Sequence-related Amplified Polymorphism Technique Research Articles

Determination of genetic diversity of edible-seeded watermelon genotypes using SRAP markers

The watermelon (Citrullus lanatus L.) is one of the most commonly grown and consumed vegetables in the world. Some genotypes of watermelon, which have significant variations, have a snack potential due to their seed characteristics. In this study, SRAP (Sequence Related Amplified Polymorphism) marker technique was used to determine the genetic relationship between some edible-seeded watermelon genotypes. A total of 166 bands were obtained in 24 genotypes and the polymorphism rate was calculated as 97.4%. Four main clusters were observed in the cluster analysis. It was determined that genotypes 2 and 7 clustered separately from the others. Structure analysis revealed that the genotypes consisted of two subpopulations. It was concluded that the edible-seeded watermelon genotypes can be genetically differentiated by the SRAP techniques. The results of this study can be used in breeding strategies for the improvement of the edible-seeded watermelon cultivars.

Molecular Characterization of Olive (Olea europaea L.) Cultivars in Duhok Governorate

The olive tree (Olea europaea L.) was considered one of the most cultivated crops in the Mediterranean region, and it had an economically important value due to it began consumed as a table olive and oil production. In this study, AFLP and SRAP techniques were used to identify 15 cultivars of olive plants that were collected from 12 different locations in the Duhok governorate, eleven primer combinations of sequence-related amplified polymorphism (SRAP) and ten primer combinations of amplified fragment length polymorphism (AFLP) were used to fingerprinting these cultivars. The results show that the SRAP marker had the least genetic distance 0.241between (Shtla falay olive and Khodeiri olive) and the highest value 0.946 was found between (Zawita olive and Sorani olive). While AFLP marker show the lowest value of 0.107 between (Nzarky olive and Dger olive) and the highest value of 0.916 between (Amedi olive and Shtla falay olive). Cluster analysis based on AFLP and SRAP combined data clearly differentiated the genotype and it showed it was a powerful tool to evaluate the genetic distance and fingerprinting of these tested cultivars. Establishing the genetic relationships between cultivars and species, and the construction of genetic maps, had the main aims for the researchers in plant breeding programs and to understand the plant evolution lingues, and as a conclusion, this study demonstrates that AFLP and SRAP markers both are powerful tools in plants tree genetics.

Exploring Genetic Variability among and within Hail Tomato Landraces Based on Sequence-Related Amplified Polymorphism Markers

Landraces are valuable sources of genetic characteristics, which are of plant breeders’ interest to include in breeding programs for crop improvement. We assess the inter- and intra-genetic variability among 96 accessions representing three Hail tomato landrace using DNA-based marker sequence-related amplified polymorphism (SRAP). Seven SRAP primer combinations generated 55 alleles with a polymorphism of 100%, and an average of 7.86 polymorphic alleles per pair of primers. All primers showed an average of 0.68 polymorphic information content (PIC) value and discrimination power (DP) of 14.29. Principal coordinate analysis (PCoA) confirmed the clustering produced via the UPGMA similarity dendrogram allowed for the grouping of the 96 accessions according to its gene bank accession numbers and showed relatively good separation between landraces. A similarity value ranged from 0.04 to 1.0 among accessions of Hail 747, 0.05 to 1.0 in Hail 1072, and from 0.16 to 0.92 in Hail 548. These results showed the landraces harbor a wide range of genetic diversity at both inter and intra-variation levels. AMOVA showed that most of the genetic variation was because of differences within populations (87%). Tomato Hail landraces have well-differentiated genetic populations and admixtures, where Hail 747 formed their separate group, and both Hail 548 and Hail 1072 were admixed, and some accessions showed more diversity pattern. We have to take the SRAP technique’s effectiveness in the study of genetic variability among and within landraces into consideration in the tomato-breeding programs through marker-assessed selection.

Development of a SCAR marker linked to fungal pathogenicity of rice blast fungus Magnaporthe Oryzae.

PCR-based molecular approaches including RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and SRAP (sequence-related amplified polymorphism) are commonly used to analyze genetic diversity. The aims of this study are to analyze genetic diversity of M. oryzae isolates using PCR-based molecular approaches such as RAPD, ISSR, and SRAP and to develop SCAR marker linked to the pathogenicity of rice blast fungus. Twenty Magnaporthe oryzae isolates were collected mainly from the south of Vietnam and assessed for genetic variation by RAPD, ISSR, and SRAP methods. The comparison of those methods was conducted based on the number of polymorphic bands, percentage of polymorphism, PIC values, and phylogenetic analysis. Then, sequenced characterized amplified region (SCAR) markers were developed based on specific bands linked to fungal pathogenicity of rice blast fungus, M. oryzae. The results indicated that SRAP markers yielded the greatest number of polymorphic bands (174) and occupied 51.7% with polymorphism information content (PIC) value of 0.66. Additionally, the SRAP approach showed stability and high productivity compared with RAPD and ISSR. The SCAR marker developed from the SRAP method identified the presence of the avirulence AVR-pita1 gene involving fungal pathogenicity that can break down blast resistance in rice cultivars. The consistency of SCAR marker obtained in this study showed its efficiency in rapid in-field detection of fungal pathogenicity. SCAR marker developed from SRAP technique provides a useful tool for improving the efficiency of blast disease management in rice fields.

Assessment of Genetic Relationships between Streptocarpus x hybridus V. Parents and F1 Progenies Using SRAP Markers and FT-IR Spectroscopy.

The genetic relationship among three Streptocarpus parents and twelve F1 hybrids was assessed using sequence-related amplified polymorphism (SRAP) molecular markers and Fourier-transform infrared (FT-IR) spectroscopy. Both methods were able to discriminate F1 hybrids and parents as revealed by cluster analysis. For hybrid identification, the type III SRAP marker was the most effective due to the presence of male-specific bands in the hybrids. Different behaviors in the biochemical variability of DNA samples have been observed by FT-IR spectral analysis, which might be attributed to the inherent nature of the genomic DNA from parents and their F1 progenies. Mantel test was also carried out to compare morphological, SRAP, and FT-IR results based on genetic distances. The highest correlation coefficient was found between morphological and SRAP marker distances (R = 0.607; p ≤ 0.022). A lower correlation was observed between the morphological and FT-IR distance matrix (R = 0.231; p ≤0.008). Moreover, a positive correlation was found between the distances generated with SRAP and FT-IR analyses (R = 0.026) but was not statistically significant. These findings show that both SRAP and FT-IR techniques combined with morphological descriptions can be used effectively for nonconventional breeding programs for Streptocarpus to obtain new and valuable varieties.

Linkage map development by GBS, SSR, and SRAP techniques and yield-related QTLs in pea

The knowledge of the genetic basis of yield-related traits provides useful information for pea (Pisum sativum L.) breeding. The aim of this work was to develop a genetic linkage map of pea using SRAP (sequence-related amplified polymorphism), SSR (simple sequence repeat), and SNP (single-nucleotide polymorphism) markers and to identify QTLs (quantitative trait loci) controlling yield-related traits. An F2 mapping population and their F2:3 families were derived from an initial cross between cvs. Explorer and DDR14. The F2 individuals were evaluated with SRAP, SSR, and GBS techniques, which proved to be efficient, generating a set of 872 polymorphic markers for linkage mapping. The resulting map consisted of 128 genetic markers distributed in 9 linkage groups (LGs), covering 655.5 cM. The length of the LGs ranged from 49.1 to 114.8 cM, with 8 to 26 markers. QTL detection was performed using the composite interval mapping (CIM) method. A total of 45 QTLs were detected through the generations and environments evaluated. All of them were major QTLs that explain more than 10% of the phenotypic variation. The comparison among QTLs detected in the F2 population and F2:3 families in the two environments allowed assessing the consistency of two QTLs for pod width. On the other hand, the analysis of previous works allowed the identification of a common region in LG 3 associated with seed weight in different crosses and environments.

Evaluation of SRAP markers for mapping of Pisum sativum L.

Linkage maps have become important tools for genetic studies. With the aim of evaluating the SRAP (sequence-related amplified polymorphism) technique for linkage mapping in Pisum sativum L., a F2 mapping population derived from an initial cross between cvs. DDR11 and Zav25 was generated. A total of 25 SRAP primer combinations were evaluated in 45 F2 plants and both parental lines, generating 208 polymorphic bands/markers. The markers were analyzed by the chi-square goodness-of-fit test to check the expected Mendelian segregation ratio. The resulting linkage map consists of 112 genetic markers distributed in 7 linkage groups (LGs), covering a total of 528.8 cM. The length of the LGs ranged from 47.6 to 144.3 cM (mean 75.54 cM), with 9 to 34 markers. The linkage map developed in this study indicates that the SRAP marker system could be applied to mapping studies of pea.

Molecular phylogeny of Lathyrus species: insights from sequence-related amplified polymorphism markers.

Sequence-related amplified polymorphism (SRAP) markers were used to evaluate the intra- and interspecific variation among 40 Lathyrus genotypes (four species) (Fabaceae). Ten SRAP primer combinations resulted in a total of 94 bands, and they exhibited high interspecific variability. The genetic differentiation among Lathyrus, estimated using AMOVA, was highly significant. The results indicated that 58% of the total genetic variation existed among species, and 42% of the differentiation was within species. This was explained by the high level of genome conservation of these species as well as the recent and slow evolution of this genus. These results were confirmed by the topology of the neighbor-joining cladogram and the results of the principal coordinate analysis. Our data support previous results based on seed protein diversity. These results make SRAP markers choice markers for the study of functional polymorphism that is directly related to the transcriptomic data. The SRAP markers used in this study provide an accurate picture of the population structure within Lathyrus germplasm, which is critically important information for the design of genetic diversity and structure analyses. Moreover, further extensive studies are necessary to fully examine other Lathyrus species and tests that adopt the SRAP technique to enrich the Lathyrus library for next-generation sequencing, thus providing a potent protocol for the study of polymorphism.

Selection of DNA Isolation Method and PCR Protocol for the Study of Endemic Astragalus exscapus L. ssp. Transsilvanicus (Schur) Nyár

Astragalus exscapus ( Fabaceae ) is a melliferous, perennial plant, which fits within a rarity type characterized by having small populations of relatively high habitat specificity (Rabinowitz, 1981). Astragalus exscapus L. ssp. transsilvanicus (Schur) Nyar . is an endemic subspecies, which has been identified in the Transylvanian basin in 24 populations. In order to establish conservation strategies and to characterize polymorphism of the threatened subspecies, genetic and morphologic analysis has to be implemented. Our study describes the application and optimalization of SRAP procedures to A . exscapus, including sampling, DNA extraction, PCR amplification and electrophoresis. Sequence Related Amplified Polymorphism (SRAP) molecular marker system is going to be used on eight different populations of A. exscapus ssp. transsilvanicus to identify the degree of polymorphism which is essential for the evolution of species. The methodology was tested and optimized successfully in two repetitive experiments. The results confirmed the suitability of the DNA extraction method to be applied and the optimization of the SRAP technique for the large-scale studies we are performing on this subspecies.

SRAP as an Informative Molecular Marker to Study the Fusarium poae Genetic Variability

AbstractFusarium poae is one of the Fusarium species isolated from grains associated with Fusarium head blight (FHB), whose occurrence has increased in the last years. In this study, a total of 105 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated using sequence‐related amplified polymorphism (SRAP) to analyse the capacity of this molecular marker to evaluate the F. poae genetic variability. The molecular analysis showed high intraspecific variability within F. poae isolates, and a partial relationship was revealed between variability and the host/geographic origin. Analysis of molecular variance (amova) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between American and European populations. The analysis of sequenced SRAP fragments targets into hypothetical proteins from different Fusarium species showing that the SRAP technique not only allows studying F. poae genetic variability, but also targets coding regions into the F. poae genome. To our knowledge, this is the first report on genetic variability of F. poae using SRAP technique and also demonstrates the efficacy of this molecular marker to amplify open reading frames in fungus.

Evaluation of bamboo genetic diversity using morphological and SRAP analyses.

Bamboo is an important member of the giant grass subfamily Bambusoideae of Poaceae. In this study, 13 bamboo accessions belonging to 5 different genera were subjected to morphological evaluation and sequence-related amplified polymorphism (SRAP) analysis. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among accessions. On the basis of morphological characteristics, the 13 accessions were distinctly classified into 2 major clusters; 3 varieties, PPYX, PGNK, and PLYY were grouped as cluster A, and 10 accessions were categorized under cluster B. Similarity coefficients ranging from 0.23 to 0.96 indicated abundant genetic variation among bamboo varieties. Approximately 38 SRAP primer combinations generated 186 bands, with 150 bands (80.65%) showing polymorphisms among the 13 accessions. Based on SRAP analysis, 13 bamboo accessions were grouped into 3 major clusters. Five species comprised Cluster I (PASL, PLYY, PTSC, SCNK, and BMAK), which belongs to genus Phyllostachys. Cluster II consisted of 5 varieties, PASL, PLYY, PTSC, SCNK, and BMAK; Cluster III included 3 varieties, PGNK, PLSY, and BMRS. Comparison of the results generated by morphological and SRAP analyses showed that the classification based on SRAP markers was more concordant to the taxonomic results of Gamble than that performed using morphological characters, thus suggesting that SRAP analysis is more efficient in evaluating genetic diversity in bamboos compared to morphological analysis. The SRAP technique serves as an alternative method in assessing genetic diversity within bamboo collections.

Comparative analysis of genetic diversity among species of Chrysanthemum and its related genera using inter-simple sequence repeat and sequence-related amplified polymorphism markers.

In this study, inter-simple sequence repeats (ISSRs) and sequence-related amplified polymorphism (SRAP) were applied to assess the genetic diversity in 38 species of Chrysanthemum and related genera. A total of 204 and 567 bands were amplified by 24 ISSR and 25 SRAP primers, of which 196 (97%) and 557 (99%) were polymorphic, respectively. The ISSR-based genetic similarity ranged from 0.016 to 0.886 and averaged 0.201, while the SRAP-based genetic similarity varied from 0.010 to 0.811 and averaged 0.122. Both the ISSR and SRAP techniques revealed similar clustering patterns and grouped species of Chrysanthemum and Ajania together. The results of principal coordinate analysis corroborated the unweighted pair group method with arithmetic average clustering. Additionally, results from ISSR and SRAP data were significantly correlated (r = 0.89, P < 0.001). Knowledge about genetic diversity among species can aid the transfer of traits of interest from the wild into cultivated chrysanthemum in future distant interspecific breeding.

Research Note Genetic diversity of endangered Polyporus umbellatus from China assessed using a sequence-related amplified polymorphism technique

Polyporus umbellatus (Pers.) Fries is an endangered medicinal fungus in China with in vivo anticancer activity, but its genetic information is lacking. Eight natural P. umbellatus strains collected from 7 provinces in China were subjected to sequence-related amplified polymorphism markers to estimate the level and pattern of genetic diversity. Forty-nine primer combinations generated 1219 highly reproducible and discernible loci, among which 1023 were polymorphic. The percentage of polymorphism varied from 35.71 to 96.30 with an average of 83.92. Genetic identity among all strains ranged from 0.15 to 0.78 with an average of 0.46. The unweighted pair group method with arithmetic mean dendrogram clustered 8 strains into 3 clusters, and the clustering pattern showed 3 groups. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and instead displayed a clustered distribution pattern. A relatively high level of genetic diversity was maintained in 8 natural P. umbellatus strains, but its abundance might be subjected to environmental heterogeneity, and the population structure of co-evolved Armillaria species may be selected by nature under the specific microenvironment.

Genetic diversity of Cynodon radiatus assessed by sequence-related amplified polymorphism markers

Abstract Sequence-related amplified polymorphism (SRAP) markers were used to assess the genetic relatedness among 33 Cynodon radiatus accessions from different regions of China. Fifteen primer combinations were used to amplify specific C. radiatus genomic sequences. A total of 382 SRAP fragments were generated, with fragment sizes ranging from 200 to 1800 bp. Genetic similarity coefficients among the 33 accessions ranged from 0.53 to 0.95, with an average of 0.70. Cluster analysis by two methods, the unweighted pair–group method with arithmetic averages and the principle coordinate analysis, separated the accessions into 3 distinct groups. This study shows that the SRAP technique is a reliable tool for differentiation of C. radiatus accessions and for determination of the genetic relationships among them.

Identification of Genetic Diversity Among Cultivars of Phyllostachys violascens Using ISSR, SRAP and AFLP Markers

Bamboo is one of the most important forest resources with a strong carbon fixation capability. To utilize genetic resource of Phyllostachys violascens, ISSR (inter-simple sequence repeat), SRAP (sequence-related amplified polymorphism), and AFLP (amplified fragment length polymorphism) techniques were used for the first time for the assessment of genetic diversity within its different cultivars. A total of 209 (136 polymorphic), 222 (152 polymorphic), and 434 (253 polymorphic) bands were detected using 15 ISSR primers, 15 primer combinations of SRAP, and 15 primer combinations of AFLP, respectively. The mean genetic similarity of Ph. violascens was 0.872, 0.867 or 0.871 for the ISSR, SRAP and AFLP analyses, respectively. Based on genetic diversity, all the cultivars of Ph. violascens could be divided into four groups, which are reflected by their morphologies. Our data demonstrated that all three methods are useful in the identification of genetic diversity in Ph.violascens, but AFLP is the most efficient.

Evaluation of genetic diversity in Piper spp using RAPD and SRAP markers

Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) analysis were applied to 74 individual plants of Piper spp in Hainan Island. The results showed that the SRAP technique may be more informative and more efficient and effective for studying genetic diversity of Piper spp than the RAPD technique. The overall level of genetic diversity among Piper spp in Hainan was relatively high, with the mean Shannon diversity index being 0.2822 and 0.2909, and the mean Nei's genetic diversity being 0.1880 and 0.1947, calculated with RAPD and SRAP data, respectively. The ranges of the genetic similarity coefficient were 0.486-0.991 and 0.520-1.000 for 74 individual plants of Piper spp (the mean genetic distance was 0.505 and 0.480) and the within-species genetic distance ranged from 0.063 to 0.291 and from 0.096 to 0.234, estimated with RAPD and SRAP data, respectively. These genetic indices indicated that these species are closely related genetically. The dendrogram generated with the RAPD markers was topologically different from the dendrogram based on SRAP markers, but the SRAP technique clearly distinguished all Piper spp from each other. Evaluation of genetic variation levels of six populations showed that the effective number of alleles, Nei's gene diversity and the Shannon information index within Jianfengling and Diaoluoshan populations are higher than those elsewhere; consequently conservation of wild resources of Piper in these two regions should have priority.

Grain yield stability and molecular characterization of durum wheat genotypes under heat stress conditions

The objectives of the present study are to determine the performance of six promising durum wheat genotypes for yield stability under heat stress conditions and to compare the application and utility of SRAP (Sequence-Related Amplified Polymorphism) and TRAP (Target Region Amplified Polymorphism ) marker techniques, for analysis of genetic diversity among durum wheat genotypes under heat stress. Field experiments were conducted for four sowing dates, over two seasons, to expose genotypes to different levels of heat stress during the grain-filling period. Grain yield and yield attributes during the grain filling period were investigated. Results indicated that significant variations were observed among different durum wheat genotypes in respect of all yield attributes. The effect of sowing date on the relative grain yield of durum genotypes was of greater magnitude than the effect of year. On the other hand, under the fourth sowing date (20th January), where heat stress was imposed, line KSUDW104 was the best performing line (3.26 ton/ha) out yielding Benysowef (2.21 ton/ha) by 47.5% and Kronos (2.41 ton/ha) by 35.3%. This line should be recognized as heat tolerant germplasm. The regression coefficients were significant for the six durum genotypes which indicated that they were highly responsive to the change in the average productivity of the growing season. SRAP and TRAP markers, were assayed to determine the genetic diversity of 6 durum wheat genotypes. In SRAP analysis, 45 out of 128 bands (35.16%) were polymorphic while in TRAP analysis, 22 out of 55 bands (40.0%) were polymorphic. The dendrogram based on SRAP markers differed from that based on TRAP markers. Key words: Durum wheat, genetic diversity, heat tolerance, SRAP markers,TRAP markers, yield stability.

Analysis of the Genetic Relationships in Chinese Ziziphus with SRAP Markers

The phylogenic and genetic relationships in germplasm resources of Chinese Ziziphus were studied at molecular levels, for providing new molecular evidences of classification, protection, and utilization of germplasm resources of Ziziphus. The sequence-related amplified polymorphism (SRAP) was assessed to analyse the genetic relationships among 14 species of Ziziphus, 11 cultivars of Z. jujuba Mill., and one outgroup. A total of 580 DNA bands were amplified by 19 selective primers, 570 of which (98.28%) were polymorphic. The average number of polymorphic DNA bands amplified by each primer was 30. The genetic similarities of 26 sample materials were between 0.22 and 0.99. UPGMA method cluster analysis showed that 26 sample materials were classified into six cluster groups with the genetic similarity of 0.28. The results showed that SRAP technique is efficient in studying genetic relationships among Ziziphus, Z. jujuba Mill. and Z. acidojujuba C. Y. Cheng et M. J. Liu which should be treated as one species, and further infraspecific classification of Z. jujuba Mill. should be classified into two subspecies; the scientific names of new taxa, Chinese jujube, and wild jujube were Ziziphus jujuba Mill. subsp. jujuba and Ziziphus jujuba Mill. subsp. spinosa (Bunge) J. Y. Peng, X. Y. Li et L. Li, respectively. Z. xiangchengensis Y. L. Chen et P. K. Chou, Z. montana W.W. Smith and Z. mairei Dode might be originated from same ancestry. Z. xiangchengensis Y. L. Chen et P. K. Chou and Z. montana W.W. Smith should be treated as one species. The infrasubspecific taxon of Z. jujuba Mill. was not suitable to set up varieties.

Genetic diversity of a germplasm collection of &lt;I&gt;Cucumis melo&lt;/I&gt; L. using SRAP markers

Genetic relationships and classifications of a set of melon accessions were analyzed to provide the experimental support for utilizing effectively genetic materials for breeding. Sequence-related amplified polymorphism technique was adopted to analyze 61 melon accessions. Sixteen primer combinations with clear band pattern and polymorphism were selected from over 42 primer combinations. Four hundred and fifty-two loci were detected by 16 pairs of SRAP primers. Among them, 265 were polymorphic, the polymorphic rate was 58.63%, and 28.56 loci and 16.56 polymorphic loci were amplified by each pairs of primers on average. The genetic similarity coefficient of the 61 accessions ranged from 0.48 to 0.93, with an average of 0.73. These results suggested that there was rich genetic diversity among the melon accessions tested. The varieties examined were clustered into two groups,which were thick-skinned melon and thin-skinned melon. Five groups were clustered according to genetic similarity coefficient of 0.74. The Nei's gene diversity index and Shannon's Information index of melon were 0.2231 and 0.3422, respectively, in Xinjiang, the highest among all the ecological regions.

Sequence‐related amplified polymorphism, an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significance

In the present study, a recently described molecular approach, namely sequence-related amplified polymorphism (SRAP), which preferentially amplifies ORFs, was evaluated for the studies of genetic variation among Fasciola hepatica, Fasciola gigantica and the "intermediate" Fasciola from different host species and geographical locations in mainland China. Five SRAP primer combinations were used to amplify 120 Fasciola samples after ten SRAP primer combinations were evaluated. The number of fragments amplified from Fasciola samples using each primer combination ranged from 12 to 20, with an average of 15 polymorphic bands per primer pair. Fifty-nine main polymorphic bands were observed, ranging in size from 100 to 2000 bp, and SRAP bands specific to F. hepatica or F. gigantica were observed. SRAP fragments common to F. hepatica and the "intermediate" Fasciola, or common to F. gigantica and the "intermediate" Fasciola were identified, excised and confirmed by PCR amplification of genomic DNA using primers designed based on sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair-group method with arithmetic averages clustering algorithm categorized all of the examined representative Fasciola samples into three groups, representing the F. hepatica, the "intermediate" Fasciola, or the F. gigantica. These results demonstrated the usefulness of the SRAP technique for revealing genetic variability between F. hepatica, F. gigantica and the "intermediate" Fasciola, and also provided genomic evidence for the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica. This technique provides an alternative and a useful tool for the genetic characterization and studies of genetic variability in parasites.