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Random amplified polymorphic DNA (RAPD) markers were used to estimate genetic relationships between two seasonal immigrant forms (namely full-run and spring-run) of Caspian trout Salmo trutta caspius. 62 fin tissue samples were collected from southern parts of Caspian Sea basin and examined with 16 oligodecamer primers to asses their genetic diversity as well as probable specific population bands .Totally 162 bands were produced, of these 71 bands (43.83%) were polymorphic and 91 fragments were monomorphic bands and no band was population specific. The percentages of polymorphic bands were comparable in full-run (42.59%) and spring run (32.72 %), suggesting nearly similar levels of polymorphism of the two populations to be used for establishing selective breeding programs. Nei's genetic identity and genetic distance between spring-run and fall-run populations were 0.9858 and 0.01430, respectively. The RAPD based data revealed that the two migratory forms of Caspian salmon were categorized in conspecific value.
Keywords: genetic diversity, RAPD, Caspian trout, fall and spring run.
Salmo trutta caspius is an endemic, valuable and commercial subspecies of Salmo trutta in Caspian Sea basin which migrates to some Iranian southern rives for spawning. The most important rivers for their spawning in Gilan and mazandaran states of Iran are: Karganrood, Navrood, Astarachiy in Gilan state and Tonekabon (Cheshmekileh), Chaloos, Sardaberood in Mazandaran state, respectively. Distribution of subspecies within the Caspian Sea occurs commonly at the western and southern coasts of the Caspian Sea, from the Terek River to Sefid-Rud and seldom found in the North of Caspian Sea. Figure 1 explain locality of Gilan and Mazandaran states. S. t. caspius spawns in the Terek, Kura, Sefid-Rud and other minor
Fig1. Locality of Rasht (city center of Gilan state) and Sari (city center of mazandaran state) in southern part of Caspian Sea of coastline states in Iran.
rivers of the western and southern coasts of the Caspian Sea. (Caspianenvironment.org) in respect to its reproductive life cycle, two immigrant forms namely fall-run and spring-run were observed. In local areas, spring-run and fall-run called Salmon and Tian, respectively. Spring-runs differ from fall-runs by a greater body depth and silver color body; they migrate with unripe gonads to rivers at the end of winter and beginning of spring; they stay in rivers until autumn for spawning; fall-runs migrate with mature gonads and spawn at the end of winter. There is no good conditions in all of these rivers for S. t. caspius migrating and natural spawning.But among these, the most stock migratory forms of S.t. caspius were found in Tonekabon river. There is a local hypothesis that these two immigrant forms may be genetically different and probably they belonged to different populations.
Determination of genetic diversity between them could have a great importance for aquacultural strains development, protection of small-endangered populations and biogeographically inferences (Hassanien, et al., 2004). Despite the commercial and conservation importance of this subspecies, there were no published data on their molecular variability, yet.
In this regard Random Amplified Polymorphic DNA technique (RAPD) was applied as a useful tool for their genetic variability. Polymorphisms in RAPD markers are inherited in a Mendelian fashion and can be used as genetic markers. (Bardakci, 1994) Thus, the genome can be scanned more randomly than with conventional techniques. The ability of genomic variation examination without previous sequence information, relatively low cost of the technique, and only nanograms of template DNA requirement, is all advantages of RAPD in population and other genetic studies (Tassanakajon et al., 1997).
RAPD has been used in several fish genomic study including , identification of largemouth bass subspecies and its intergrades (Williams et al., 1998), study of Rutilus rutilus caspius populations in Caspian sea (Keyvanshokoh and Kalbassi, 2006) and genetic relationships in Salmonidae species including brown trout and Atlantic salmon (Elo et al., 1997).Thus, RAPD has been used in population studies of fish, and can be used efficiently for differentiate two migratory forms of endangered S. t. caspius in spring and autumn seasons.
The objective of present study was to investigate the genetic diversity and similarity between spring-runs and fall-runs in Iran coastlines with RAPD technique; detecting different populations in S.t. caspius for better broodstocks management and conservation of endemic populations.
Material and Methods
Fish sampling and DNA extraction
Between October 2005 till November 2007, 31 individuals of fall-run S.t. caspius were obtained from Shahidbahonar center of breeding which were collected from Tonekabon River in Mazandaran state. For preventing breeding depression; different couple samples were mated together in this center. 31 individual of spring-run were obtained in local areas (Tonekabon River) by morphologically detection of spring and autumn migratory forms in mentioned period. Fin tissue samples were preserved in 96% ethanol until DNA extractions.
Total DNA was extracted from 50 mg of fin tissue using Roche DNA Extraction Kit (Roche, Germany).Resting of DNA extraction was done according to Roche manual kit. The quality and concentration of DNA from samples were assessed by 1% agarose gel electrophoresis. The samples were stored at 4°C until use.
Sixteen commercially available oligodecamers random primers from Bioneer Company (Table 1) were used in the detection of polymorphism in this study.
Table 1: Sequence and name of primers used in this study were applied from Bioneer Company.
RAPD-PCR was performed in 25 Âµl reaction volumes containing 25 ng of S.t. caspius DNA, 20 pmoles of primer ,0.1 each dNTP, 4 mM MgCl2, 2.5 Âµl PCR buffer and 1.25 unit Taq DNA polymerase(Cinnagen). DNA was amplified by Eppendorff thermalcycler (Germany) programmed to provide a first denaturation of 5 min at 94ï‚°C, followed By 45cycles of 1 min at 94ï‚°C, 1 min at 36ï‚°C and 3 min at 72ï‚°C, and, finally, one cycle at 72ï‚°C for 6 min. Amplification products were resolved by electrophoresis in 2% agarose gels with TAE buffer (40 mMTris-Acetate, 1 mM EDTA pH 8.0) containing ethidium bromide. A 100 bp ladder Fermentas and markerÎ™Î™ Roche (Germany) were used as molecular standard size markers.
The genotypes were detected by scoring the presence (1) or absence (0) of distinct reproducible bands and faint bands were neglected. The index of similarity (Band Sharing Frequency) between individuals was calculated according to Nei and Li (1979) formula:
F =2NXY/ (NX+NY)
Where NXY is the number of bands shared in common between individuals X and Y, and NX and NY are the total numbers of bands scored for individuals X and Y respectively. Thus, F reflects the proportion of bands shared between two individuals and ranges from 0 (no common bands) to 1 (all bands identical). The genetic distance (d) was calculated as:
The analysis of the data was carried out using the POPGENE version 2.6.2 program.
A total of 162 bands were produced from 16 primers. Of these, 71 bands (43.83%) were polymorphic and 91 fragments were monomorphic band. The number of fragments generated per primer varied between 5 and 17. All primers gave RAPD patterns, (Fig.2, 3, 4) the average number of bands per primer was 10.125. The bands ranged in molecular size from approximately100 to 2500 bp. No band was population specific. Primer OPA-05 produced the highest number of fragments among the primers used with an average of 17 fragments, and primer P-07 produced the lowest number of fragments with an average of 5 bands). The total number of RAPD bands produced in spring and fall-run were 162 bands, of which 69 and 53 bands were polymorphic for each population. (42.59% and 32.72%, respectively). Nei's genetic identity and genetic distance between spring and fall-run were 0.9858 and 0.01430, respectively.
Fig2.RAPD pattern of primer OPA8 in 2% Agarose gel,
15 reproducible band were generated which none of them was
Populations' specific. From left to right 2-4 (Spring-run), 5-7 (Fall-run).
In these samples all of generated bands didn't show any polymorphism.
lane 1and 8 are 100 bp Ladder Fermentas company.
Fig3.RAPD pattern of primer OPA12 in 2% Agarose gel,
10 reproducible band were generated which none of them was
Populations' specific. From left to right S1-S3 (Spring-run), A1-A3 (Fall-run).
Two bands which were shown, have polymorphism in two Spring and fall-run populations, but not population specific .M:marker II Roche company.
Fig4.RAPD pattern of primer P07 in 2% Agarose gel,
5 reproducible band were generated which none of them was
populations specific. From left to right L:100 bp ladder Fermentas company,M:markerII Roche,1-3 (Spring-run), 1-6 (Fall-run),two
bands were shown,have polymorphisms in two poplation in these samples.
Degradation of the natural spawning areas environment of Salmo trutta caspius, through industry, municipal and agricultural pollution of rivers, amelioration works and forestry activities, lead to reduction of its normal reproduction. Also over-fishing due to excellent flesh quality of trout cause reduction in population size.
Although previous study based on hatchery stocks, confirm considerable population diversity between fall run forms(Rafiee,2006), but there is no study on populations genetic diversity between two migratory forms in Iranian coastlines and this study is the first report on two seasonal migratory forms of Caspian trout based on RAPD -PCR marker.
RAPD technique is suggested to be more useful in closely related populations (Smith & Williams, 1994; Borowsky et al., 1995). This technique has been used as a molecular tool for detecting genetic variation and genetic similarity in several fish species including tilapia species and subspecies identification (Bardakci and Skibinski,1994),brown trout and Atlantic salmon (Elo, et al. 1997),largemouth bass(Williams, Kazianis & Walter 1998),Spanish Barbus (Callejas and Ochando,2002) common carp(Bartfai, et al 2003), Indian carps (Barman, et al. 2005) and tilapia (Hassanien, et al. 2004).In this study we didn't find any population-specific marker between two populations and Nei's genetic distance (Nei 1972; Lynch, 1991) for the populations (0.01430) was very low. Thorpe and Sol-Cave (1994) showed that average genetic distance for conspecific populations is 0.05 (range: 0.002- 0.07) and for congeneric species is 0.30 (range: 0.03-0.61). Therefore the genetic distance of our study revealed that, the two migratory forms of Caspian salmon were categorized in conspecific value. As the RAPD markers show dominant markers, therefore it couldn't differentiate heterozygous and homozygous fish (Weising et al.,1995) and this is disadvantage of RAPD marker, but the randomly amplified polymorphic DNA technique is one of the most frequently used molecular methods for taxanomic and systematic analyses of various organisms (Bartish, et al., 2000).Although S.t. caspius spring-run and fall-run populations differ in observed forms (body depth and color), but analysis of the RAPD data showed very low genetic variation.
Artificial breeding of adults and subsequent release of juveniles to the wild is commonly performed in conservation programs to avoid the short-term extinction probability of endangered species since. Probably no selective programs in their artificial breeding, cause low genetic variation between them. Also, in conservation genetics programs this subspecies is being bred and released into the Caspian Sea Rivers for restocking management, but exact monitoring of genetic similarity and genetic distance among bred fishes is essential to avoid inbreeding problems.
This study represents a first step towards the initial assessment of genetic variation among two migratory forms of Salmo trutta caspicus from Iranian coastline of the Caspian Sea. Other genetic markers such as microsatellites, mtDNA and AFLP, may be serve as a better marker to detect genetic variation among them.
The authors express thanks to College of Natural Resources and Marine Sciences of Tarbiat Modares University and also Department of Animal Science in National Institute of Genetic Engineering and Biotechnology for their assistance.