DNA extraction and SSRs analyses

For extraction of the mycelia mass, liquid cultures were initiated by adding 2–4 mm² pieces of fungus plague to 250-mL Erlenmeyer flasks containing 100 mL PDB medium (Potato Dextrose Broth). Flasks were incubated at room temperature approximately 25 °C on a rotary shaker for 6–8 days. Mycelium was collected by filtration through sterile filter paper with a vacuum funnel. Mycelia were harvested, frozen and stored at −20°C. DNA was extracted using a modified hexadecyl trimethyl-ammonium bromide (CTAB) procedure (Doyle & Doyle 1987). Genomic DNA was isolated from single spore culture of each isolate (Murray & Thompson 1980). Mycelia were ground in liquid nitrogen and suspended in 2% CTAB extraction buffer (1.4 M NaCl, 0.1 M Tris-HCl, pH 8.0, 20 mM EDTA, 0.2% β-mercaptoethanol). Samples were treated with 5 units RNase at 37°C for 30 min. and then extracted with Chloroform Isoamyl alcohol 24:1 (v/v). DNA in the supernatant was precipitated with isopropanol, rinsed with ethanol, and adjusted to a final concentration of 20 ng.μl^-1 in TE (pH 7.4). The quality of the extracted DNA was visually checked on 0.8% agarose gels.

A set of five locus-specific primers pairs for SSRs (Table 2) described by Bogale et al. (2005), were selected. Primer aliquots for each marker were prepared by mixing equimolar amounts of appropriate forward and reverse primer in 1× TE (1mM EDTA, 10 mM Tris–HCl, pH 8.0) and used for the amplification of individual microsatellite loci. PCR amplification was performed in a 25 μl reaction volume containing 1.5 mM MgCl₂, 0.2 mM of dNTPs mix (100 mM of each dNTPs), 1 μl of each forward and reversed primer, 0.6 U of Taq polymerase with 25 ng of template DNA. Amplification was performed using Biometra thermal cycler (USA), PCR conditions for SSR were as Follows, the PCR programmed had one initial denaturation step at 94°C for 3 min Followed by 30 cycles of 94°C for 60 s, annealing for 60 s (appropriate annealing temperature was used for each primer set, Table 2) and 72°C for 2 min. The thermal cycles were terminated by a final extension of 7 min at 72°C. Amplified products were resolved in 2 % agarose gel at 60 V cm-1 using in 1X TBE buffer and stained with DNA Safe Stain at 0.5mg.ml^-1 and photographed under UV Transilluminator with Gel Doc. Intas. A 1 kbp ladder (Gene Ruler TM, Fermantas,) was used as a molecular size standard.

Table 2. SSR primer sequences used in this study (Bogale et al. 2005).

RESULTS

Isolates collection and pathogenicity test

A total of 114 isolates of FOC were isolated and purified from infected chickpea plants (Table 1). Based on morphological characteristics, the isolates were identified as FOC. FOC isolates were identified according to their morphological and microscopic characters as described by (Jens et al. 1991, Nelson et al. 1983, Barnett and Hunter 1972, Leslie et al. 2006). Isolates showed significant variations in cultural characteristics, production of microconidia, macroconidia and chlamydospores. Our morphological identifications were further confirmed by molecular method (PCR) using specific SSR primers used in this study for FOC isolates.

The result of the pathogenicity test indicates that FOC isolates were virulent and caused wilting and death of the seedlings in 20-22 days. Characteristic wilt symptoms such as drooping of leaflets and yellowing of the leaves starting from the apical part, progressing downward and final wilting of the whole plant were observed. Internal discoloration of the root vascular system was visible in wilted plants.

Allele’s frequency and primers information

Five pair SSR primers were used for amplification of SSR loci of 114 FOC isolates. A total of seventeen alleles were produced by SSR primers with an average of 3.4 alleles in all loci. All isolates amplified a single band ranging from 100 to 1000 bp. Five microsatellite loci were polymorphic when considered over all isolates. The average of allelic variability per locus (H) was the highest (0.281) in Badreh population and the lowest inDareh Shahr with 0.242 alleles. The allele’s frequency (No. allele) is the highest in Eivan, Badreh and Chardavol with 15 and lowest in Sarableh with 12 alleles (Table 3).

A summary of the genetic diversity data of five microsatellite loci in populations is given in Table 3. Observed allele number (Na = 1.765) was higher in Eivan, Badreh and Chardavol, effective numbers (Ne = 1.533) of alleles was higher in Asemanabad compared to other populations. The amount of genetic diversity (H)and Shannon's Information Index (I) were also higher in Badreh (H= 0.481, I= 0.430) but lower values were estimated for Dareh Shahr (H= 0.242, I= 0.376).

On basis of microsatellite data, the polymorphic information content (PIC) varied from 0.266 (Primer MB18) to 0.384 (Primers MB11), with an average of 0.342, which reflects the informative content of the used primers. EMR(effective multiplex relationship ratio) varied from 2 to 4. The marker index (MI), which incorporates the informative content of the marker (PIC), the number of fragments per primer pair and the fraction of poly­morphic fragments (EMR), varied from 0.76 (MB11) to 1.49 (MB5) (Table 2).

Genetic variability of populations

The average genetic distance was calculated among the six populations. Nei’s pairwise genetic distances between the populations varied from 0.0005 to 0.0233. The lowest genetic distance was found between Chardavol and Dareh Shahr, while the highest genetic distance was revealed between Eivan and Sarableh (Table 4).

Table 3. Genetic diversity estimates in FOC populations based on SSR loci.

I = Shannon's Information Index H = Nei's (1973) gene diversity, Ne = Effective number of alleles, Na = Observed number of alleles, P = Polymorphism %

Table 4. Information about genetic distance between pairs of FOC populations.

Cluster analysis (UPGMA) was used to produce a dendrogram showing the genetic relationships between the populations based on the SSR data, the dendrogram showed a distinction between Asemanabad population and the five remaining populations (Fig. 2). 

Fig 2. Dendrogram of genetic relationships between each FOC population constructed by UPGMA.

 The total gene diversity (Ht) and gene diversities between subpopulations (Hs) were estimated to be 0.2004 and 0.1880, respectively. Gene diversity attributable to differentiation among populations (Gst) was 0.0618, while gene flow (Nm) was 7.589 (data not shown). The genetic similarity varied from 97% to 99%, in isolates with different geographical origins. A dendrogram based on UPGMA analysis showed three major groups containing isolates with different origins.

 The AMOVA of genetic variation in FOCpopulationsrevealed that 8% of the variance occurred among populations and 92% within populations (Table 5). PCA (Principal Component Analysis) using SSR data showed the genetic differences among isolates within populations and gene flow between different populations (Fig. 3), the first and second principal coordinates account for 25.07% and 19.52% of the variation, respectively. There was no clear separation among individuals from different populations, however, isolates in the same populations tended to gather together. This suggests the geographical regions of sampling play an important role in the formation of populations. PCA allows for visualizing the patterns of a genetic relationship without altering the data itself and finds patterns within a multidimensional data set.

DISCUSSION

 The present work was carried out to discover the possible utilization of SSR marker for identifying genetic variation in Iranian FOC isolates. In conclusion, the present study generated significant information about genetic diversity among FOC isolates, collected from different regions. The study also highlighted the fact that genetic analysis using SSR marker is a useful tool and have high discriminatory power for studying the diversity in F. oxysporum f. sp.ciceri.

The advantage of microsatellite markers are their high specificity, high polymorphism, good reproducibility and unambiguous scorability (Tenzer et al. 1999, Sahran & Naef 2008). Alleles vary according to the number of repeat units present but other mutations have also been shown to be responsible for allele length variation in SSR analysis (Burgess et al. 2001, Slippers et al. 2004b). 

SSRs provides a powerful tool for taxonomic and population genetic studies (Britz et al. 2002). In this research, SSR markers divided all the FOC population into two major groups. Earlier, 64 FOCIndian isolates placed into two major categories with ISSR and RAPD markers (Dubey & Singh 2008), similar studies on other plant pathogenic fungi have emphasized the importance of molecular approaches to characterize genetic diversity within and between isolates (Bentley et al. 1995, Nourollahi et al. 2011, Sivaramakrishnan et al. 2002, Belabid et al. 2004). By SSR markers, different allele sizes were recorded in FOC genome, the smallest allele size was detected as 120 bp and the largest one was 1000 bp as reported by Mohammadi et al. (2004). In this study the mean number of PIC value of SSR markers was 0.342, This PIC value indicates that the isolates had a low degree of biodiversity. The variable numbers of alleles per loci is an indication of the high level of polymorphism, and Polymorphism has been observed in other fungi as a direct record of genetic evolution (Sanders, 2002, Mwang`Ombe et al. 2007). The polymorphic character of SSRs produces highly discriminating fingerprints that often allow characterization of fungi at a strain level (Migheli et al. 1998). The analysis performed by AMOVA indicated that most of the variation in the populations is accounted for among individual FOC isolates (98%) and low proportion (2%) of the total genetic variations were accounted for differences among populations.

The results showed that there was a low level of genetic variation among the isolates of FOC populations in sampling regions. The Nm (7.589) indices demonstrated high gene flow and low genetic differentiation among populations. This study suggests that frequent gene flow and recombination between populations of FOC are significantly influenced on the evolution and development of this fungus. This subject is in accordance with previous results of Jimenez-Gasco et al. (2002) who stated that F. oxysporum f. sp. ciceri is a monophyletic group that derived from a small population or a single individual. Molnar et al. (1990) demonstrated that somatic recombination occurred through parasexuality in F. oxysporum. High degrees of pathogenic and genetic variation in asexual fungi such as F. oxysporum f. sp. ciceri are also observed with accumulation of genetic mutations over time (Gordon & Martyn 1997). In this study, FOC isolates showed a different level of diversity within and between populations in comparison with the worldwide isolates in different researches. Similar results were also reported in F. oxysporum f. sp. phaseoli (Woo et al.1996), F. oxysporum f. sp. ciceri (Jimenez-Gasco et al. 2001), and F. oxysporum f. sp. lentis (Nourollahi & Jalali 2017). Similar findings have also been reported in the Ethiopian F. oxysporum isolates by AFLP, SSR and ITS sequence analysis (Bogale et al. 2006). In Iranian populations, over 92% of the gene diversity was distributed on a local level within populations. However, there was a high degree of genetic similarity among populations separated by low geographical distances. The low level of gene diversity (Gst = 0.0618) was detected among all populations. The low Gst value indicated little genetic differentiation among the populations and showed little evidence for geographical subdivision among populations (Bayraktar et al. 2010).