Genomic DNA Fingerprinting of Oenococcus oeni Strains by Pulsed-Field Gel Electrophoresis and Randomly Amplified Polymorphic DNA-PCR

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking. Received: 3 November 1999 / Accepted: 8 December 1999     

Rapid molecular identification and characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S–23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)₅ primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2–3 d (in comparison to the standard methods).     

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed‐field gel electrophoresis and repetitive element PCR typing

Aims: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. Methods and Results: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed‐field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R‐I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. Conclusions: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. Significance and Impact of the Study: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.     

Intraspecies Genomic Groups in Enterococcus faecium and Their Correlation with Origin and Pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Characterization of Yersinia enterocolitica 4/O:3 isolates from tonsils of Bavarian slaughter pigs

Aims: Yersinia enterocolitica 4/O:3 isolates of slaughter pigs originating from different farms were characterized to study the distribution of different genotypes at farm. A correlation between the genotypes and the resistance patterns was also examined. Methods and Results: Hundred and eighty‐seven ail‐positive Y. enterocolitica 4/O:3 isolates recovered from pigs originating from 31 Bavarian farms in 2000, 2003 and 2004 were characterized. PFGE using NotI, ApaI and XhoI enzymes revealed 31 genotypes. The most common genotype was found in 13% of the pigs. From most farms (71%), only one genotype was found. Some genotypes were found during different years. Low resistance was noted to streptomycin (9%), sulphamethoxazole (9%), amoxicillin/clavulanic acid (5%) and tetracycline (1%) by agar disc diffusion method. Conclusions: Several genotypes were found. Some genotypes were widely distributed and persisted for years. Farm‐specific genotypes may exist. No clear relation between the genotypes and antimicrobial patterns was found. Significance and Impact of the Study: This study provides data on the genetic diversity of Bavarian pig strains and antimicrobial resistance. It may be of interest for other countries where Y. enterocolitica strains are genotyped to get more information about the strain distribution of this pathogen.     

Development of simple and rapid PCR‐fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron

Aims: To develop simple and rapid PCR‐fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. Methods and Results: PCR‐restriction fragment length polymorphism (PCR‐RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat‐amplified fragment length polymorphism (VCR‐AFLP) assay was also developed. In the PCR‐RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR‐AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed‐field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). Conclusions: The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. Significance and Impact of Study: This newly developed method is more discriminatory, simple, rapid and cost‐effective in comparison with PFGE, and thus can be widely applicable.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Typing of clinical and environmental strains of Aeromonas spp. using two PCR based methods and whole cell protein analysis

Two PCR based typing methods i.e. random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR were evaluated for typing of 42 Aeromonas isolates from clinical and environmental sources and whole cell protein (WCP) profiles were analyzed. Both RAPD and ERIC-PCR showed a high level of genetic diversity. Numerical index of the discriminatory (D) values were 0.94 and 0.96 (>0.90) for RAPD and ERIC-PCR, respectively. No correlation in banding pattern and evidence of genetic similarity was found between Aeromonas isolates from environmental and clinical sources. Therefore these techniques are highly reproducible and sensitive methods for typing the Aeromonas isolate from different sources. WCP profile showed two major variable regions i.e. 20 kDa to 45 kDa region and 70 kDa to 85 kDa region. Though WCP profiling had less discriminatory power, use of this method in combination with other established typing methods such as RAPD and ERIC-PCR may be helpful for reliable typing of Aeromonas isolates or to identify new proteins with pathogenic potential.     

Characterization of isolates of Listeria monocytogenes from sludge using pulsed‐field gel electrophoresis and virulence assays

Aims: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. Methods and Results: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque‐forming assay and by mouse virulence assay. Conclusions: Our findings suggest that L. monocytogenes strains found in non‐sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. Significance and Impact of the study: This is the first study dealing with the characterization of L. monocytogenes isolates from non‐sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.     

Strain typing of shiitake (<Emphasis Type="Italic">Lentinula edodes</Emphasis>) cultivars by AFLP analysis,focusing on a heat-dried fruiting body

To validate strain typing by amplified fragment length polymorphism (AFLP) analysis in shiitake (Lentinula edodes) cultivars, the reproducibility of AFLP markers with DNA extracted from the heat-dried fruiting body was evaluated. DNAs were extracted from three different portions of the heat-dried fruiting body – the stipe, pileus, and gill – and AFLP analysis of all parts was carried out using two combinations of selected amplification primer pairs. AFLP profiles of DNA from the gill tissue of heat-dried fruiting body were almost identical to those of cultured mycelia in the same strains, although it was difficult to detect reproducible AFLP profiles from stipe and pileus DNA. These results indicated that AFLP analysis would be applicable for strain typing with heat-dried fruiting bodies of L. edodes by using the DNA extracted from gills.Contribution No. 364 of the Tottori Mycological Institute     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

Analysis of Swedish Bordetella pertussis isolates with three typing methods: characterization of an epidemic lineage

Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and the Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.     

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

Improvement of strain discrimination by combination of RAPD with PFGE for the analysis of the swine isolates of Salmonella enterica serovar Choleraesuis

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) can cause salmonellosis in pigs and humans. Currently, the most common method used for the subtyping of this Salmonella serovar is pulsed field gel electrophoresis (PFGE) using XbaI as a DNA digestion enzyme. In this study, we compared and combined PFGE with the randomly amplified polymorphic DNA method, for the typing of 95 S. Choloraesuis strains isolated from diseased pigs. Using PFGE with XbaI, with AvrII, and with SpeI digested DNA, 29, 74, and 40 patterns, respectively, were obtained. Also, 53, 15, and 35 strains, respectively, belong to the major patterns X1, A1, and S1. When these three digestion patterns were combined, 83 PFGE pattern combinations were obtained. On the other hand, using RAPD with selected primer alone generated 76 patterns, and 11 strains which fell within a single X1A1S1 PFGE combination pattern were discriminated into 10 patterns. Thus, for S. Choloraesuis, PFGE with AvrII allowed higher discrimination than PFGE with XbaI, and some of the PFGE groupings obtained by combining the XbaI, AvrII and SpeI digestion patterns were further subdivided by the RAPD method.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.     

Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources

Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia, and the Americas as a source of high quality vegetable and industrial oil. Twenty-two RAPD primers, 18 SSR primers, and 10 AFLP primer combinations were used to assess: (1) the genetic diversity of 85 accessions (originating from 24 countries) representing global germplasm variability of safflower and (2) the interrelationships among safflower ‘centers of similarity’ or ‘regional gene pools’ proposed earlier. The RAPD and SSR primers and AFLP primer combinations revealed 57.6, 68.0, and 71.2% polymorphism, respectively, among 111, 72, and 330 genetic loci amplified from the accessions. The sum of effective number of alleles (66.44), resolving power (59.16), and marker index (51.3) explicitly revealed the relative superiority of AFLP as a marker system in uncovering variation in safflower. Overall, AFLP markers could recognize ‘centers of similarity’ or ‘regional gene pools’. Analysis of molecular variance and Shannon’s information index provided corroborating evidences for the present and previous studies that concluded fragmentation of safflower gene pool into many gene pools. Divergent directional selection is likely to have played an important role in shaping the diversity. From the practical applications standpoint, the diversity of Iran–Afghanistan gene pool is very high, equivalent to the total diversity of the species. The Far East gene pool is the least diverse. The present comprehensive input, first of its own kind in safflower, will assist marker based improvement programmes in the crop.