genetic relatedness among Cladosporium spp., isolates. The obtained dendrogram (Fig. 9) depicts that all isolates were separated from each other into two distinct groups (similarity are above 40%). The genetic similarity between Cladosporium spp., isolates ranged from 40-48% for inter-specific and 50-100% for intra-specific comparisons. The two Cladosporium herbarum isolates 18 and 19 share high genetic similarity value (100%).

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with T3B primer: Two major groups were observed in the dendogram (Fig. 10), which was divided into four subgroups. The average between-group genetic similarity was observed to be 50% between all Cladosporium spp. groups. Between Cladosporium spp., similarities ranged from 50-52% for inter-specific comparisons. The genetic similarities among all taxa ranged from 52-95% for intra-specific comparisons.

Microsatellite-primed PCR (MP-PCR) patterns of 23 Cladosporium isolates obtained with M13 primer: An UPGMA dendrogram produced by using BandMap software analysis, between all studied individuals is shown in Fig. 11.

The tree obtained indicated that the populations of Cladosporium spp., formed a distinct group and genetic similarity between these isolates was high (48%). There were no clear-cut kinships between clustering in the M13 dendrogram and morphological characterization of Cladosporium spp.

DISCUSSION

There has been an increasing interest in the application of Polymerase Chain Reaction (PCR) technology for the identification of pathogenic fungi. This technique offers the advantage of reducing or eliminating the need for lengthy culturing and difficult morphological identification procedures¹². The DNA-based techniques provide practical markers for molecular typing of a range of fungal species. These methods are commonly used as tools in fungal taxonomy, allowing the discrimination of isolates from intrageneric to strain levels¹³. The PCR-based genomic fingerprinting is a good alternative to methods that rely on specifically targeted primers.

These techniques, which analyze the whole genome, have been shown to be relatively robust and discriminatory¹⁴.

Nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) have been used successfully to analyze intraspecific and interspecific relationships in various fungi¹⁵. The anamorph genus Cladosporium is one of the most widespread and prevalent of all fungal genera¹⁶. It lacks morphological structures that would firmly place species in an evolutionary context. Molecular data are therefore important to reveal phylogenetic relationships within this genus. In an earlier study reported by Curtis et al.¹⁷, partial rDNA sequences of several independent isolates of C. fulvum (syns. Fulvia fulva, Mycovellosiella fulva) and single isolates of C. herbarum, C. oxysporum, C. cladosporioides and C. sphaerospermum indicated that these species together might form a monophyletic clade.

In the present study, the Internal Transcribed Spacer (ITS) was used to compare the ITS in length and restriction patterns. The Internal Transcribed Spacer (ITS) was amplified using PCR combining primers ITS4 and ITS5. The PCR products were digested with three restriction enzymes and separated by agarose electrophoresis. Restriction patterns generated by CfoI and MspI and RsaI were unique for most species assayed. Clear results were obtained by using ITS-PCR in the present study. The results were consistent with those based on biological characteristics and morphological features. The ITS-PCR fingerprinting methods presented here led to clear differentiation of the isolates at the species level.

Fingerprinting profiles generated discriminated between each of the 6 species. Cluster analysis further supported this observation and clusters corresponding to each species could be identified in the dendrograms. Our results support an earlier report that ITS molecular marker technique represents a possible method for the classification of Cladosporium spp.¹¹. Additionally, the ITS restrictions profiles showed a great genetic similarity between C. cladosporioides and almost all of C. sphaerospermum isolates.

Microsatellites (tandem repeats of 1-5 base pairs) and minisatellites (tandem repeats of a basic motif 10-60 bp long) are ubiquitous components of eukaryotic genomes. The fingerprinting methods referred to as microsatellite-primed polymerase chain reaction (MSP-PCR) and repetitive-sequence based polymerase chain reaction (rep-PCR) are used to discriminate between fungal species¹⁸.

Primer (AG)₈C could not discriminate between the two Cladosporium spp., isolates 12 and 13 as they share high genetic similarity value (100%). The differentiation power of (AGG)₅ and (CAG)₅ primer profiling for identification of Cladosporium spp., isolates was relatively poor. Six microsatellites primers from eight primers were unable to generate visible DNA fingerprints. It is possible that these repeats are not present in the genomes of these fungi. Another possibility is that the primer annealing sites are at such a distance that amplification by Taq polymerase is impossible. Amplification experiments demonstrate that microsatellite primer, T3B and (GTG)₅ are technically simple tools for assaying genetic variability in Cladosporium spp.

CONCLUSION

To conclude, in the present study, different primers provided varying levels of discrimination between morphological group representatives. Cluster analysis demonstrated that there is a genetically distinct variation among Cladosporium spp. These data confirmed the hypervariability of the PCR-based markers.

REFERENCES