DEVELOPMENT OF MICROSATELLITE LOCI FOR PINUS KORAIENSIS (PINACEAE)

Premise of the study: Microsatellite markers were developed for Pinus koraiensis to characterize its genetic diversity and understand its population structure. Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 20 primer sets were developed in Chinese populations of P. koraiensis. Three of the markers showed polymorphism with two alleles per locus when assessed in a sample of two populations of P. koraiensis from the Changbai Mountain in the Jilin Province of China. Five and three loci were successfully amplified in P. taiwanensis and P. massoniana, respectively. The amplification size of these loci matches those in P. koraiensis. Conclusions: These markers may be useful for further investigation of population genetics of P. koraiensis.

Population genetic structure of Rutilus frisii kutum in Caspian Sea using biometry and microsatellite molecular technique

In order to carry out Biometric studies, 75 samples were caught from 3 locations ( Tajan river, Sefidrud and Shirud) using Salic and the length (±1 mm) and weights (± 5 gr) of samples were determined. Using One-way ANOVA by SPPSS software, there wasn’t significant difference between locations in length and fecondity (P ≥0.01(, but there was significant difference between Shirud and tajan samples with sefidrud in weight ) P≤0.01(. In order to carry out genetic variation studies, 210 fish were caught from 3 different regions of the Iranian coastline (Khoshkrud, Tonekabon, Gorganrud) and 1 region in Azerbaijan (Waters of the Caspian Sea close to Kura River mouth) during 2008-2009 . Genomic DNA was extracted of fin using the phenol-chloroform. The quantity and quality of DNA from samples were assessed by spectrophptometer and 1% agarose gel electro-phoresis. PCR was carried out using 15 paired microsatellite primers. PCR products were separated on 8% polyacrylamide gels that were stained using silver nitrate. Molecular weight calculate using UVTech software. The recorded microsatellite genotypes were used as input data for the GENALEX software version 6 package in order to calculate allele and genotype frequencies, observed (Ho) and (He) expected heterozygosities and to test for deviations from Hardy-Weinberg equilibrium. Genetic distance between two populations was estimated from Nei standard genetic distance and genetic similarity index (Nei, 1972). Genetic differentiation between populations was also evaluated by the calculation of pairwise estimates of Fst and Rst values. From 15 SSR markers were used in this investigation, 9 of them were polymorph. Average of expected and observed heterozygosity was 0.54 and 0.49 respectively. Significant deviations from Hardy-Weinberg expectations were observed in all of location except Anzali lagoon- autumn in AF277576 and EF144125, Khoshkrud in EF144125 and Gorganrud and Kura in AF277576. Using Fst and Rst there was significant difference between locations ) P≤0.01(. According to Fst , the highest population differentiation (Fst= 0.217) was between Gorganrud and Khoshkrud that have the lowest Nm and the lowest (Fst= 0.086) was between Gorganrud and Tonekabon that have the highest Nm. Using Rst the highest population differentiation (Rst= 0.271) was between Tonekabon and spring Anzali lagoon and the lowest (Rst= 0.026) was between Tonekabon and Autumn Anzali 159 lagoon. Also the difference between Spring Anzali lagoon and Autumn Anzali lagoon was noticeable (Fst=0.15). AMOVA analysis with consideration of 2 sampling regions (Iran and Azerbaijan) and 7 sampling locations (Iran: Khoshkrud, Tonekabon, Gorganrud, Spring Anzali lagoon and Autumn Anzali lagoon ; Azerbaijan: the Kura mouth) revealed that almost all of the variance in data namely 83% )P≤0.01( was within locations, Genetic variances among locations was 14% )P≤0.01( and among regions was 3% )P≤0.01(. The genetic distance was the highest (0.646) between Gorganrud and Autumn Anzali lagoon populations, whereas the lowest distance (0.237) was between Gorganrud and Tonekabon River. Result obtained from the present study show that at least 2 different population of Rutilus frissi kutum are found in the Caspian sea,which are including the kura river population and the southern Caspian sea samples and it appears that there is more than one population in southern Caspian sea that should be attantioned in artifical reproduction Center and stoke rebilding.

The genetic diversity of Acipenser stellatus populations in the Caspian Sea was studied using microsatellite technique

A total of 361 caudal fin samples were collected from adult A. stellatus specimens caught in the north Caspian Sea, including specimens from Kazakhstan (Ural River), Russia (Volga River), Azerbaijan (Kura River), specimens caught in the south Caspian Sea including specimens from Fishery Zone 1 (from Astara to Anzali), Fishery Zone 2 (from Anzali to Ramsar), Fishery Zone 3 (from Nowshahr to Babolsar), Fishery Zone 4 (from Miyankaleh to Gomishan) as well as from specimens caught in Turkmenistan (all specimens were collected during the sturgeon stock assessment survey). About 2 g of fin tissue was removed from each caudal fin sample, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using phenol-chloroform method. The quality and quantity of DNA was assessed using 1% Agarose gel electrophoresis and Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 paired microsatellite primer. PCR products were electrophoresed on polyacrylamide gels (6%) that were stained using silver nitrate. Electrophoretic patterns and DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected heterozygosity and observed heterozygosity allele number, and the effective allele number, genetic similarity and genetic distance, FST and RST were calculated. The Hardy Wienberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendrogram for genetic distances and identities were calculated using TFPGA program for any level of the hierarchy. It is evident from the results obtained that the 15 paired primers studied, polymorphism was observed in 10 pairs in 12 loci, while one locus did not produce DNA bands. Mean allele number was 13.6. Mean observed and expected heterozygosity was 0.86 and 0.642, respectively. It was also seen that specimens from all regions were not in Hardy Wienberg Equilibrium in most of the loci (P≤0.001). Highest Fst (0.063) was observed when comparing specimens from Fishery Zone 2 and Fishery Zone 4 (Nm=3.7) and lowest FST (0.028) was observed when comparing specimens from the Volga River and those from the Ural River (8.7). Significant differences (P<0.01) were observed between RST recorded in the specimens studied. Highest genetic distance (0.604) and lowest genetic resemblance (0.547) were observed between specimens from Fishery zones 2 and 4. Lowest genetic distance (0.311) and highest genetic resemblance (0.733) was observed between specimens from Turkmenistan and specimens from Fishery zone 1. Based on the genetic dendrogeram tree derived by applying UPGMA algorithm, A. stellatus specimens from Fishery zone 2 or in other words specimens from the Sepidrud River belong to one cluster which divides into two clusters, one of which includes specimens from Fishery zones 1, 3 and 4 and specimens from Turkmenistan while the other cluster includes specimens from Ural, Volga and Kura Rivers. It is thus evident that the main population of this species belongs to the Sepidrud River. Results obtained from the present study show that at least eight different populations of A. stellatus are found in the north and south Caspian Sea, four of which are known populations including the Ural River population, the Volga River population, the Kura River population and the Sepidrud River populations. The four other populations identified belonging to Fishery zones 1, 3, and 4 and to Turkmenistan are most probably late or early spawners of the spring run and autumn run of each of the major rivers mentioned. Specific markers were also identified for each of the populations identified. The Ural River population can be identified using primers Spl-68, 54b and Spl-104, 163 170, 173, the Volga River population can be identified using primers LS-54b and Spl-104, 170, 173 113a and similarly the population from the Kura River can be identified using primers LS-34, 54b and Spl-163, 173 and that from the Sepidrud River can be identified using primers LS-19, 34, 54b and Spl-105, 113b. This study gives evidence of the presence of different populations of this species and calls for serious measures to be taken to protect the genetic stocks of these populations. Considering that the population of A. stellatus in Fishery zone 2 is an independent population of the Sepidrud River in the Gilan Province, the catch of these fishes in the region needs to be controlled and regulated in order to restore the declining stocks of this species.

Microsatellite marker isolation and cultured strain identification in Carassius auratus gibelio

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.

Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the "Fast Isolation by AFLP of Sequences Containing repeats" (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosi ties were 0.300-0.633 (mean 0.496) and 0.473-0.804 (mean 0.659), respectively.

Isolation and characterization of polymorphic microsatellite loci in Wuchang bream (Megalobrama amblycephala)

Wuchang bream (Megalobrama amblycephala) is an economically important fish in China. From a (GT)(13)-enriched genomic library, 20 microsatellites were developed. Nine of these 20 loci were polymorphic in a test population with allele numbers ranging from two to four, and the observed and expected heterozygosities ranging from 0.2609 to 0.7826 and from 0.3739 to 0.7546, respectively. In the cross-species amplifications, six of these nine loci were also polymorphic in white amur bream (Parabramis pekinensis). These polymorphic microsatellite loci are potentially useful for population genetics of Wuchang bream and its closely related species.

Characterization of novel microsatellite loci in rare minnow (Gobiocypris rarus) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2-7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.

Microsatellite diversity and population genetic structure of redfin culter (Culter erythropterus) in fragmented lakes of the Yangtze River

Redfin culter (Culter erythropterus) is a small lethic freshwater fish and widely distributed in the adjacent lakes of the Yangtze River of China. Five microsatellite loci were applied to investigate the genetic variation and population structure of redfin culter from seven lakes in the middle-and-lower reaches of the Yangtze River. The gene diversity was high among the populations (H > 0.9), the average number of alleles among seven populations was low with a range from 2.00 to 3.87. The mean observed (H-O) and expected (H-E) heterozygosity ranged from 0.111 to 0.419 and from 0.162 to 0.750, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found in 50% of the total locus-population combination tests in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percentage of variance among and within these populations were 6.18 and 93.82, respectively. The Fst values (0.062, P < 0.001) among studied populations indicated that there were significant genetic differentiations among redfin culture populations from the scattered lakes with different connections to the Yangtze River. These results are useful for the evaluation and conservation of small freshwater fishes. The factors that may be involved in low intra-population polymorphism and the pattern of the population genetic structure of redfin culter from the Yangtze River were discussed.

Isolation and characterization of 18 polymorphic microsatellite markers in Chinese mandarin fish Siniperca chuatsi (Basilewsky)

Eighteen microsatellite markers were isolated and characterized using an enrichment protocol in the Chinese mandarin fish Siniperca chuatsi (Basilewsky), a commercially important piscivorous fish in China. Out of 48 pairs of primers designed, 18 loci exhibited polymorphism with three to six alleles (mean 4.4 alleles/locus) and average observed heterozygosity ranged from 0.633 to 0.833 (mean 0.748) in a test population from Dongting Lake of China. Except for two loci, all other 16 loci were in the Hardy-Weinberg equilibrium. These markers would be useful for such studies as population genetics, ecology and selective breeding of the Chinese mandarin fish in future.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H (O)) and expected (H (E)) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Isolation and characterization of six microsatellite markers in the large yellow croaker (Pseudosciaena crocea Richardson)

Six polymorphic microsatellite markers were isolated and characterized using an enriched library technique in the large yellow croaker (Pseudosciaena crocea Richardson, 1864), a commercially important marine fish in China. They showed PIC (polymorphism information content) ranging from 0.064 to 0.885 (average of 0.580) and allele numbers ranging from two to 13 (average of 7.5), which were useful for the studies on population genetics and selective breeding of the large yellow croaker.

Characterization of a highly conserved microsatellite marker with utility potentials in cyprinid fishes

A microsatellite locus, MFW1, originating from common carp is highly conserved in flanking nucleotides but variable in repeat length in some fishes from different families of the Cypriniformes. This orthologous locus is polymorphic in approximately 58% of the species tested in the order and is inherited by Mendelian law. It proved to be a potentially good marker in population genetics and in the cyprinid species-breeding programme in which no microsatellite markers were available.