Genetic variation inferred from RAPD fingerprinting in three species of tilapia

This study used random amplified polymorphic DNA (RAPD) fingerprinting for estimating genetic variation and species differentiation in three species of tilapia. A 16-mer random primer generated RAPD markers ranging from 250 to 2400 base pairs (bp). Genetic similarity estimates obtained by pairwise comparisons based on the method of Nei and Li (1979) indicated high genetic similarity (mean genetic similarity (± sd), 0.73 (± 0.15) for Nile tilapia; 0.78 (± 0.12) for Mozambique tilapia; and 0.87 (± 0.07) for Aureus tilapia) within each of the tilapia species. The average interspecies genetic similarities obtained among the three species were 0.59 (± 0.07) for Mozambique/Nile tilapia, 0.46 (± 0.09) for Aureus/Nile tilapia and 0.38 (± 0.07) for Aureus/Mozambique tilapia pair. DNA profiles generated in each species of tilapia were unique. A total of 13 RAPD markers differentiating the three species of tilapia were detected. Our study presented RAPD markers as a new class of useful genetic markers for assessment of genetic diversity and species differentiation in tilapia.     

Phytase-producing bacteria in the digestive tracts of some freshwater fish

Isolation and enumeration of phytase‐producing bacterial flora in the foregut and hindgut regions of the gastrointestinal tracts of 10 culturable freshwater teleosts of different feeding habits, namely rohu (Labeo rohita), catla (Catla catla), mrigal (Cirrhinus mrigala), bata (Labeo bata), kalbasu (Labeo calbasu), Nile tilapia (Oreochromis niloticus), climbing perch (Anabas testudineus), common carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix) and grass carp (Ctenopharyngodon idella), have been carried out. Microbial culture of the gut mucosa on selected nutrient media following the enrichment culture technique was performed for bacterial isolation. The bacterial isolates were screened on the basis of their enzyme‐producing ability. The bacterial population on the tryptone soya agar (TSA) plate was maximum in the hindgut region of bata, followed by mrigal and minimum in the foregut region of Nile tilapia. In modified phytase screening medium (MPSM), phytase‐producing strains were recorded at higher densities in the foregut region of mrigal and grass carp and minimum in the foregut region of bata. In case of the hindgut, maximum phytase‐producing strains were present in grass carp and mrigal and minimum in rohu. In general, in MPSM, the bacterial population was lower in the hindgut region of all the 10 species of fish examined. The phytase‐producing ability of the selected 31 strains (16 from the foregut and 15 from the hindgut region) was determined by clearing zones on phytate‐containing plates. Among these isolates, 22 strains (12 from the foregut and 10 from the hindgut region) were selected as potent phytase producers according to a quantitative enzyme assay. The highest phytase activity was observed in the bacterial strains LF1 and LH1 isolated from the fore and the hindgut regions of rohu respectively. Both the strains were identified as Bacillus licheniformis on the basis of phenotypic characteristics as well as 16S rDNA sequence analysis.     

RAPD fingerprinting of the eel-loaches Pangio filinaris and Pangio piperata: preliminary evaluation

Random amplified polymorphic DNA (RAPD) analysis was applied to two morphologically similar species of the increasingly popular ornamental eel‐loaches, Pangio filinaris and P. piperata, collected from Kemat River, Terengganu and Padang Sanai River, Kedah, West Malaysia. Forty primers were tested in a preliminary screening. Of these, five (OPA‐03, OPA‐09, OPA‐11, OPA‐13 and OPA‐18) were chosen for their ability to provide consistent amplification. RAPD analysis differentiated the two species, with each showing different RAPD patterns for each primer used. The five primers generated a total of 82 scorable loci with up to 83% and 60% polymorphism in P. piperata and P. filinaris respectively. The RAPD banding patterns of these two species ranged from 2 to 14 fragments, with a size range of 250–2000 bp. Genetic similarity within species ranged from 0.610 to 0.985 (mean 0.799±0.14) for P. piperata and from 0.824 to 0.934 (mean 0.885±0.04) for P. filinaris respectively. The interspecies genetic similarity ranged from 0.386 to 0.486 with an average value of 0.434±0.040. Our study revealed RAPDs as useful genetic markers for the taxonomic clarification and assessment of genetic variability of the eel‐loaches.     

Application of the random amplified polymorphic DNA technique in a study of heterosis in common carp, Cyprinus carpio L.

Random amplified polymorphic DNA (RAPD) analysis was applied to three varieties of common carp, Cyprinus carpio L.: Xingguo red carp, German mirror carp and Russian mirror carp. Twenty-seven decamer primers pre-screened from 40 random primers were used to assay polymorphisms within and between the populations. One hundred and ninety-nine fragments were generated in the three populations; 155 of these fragments were polymorphic. The similarity indices and genetic distances within and between the three populations of carp were calculated. The results indicated that the highest value of similarity within populations was obtained for German mirror carp, and the genetic distance between Xingguo red carp and Russian mirror carp was the farthest. It can be presumed that the heterosis between Xingguo red carp and Russian mirror carp is the highest within these three populations.     

Analysis of DNA relationships among Aeromonas species by RAPD (randomly amplified polymorphic DNA) typing

Genetic differences between a collection of aeromonads were studied in two laboratories by analysis of randomly amplified polymorphic DNA (RAPD). A single randomly designed primer, generated reproducible profiles of genomic DNA in both laboratories for Aeromonas salmonicida subspecies salmonicida, although the profiles differed between laboratories. Analysis of atypical strains of A. salmonicida and isolates of the A. hydrophila group produced scattered profiles in both laboratories. The uniform fingerprints produced for A. salmonicida subspecies salmonicida indicate genomic homogeneity. The scattered RAPD profiles of the motile aeromonads demonstrate the genomic diversity of this group. A group of unspeciated motile aeromonads gave uniform fingerprints, suggesting the possibility of a genomically homogeneous species. Although the RAPD technique is susceptible to the effects of minor technical variations, this study has demonstrated that where there is DNA similarity, it can be recognized, and where there is diversity, differentiation can be made. RAPD promises to be useful in epidemiological studies for rapid identification of bacteria where a source of reference DNA is available and may be useful in preliminary investigations of relatedness within groups.     

Genetic variation of Rutilus rutilus caspicus (Jakowlew 1870) populations in Iran based on random amplified polymorphic DNA markers: a preliminary study

Rutilus rutilus caspicus is regarded as a valuable fish species both for angling and commercial food in Iran. This fish is also considered as a significant food source for beluga sturgeon. The genetic diversity of this fish species collected from two geographical areas (Gorgan Bay and Anzali Wetland) along the Iranian coastline of the Caspian Sea was examined using the analysis of randomly amplified polymorphic DNA (RAPD). Using 10 decamer primers, the total number of RAPD bands produced in both Gorgan Bay and Anzali Wetland populations were 94 bands. The percentages of polymorphic bands were comparable in Anzali Wetland (41.48%) and Gorgan Bay (43.61%), suggesting similar levels of polymorphism of the two populations to be used for establishing selective breeding programmes. The value of Nei's genetic distance (d=0.04) among populations was small, despite the large geographic separation. The data serve as a baseline analysis of current genetic diversity found among R. rutilus caspicus populations in Iran.     

Genetic variation of the razor clam Ensis siliqua (Jeffreys, 1875) along the European coast based on random amplified polymorphic DNA markers

Ensis siliqua is regarded as an increasingly valuable fishery resource with potential for commercial aquaculture in many European countries. The genetic variation of this razor clam was analysed by randomly amplified polymorphic DNA (RAPD) in six populations from Spain, Portugal and Ireland. Out of the 40 primers tested, five were chosen to assess genetic variation. A total of 61 RAPD loci were developed ranging in size from 400 to 2000 bp. The percentages of polymorphic loci, the allele effective number and the genetic diversity were comparable among populations, and demonstrated a high level of genetic variability. The values of Nei's genetic distance were small among the Spanish and Portuguese populations (0.051–0.065), and high between these and the Irish populations. Cluster and principal coordinate analyses supported these findings. A mantel test performed between geographic and genetic distance matrices showed a significant correlation (r=0.84, P<0.05), suggesting an isolation by distance process.     

Genetic relationship between Litopenaeus setiferus (L.) and L. schmitti (Burkenroad) determined by using 16S mitochondrial sequences and enzymatic analysis

Genetic differentiation and variability data of two populations of two species of shrimp (Litopenaeus setiferus (L.) and L. schmitti (Burkenroad)) have been obtained by electrophoretic analysis and by analysis of 16S mitochondrial DNA. Using eight polymorphic enzymes, the genetic distance (GD) between the two species was 0.165. The GD between L. setiferus populations was 0.0057 and between L. schmitti populations it was 0.0034. The greatest differentiation was found within, rather than between, populations, although the differentiation value between Mexican and Cuban populations varied in accordance with the geographic distance and ecological characteristic of each. We found a high similarity between these two species with a bimodal distribution of the loci with respect to genetic identity. The homology percentages for gene 16S fragments were compared with those from six different shrimp species (L. vannamei, L. stylirostris, Farfantepenaeus notialis, Metapeneopsis lamellata) and Artemia salina. Ninety‐seven percent of identity was found by analysis of a 409 bp of 16S mitochondrial DNA. With these values a phylogenetic tree was made using parsimony criteria. The GDs obtained with this method confirm the classification proposed by Pérez‐Farfante & Kensley (1997) .     

A comparative account of the structure of the growth hormone encoding gene and genetic interrelationship in six species of the genus <Emphasis Type="Italic">Labeo</Emphasis>

The growth hormone (GH) gene isolated and cloned from various Labeo species (L. rohita, L. calbasu, L. fimbriatus, L. gonius, L. bata, and L. kontius) is shown to contain a single copy in the haploid genome, with an overall size of ∼2.5 kb. The GH gene in all the Labeo species studied has five exons and four introns of various sizes with the exon/intron boundary sequence of GT/AG. The length variation of the GH gene between the species is found to be due to length variation in the form of several deletions in the third intron. The length of individual exons is the same in all the species with an open reading frame (ORF) of 630 bp (210 amino acids) except in L. rohita, which has a 9 bp deletion in the fourth exon, resulting in a shorter GH of 621 bp (207 amino acids). The similarity in the nucleotide and amino acid sequences between the different Labeo species is greater than 97%, in spite of eight amino acids being altered in the GH protein of Labeo that reside outside the conserved domain sequence required for its function. Nucleotide substitutions are seen in the form of 20 transitions and three transversions in the ORF of the GH gene. Both types of transitions (A–G; T–C) and only one type of transversion (A–C) are detected in the GH gene. Codon preference in GH gene shows a strong preference for G and C in the wobble position of the codons. Genetic interrelationships determined between Labeo and other species of fishes using nucleotide sequence of GH cDNA supports the overall teleost classification of Nelson (Fishes of the World. Wiley, New York, 1984) with separate clades for Ostariophysi, Protacanthopterygii, and Acanthopterygii. Besides, the unweighted pair group method with arithmetic means (UPGMA) analysis clearly distinguishes between the species having five exons and four introns in the GH gene from the species having six exons and five introns in the same gene. The Labeo species analyzed in the present study could be clustered into two groups using the maximum-parsimony method on the intron sequences data of the GH gene.     

Random amplified polymorphic DNA markers for three Japanese laminarian species

ABSTRACT: Random amplified polymorphic DNA (RAPD) was studied to detect genetic markers for three economically important Japanese laminarian species, Laminaria japonica Areschoug, L. religiosa Miyabe and L. ochotensis Miyabe, which were sampled from their representative localities on the coasts from Tsugaru Straits to the Sea of Japan. DNA templates for RAPD were extracted from lamina using a DNA extraction kit and were purified with glassmilk. Reproducible RAPD markers for these three species were detected using three random primers from a total of 15 tested. In L. japonica and L. religiosa , these RAPD markers were confirmed to be useful for populations from other localities. The three species studied showed high intraspecific and interspecific band sharing index (BSI) values. Like annual typical L. religiosa from other localities, biennial individuals identified as putative L. religiosa from Atsuta could be discriminated from L. japonica but not from L. ochotensis by any of the RAPD genetic markers studied so far.     

Genetic variability of Channa punctatus populations using randomly amplified polymorphic DNA

Genetic variations between Channa punctatus populations collected from three rivers of south India were examined using randomly amplified polymorphic DNA (RAPD). Sixty samples from each population were collected. Genomic DNA was isolated from the blood samples. Out of the 20 primers tested, six primers were used for genetic variation studies. A total of 42 RAPD bands were amplified in C. punctatus by the six primers. The total number of bands observed ranged from 34 in the Quilon population to 37 in the Thirunelveli population. Among the three populations, the highest genetic identity (0.9231) was found between Thirunelveli and Quilon populations. The results of the present study demonstrated that Thirunelveli and Quilon populations are more related to each other than to the Coimbatore population.     

Estimation of genetic variation in green tiger prawn,Penaeus semisulcatus by using random amplified polymorphic DNA,inter simple sequence repeat and simple sequence repeat markers

In this study, three molecular markers including random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) were used to evaluate genetic variation of green tiger prawn Penaeus semisulcatus collected from two geographically isolated environments; located in the Manifa, Arabian Gulf, Saudi Arabia and Ataka, Suez Gulf, Egypt. Genetic parameters included the percentage of polymorphism (P%), effective alleles (Ne), Nei genetic diversity (H) and Shannon index (I), which were calculated based on molecular data. All three marker systems distinguished genetic variation of P. semisulcatus in various levels. The highest polymorphism (91.30%) was obtained with SSR, followed by ISSR (82.26%) and RAPD markers (62.04%), respectively. Our results indicate that SSR appeared to be the best suited molecular assay for assessing the genetic variation between genotypes of P. semisulcatus. The present study indicated that Manifa and Ataka genotypes were closely related. Moreover, the analysis of variability could require more than one DNA‐based molecular marker techniques.     

Genetic structure of Spanish white‐clawed crayfish (Austropotamobius pallipes) populations as determined by RAPD analysis: reasons for optimism

• 1. Spanish populations of the white‐clawed crayfish have declined sharply over the last three decades. Although Austropotamobius pallipes was once widely distributed and very abundant in most of the limestone basins of the country, outbreaks of crayfish plague since 1978 have reduced its populations, and now only some 500–600 small populations are left.
• 2. Consequently, the species now enjoys protection under national legislation. Management decisions regarding the conservation of a threatened species require an understanding of the genetic structure of its populations.
• 3. Using random amplified polymorphic DNA (RAPD) fingerprinting the genetic variability of 11 populations of A. pallipes was assessed over the species' range in Spain, and their phylogenetic relationships determined.
• 4. Substantial genetic differentiation was detected among the populations tested; no clear relationship was found between patterns of genetic variability and hydrological basin. The RAPD markers showed the degree of genetic variability of these populations to be similar to, and in some cases slightly higher than, that reported in previous studies on other Spanish and European populations of A. pallipes.
• 5. The results offer hope for the recovery of this species in Spain, and provide information that might be useful in the management of crayfish reintroduction programmes.

Copyright © 2007 John Wiley & Sons, Ltd.     

Genetic diversity of Nile tilapia populations revealed by randomly amplified polymorphic DNA (RAPD)

Genetic diversity of the Nile tilapia Oreochromis niloticus collected from the river Nile (Cairo, Assuit and Qena) and two Delta lakes (Burullus and Manzalla) in Egypt was examined by the analysis of randomly amplified polymorphic DNA (RAPD). Of 25 primers examined,21 primers produced 230 RAPD bands. The percentage of polymorphic bands in Manzalla (29.4%) and Burullus (24%) populations was low compared with Assuit (30.54%), Cairo (33.5%) and Qena (44.84%) populations. The highest percentage of polymorphic bands was observed in the Qena population,suggesting a greater potential for use in breeding programs. The molecular phylogenetic tree constructed by unweighted pair‐group method of analysis shows Manzalla and Burullus populations strongly linked and separate from the Assuit and Cairo populations, with Qena population as outgroup. The data serve as a baseline analysis of the current genetic diversity found among O. niloticus populations in Egypt.     

Genetic diversity and species identification of Argulus parasites collected from major aquaculture regions of India using RAPD‐PCR

Argulus is one of the most important fish parasites that cause heavy economic loss to aquaculture industry. The present investigation was undertaken to study the genetic diversity of the Argulus sp. collected from 13 locations representing major aquaculture zones in India by RAPD analysis and to develop species‐specific markers. Thirteen random decamer primers were used to amplify DNA fragments from three individual parasites of each location. Of the 172 bands scored by the primers, 168 were polymorphic. The per cent polymorphic loci and gene diversity values varied within a range of 8.14–43.02 and 0.0342–0.1727 respectively. Nei's genetic similarity between populations across all the primers ranged from 0.363 to 0.969. The dendrogram based on Nei's genetic distance showed two clusters; Bangalore and Mandi populations forming one cluster, and the rest in another cluster. The clusters also revealed strong correlation with the species identified as A. japonicus and A. siamensis respectively by morphological method. The study thus indicated A. siamensis as the major prevalent species in carp culture farms in India. Species‐specific primers were designed from unique sequences cloned from RAPD fragments that could able to identify A. siamensis and A. japonicus separately.     

三个不同养殖群体金鱼遗传多样性的RAPD分析

采用随机扩增多态性DNA(RAPD)方法对金鱼3个不同地区养殖群体-天津塘沽(TJ)、江苏苏州(JS)及福建福州(FZ)群体各20ind的DNA多态性进行了分析。在事先优化的反应条件下,所使用的100个随机引物中,有14个引物扩增出清晰稳定的片段,共计103条,大小在200~2500bp,其中多态性片段27条。金鱼总的DNA多态位点百分率为26.21%。数据分析结果显示,3个金鱼群体内遗传多样性偏低,遗传相似度为0.9519~0.9785。金鱼3个群体总的Nei基因多样性指数为0.1061,Shannon信息指数为0.1547。金鱼3个群体间平均遗传距离为0.0375,基因分化系数Gst为0.243,表明3个养殖群体间产生了一定程度的遗传分化。UPGMA法和NJ法的聚类分析显示,两者结果一致。TJ群体和JS群体优先聚类,再与FZ群体聚类。     

Genetic characterization of populations of the ectoparasitic caligid, Lepeophtheirus salmonis (Krøyer 1837) using randomly amplified polymorphic DNA

Genetic variability within salmon louse, Lepeophtheirus salmonis (Caligidae: Copepoda), populations parasitizing farmed and wild Scottish Atlantic salmon (Salmo salar) was investigated using analysis of randomly amplified polymorphic DNA (RAPD) fragments. Seven individual decamer primers were used to analyse samples of salmon lice collected from 15 different locations in Scotland. The polymerase chain reaction products were separated using agarose gel electrophoresis and the resulting band patterns were analysed using a semi‐automated analytical scoring system. Dendrograms were produced using the unweighted pair‐group average (UPGMA) method using Dice similarity values. The summary dendrogram of the analysis of all RAPD bands showed two separate clusters of salmon lice, the larger being sub‐divided into a further two sections. The collections of lice occupying each of these sub‐divisions, however, were a mix of sites, which did not exhibit a structured geographical pattern.     

Sequence characterized amplified regions (SCAR) reveal candidate markers linked to growth traits in the Chinese shrimp Fenneropenaeus chinensis

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different (P < 0.05) according to a χ ² test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A₁ was only found in the population of shorter shrimps, it may be a negative growth marker.     

Molecular characterization and induced expression analysis of the terminal complement component C9 in rohu,Labeo rohita

The complement component 9 (C9) plays a significant role in the formation of membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species in India. An open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids having a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. The SMART domain architecture analysis revealed two thrombospondin type‐1 domains (TSP1), a low‐density lipoprotein receptor domain class A (LDLa), a membrane attack complex and perforin (MACPF) domain, and an epidermal growth factor (EGF)‐like domain. Multiple sequence alignment and evolutionary analysis revealed a primitive C9 sequence of rohu with maximum similarity and clustering with common carp, grass carp and zebrafish. C9 was highly expressed in liver and constitutively expressed in wide array of tissues except in eye of rohu juveniles. It was expressed during early developmental days of rohu including in milt. A variable level of up‐regulation in C9 expression was noticed upon poly I:C induction, Aeromonas hydrophila and Argulus siamensis infections in liver, spleen and gill tissues of rohu at different time points. A constitutive expression of C9 in different stages of rohu during the ontogeny and in response to pathogen exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.     

Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases

Streptococcus iniae is an important bacterial pathogen of fish, causing up to 50% mortality in stocks, which has recently been associated with human infections. To determine whether S. iniae isolates from humans and fish are similar, the present authors examined the biochemical profiles and genetic relatedness of these isolates by random amplified polymorphic DNA (RAPD) analysis and repetitive primer polymerase chain reaction(REP PCR). The biochemical profiles differentiated between the human and fish isolates of S. iniae using pyrrolidonyl arylamidase, arginine dehydrogenase, ribose, β-glucoronidase and glycogen as markers. These biochemical results suggest that the fish and human S. iniae isolates are genetically different. However, RAPD and REP PCR do not have the discriminatory power to differentiate between these streptococcus isolates using five different RAPD primers and BoxA primer.