Random Amplified Polymorphic DNA Fragments Research Articles

Mapping of tms8 gene for temperature‐sensitive genic male sterility (TGMS) in rice (Oryza sativa L.)

With 3 figuresAbstractGenetic analysis of F2 and backcross populations of an induced temperature‐sensitive genic male sterility (TGMS) mutant source F 61 with normal pollen parents revealed that TGMS trait was controlled by a single recessive gene. Molecular tagging of TGMS trait was attempted using polymorphic randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers through bulked segregant analysis. The RAPD primers UBC 345830, UBC 313927, microsatellites RM224 and RM21 produced putative markers, which differentiate parents and bulks from sterile parent and sterile bulks. The RAPD analysis of individual F2 plants with the primer UBC345830 showed perfect marker–phenotype cosegregation. The 830‐bp RAPD fragment, which segregated with TGMS locus at a distance of 1.33 cM, was eluted and cloned, and sequence information was used for designing sequence‐characterized amplified region (SCAR) primer, which cosegregated with TGMS locus at a distance of 0.8 cM. TGMS locus was mapped onto chromosome 11 using RM21 and RM224, flanking it at a distance of 4.3 and 3.0 cM, respectively. The DNA markers tightly linked to TGMS gene (tms8) in F 61 can be cost effectively used for marker‐assisted selection of TGMS trait.

국내에서 분리된 Verticillium dahliae의 유전적 유연관계 분석

국내 Verticillium dahliae 균주를 공시하여 다른 Verticillium 종과의 유전적 차이와 다양성을 밝히고자 국내 두 지역으로 부터 분리한 V. dahliae를 포함한 7종의 Verticillium속 14균주를 대상으로 mitochondrial small subunit rRNA gene (rns) 영역 염기서열 분석과 random-amplified polymorphic DNA(RAPD)를 실시하였다. rns 영역 염기서열의 분석에서 국내 분리균인 5개의 V. dahliae균주는 Verticillium속 내 다른 종들과 달리 하나의 그룹을 형성하였고, 외국 균주들과도 동일한 그룹을 이루었다. rns 영역 염기서열 분석뿐만 아니라 RAPD 분석에서도 Verticillium속의 다른 종들과 구별되어 V. dahliae가 한 그룹을 형성하였으나 V. dahliae 균주간에 형성된 세 개의 소그룹을 통하여 동일한 국화과 작물로 부터 분리한 V. dahliae 간에 분리된 지역에 따라 유전적 다양성이 존재함을 확인하였다. 이러한 결과는 국내 V. dahliae의 유전적 다양성연구와 유연관계분석에 기초적인 자료로 사용될 것이다. To provide basic information for Verticillium spp., molecular methods were applied to analyze genetic characteristics within Verticillium spp. including Verticillium dahliae, isolated from diseased plants in two regions of Korea. Five Korean isolates of V. dahliae causing Verticillium wilt on chrysanthemum were analyzed, together with six other Verticillium spp., using mitochondrial small subunit rRNA gene (rns) sequence and random-amplified polymorphic DNA (RAPD). In a phylogenetic tree based on rns region sequences, Korean V. dahliae isolates formed a single clade with foreign isolates, whereas the other Verticillium spp. formed separate groups. In addition to rns sequence analysis, a dendrogram based on RAPD fragment patterns also showed clustering of all V. dahliae isolates into one group, separate from the six different Verticillium spp., and the V. dahliae isolates formed three subgroups which corresponded to the regions of origin, Kumi, Busan city and Canada. This indicates that high genetic variation exists between regions, although the fungus was isolated from the same host plant, chrysanthemum. These results provide the foundation for the study of genetic diversity and relationships among V. dahliae isolates in Korea.

A SCAR molecular marker to distinguish Bursaphelenchus mucronatus from the pinewood nematode, B. xylophilus

SummaryBursaphelenchus mucronatus is closely related to the pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. B. xylophilus became a devastating pest when it was introduced in the Far East; however, B. mucronatus is considered to have low virulence. Morphological similarities between B. xylophilus and B. mucronatus make the accurate morphological identification of both species difficult. Thus, it has become important to pay attention towards B. mucronatus impact and the need of discrimination of these two species. To distinguish among the two species, a B. mucronatus‐specific sequence‐characterized amplified region (SCAR) marker has been developed. The specific Random amplified polymorphic DNA (RAPD) fragment of B. mucronatus, OPY01‐M850 was excised from agarose gels and purified. The gel‐purified fragment was cloned into the pGEM®‐T Vector and subjected to sequencing. Based on the sequenced RAPD fragments, a number of SCAR primers were designed. It is demonstrated that OPY01‐M850 through primers Y01F/R can be transformed into a B. mucronatus‐specific SCAR‐Y01‐M609 marker. Primers set Y01F/R had high specificity that could be used for the discriminative identification of B. mucronatus versus B. xylophilus.

Molecular tagging and candidate gene analysis of the high gamma-tocopherol trait in safflower (Carthamus tinctorius L.)

Genetic control of the synthesis of high gamma-tocopherol (gamma-T) content in the seed oil of safflower (Carthamus tinctorius L.) and development of highly reliable molecular markers for this trait were determined through molecular tagging and candidate gene approaches. An F2 population was developed by crossing the high gamma-T natural mutant IASC-1 with the CL-1 line (standard, high alpha-T profile). This population segregated for the partially recessive gene Tph2. Bulked segregant analysis with random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers revealed linkage of eight RAPD and one SSR marker loci to the Tph2 gene and allowed the construction of a Tph2 linkage map. RAPD fragments closest to the Tph2 gene were transformed into sequence-characterized amplified region markers. A gamma-T methyltransferase (gamma-TMT) locus was found to co-segregate with Tph2. The locus/band was isolated, cloned and sequenced and it was confirmed as a gamma-TMT gene. A longer partial genomic DNA sequence from this gene was obtained. IASC-1 and CL-1 sequence alignment showed one non-synonymous and two synonymous nucleotide mutations. Intron fragment length polymorphism and insertion-deletion markers based on the gamma-TMT sequence diagnostic for the Tph2 mutation were developed and tested across 22 safflower accessions, cultivars, and breeding lines. The results from this study provide strong support for the role of the gamma-TMT gene in determining high gamma-T content in safflower and will assist introgression of thp2 alleles into elite safflower lines to develop varieties with improved tocopherol composition for specific market niches.

Genetic Variability Analysis and Molecular Detection of Fusarium oxysporum f.sp. eustomae Isolated from Eustoma grandiflorum in Northern Italy

AbstractA total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiflorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505‐bp SCAR (sequence characterized amplified region) marker (SCAR505) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature‐controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development.

Genetic Diversity Analysis in Piper Species (Piperaceae) Using RAPD Markers

The genetic diversity of eight species of Piper (Piperaceae) viz., P. nigrum, P. longum, P. betle, P. chaba, P. argyrophyllum, P. trichostachyon, P. galeatum, and P. hymenophyllum from Kerala state, India were analyzed by Random amplified polymorphic DNA (RAPD). Out of 22 10-mer RAPD primers screened, 11 were selected for comparative analysis of different species of Piper. High genetic variations were found among different Piper species studied. Among the total of 149 RAPD fragments amplified, 12 bands (8.05%) were found monomorphic in eight species. The remaining 137 fragments were found polymorphic (91.95%). Species-specific bands were found in all eight species studied. The average gene diversity or heterozygosity (H) was 0.33 across all the species, genetic distances ranged from 0.21 to 0.69. The results of this study will facilitate germplasm identification, management, and conservation.

Efficiency of RAPD and ISSR Markers in Differentiation of Homo- and Heterokaryotic Protoclones of Agaricus bisporus

Morphologically, nine different slow-growing protoclones were screened from regenerated protoplast of heterokaryotic Agaricus bisporus. The present study is the first report of fingerprinting on differentiating homo- and heterokaryotic protoclones using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Among 80 primers tested, the seven ISSR and seven RAPD primers selected for the analysis generated a total of 94 ISSR and 52 RAPD fragments, respectively. ISSR fingerprinting detected more polymorphic loci (38.29%) than RAPD fingerprinting (34.61%). Principal coordinate analysis (PCA) was employed to evaluate the resolving power of the markers to differentiate protoclones. The mean polymorphism information content (PIC) for each of these marker systems (i.e., 0.787 for RAPD and 0.916 for ISSR, respectively) suggests that the ISSR marker system was more effective in determining polymorphisms. The dendrograms constructed using RAPD, ISSR, and integrated RAPD, and ISSR marker systems were highly correlated with each other as revealed by the high Mantel correlation (r = 0.98). Pair-wise similarity index values ranged from 0.64 to 0.95 (RAPD), 0.67 to 0.98 (ISSR), and 0.67 to 0.98 (RAPD and ISSR), and mean similarity index values of 0.82, 0.81, and 0.84 for RAPD, ISSR, and combined data, respectively, were obtained. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment and accurate differentiation of homo- and heterokaryotic protoclones of A. bisporus.

Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers

Biochemical and molecular markers have been used on eleven species of Cucurbitaceae collected from lower Gangetic plains. Six enzyme systems were selected. Among 40 primers examined, 14 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) primers were selected for the analysis. Generated RAPD (100) and ISSR (100) fragments showed high variations among the species. Jaccard similarity coefficients were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, principal coordinate analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.

Genetic diversity and population structure of Fusarium oxysporum f. sp. cepae, the causal agent of fusarium basal plate rot on onion, using RAPD markers

A total of 116 single spore isolates of Fusarium oxysporum f. sp. cepae representing seven populations from Turkey and one population from Colorado, USA were subjected to molecular marker analysis using Random amplified polymorphic DNA (RAPD) technique. Using eleven polymorphic primers, 110 RAPD fragments were obtained with an average of 10 polymorphic bands per primer. Cluster analysis with UPGMA revealed five distinct lineages at arbitrary level of 65% similarity. The majority of F. oxysporum f. sp. cepae isolates from Turkey were observed to derive probably from the same clonal lineage. Genetic estimates and population differences demonstrated that the isolates from Turkey were significantly distinct from Colorado isolates and that Bursa population was the most divergent among Turkish populations. Cluster analysis of Nei's genetic distances supported populations grouping according to the geographical regions. Comparison of genetic differentiation estimates (H T : 0.140, H S : 0.124) revealed low levels of genetic differentiation among Turkish populations. Only, 11.4% of total genetic diversity (G ST ) attributed to differentiation among the geographical populations. Analysis of molecular variance (AMOVA) confirmed that there was low genetic differentiation among populations. The results suggest that RAPD-PCR is a useful method for analyzing genetic variation within and between populations of F. oxysporum f. sp. cepae .

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Clonal diversity along a water depth gradient in a declining reed stand as detected by three different genetic methods

Isozyme, RAPD (random amplified polymorphic DNA) and microsatellite techniques were applied to determine the genetic diversity of a reed stand declining at the open water fringe for several decades and the accuracy of these methods was evaluated. Sampling was carried out along three transects parallel with and one transect at right angle to the lakeshore. In isoenzyme investigation, esterase and further 11 polymorphic loci of seven isozymes (with a total of 26 alleles) were analysed and 39 clones were distinguished. RAPD analysis involved 11 random decamer primers and, based on 141 repeatably amplifying and polymorphic RAPD fragments, 52 multilocus genotypes were differentiated. In microsatellite investigation, four primer pairs provided 47 polymorphic allelic variants that determined 37 multilocus phenotypes. Besides the differences, all the three methods revealed high number and intermingling (mixed) arrangement of clones at lower water depths, and low clone number and successive structure at the open water fringe of the stands. Only microsatellite analysis revealed different stages of colonization: old successful clone (colonizing the deeper water but almost completely disappearing at the lakeshore edge) and successful but young clone (with large extension at the lakeshore and just reaching the open water edge of the stand) were also distinguished. Finally, the present paper demonstrated that an appropriate data evaluation could make the result of RAPD comparable to that of microsatellite studies.

Assessment of Genetic Relationship Among Landraces of Bangladeshi Ridge Gourd (Luffa acutangula Roxb.) Using RAPD Markers

Information on genetic relatedness among ridge gourd (Luffa acutangula) genotypes from Bangladesh is currently not reported. Twenty eight accessions collected from different parts of Bangladesh were studied using random amplified polymorphic DNA (RAPD) technique. Four selected decamer primers, out of sixteen screened, could generate a total of 27 RAPD fragments of which 22 were polymorphic (81.5%). The bands ranged from 50 to 1500 bp in size. Genetic variation statistics for all loci estimated the average gene diversity (h) value as 0.278 and the Shannon’s Information Index (I) as 0.415. Dendrogram based on unweighted pair-group method with arithmetic averages (UPGMA) segregated the accessions into five clusters. Cluster III was the largest with 13 members followed by cluster II, V, I and IV with 6, 4, 3 and 2 members, respectively. Accession LA27 and LA29 were found very close to each other with the highest inter-variety similarity index (96.05%) and the lowest genetic distance (0.077); whereas accession LA40 and LA72 were more distant to each other with the lowest inter-variety similarity index (44.43%) and the highest genetic distance (0.73). A DNA extraction method has been standardized. The marker was found to be useful tool for assessing genetic variations in Luffa acutangula. Keywords: Genetic relationship; Germplasm; Ridge gourd; Luffa acutangula; RAPD. © 2009 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v1i3.1968 J. Sci. Res. 1 (3), 615-623 (2009)

Genetic Characterization of a New Growth Habit Mutant in Tomato (Solanum lycopersicum)

A mutant tomato cultivar (HT10-3-2) with indeterminate growth habit was obtained from a cultivar (T10-3-2) with determinate growth habit. The character of indeterminate growth habit in HT10-3-2 could be inherited stably. Unlike other normal growth habit cultivars, which are controlled by the SELF PRUNING gene, it was shown here that HT10-3-2 has no mutation in the sp gene. Two hundred random amplified polymorphic DNA (RAPD) primers were used to screen for polymorphism between the two genotypes from genomic DNA, and a polymorphic fragment (S1681458) was obtained and subsequently sequenced. However, Basic Local Alignment Search Tool searches indicated that the sequence of this RAPD fragment shares no significant homology with known sequences in GenBank. The RAPD marker (S1681458) was converted to a sequence characterized amplified region (SCAR) marker, SCA1681453. The SCAR marker was tested using an F2 population derived from the cross of T10-3-2 and HT10-3-2 and the results showed that this marker was closely linked with the unknown factor influencing the growth habit in HT10-3-2.

Identification of rice hybrids using microsatellite and RAPD markers

Having produced a series of hybrid rice varieties especially the first Iranian hybrid rice (IRH1), fingerprinting and genetic purity determination of hybrid seeds were conducted using microsatellite (SSR) and random amplified polymorphic DNA (RAPD) markers. Sixteen rice genotypes including 3 cytoplasmic male sterile (CMS) lines, 5 restorer lines and their 8 hybrid combinations were used in this study. Ten out of fourteen SSR markers produced polymorphic bands in the 16 rice genotypes. The first Iranian hybrid rice, IRH1, could be differentiated from other hybrids possessing a unique SSR fingerprint. Although the SSR markers amplified the unique fingerprints for the restorer lines, they could not differentiate between 2 CMS lines (Neda-A and Nemat-A) and their resultant hybrids. The rice CMS lines and restorer lines could be uniquely identified by RAPD multilocus amplified profile at 7 informative loci. Cluster analysis based on shared alleles and Jaccard’s similarity coefficient using UPGMA algorithm, grouped the rice genotypes into 3 and 4 major clusters according to their microsatellite and RAPD fragment similarities respectively. Non-parental bands were also observed in addition to parental bands which probably correspond to heteroduplex molecules formed by two allelic sequences of different amplified fragments. This demonstrates that heteroduplex bands can be an indicative feature for identifying hybrid individuals.

Genetic characterization of two Sudanese goat breeds ( Capra hircus ) using RAPD molecular markers

Seven primers of randomly amplified polymorphic DNA (RAPD) were selected to study the genetic variations among 14 individuals of goat (Capra hircus) from two domestic Sudanese goat breeds (Nilotic and Nubian). The test generated 59 entirely repeatable RAPD fragment bands and the statistical analysis showed 55 polymorphic bands among the 14 individuals. The genetic distances among the population range from 8 to 72%. The highest dissimilarity coefficient was between individuals within the Nilotic breeds while there was a comparatively low degree of differentiation among the Nubian population. The constructed UPGMA dendrogram of the coefficient of similarity showed that the Nubian clustered together while the individuals from the Nilotic form 4 groups. It was clearly seen that the link between the individual of the Nilotic is quite weak and some of them linked to the Nubian. The results of the study offer useful information about some Sudanese goat breeds.

Generation of a Sequence Characterized Amplified Region Probe for Authentication of Mainland Serow (Capricornis sumatraensis)

Mainland serow is an endanged artiodactyl of southern Anhui province, China, that is often subject to poaching. To provide an easy, rapid and reliable marker for identification of bushmeat, skin and other tissues of the species, we developed a sequence characterized amplified region (SCAR) based on a species-specific random amplified polymorphic DNA (RAPD) marker. Initially, a 1012-bp species-specific DNA fragment of mainland serow was detected by a RAPD primer S1193. Then, a serow-specific primer pair (SCF/SCR) was designed according to the specific RAPD fragment, resulting in a 438-bp SCAR for the species. Finally, the reliability of the SCAR primers was tested by a common multiplex polymerase chain reaction using the combination of the SCAR and cyt b universal primers. The results that all mainland serow samples presented two target bands but the others failed to produce the SCAR indicated that the designed primers were highly diagnostic. Therefore, the SCAR probe developed in this study will be useful for quick authentication of mainland serow tissue samples for conservation biology and bushmeat regulation.

Mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis)

The mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean+/- s.e. t(m) = 0.86+/-0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%-70%), followed by among pods within inflorescence (29%-37%), and the least variation among inflorescences within tree (1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.

RAPD markers associated with quercetin accumulation in Psidium guajava

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.

A contribution to characterisation of genetic variation in some natural Polish populations ofElymus repens(L.) Gould andElymus hispidus(Opiz) Melderis (Poaceae) as revealed by RAPD markers

To determine the relative importance of clonal growth and sexual reproduction, the Randomly Amplified Polymorphic DNA (RAPD) method was used to study genetic diversity and clonal structure of six populations of Elymus repens and four populations of Elymus hispidus from Poland. These outbreeding species are virtually self-sterile and form widely spreading and long-lived rhizomes. Using 12 primers, a total of 150 unambiguous RAPD fragments were amplified and scored. Results of AMOVA showed no significant genetic distinction between morphologically distinguished varieties of E. repens and E. hispidus. E. repens had slightly higher intra-specific genetic polymorphism than E. hispidus; the percentage of polymorphic bands per population ranged from 38 to 49 and from 19 to 38 respectively. Clonal diversity measured using the Simpson diversity index (D) indicated different contributions of clonal reproduction in particular populations of E. repens (D: 0.20-0.72). Populations of E. hispidus were dominated by one or a few clones, which were generally restricted to a single population (D: 0.00-0.22). RAPD revealed that most genetic diversity resided within populations of the two studied species, suggesting that, despite their clonal character, propagation by seeds contributes considerably to reproduction of E. repens and E. hispidus.

Development of RAPD‐SCAR and RAPD‐generated PCR‐RFLP markers for identification of four Anguilla eel species

Discriminating between eel species of the genus Anguilla using morphological characteristics can be problematic, particularly in the glass eel and elver stages. In this study, sequence‐characterized amplified region (SCAR) and polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) markers were developed for the identification of Anguilla japonica, Anguilla bicolor bicolor, Anguilla rostrata, and Anguilla anguilla. Random amplified polymorphic DNA (RAPD) fragments from A. japonica (362 bp), A. bicolor bicolor (375 bp), A. rostrata (375 bp), and A. anguilla (375 bp) were isolated, sequenced, and converted to SCAR markers. The principal difference between the SCARs of A. japonica and the three other species is the absence of a 13 bp deletion in the A. japonica SCAR. Specific PCR primers amplified a 290 bp fragment for A. japonica and 303 bp fragments for A. bicolor bicolor, A. rostrata, and A. anguilla. Restriction enzyme digestion with TaqI, MaeI, and Tru9I yielded PCR‐RFLP patterns with differences that, when analyzed together, are sufficient for distinguishing each of the four eel species. In addition, RAPD fragments for A. japonica (577 bp), A. bicolor bicolor (540 bp), A. rostrata (540 bp), and A. anguilla (509 bp) were also isolated and sequenced. The A. japonica, A. bicolor bicolor, A. rostrata, and A. anguilla PCR products contain ten, nine, nine, and eight tandem repeats, respectively, of a 37 bp sequence. These results suggest that SCAR and PCR‐RFLP markers and repeat numbers for specific loci will be useful for the identification of these four Anguilla eel species.