Background and Objective: Morphological traits of the genus Colletotrichum are extremely variable and hosts might be infected by a single or multiple species of the pathogen, hence development of species-specific primers has provided a powerful tool for the detection of plant pathogens. The objective of the study was to assess efficiency of Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) primers against C. gloeosporioides in terms of genetic variability. Materials and Methods: Random amplified polymorphic DNA and inter simple sequence repeat primers are simple PCR-based assays targeting microsatellite regions of the genome. Intra and interspecific polymorphisms among the twentysix isolates of C. gloeosporioides, causing anthracnose on mango were evaluated by these markers. Results: In the present research, RAPD primers generated 519 amplicans with 82 alleles of which 77 were polymorphic with 7.4 assay efficiency index; ISSR markers produced 1469 amplicans with 189 amplification products, out of which 180 were polymorphic with 11.81 assay efficiency index. Although both the techniques were efficient and reproducible, ISSR indicated higher genetic variability in terms of percent polymorphism, polymorphism information content and effective multiplex ratio compared to RAPD analysis. Among 16 ISSR primers (CAG)5, (TGTC)4, (AGG)5, (TCC)5, (CAG)3, (AG)8 T, (GA)8 T, (TG)8 A, (GA)8 YG, (GT)8 YC recorded highest percent polymorphism and highest PIC value of 0.97 by (GA)8 T and EMR of 23.04 by (GACA)4 was recorded. Conclusion: It is concluded that well-chosen ISSR primers could result in quick estimate in terms of their efficiency in detecting polymorphisms among the isolates.
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Colletotrichum gloeosporioides, anthracnose diseases of mango is more prevalent and severe in humid areas, affecting mango tress with maximum disease incidence. Genome sequencing has virtually opened a door for characterization and evaluation of genetic diversity within and between species and populations using molecular markers. It has been showed that different markers might reveal various classes of diversity1,2. It is correlated with the genome fraction surveyed by each kind of marker, their distribution throughout the genome and the extent of the DNA target which is analyzed by each specific assay3. Though, genetic variability among the fungal population limited by the proteins and isozymes, correlation of molecular markers to morphological, cultural and virulence characters provide us requisite landmarks for the elucidation of genetic variation. An ideal molecular marker should be highly polymorphic in nature with consistent distribution throughout the genome, also provide adequate resolution of genetic differences, have linkage to distinct phenotypes and without necessitate evidence about the genome of an organism4,5.
The RAPD and ISSR markers does not require radioactive compounds and it could be revealed a high degree of polymorphism. In the recent era, the efficiency of different molecular marker systems has been studied in assessing genetic diversity using various statistical parameters6.
By using these markers, the correlation between different polymorphism assays may vary between as well as within species. Therefore, careful evaluation is needed for each PCR based marker system before being wellgrouped7. In addition, primers with more than 50% GC content are desired because during polymerization process, it could be strong enough to form a duplex. Kumar et al.8 compared three marker system viz., Random Amplified Polymorphic DNA (RAPD), Universal Rice Primers (URP) and Inter Simple Sequence Repeat (ISSR) markers to characterize twenty C. falcatum isolates and among three markers, URP markers could successfully assess genetic diversity in C. falcatum as it produced more number of total bands amplified (TB) and Polymorphic Bands (PB). In addition, the parameters measuring the efficiency of markers viz., expected heterozygosity (Hn), Effective Multiplex Ratio (EMR), resolving power (Rp) and Marker Index (MI) were more in URP markers. Studies by Tymon and Pell9 clearly depicted that usage of ISSR, ERIC and RAPD markers for the estimation of genetic diversity of 30 isolates of the entomopathogenic fungus Pandora neoaphidis of different geographic origins produced larger number of polymorphic bands obtained from ISSR. In addition, ISSR marker is a simple and rapid technique, requires no sequence information and using a single primer for detection and random amplification of the DNA. Hence, comparing RAPD and ISSR amplification will be clearly depicted the polymorphism among the C. gloeosporioides isolates of mango separately by each primer, which was further used for genetic variability.
Mirmajlessi et al.10 assessed the genetic diversity, by comparing RAPD and ISSR markers in twenty three isolates of Rhizoctonia solani from root, crown and rhizosphere of cucumber, pumpkin, watermelon and melon plants and reported that ISSR profiles showed the highest levels of polymorphism (0.37) in R. solani AG4, while the lowest levels of polymorphism (0.32) was detected with RAPD profiles. A comparative analysis of genetic variation from different places of Tamil Nadu will help explain the overall structure of the C. gloeosporioides population and the diversity within each location will better define against potentially damaging strains. The present study objectives were designed to analyse the genetic diversity of C. gloeosporioides pathogen of mango anthracnose disease using two molecular markers viz., RAPD and ISSR.
MATERIALS AND METHODS
Isolation of C. gloeosporioides: Twenty six isolates of C. gloeosporioides were isolated from anthracnose disease infected mango samples during the period of 2011-2012 from different districts of Tamil Nadu, India and their pathogenecity, morphological variation and virulence of all isolates were previously studied by Archana et al.11.
Molecular identification: Genomic DNA was extracted from the mycelial mat of C. gloeosporioides isolates by Cetyl Trimethyl Ammonium Bromide (CTAB) method as described by Knapp and Chandlee12. The ITS1-5.8S-ITS2 region of ribosomal DNA from twenty six isolates of C. gloeosporioides was amplified with ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) primers and further it confirmed with the specific primer (CgInt) (5-GGCCTCCCGCCTCCGGGCGG-3) coupled with ITS 4 (5-TCCTCCGCTTATTGATATGC-3).
Comparison of marker systems: A set of 10 RAPD (OPB 07, OPF 11, OPF14, OPF 07, OPL 12, OPL 05, OPD 07, OPA 09, OPG 16, OPC 08) and 16 ISSR ((CAG)5, (GACA)4, (GACAC)3, (TGTC)4, (AGG)5, (ACTG)4, (TCC)5, (GTG)5, (CAG)3, (CAC)5, (AG)8 T, (AG)8 C, (GA)8 T, (TG)8 A, (GA)8 YG, (GT)8 YC) primers (Chromous Biotech Pvt. Ltd., Bangalore) were used to identify the molecular variability of twenty six C. gloeosporioides isolates.
Data analysis: For data analysis, each band with a different electrophoretic mobility was assigned a position number and a mark of 1 or 0 based on the presence or absence of the band. Only reproducible bands were considered for analysis. Bands common to all isolates were incorporated into the analysis. The parameters of banding pattern viz., total number of bands, number of monomorphic, polymorphic bands per primer and percent polymorphism were calculating by counting the number of bands produced by each primer in all the twenty six isolates. In addition, assay efficiency index (AEI = Polymorphic bands/Total number of primers) of RAPD and ISSR markers were documented.
Polymorphism Information Content (PIC) was calculated using the formula developed by Anderson et al.13. A PIC value of each locus was calculated as:
where, Plj is the relative frequency if the lth allele for the locus j and is summed across all the alleles (L) over all lines. The PIC provides an estimate of the discriminatory power of a locus by taking into account, not only the number of alleles that are expressed but also the relative frequencies of those alleles. The PIC values may range from 0 (monomorphic) to 1 (very highly discriminative), with many alleles in equal frequencies.
Genotypic gene diversity was calculated as described by Mariette et al.14:
where, pi2-qi2 are the frequencies of the dominant and null alleles, respectively. Here, allele frequencies were calculated based on the frequency of the null allele (i.e., the number of individuals without the band). Where, pi (pi = 1-qi) represents the frequency of the dominant allele and qi represents the frequency of the null allele:
Marker Index (MI) was determined as the product of PIC and the number of polymorphic bands per assay unit and Effective Multiplex Ratio (EMR) is the product of the fraction of polymorphic loci and the number of polymorphic loci for an individual assay:
where, np is the number of polymorphic loci and n is the total number of loci1.
Statistical analysis: The banding patterns were scored for RAPD and ISSR primers in each C. gloeosporioides isolate starting from the small size fragment to large sized one. Presence and absence of each band in each isolate was coded as 1 and 0 respectively. The scores were used to create a data matrix to analyse genetic relationship using the NTSYS-pc program version 2.02 (Exeter Software, New York, USA) described by Rohlf 15.
RESULTS AND DISCUSSION
In the present study analysis of the ITS sequence of the ribosomal DNA, all the twenty six isolates amplified with the primer pairs of ITS1 and ITS4 and CgInt and ITS4, confirming that they pertained to C. gloeosporioides by producing the amplicons at 560 and 450 bp, respectively.
To compare the utility of the two marker systems, twenty six isolates of C. gloeosporioides were analyzed with 10 RAPD and sixteen ISSR primers. Various parameters viz., total number of alleles, number of polymorphic bands per assay unit, mean percentage of polymorphism per assay, number of monomorphic bands per assay, Polymorphic Information Content (PIC) value, genotypic gene diversity, Marker Index (MI), Effective Multiplex Ratio (EMR) and Assay Efficiency Index (AEI) were recorded as criteria to differentiate their efficacy.
RAPD analysis: All the twenty six C. gloeosporioides isolates had polymorphic fragments which were generated by 10 oligonucleotides decamers. The selection of primers was based on clear, scorable and reproducible amplified banding patterns. The number of amplification products obtained in all isolates was specific to each primer and the size was varied from 100 to 2000 bp. Of the 10 primers used, six primers viz., OPB 07, OPF 14, OPF 07, OPL 05, OPD 07 and OPG 16 were found to show 100 per cent polymorphism which is presented in (Table 1, Fig. 1). Of the 82 total alleles observed, 77 alleles were polymorphic and maximum numbers of 14 alleles were obtained with primer OPL 12, followed by primer OPF 14 with 12 alleles. Minimum numbers of 5 alleles were generated with primer OPL 05. Thus, amplifications varied across the primer employed. Among the 10 RAPD primers, the Polymorphism Information Content (PIC) was in the range of 0.69 to 0.90 and the Marker Index (MI) were 3.47 to 10.82. In addition, the Effective Multiplex Ratio (EMR) was in the range of 4.17-12.00.
|Fig. 1(a-h):|| |
RAPD fingerprints of C. gloeosporioides obtained by arbitrarily selected RAPD primers (a) OPB 07, (b) OPF 07, (c) OPL 06, (d) OPD 07, (e) OPA 09, (f) OPF 14, (g) OPG 16 and (h) OPL 12
1: MCG 1, 2: MCG 2, 3: MCG 3, 4: MCG 4, 5: MCG 5, 6: MCG 6, 7: MCG 7, 8: MCG 8, 9: MCG 9, 10: MCG 10, 11: MCG 11, 12: MCG 12, 13: MCG 13, 14: MCG 14, 15: MCG 15, 16: MCG 16, 17: MCG 17, 18: MCG 18, 19: MCG 19, 20: MCG 20, 21: MCG 21, 22: MCG 22, 23: MCG 23, 24: MCG 24, 25: MCG 25, 26: MCG 26, M: Marker (100 bp) and M1: Marker (1 kb)
The minimum PIC value, MI and EMR was observed in the primer OPA 09. By using RAPD markers a total of 519 amplicons were observed and the Assay Efficiency Index (AEI) was 7.4. By analyzing these parameters, the most informative RAPD primers were OPL 12 and OPF 14.
ISSR analysis: Sixteen ISSR markers that generated 189 alleles were used to estimate the genetic diversity of twenty six C. gloeosporioides isolates, among that 180 were polymorphic bands and 9 were monomorphic bands. Clearly detectable amplified ISSR fragments of allele ranged from 100-2500 bp in size (Table 2, Fig. 2).
|Table 1:||Polymorphism detected by RAPD markers|
MB: No. of monomorphic bands, PB: No. of polymorphic bands, MM (%): Monomorphism percentage, PM (%): Polymorphism percentage, PIC: Polymorphism information content, MI: Marker index, EMR: Effective multiplex ratio, AEI: Assay efficiency index
|Table 2:||Polymorphism detected by ISSR markers|
MB: No. of monomorphic bands, PB: No. of Polymorphic bands, MM (%): Monomorphism percentage, PM (%): Polymorphism percentage, PIC: Polymorphism information content, MI: Marker index, EMR: Effective multiplex ratio, AEI: Assay efficiency index
The number of alleles revealed by each marker ranged from 3 alleles in (AG)8 T to 25 alleles in (GACA)4, with an average of 11.81 alleles per locus. With the average of 96.91 per cent polymorphism produced by sixteen ISSR primers, percent polymorphism was detected by the primers (CAG)5, (TGTC)4, (AGG)5, (TCC)5, (CAG)3, (AG)8 T, (GA)8 T, (TG)8 A, (GA)8 YG and (GT)8 YC. Polymorphism Information Content (PIC), a measure of gene diversity was an average of 0.86 with a range of 0.66 by (AG)8 T to 0.97 by (GA)8 T primer. A convenient estimate of marker utility may therefore, be devised from the product of information as measured by PIC and the number of polymorphic bands per assay unit. The maximum marker index value of 22.85 was observed in the primer (GACA)4 and the minimum marker index of 1.98 was observed in the primer (AG)8 T. Among the sixteen ISSR primers used, (GACA)4 produced highest EMR of 23.04 and the primer (AG)8 T produced lowest EMR of 3.00.
Comparison of RAPD and ISSR marker systems for their efficacy in assessing the genetic diversity of C. gloeosporioides isolates: The mean number of allele per assay unit, number of polymorphic and monomorphic bands per assay unit in ISSR analysis was 11.81, 11.25 and 0.56 respectively, which were superior over RAPD primers accounting 8.20, 7.70 and 0.50 alleles (Table 3). The ISSR marker index (10.13) indicative of marker utility and mean polymorphic information content per assay (0.86) was greater than the value of RAPD, which has 6.32 and 0.81 respectively, is due to ISSRs higher effective multiplex ratio (10.74). The mean genotypic gene diversity was 1.80 for RAPD analysis, while for ISSR it was 1.01, despite the higher multiplex ratio and marker index. Further, the higher percentage of polymorphic bands obtained from ISSR analysis (96.91%) compared to RAPD (93.81%).
|Fig. 2(a-f):|| |
DNA fingerprint of C. gloeosporioides isolates by ISSR primers (a) (GACA)3, (b) (GTG)5, (c) (CAC)5, (d) (GACA)4, (e) (CAG)5 and (f) (ACTG)4
M: Marker (100 bp), 1: MCG 1, 2: MCG 2, 3: MCG 3, 4: MCG 4, 5: MCG 5, 6: MCG 6, 7: MCG 7, 8: MCG 8, 9: MCG 9, 10: MCG 10, 11: MCG 11, 12: MCG 12, 13: MCG 13, 14: MCG 14, 15: MCG 15, 16: MCG 16, 17: MCG 17, 18: MCG 18, 19: MCG 19, 20: MCG 20, 21: MCG 21, 22: MCG 22, 23: MCG 23, 24: MCG 24, 25: MCG 25 and 26: MCG 26
|Table 3:|| |
Comparative analysis of banding patterns generated by RAPD and ISSR
RAPD: Random amplified polymorphic DNA, ISSR: Inter simple sequence repeats
The ISSR marker system outperformed in all marker parameters i.e., higher allele number, total amplified bands, EMR, MI as well as PIC except the mean genotypic gene diversity revealed best by the RAPD marker system. Considering these parameters, it confirmed that the ISSR technique is very potent to evaluate genetic diversity among the C. gloeosporioides isolates, also it could be highly polymorphic and have a significant discriminative power when compared to RAPD. Further, from the studies it was observed that ISSR primers could clearly distinguish the isolates according to their virulent nature but it was not so in RAPD. Thus, microsatellites are ideal markers for assessing the genetic diversity of C. gloeosporioides isolates.
The present study was aimed to analyse the molecular diversity of C. gloeosporioides isolates, causing anthracnose disease of mango from various places of Tamil Nadu, India. Variability in terms of morphological, physiological and genetical was important to study the biology of the pathogen in an endemic area. Hence, in this paper, RAPD primers and highly efficient microsatellite ISSR primers are used for the genetic diversity of C. gloeosporioides with aim of identifying the ideal marker.
Random amplified polymorphic DNA (RAPD): Molecular methods have been employed successfully to differentiate between populations of Colletotrichum from many hosts in general, according to the study of Agwanda et al.16. Random Amplified Polymorphic DNA (RAPD) markers, in which short oligonucleotides of random sequence are used as primers to arbitrarily amplify segments of a target genome, are used widely to detect genetic variation4.
In this study, the suitability of RAPD technique for rapid molecular characterization of C. gloeosporioides isolates was assessed. Based on the results it was observed that six primers (OPB 07, OPF 14, OPF 07, OPL 05, OPD 07 and OPG 16) produced 100 per cent polymorphism with the allele range of 100-2000 bp, although majority was below 1000 bp. It was observed that there was a variation in the number of alleles and their intensity, number of monomorphic (5) and polymorphic bands (77), polymorphism information content (80.06), marker index (63.21) and effective multiplex ratio (72.76) among the ten RAPD primers used. The EMR was ranged from 4.17 (OPA 09) to 12.00 (OPF 14). This research was similar to results of Kumar et al.8, where the EMR ranged from 7 (OPA07) to 12 (OPA04) in Colletotrichum falcatum isolates. Application of RAPD markers to fungal isolates would be useful in providing information regarding polymorphisms within the reference isolates of C. gloeosporioides and established DNA fingerprints which was useful for race characterization17.
Inter simple sequence repeat (ISSR): The ISSR primers are based on di-, tri-, tetra- or pentanucleotide repeats with 5 or 3 anchored base(s). To access the variation around the diverse microsatellite regions, Inter Simple Sequence Repeat (ISSR) markers are powerful tools which can be utilized as molecular tools5 for the characterization of genetic variations within fungi18. In the present study, all the 16 primers generated amplification products of C. gloeosporioides isolates for a total of 189 allelles, 180 of which were polymorphic with the allele range of 100-2500 bp. Out of sixteen ISSR primers, ten primers viz., (CAG)5, (TGTC)4, (AGG)5, (TCC)5, (CAG)3, (AG)8 T, (GA)8 T, (TG)8 A, (GA)8 YG, (GT)8 YC were shown percent polymorphism. The distribution of different microsatellite sequences in all the genomes determines the possibility of using this method for DNA fingerprinting. This study was in accordance with Mahmodi et al.19 where he reported the ISSR primers viz., UBC 808, UBC 810, UBC 820, UBC 834, UBC 841, UBC 864 and UBC112 produced 16.5 average numbers of bands per primer which ranged in size from 300-2600 bp with 100% polymorphism in Colletotrichum spp., obtained from cowpea. Further, Kumar et al.8 reported, two ISSR markers, ISSR 02 (ACTG4) and ISSR10 (CAC5) differentiated twenty five C. falcatum isolates with alleles ranging from 250-3500 bp witheight average number of bands per primer (100% polymorphism). In the current research, primer (GACA)4 produced highest total number of alleles (25 alleles), total number amplicons (250), effective multiplex ratio (23.04) and marker index (22.85) revealed more heterozygosity as tetranucleotide repeats are more abundant in the genome and was thus better able to characterize the polymorphism in Colletotrichum isolates. The overall eficiency of a primer can be judged by the higher value of marker index. This study was in accordance with the findings of Abadio et al.20. Further it produced higher mean PIC value as 0.86 with 16 primers, it was supported by Chadha and Gopalakrishna21, where the mean PIC value was 0.27 with 17 ISSR primers in Magnaporthe grisea.
Comparison of markers: Molecular markers are useful for assessing the genetic variation rapidly within and among species22. In this study, comparison of two different molecular marker systems RAPD and ISSR was carried out to define genetic relationships and polymorphism among the twenty six isolates of C. gloeosporioides causing mango anthracnose and to investigate which marker system can be more effectively used. The foregoing study compared two marker systems by estimating discriminatory power of matrices viz., number of total allele, allele range, per cent polymorphism, Polymorphism Information Content (PIC), Marker Index (MI), Effective Multiplex Ratio (EMR) and Assay Efficiency Index (AEI).
Among the sixteen ISSR markers assessed across the C. gloeosporioides isolates, ten markers possessed more than ten alleles indicating better resolving power of the ISSR markers. This is because of polyallelic nature of ISSR markers. Comparison of PIC values for two marker systems (a parameter associated with the discriminating power of markers) indicated that the range of PIC values for RAPD primers was from 0.69-0.90 with an average of 0.81 and for ISSR primers it was from 0.66 to 0.97 with an average of 0.86. Marker Index (MI) is the marker attribute used to calculate the overall utility of a marker system and the mean of marker index was higher in ISSR (10.13) than RAPD (6.32) markers. In this study, a comparison of the PIC values and MI between the RAPD and ISSR data clearly demonstrated the stronger discriminatory power of ISSR primers. Mean of effective multiplex ratio for each of these marker systems in this study (7.28 for RAPD and 10.74 for ISSR) suggested that ISSR marker systems were effective in determining polymorphism. It may be due to highly polymorphic, abundant nature of the microsattelites due to slippage in DNA replication23. The ISSR technique has specific and higher levels of polymorphism with more reproducibility than RAPD technique due to the use of longer oligonucleotide sequences, allowing more stringent annealing conditions in PCR amplification24.
Cluster analysis was carried out on two sets of marker profiling data based on RAPD and ISSR. The results based on the two DNA marker profiles broadly grouped twenty six isolates into two clusters. However, formation of subclusters within the main cluster varied between RAPD and ISSR. The similarity coefficients of C. gloeosporioides based on 10 RAPD markers and sixteen ISSR markers ranged from 65.00-88.00 and 59.50-78.00%, respectively. The cluster formation was observed at the minimum of 59% similarity value, indicating the presence of considerable divergence between the isolates. The cluster analysis of the RAPD data confirmed the presence of high diversity at molecular level among the C. gloeosporioides isolates under the study. The results of ISSR analysis demonstrated, not only high diversity among studied isolates but also ISSR markers can be highly polymorphic.
Comparison of the DNA profile performance of current experiment indicated that ISSR technique was more informative in detecting genetic diversity in C. gloeosporioides than RAPD technique and it was supported by Mirmajlessi et al.10 in Rhizoctonia solani. Similar results have been found by Mahmodi et al.19, that high level of polymorphism detected by ISSR-PCR technique, suitable for the discrimination of Pseudocercospora griseola causing angular leaf spot of bean. The utility of a marker system is a balance between the level of polymorphism detected and the extent of an assay which can identify multiple polymorphisms25. Comparative studies in different species using various marker systems were successfully conducted by other researchers and concluded that ISSR would be a better tool than RAPD for phylogenetic studies26. While considering all, the results of the present study clearly depicted that the ISSR primer can be well adopted for the molecular variability studies.
The diversity of 26 isolates of C. gloeosporioides isolates were analyzed using the primers of RAPD and ISSR using the molecular parameters. The valuing parameters clearly showed that the polymorphism percentage, assay efficiency index and marker utility was higher in ISSR primers.
Analyzing morphological and molecular diversity of the pathogen C. gloeosporioides, causing mango anthracnose disease is important to control the disease. Here, this study revealed that ISSR was the best choice to study the genetic diversity compare to RAPD primer.
The authors would like to thank the University Grants Commission (No. F.14-2 /2010/ (SA-III)), GOI, New Delhi, India for providing finacial support.
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