Study of inter species diversity and population structure by molecular genetic method in Iranian Artemia

Artemia is a small crustacean that adapted to live in brine water and has been seen in different brine water sources in Iran. Considering the importance of genetic studies manifest inter population differences in species, to estimate genetic structure, detect difference at molecular level and separate different Artemia populations of Iran, also study of phylogenic relationships among them, samples of Artemia were collected from nine region: Urmia lake in West Azerbaijan, Shoor and Inche-Borun lakes in Golestan, Hoze-Soltan and Namak lakes in Qom, Maharloo and Bakhteghan lakes in Fars, Nough pool in Kerman and Mighan pool in Markazi and DNA extracted by phenol-chloroform method. Primers designed on a ribosomal fragment (16s rRNA) of mt DNA sequence and PCR was done. Digestion of the 1566 bp segment PCR product by 10 restriction endonuclease (Alu I, EcoR I, Eco47 I, Hae III, Hind III, Hinf I, Mbo I, Msp I, Rsa I, TaqI) showed 25 different haplotypes: 9 in Urmia, 4 in Shoor and Inche- Borun, 1 in Namak and Hoze-Soltan, 3 in Mighan, 1 in Bakhtegan Maharlo, 3 in Maharloo and 4 in Nough. Measurement of haplotype and nucleotide diversity intra population and nucleotide diversity and divergence inter populations and evolutionary distance between haplotypes showed a high diversity in mitochondrial genome of Artemia in studied regions whose results are similar to those explained for highly geographic expansion organism. In addition, results showed considerable heterogeneity between different populations and there are enough evidences in haplotypic level for separation of studied samples and division of Iranian Artemia to seven populations including Urmia, Shoor and Inche-Borun, Hoze-Soltan and Namak, Maharloo, Bakhteghan, Nough and Mighan. Phylogenetic analysis of the 16S rRNA data set resulted strict consensus and neighbor joining distance trees, demonstrated that all samples were monophyletic and parthenogenetic form derivation from bisexual populations and genetically high resemblance to those of A. urmiana. Study of 270 specimens from different region showed the genus Artemia in Iran clustered into three clades including: 1- Shoor, Inche-Burun, Hoze-Soltan, Namak, Bakhtegan and Maharloo 2- Nough and Mighan 3- Urmia. Totally, obtained results indicated to ability of used techniques for study of inter species diversity, population structure, reveal of phylogenic relationship and dividing of different populations of Artemia in Iran.

Comparative study of seasonal changes in some blood parameters and genetic polymorphisms of serum antibodies and antigens on red blood cells surface in immature Caspian salmon

Seasonal sampling from 40 immature Caspian salmon were performed in summer, autumn, winter and spring. The maximum ranges of RBC counts, Hct, Hb, WBC count and clotting times were observed in spring, summer, spring, spring and winter, respectively. The minimum amounts of these factors were counted in summer, winter, winter, winter and winter, respectively. Blood Samples were taken from healthy smolt, immature and adult Caspian salmon in spawning time. Hematological determinations and biochemical serum analysis were performed in 101 fish in the three samples. The ranges of hematological values for sample mean were counted. Red blood cell counts were 866600 mm3 and 1259400 mm3 in smolt and adult respectively. Hematocrit was 48.39% in smolt and 44.29% in adult. Hemoglobin was 8.85 gr/dl in smolt and 10.91 gr/dl in adult. White blood cell count was 8781.58 mm3 in smolt and 5217.55 mm3 in adult and mean were differential of WBC, Lymphocyte 90.57%in smolt and73.22% in adult. Neutrophil was 5.12% in smolt and 16.92% in adult, Monocyte were 1.27% in smolt and 4.24% in adult, Clotting time was 282.34 Seconds in smolt and 291.47 seconds in adult MCV, MCH and MCHC also meagered in smolt and adult. Biochemical parameter in immature and mature Caspian salmon meagered .Glucose concentration was 2.97 mmol.l- in immature and 1.99 mmol.l- in mature .Cholesterol concentration was 4.26 mmol.l- in immature and 7.06 mmol.l- in mature. Triglyceride amount was 2.35 mmol.l- in immature and 2.47 mmol.l- in mature and Calcium was 2.47 in immature and 2.61 mmol.l- in mature. An in situ study was made on erythrocytic isoantigens and hetero-antigen and their corresponding iso-and hetero-antibodies of sera by means of hemoagglutination tests on the blood sample, of 450 immature and 50 mature Caspian salmon. The absence of erythrocyte iso-antigens and hetero-antigen and their corresponding iso-and hetero-antibodies were shown by the experimental. It could be indicated an intra-specific variation and differences in species for kelardasht hatchery.

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.

The genetic structure and phylogenetics of pikeperch (Sander lucioperca) Anzali and Amirkolaye wetlands and perch (Perca fluviatilis) populations in Aras Dam and south-west of the Caspian Sea

The genetic structure of pikeperch (Sander lucioperca) and perch (Perca fluviatilis) populations was studied using microsatellite technique. A total of 207 specimens of adult pikeperch were collected from Aras dam (57 specimens), Anzali wetland (50 specimens), Talesh (50 specimens) and Chaboksar (50 specimens) coasts. Also a total of 158 specimens of adult perch were collected from Anzali (Abkenar (50 specimens)and Hendekhale(48 specimens)) and Amirkolaye(60 specimens) wetlands. About 2 g of each specimen's dorsal fin was removed, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using ammonium-acetate method. The quality and quantity of DNA was assessed using 1% agarose gel electrophoresis. Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 pairs of microsatellite primers. PCR products were electrophoresed on poly acryl amide gels (6%) that were stained that were stained using silver nitrate. DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected and observed heterozygosity , allele number and the effective allele number, genetic similarity and genetic distance, Fst, Rst, Hardy Weinberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendogram for genetic distances and identities were calculated using TFPGA program for any level of hierarchy. The results for P. fluviatilis showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 4.1±1.1 and mean observed and expected heterozygosity was 0.56±0.12 and 0.58±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.095) with Nm=2.37 was observed between Hendekhale and Amirkolaye and the lowest Fst (0.004) with Nm=59.31 was observed between Abkenar and Hendekhale. According to AMOVA Significant difference (P<0.05) was observed between recorded Rst in the studied regions in Anzali and Amirkolaye lagoons. In another words there are two distinct populations of this species in Anzali and Amirkolaye lagoons. The highest genetic distance (0.181) and lowest genetic resemblance (0.834) were observed between specimens from Hendekhale and Amirkolaye and the lowest genetic distance (0.099) and highest genetic 176 resemblance (0.981) were observed between specimens from Abkenar and Hendekhale. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Anzali and Amirkolaye wetlands have the same ancestor. On the other hand there is no noticeable genetic distance between the specimens of these two regions. Also the results for S. lucioperca showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 3.0±0.6 and mean observed and expected heterozygosity was 0.52±0.21 and 0.50±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.093) with Nm=2.43 was observed between Aras dam and Anzali wetland and the lowest Fst (0.022) with Nm=11.27 was observed between Talesh and Chaboksar coasts. Significant differences (P<0.05) were observed between recorded Rst in the studied regions exept for Talesh and Chaboksar Coasts. In another words there are three distinct populations of this species in Caspian sea, Anzali wetland and Aras dam. Highest genetic distance (0.110) and lowest genetic resemblance (0.896) were observed between specimens from Aras dam and Anzali wetland and the lowest genetic distance (0.034) and highest genetic resemblance (0.966) were observed between specimens from Talesh and Chaboksar coasts. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Talesh and Chaboksar coasts have the lowest genetic distance. On the other hand the main population of this species belongs to Anzali wetland. Phylogenetic relationship of these two species was inferred using mitochondrial cytochrome b gene sequencing. For this purpose 2 specimens of P. fluviatilis from Anzali wetland, 2 specimens of S. lucioperca from Aras dam and 2 specimens of S. lucioperca from Anzali wetland were sequenced and submitted in Gene Bank. These sequences were aligned with Clustal W. The phylogenic relationships were assessed with Mega 4. The results of evolutionary history studies of these species using Neighbor-Joining and Maximum Parsimony methods showed that the evolutionary origin of pikeperch in Aras Dam and Anzali wetland is common. On the other hand these two species had common ancestor in about 4 million years ago. Also different sequences of any region specimens are supposed as different haplotypes. 177 As a conclusion the results of this study showed that microsatellite and mtDNA sequencing methods respectively are effective in genetic structure and phylogenic studies of P. fluviatilis and S. lucioperca.

Genetic study on breeding stock of the Indian mackerel along the Indian coast

The study included: sample collection; microsatellite genotyping and analysis; and preliminary results

The genetic garden

The article discusses some strategies in the establishment of a mangrove genetic garden where species could be maintained. The genetic garden is a sustainable way to prevent further damage of the remaining mangroves. Its prime function is the protection and conservation of mangroves for sustainable use.

Report on BOBLME Indian mackerel genetic data analysis including final workshop, Phuket, Thailand, 17-19 February 2015

This workshop followed on from two previous workshops held in Colombo, Sri Lanka, 2012 and Kochi, India in 2013. The 14 microsattellite markers had previously been developed for Indian Mackerel (Rastrelliger kanagurta) were used on 31 tissue collections from all eight countries were genotyped in India.

Survey of exist specifications morphometric, meristic and inter species molecular genetic on Bream (Abramis brava orientalis) in Anzali wetland, southern coast of Caspian Sea, Aras Dam Lake and Azerbijan Republic

Bream (Abramis brava orientalis) is one of Cyprindae the Caspian Sea and its basin which has a special ecological, biological and economical role. Stock of this fish in the Caspian Sea has reduced during several years for different reason the over fishing, different industrial, agriculture, urban pollution and destroy of the spawning habitat. So that fishery company decided to recover the stock of this fish by the way of artificial reproduction of a Bream couple hunted from south coast of the Caspian Sea (Iran) and setting the fingerling to the rivers and inflow wetlands of the Caspian Sea.This activity has due to 20 tons Bream annual fishing in the Iranian South coast of the Caspian Sea (Gilan province coast and Anzali wetland), The artificial reproduction has decreased Bream population diversity of Caspian sea and Anzali wetland.So it has been declined to improve Braem population diversity by the entrance of Azerbijan republic Bream and encounter to the Caspian sea Bream. Meanwhile there is Bream in the Aras Dam Lake which had been forgotten by the Fishery Company of Iran .For this reason specifications morphometric, meristic and inter species Molecular Genetic have been surveyed in Anzali wetland,Southern coast of Caspian Sea ,Aras Darn Lake and Azerbijan republic during 2003-2005. According to the research on specifications of Morphometric and Meristic of Anzali wetland(120 species),Southern coast of Caspian Sea(90 species), Aras Dam Lake(110 species) and Azerbijan Republic(125 species)has Morphometric and Meristic differences. So that average weight and total length of Anzali wetland Bream respectively was 167 g and 23/76 cm, 102 g and 27/62 cm in Caspian Sea , 461 g and 3 5/38 cm in Aras Darn Lake and 3 4189 g and 15/21 cm in Azerbijan republic (We forced to use 1 year Bream of artificial reproduction in Iran). Also variation coefficient average Morphometric, Morphometric specification Ration and meristic in Anzali wetland Bream was 17/45, 21/56 and 4/63, in Caspian Sea bream 22/58, 15/27 and 3124, in Aras Dam lake Lake 17145. 1.5/27 and 3/57 and Azerbaijan republic Bream 22/29, 19/66 and 4/22. Also Bream of these four regions in general status had Morphometric significant differences based on One Way ANOVA Analysis. Meanwhile Anzali wetland Bream with Caspian Sea Bream from 41 Morphometric surveyed factors in 33 factors, with Aras Darn Lake Bream in 41 factors, with Azerbkjan republic Bream in 41 factors,Caspian Sea Bream with Aras Darn Lake Bream in 36 factors,with Azerbijan republic B ream in 40 factors and A ras Dam L ake Bream with Azerbijan republic Bream in 38 factors had significant statistical differences. These four regions Bream had differences according to the Morphomertric specification ration based on One Way ANOVA Analysis. Also Anzali wetland Bream was surveyed with Caspian Sea Bream from 37 factors i n 27 factors, Anzali wetland Bream with Aras Dam 1ake in 37 factors Anzali wetland Bream with Azerbijan republic Bream in 32 factors,Caspian sea bream with Arsa Dam Lake Bream in 26 factors, Caspian Sea Bream with Azerbijan republic Bream in 29 factors and Aras Dam Lake Bream with Azerbijan republic Bream in 34 factor had significant statistical differences. Based on Meristic factor of four regions bream in 16 surveyed factors in 10 factors had meaningful differences according to the One Way ANOVA Analysis. While Anzali wetland Bream was surveyed with Caspian Sea Bream from in 3 factors,Anzali wetland Bream with Aras Dam lake in 8 factors,Anzali wetland Bream with Azerbijan republic B ream in 6 factors,Caspian Sea bream with Arsa Dam Lake Bream in 6 factors,Caspian sea Bream with Azerbijan republic Bream in 3 factors and Aras Dam Lake Bream with Azerijan republic Bream in 8 factor had significant statistical differences.Meanwihle based on Factor Analysis and Discriminant Breams had differences. Also according to the resrarchs Anzali wetland Bream in 0+ age group till 5+ (6 age groups),Caspian Sea bream in 1+ - 5+(5 age groups),Aras Darn Lake Bream in 1+ - 7+ (7 age groups) and Azerbijan republic Bream for Morphometric and Meristic studies in 1+age group and for molecular Genetic reaserch were in 8+and 9+ age groups. According to the research 4 ecosystems Bream in status of same age, Aras lake Bream were bigger according to weight and length.Also in this research genetic diversity between four population was researched by PCR-RFLP technic on a piece of mitochondrion genome with the length of 3500bp contain of tRNA-leu,tRNA-glu,ND5/6,Cytb. Between 17 used enzyme. 4 enzyme, Dral, Bc11, Haefll and Banff showed diversity in totally 6 composite haplotype was detected. Maximum nucleotide diversity by the value% 0/58 in Azerbijan republic Bream by all haplotype. Aras darn Lake Bream had 2 haplotype and nucleotide diversity of %0/35.Anzali wetland and Caspian Sea Bream had no diversity. Statistical analysis by the usage of Monte Carlo with 1000 repeat showed significant differences between Azerbaijan Bream and other Bream(P<0/0001) but there was no significant difference between 3 regions Bream(P>0/5).

Genetic variation survey of intra-specific (Sepia pharaonis) in the Persian Gulf and the Oman Sea using PCR-RFLP

This research was conducted to identify Cuttlefish population (Sepia pharaonis) in The Persian Gulf and the Oman Sea using PCR-RFLP. Specimens were collected from )0 different stations. Bottom trawling method was used for sampling from different zones of the Persian Gulf and the Oman Sea, and finally specimens from S. Pharaonis were collected at each station . DNA was extracted by phenol—Coloroform method. One pair primer was designed based on 1As rRNA gene nucleotide sequences. The results obtained from 1 As rRNA gene RFLP, which was reproduced by PCR technique, were analyzed and utilized for study of diversity of the Cuttlefish population. PCR product with o pair base in length achieved for all specimens, which was subjected to enzymatic digestion by A restriction action enzymes: Alu I-Taq I-Mnl I-Rsa I-Hind III-Dra I-vu II and Hae II DNA bands patterns in all specimens digested by those enzymen showed similarity with no any polymorphism. From this result, it can be concluded that there is not any possibility to isolate different populations in the studied Cuttlefish species under exploitation of rRNA gene.

Genetic diversity of Saccostrea cucullata population in the northern coast in Persian Gulf and Oman Sea

Genetic diversity of Saccostrea cucullata in the northern coast lines of the Persian Gulf and the Sea of Oman were determined using DNA extraction and RAPD - PCR. A total of 300 samples were collected from 6 station along the coastline. Two out of six primers showed positive results namely GCG - ATC - CCC - A (Primer 1) and GTC - CAC - ACG - C (Primer 5) which were in accordance with morphometric analysis. The number of bands in the two above - mentioned primers in Khor - Tang and Chabahar station (Province of Sistan and Balouchestan) was significantly different from the number of produced bands in Dayer and Bushehr station (Province of Bushehr) as well as Gheshm and Bandar - Lengeh station (Province of Hormozgan). The cluster analisys was used to confirm the above variations. The results showed that the oyster population can be divided into two separate clusters. The first cluster included Bushehr Dayer Gheshm and Bandar - Lengeh species. The second cluster included Khor - Tang and Chabahar species. The analysis also showed that the first cluster can be divided into two Sub — cluster. Bushehr and Dayer belong to one Sub - cluster whereas Gheshm and Bandar - Lengeh form the other Sub — cluster. The formation of different vluster can be related to Physico - Chemical properties of water and climatic variations in different habitats along the Persion Gulf and the Sea of Oman. Key words: Molecular genetics, Population, RAND, PCR' Saccostrea cucullata

Genetic diversity of the black crested gibbon (Nomascus concolor) from the Wuliang Mountains of Yunnan, China: preliminary results.

The authors thank Peng Shi, Scott Groom, and two anonymous reviewers for helpful comments. This work was supported by grants from the National Basic Research Program of China (973 Program, 2007CB411600), National Natural Science Foundation of China, and Bureau of Science and Technology of Yunnan Province (to Y.-P.Z.).

Genetic variation and population structure of Indian mackerel in the southern part of BOBLME region, Indonesia

Objectives included measuring genetic variation and population structure of Indian mackerel (Rastrelliger kanagurta) in order to ascertain whether current exploitation is sustainable. .

Genetic relationship of Chinese and Japanese gamecocks revealed by mtDNA sequence variation

Cockfighting has a very long history dating back to as early as 2500 years ago in China. Cockfighting was intertwined with human cultural traditions, helped disperse chickens across the world, and influenced the subsequent breed selection. Therefore, trac