Random Amplified Polymorphic DNA-polymerase Chain Reaction Research Articles

Random amplified polymorphic DNA-based molecular heterogeneity analysis of Salmonella enterica isolates from foods of animal origin.

Aim:This study aims to study the significance of random amplified polymorphic DNA (RAPD) typing in heterogeneity analysis of Salmonella serovars, isolated from foods of animal origin.Materials and Methods:Salmonella serovars isolated and identified from different foods of animal origin such as meat, milk, and egg by standard bacteriological methods. DNA isolated from all 10 isolates which are confirmed by biochemical and serotyping methods and then RAPD was performed using the primers OPB 10, primer 1290, NSC I, NSC II, and primer 3. Then, RAPD data were analyzed using the BioNumerics software, Belgium, Germany.Results:RAPD polymerase chain reaction (PCR) using five primers, namely OPB 10, primer 1290, NSC I, NSC II, and primer 3, classified the 10 isolates into 9, 10, 10, 7, and 10 RAPD-PCR types with discriminating powers of 0.1987, 0.423, 0.50889, 0.1842, and 0.2582, respectively. The phylogram constructed with NSC I profile classified isolates based on geographical origin. Primer 1290, NSC II, and primer 3 produced some uniform bands in all isolates indicating their binding ability in conserved genomic region. This study revealed that RAPD profile can be best used for finding out the heterogeneity at molecular level of Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support earlier observation of its significance by different workers on different Salmonella serotypes.Conclusion:Repeatability of RAPD-PCR is insufficient to distinguish genetic differences among Salmonella serovars.

Analyses of the genetic diversity and population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay.

We studied the genetic diversity and the population structure of human isolates of Histoplasma capsulatum, the causative agent of histoplasmosis, using a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assay to identify associations with the geographic distribution of isolates from Mexico, Guatemala, Colombia and Argentina. The RAPD-PCR pattern analyses revealed the genetic diversity by estimating the percentage of polymorphic loci, effective number of alleles, Shannon's index and heterozygosity. Population structure was identified by the index of association (IA) test. Thirty-seven isolates were studied and clustered into three groups by the unweighted pair-group method with arithmetic mean (UPGMA). Group I contained five subgroups based on geographic origin. The consistency of the UPGMA dendrogram was estimated by the cophenetic correlation coefficient (CCCr = 0.94, P = 0.001). Isolates from Mexico and Colombia presented higher genetic diversity than isolates from Argentina. Isolates from Guatemala grouped together with the reference strains from the United States of America and Panama. The IA values suggest the presence of a clonal population structure in the Argentinian H. capsulatum isolates and also validate the presence of recombining populations in the Colombian and Mexican isolates. These data contribute to the knowledge on the molecular epidemiology of histoplasmosis in Latin America.

Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms.

Lactococcus garvieae is now recognized as a species with clinical significance for human and veterinary medicine. The aim of this study was to evaluate the presence of this pathogen in sand bedding and milk samples. Two farms in Minnesota with problems of clinical and subclinical mastitis due to streptococci-like organisms were selected. Twenty-four Lactococcus garvieae isolates from sand bedding and 18 isolates from quarter milk were comparatively studied using a genotypic approach. RAPD (random amplification of polymorphic DNA) PCR and REP (repetitive element palindromic) PCR experiments highlighted a similar electrophoretic profile. When genes belonging to the core genome of L. garvieae were tested through a MLRT (multilocus restriction typing), we again observed that all L. garvieae isolates coming from sand bedding and milk shared a common profile, distinguishable from previously studied representative L. garvieae strains. These data indicate that the L. garvieae isolated from sand bedding and milk originated from a few strains adapted to persist in the same habitat. This supports the hypothesis that sand bedding can represent a reservoir of L. garvieae strains and be a potential vehicle for their dissemination in dairy farms.

Morphological and Pcr Differentiation between some Phages from Bacillus thuringiensis Strain C18

Bacillus thuringiensis (Bt)-based pesticides hold the biggest share of the biopesticides market. This industry suffers huge economic losses due to the activation of their lysogenic phages during fermentation process. The locally isolated wide-spectrum BtaC18 is currently used for commercial production and found to harbors many of these integrated prophages. Many phages were isolated from a laboratory-grown BtaC18; from amongst them three belonged to the order Caudovirales were characterized. Traditional and morphological examination found that one of the three viruses has tail: ACTP-01 and two are non-tailed phages: ACTP-02 and ACTP-03. The ACTP-01 phage showed an isometric head (92.6 nm) with long non-contractile, flexible tail (321.33 x 12.5 nm). While, the ACTP-02 phage has a hexagonal head (50.82 nm) and the ACTP-03 phage possesses an icosahedral head (61.3 nm). Data suggested that ACTP-01 phage belongs to Siphoviridae family,while ACTP-02 and ACTP-03 belonged to the Tectiviridae family. Random Amplified Polymorphic DNA polymerase chain reaction (RAPD-PCR) showed a strong differentiating power between the isolated phages. In conclusion, the numerous phages hosted by this bacterium complicate the derivatization of phage resistant BtaC18 strain.

Genetic Analysis with Random Amplified Polymorphic DNA of the Multiple Enterocin-Producing Enterococcus lactis 4CP3 Strain and Its Efficient Role in the Growth of Listeria monocytogenes in Raw Beef Meat.

In this manuscript, a multiple enterocin-producing Enterococcus lactis strain named 4CP3 was used to control the proliferation of Listeria monocytogenes in refrigerated raw beef meat model. Also, the intraspecific genetic differentiation of 4CP3 strain was assessed by Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) analysis. E. lactis 4CP3 strain was found to produce the enterocins A, B, and P. It displayed activity against L. monocytogenes EGDe 107776 by agar-well diffusion method. The application of E. lactis 4CP3 culture at 107 CFU/g in raw beef meat was evaluated using both ANOVA and ANCOVA linear models in order to examine its effect on the growth of the pathogen L. monocytogenes during refrigerated storage. Hence, a very interesting result in decreasing (P<0.05) and suppressing the growth of L. monocytogenes in refrigerated raw beef meat was shown during 28 days of storage. In conclusion, E. lactis 4CP3 strain might be useful for prevention of the proliferation and survival of L. monocytogenes in raw meat during refrigerated storage.

DNA Sequencing Analysis of African Xanthomonas oryzae pv. oryzae Virulence Gene (AXaVrg) DNA Marker

Abstract Global rice production is constrained by bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo). BLB disease incidence in West Africa was between 70–85% and yield loss in farmers’ fields was in the range of 50–90% from 2005 to 2010. In the present study, African Xoo virulence gene OPP-172000 DNA marker was identified and purified using randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) products from 50 Xoo isolates. Genomic DNA of 50 Xoo isolates were analyzed using OPP-17 primer in RAPD-PCR during which African Xoo virulence gene OPP-172000 DNA marker was identified, purified, cloned, and sequenced. Cloning and DNA sequencing of African Xoo virulence gene OPP-172000 DNA generated a 1953 bp nucleotide sequence consequently tagged as AXaVrg-1953. BLAST homologous analysis of the AXaVrg-1953 sequence provides comprehensive identification of the type II secretion genes and secreted proteins, type III secretion genes and secreted proteins in African Xoo virulence gene. Phylogenetic unweighted pairgroup method arithmetic (UPGMA) analysis revealed the African AXaVrg-1953 sequence was distinct from the other Xoo virulence gene sequences from China, Japan, Korea, Germany, and the United States. This information is potentially useful for effective management of BLB disease in West Africa.

Identification of Lactobacillus Species Isolated From Traditional Dairy Products Using RAPD-PCR

Background: Probiotics are non-pathogenic useful microorganisms having positive effects on the host health. The aim of the present study was to discriminate Lactobacillus species extracted from traditional dairy products. Methods: This study was conducted on 26 specimens collected from traditional dairy products in Bukan. Lactobacillus species were separated and purified employing biochemical tests. Then, the intra/inter-species diversity was investigated using RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) technique. Results: Polymorphism information content (PIC) value varied between 15.9% and 34.4% with a maximum value of 34.4% associated with primer 1254. The mean of and Marker index (MI) for the 6 primers was 4.52, in which the maximum and minimum values belonged to the primers 1254 and OPA-02, respectively. The isolates were categorized into 4 main clusters according to Jaccard similarity coefficient using UPGMA (unweighted pair group method with arithmetic means) clustering method. Principle coordinates analysis (PCoA) demonstrated that the first and second components explained 30.59% and 22.48% of variances, i.e. 53.07% of variances in total. The results of RAPD marker indicated that the intra-species diversity was greater than inter-species diversity. The intra-group variance explained 94% of the all variance, while inter-group variance explained only 6% of the all variance. Moreover, the results of analysis of molecular variance (AMOVA) indicated that the highest level of discrimination occurred at the 16 groups cut-off point with a similar coefficient of 0.56. Conclusions: From the results of the present study, it can be concluded that traditional dairy products are enriched sources of probiotic bacteria which can ensure the health of general population and enhance their immunoe systems. Moreover, RAPD-PCR is an appropriate method for detection and calssification of lactobassiluses.

An Increasing Threat in an Iranian Referral Children's Hospital: Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii has emerged as an important nosocomial pathogen in the past decades. Due to the prevalence of A. baumannii across the world, suitable typing methods to investigate the epidemiological distribution of the organism have been developed. The aim of this study was to investigate the epidemiological and molecular diversity of A. baumannii strains isolated from nosocomial infections of hospitalized children in Children Medical Center Hospital (CMC), an Iranian referral hospital, in Tehran, Iran. A total of 27 non-duplicate clinical A. baumannii isolates were collected during October 2013 to March 2014 and tested for antimicrobial resistance to several antibiotic agents. The genetic similarity of the strains was investigated by amplification of Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. One predominant RAPD profile (type B) was identified in 15 strains (56% of all typed isolates). Other clusters depicted in the dendrogram, namely A, C, and D comprised 6 (22%), 5 (19%) and 1 (3%) isolates, respectively. All A. baumannii strains were resistant to all antibiotics except colistin. This study highlights the clonal spread of multidrug-resistant A. baumannii in our hospital. Therefore, the factors responsible for dissemination of such isolates need to be identified, controlled, and prevented to avoid major outbreaks.

Molecular Investigation of Genetic Polymorphisms in Type 2 Diabetic Patients Using Random Amplified Polymorphic DNA (RAPD-PCR)

To find DNA markers associated with Type 2 diabetes mellitus (T2D) using RAPD-PCR (Random amplified polymorphic DNA-Polymerase Chain Reaction). Peripheral blood samples were collected from 12 unrelated Iraqis with type 2 diabetes mellitus and 10 apparently healthy individuals (control subjects). DNA was extracted and amplified using RAPD-PCR. Out of the 16 primers used, 3 did not produce amplification patterns. Seven primers produced monomorphic bands while 6 primers namely A10, A18, C5, D20, R3 and R4 produced polymorphic DNA profiles. The highest discriminatory power was produced by primers A18 and D20 reaching 25%. Primer D20 produced the highest number of bands (16) and largest molecular weight band 2.470Kb whilst primer A10 produced the lowest number of bands (3) and primer R4 the smallest molecular weight band of 0.296Kb. Furthermore a band of 1.056 Kb was produced by primer R4 with frequency of 100% in diabetic patients and total absence in control subjects. The total length of the genome screened was approximately 118.661Kb, 40% of which represent polymorphisms. In conclusion Polymorphisms between diabetic patients and control subjects can be detected by RAPD-PCR and DNA markers associated with type 2 diabetes mellitus can be found using the same technique

Developing novel species-specific DNA markers for PCR-based species identification of the Lactobacillus sakei group.

Neither phenotypic nor the most commonly used genotypic method (16S rRNA gene sequencing) provides sufficient resolution for accurate identification of the Lactobacillus sakei group. A sequence-characterized amplified region method developed in this study provides a rapid, cost-effective way to detect the member of the L. sakei group in meat sample.

Ceftriaxone promotes the emergence of AmpC-overproducing Enterobacteriaceae in gut microbiota from hospitalized patients.

Epidemiological data suggest that ceftriaxone may promote the emergence of commensal AmpC-overproducing Enterobacteriaceae because of a high biliary excretion. We tested this hypothesis in hospitalized patients either treated by ceftriaxone alone or receiving no antibiotics. Hospitalized patients with no previous antibiotics or hospitalization in the last 3months, treated only with ceftriaxone, were prospectively included. For each ceftriaxone-treated patient, a control patient receiving no antibiotics was included. Clinical data and stools were collected at T0 (before antibiotics) and T1 (at the end of ceftriaxone treatment or at discharge) and T2 (3-6months after T1) for the ceftriaxone-treated patients and at T0 and T1 for control patients. Third-generation cephalosporin-resistant Enterobacteriaceae were detected, identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and characterized genetically. Clonal relatedness was evaluated by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Fifteen ceftriaxone and 22 control patients were included. Patients' characteristics did not differ. At T0, 2/15 ceftriaxone-treated versus 1/22 control patients carried third-generation cephalosporin-resistant Enterobacteriaceae (p = 0.6). At T1, 4/15 (27%) ceftriaxone-treated patients carried AmpC producers versus 0/22 control patients (p = 0.02). Additionally, two and three subjects carried extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in the ceftriaxone and control groups, respectively (p = 1). At T2, three ceftriaxone-treated patients still carried AmpC-producing Enterobacteriaceae with the same RAPD profile as at T1. In hospitalized subjects with no other selective pressure, treatment by ceftriaxone alone promotes the gut colonization by AmpC-overproducing Enterobacteriaceae in over a quarter of patients, with a persistent carriage after the end of antibiotic exposure. The ecological impact of ceftriaxone should not be underestimated.

Genetic Diversity in the Camel Tick &amp;lt;i&amp;gt;Hyalomma dromedarii&amp;lt;/i&amp;gt; (Acari: Ixodidae) Based on Mitochondrial Cytochrome c Oxidase Subunit I (COI) and Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

Hyalomma dromedarii ticks are important disease vectors to camels in the UAE and worldwide. Ticks can be identified using DNA-based techniques. In addition, such techniques could be utilized to study the intraspecific genetic diversity in tick populations. In this study, the genetic diversity of four H. dromedarii populations was investigated using mitochondrial cytochrome c oxidase subunit I (COI) gene and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The results showed that both of the aforementioned techniques produced similar grouping patterns. Moreover, they revealed that the four tick populations had high levels of genetic similarity. However, one population was slightly different from the three other populations. The current study demonstrated that H. dromedarii ticks in the UAE are very similar at the genetic level and that investigating more locations and screening larger numbers of ticks could reveal larger genetic differences.

Variability of Deltamethrin-Resistant &amp;lt;i&amp;gt;Metarhizium anisopliae&amp;lt;/i&amp;gt; Aggressive Strains Group to a Population of the Cossid Moth from &amp;lt;i&amp;gt;Eucalyptus nitens&amp;lt;/i&amp;gt;

Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used to examine the genetic variability among Metarhizium anisopliae isolates tested to the cossid moth, Coryphodema tristis. All the isolates tightly clustered into one or the other of two groups that diverged at 12%. Results suggested that certain genotypes of the fungus, that grouped together, were able to infect moth larvae while others did not. A fragment of 760 bp, which presents high homology with a host-adaptation related protein coding gene, distinguished between aggressive and non-aggressive isolates. Neither mycelial growth nor sporulation rate or presence of known virulence genes was correlated with mortality values. Some isolates, including the most aggressive isolate ARSEF2518, were compatible with deltamethrin. Deltamethrin treatment killed all the larvae after seven days whereas fungal and mixed treatments respectively reached the same mortality after 28 and 21 days.

Rapid screening of starter cultures for maari based on antifungal properties

Forty Bacillus isolates obtained from maari (used as condiment in Burkina Faso) including 17 B. subtilis, 4 B. circulans, 7 B. pumilus and 6 B. licheniformis were investigated for use as starter cultures in maari production. The isolates were screened by PCR for the sfp gene responsible for the production of the lipopeptide biosurfactant, surfactin. The sfp gene was detected in all of the seventeen B. subtilis isolates, in 2 out of 7 B. pumilus, in 4 out of 6 B. licheniformis whereas no B. circulans was positive for the sfp gene by PCR screening. Furthermore, all the 40 Bacillus spp. were screened for biosurfactant production and inhibitory activity against Aspergillus flavus, A. niger, A. versicolor and Rhizopus oryzae. Results demonstrated a relationship between the presence of the sfp gene and the antifungal activity and biosurfactant production of Bacillus isolates. In addition, molecular typing of the 17 B. subtilis isolates by Multilocus Sequence Typing (MLST) resulted in 15 Sequence Types, one of them included three strains. Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), used for B. licheniformis, B. megaterium, B. circulans and B. pumilus revealed that the inhibitory activity and biosurfactant production were strain-dependent. Finally, the detection of chitinase (chi) and β-glucanase (glu) biosynthesis genes was found to be associated with the antifungal activity for 16 B. subtilis isolates. The present work provides a greater understanding of the antifungal activity and biosurfactant production ability within the Bacillus spp. isolated from maari and contributes to the selection of Bacillus isolates to be used as starter cultures for controlled production of maari.

Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Genetic diversity of Tor douronensis (Pisces: Cyprinidae) in West Sumatra, Indonesia

Roesma DI, Tjong DH, Munir W, Agesi AV, Chornelia A. 2017. Genetic diversity of Tor douronensis (Pisces: Cyprinidae) in West Sumatra, Indonesia. Biodiversitas 18: 1018-1025. A random amplified polymorphic DNA-polymerase chain reaction (RAPDPCR) analysis was applied to determine the genetic diversity within and among the populations of Tor douronensis in West Sumatra river system. The results provided information for proper breeding, maintenance, and conservation of T. douronensis. The highest and the lowest values of genetic diversity were the population of Batang Matur (H = 0.1571; I = 0.2411) and Batang Malalo (H = 0.0880; I = 0.1353), respectively. The value of genetic differentiation (GST) was 0.4266 while that of heterozygosity between populations (DST) and within a population (HS) was, respectively, 0.088 and 0.1184. The value of gene flow between populations (Nm) was 0.6721. Grouping of individuals in a population based Principal Coordinates Analysis (PCO) and UPGMA was in line with the genetic distance values. Matur and Malalo were the most closely related populations (0.0174) while Batang Gumanti and Batang Sinuruik were the most distantly related populations (0.1918). The genetic variation between populations was lower than the genetic variation within the population of T. douronensis and population of Matur is recommended to be used as brood stock in the procurement of stock.

In vitro study of antioxidant and antibacterial activities of Lactobacillus probiotic spp.

This study investigated the influence of aeration and minimal medium conditions on antioxidant and antibacterial activities of 21 probiotic Lactobacillus strains isolated from dairy products. The probiotic potential of the isolates was evaluated by pH and bile tolerance. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm the phenotypic identification of isolates. Antioxidant producer isolates were screened by resistance to reactive oxygen species (ROS). The antioxidant and antibacterial activities of extracellular materials after 48h fermentation with antioxidative strains were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and broth microdilution assays, respectively. The results indicate that the antioxidant capacity of supernatants was increased by using of both minimal medium and agitation. The antibacterial activity was increased in minimal medium, but there has nearly no change in the antibacterial properties by using both agitation and minimal medium. The maximum antibacterial activity was observed during mid-exponential phase until the beginning of the early-stationary phase, but the maximum antioxidant activity was detected at the stationary growth phase. There is a significant relationship between antioxidant and antibacterial activities of the cell-free probiotic extracts, and their production rates are closely related to the fermentation type. The bioactive materials from probiotics could be extracted in a large amount at an appropriate time under a suitable condition.

English

The aim of the present study was to determine the unique molecular markers among, three species of the genus Anthemis and the construction of phylogenetic tree using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique. Genetic diversity was analyzed among 15 populations of three species of Anthemis (Anthemis melampodina, Anthemis, pseudocotula and Anthemis, bornmuelleri), collected from different locations at Saudi Arabia by using RAPD primers. Pairwise genetic distance was calculated based on Nei and Li coefficient. Unweighted pair group method with an average (UPGMA) was used for construction of dendrogram, based on the similarity matrix data. Results showed wide variations among A. bornmuelleri and other two species. A wide close genetic relation was observed between A. melampodina and A. pseudocotula. RAPD-PCR technique was shown to be an accurate tool, in other to ascertain plant relationships among species of genus Anthemis. Key words: Genetic diversity, random amplified polymorphic DNA-polymerase chain reaction (RAPD- PCR), A. melampodina, A. pseudocotula, A. bornmuelleri.

Occurrence of Carangid FIsh &lt;i&gt;Uraspis helvola&lt;/i&gt; (Forster, 1801) from the Iraqi Marine Waters, Arabian Gulf

The white tongue jack, Uraspis helvola (Forster, 1801) was recorded for the first time in the Iraqi marine waters, northwest Arabian Gulf. The samples were collected during the period from January 2014 to June 2015. U. helvola differed from other carangid fish species by tongue, roof and floor of mouth are white to yellowish and other parts are dark, and the anal fin spines embedded. Gill rakers ranged from four to five on upper limb and 12 to 13 on lower limb of first gill arch. The DNA fingerprint was identified of U. helvola by using Polymerase Chain Reaction-Random Amplified Polymorphic DNA (PCR-RAPD) with six primers: P1 (212), P2 (239), P3 (244), P4 (250), P5 (265), and P6 (347). The number of bands generated by primers was 51 and the size of bands ranged from 110 to 1440 bp.

Identifying the geographical origin of protected sea cucumbers (Apostichopus japonicus) in China using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR).

Dalian sea cucumber, Yantai sea cucumber, and Weihai sea cucumber, which belong to Apostichopus japonicus, are protected as geographical indications in China based on their high nutritional values and medical propertys. The 26 samples, including Dalian sea cucumbers (9 samples) in Liaoning province, Yantai sea cucumbers (9 samples), and Weihai sea cucumbers (8 samples) in Shandong province, were individually collected from the designated geographical sea areas and the genetic relationships and DNA polymorphisms were evaluated by random amplified polymorphic DNA technology and gene segments sequencing. The RAPD dendrogram showed that the genetic diversity of the three types of sea cucumbers was rich. The neighbor-joining tree shows that the genetic relationship of the samples from the adjacent sea areas is closer. It demonstrates that the gene characteristics of sea cucumbers from different sea areas were obvious and the genetic diversity analysis by RAPD-PCR can be used as a rapid method for geographical discrimination.