Prolactin I microsatellite as genetic markers for characterization of five Oreochromis tilapia species and two Oreochromis niloticus strains

Tilapia is an important aquacultural fish species globally because of its large size, high reproductive rate, rapid growth and palatability. According to the Food and Agriculture Organization (FAO), global tilapia production exceeded more than 4.2 million metric tons with a sales value of more than $5 billion in 2012. Because tilapia products are known for highly palatable taste and hygienic quality, therefore they have become popular worldwide. As a result, tilapia has become the one of the most important exported fish species and is a major source of income in developing countries. In general, high-quality tilapia products are derived from hybrids of superior tilapia strains that have been developed using advanced aquacultural technology. Most of these superior tilapia strains are bred using traditional generational selection. The characterization and maintenance of these superior tilapia strains are important to enhance the competitive advantage in aquaculture industry. Furthermore, food contamination occurs frequently in fish and fishery products. Consumers are increasingly concerned about what they eat and whether their foods are from high-quality and safe sources. A traceability system for the fishery chain is the most important tool to manage food safety [1]. Such a system can trace fish and fishery products to their farm of origin if toxins, diseases or unsatisfactory qualities are detected in the market. Most traceability schemes involve documentation at every processing step. However, it is difficult to verify theses records in the event of fraudulent activities. The development of molecular marker technology is needed to facilitate conventional selective breeding, sustain the tilapia aquaculture industry, promote tilapia quality and confirm traceability documentation [2].


Introduction
Tilapia is an important aquacultural fish species globally because of its large size, high reproductive rate, rapid growth and palatability. According to the Food and Agriculture Organization (FAO), global tilapia production exceeded more than 4.2 million metric tons with a sales value of more than $5 billion in 2012. Because tilapia products are known for highly palatable taste and hygienic quality, therefore they have become popular worldwide. As a result, tilapia has become the one of the most important exported fish species and is a major source of income in developing countries. In general, high-quality tilapia products are derived from hybrids of superior tilapia strains that have been developed using advanced aquacultural technology. Most of these superior tilapia strains are bred using traditional generational selection. The characterization and maintenance of these superior tilapia strains are important to enhance the competitive advantage in aquaculture industry. Furthermore, food contamination occurs frequently in fish and fishery products. Consumers are increasingly concerned about what they eat and whether their foods are from high-quality and safe sources. A traceability system for the fishery chain is the most important tool to manage food safety [1]. Such a system can trace fish and fishery products to their farm of origin if toxins, diseases or unsatisfactory qualities are detected in the market. Most traceability schemes involve documentation at every processing step. However, it is difficult to verify theses records in the event of fraudulent activities. The development of molecular marker technology is needed to facilitate conventional selective breeding, sustain the tilapia aquaculture industry, promote tilapia quality and confirm traceability documentation [2].
Several molecular markers have been used to characterize tilapia species and permit tilapia traceability, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs) and microsatellites [3][4][5][6][7][8]. Among these molecular markers, microsatellites have been commonly used because they are highly reproducible, polymorphic, co-dominant and widely distributed throughout the genome [9]. Microsatellites are repeated sequences of DNA throughout the genome. Most of microsatellite loci are associated with anonymous genomic segments [9]. However, an increasing body of research has demonstrated that some microsatellites are located with promoter and transcribed regions of genome and may be involved in regulating gene expression and function [10][11][12][13]. These functional microsatellites may cause distinct phenotypes or influence physiological functioning by directly or indirectly affecting the expression and function of a given gene [11,14]. Therefore, functional microsatellites can be used as genetic markers to characterize the phenotypes that result from the expressional variation of a given gene.
Nile tilapia can live longer than 10 years. Food availability and water temperature appear to be the limiting factors to growth for O. niloticus. Optimal growth is achieved at 28-36°C and declines with decreasing temperature. The ability to vary their diet may also result in variation in growth. In aquaculture ponds, O. niloticus can reach sexual maturity at the age of 5-6 months. Growth enhancement is a desired phenotype for tilapia in the aquaculture industry. Several growth-related genes are involved in cell proliferation and somatic growth, including growth hormone (GH), insulin-like growth factors-2 (IGF-2), prolactin-I (PRL-I) and insulin gene [15][16][17][18]. In the present study, we elucidate the ability of six microsatellite markers within these growth-related genes to characterize several tilapia species. We found that five Oreochromis species and two strains of O. niloticus can be differentiated by the microsatellite markers PRL I-MS01 and PRL I-MS02. The parentage of hybrid tilapia can also be traced by PRL I-MS01. We conclude that PRL I microsatellite markers are useful genetic markers for traceability systems and for breeding superior strains in the tilapia aquaculture industry.

Genotypic analysis of tilapia using growth-related microsatellite markers
Genomic DNA of diverse Oreochromis tilapia species was extracted from 20 mg of muscle tissue using the Master Pure TM Complete DNA Purification Kit (Epicentre Biotechnologies, Wisconsin, WI, USA) according to the manufacturer's protocol. Each genomic DNA sample was quantified using a Nano Drop ND-1000 spectrophotometer (Nano Drop Technologies, Inc., Wilmington, DE, USA). Genomic DNA samples were adjusted to a final concentration of 100 ng/L. Primer sequences for growth-related microsatellite loci were designed according to the methods of Yue and Orban [19] and are listed in Table  1. PCR was performed in 50-μl volumes containing 1×PCR buffer, 2 mM MgSO 4 , 0.2 mM dNTP, 200 nM of primers, 1.0 unit of Platinum Taq high-fidelity DNA polymerase and 100 ng of genomic DNA. PCR was carried out as follows: 94°C for 2 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s; and a final cycle at 72°C for 10 min. A 1-μl aliquot of each PCR product was used for genotypic analysis in a 2100 Bioanalyzer equipped with Expert version B.01.02.S1136 software and a DNA 1000 Lab Chip kit (Agilent Technologies, Palo Alto, CA, USA). The lengths of PCR products were calculated by the software based on the product retention times compared to size standards. A total of 25 samples (n=25) of each tilapia species were genotyped.
Bardakci [5] reported that feed treated with 17 alpha methyl testosterone (MT) is used to manipulate the gender of early tilapia fry. A series of experiments demonstrated that the analytical procedure for detecting MT in feeds lacked high precision and on average biased results. Low MT was uniformly mixed with feed by spraying an MTalcohol solution on feed while it was blended in an industrial ribbon mixer. Alcohol volumes ranging from 15 to 150 ml/kg were equally effective at dispersing MT in feed. The concentrations of MT in feeds consistently declined over time if the storage temperature was 25 o C or higher. Freezing preserved the MT in feed, and the refrigeration of feed fabricated to contain 60 mg MT/kg maintained acceptable MT concentrations during 6 months of storage. were used to evaluate growth performance. Larvae from each group were grown for one month in temperature-controlled 20-L plastic aquaria. Subsequently, 120 juvenile fish from each group were shifted to a pond (5 m x 3 m x 1 m) with a circulating water system and fed a commercial fodder (23% protein content) at a rate of approximately 1.5-5% of fish body weight twice daily for 20 weeks (n=120; duplicate). Thirty fish from each experimental group were randomly sampled on one monthly basis to determine body length, total body length and body weight. Body length and total body length were measured as the distance from the tip of the mouth to the hypural bone and from the tip of the month to the caudal fin, respectively [20]. Specific growth rate (SGR) was calculated using the following equation: SGR (%/day)=[(ln W 2 -ln W 1 )/t 2 -t 1 ]×100, where t 1 is the time at the beginning of growth, t 2 is the time at the end of growth, W 1 is the average body weight or body length at t 1 , and W 2 is the average body weight or body length at t 2 .

Parentage assignment
Hybrid tilapia population obtained by mating O. niloticus strains (N or N F ) to O. aureus (A) (NxA or N F xA) were used to study parentage assignment with PRLⅠmicrosatellite markers. Genomic DNA aliquots (100 ng) from hybrid tilapia and their parental strains were used as

Identification of tandem repeats in PRL I microsatellite loci
Genomic DNA was extracted from the progeny of NxN and N F xN F tilapia as described above (Genotypic analysis of tilapia using growthrelated microsatellite markers). Genomic DNA from both populations was used in PCR reactions with PRL I-MS01 and PRL I-MS02 primers. The major PCR product bands were separated by electrophoresis on a 3% agarose gel and then purified using a QIAquick gel extraction kit (Qiagen, Düsseldorf, Germany). PCR products containing tandem repeats were ligated into the pGEMT-T Easy vector (Promega, Madision, WI, USA) for sequencing.

PRLⅠmicrosatellites can be used to discriminate between tilapia species
Tilapia species differ in growth performance due to their distinct genetic backgrounds. We elucidate the ability of six growth-related microsatellites regions found in the growth-related gene IGF-II, PRL I, GH and insulin to discriminate between five Oreochromis tilapia species. Of these microsatellites loci, only two PRL I microsatellites were able to distinguish between different tilapia species.  Figure 1B). A few additional genotypes were observed in each species, but they did not affect our ability to distinguish each species. These results suggest that the combination of PRL I-MS01 and PRL I-MS02 can efficiently discriminate between the five tilapia species studies.
The acclimatizing the fry was done at 20 o C for 48 hours. After that aquarium water temperature was monitored each hour from the beginning to the end of the experiment. The temperature measurements were done hourly, while the dissolved oxygen (DO), pH, total ammonia, nitrate and nitrite were measured once a day, using a WTWR multi 340i meter and HACH kits. Dissolved oxygen ranged between 6.  and 2.44% g/day for A×A. SGR values based on body length were also calculated. The N F ×N F also exhibited the highest length-based SGR value. These results suggest that the rapid growth performance of N F compared N is due to differences in genetic background.

PRL I microsatellite markers are useful to distinguish between O. niloticus strains
To distinguish between the two strains of O. niloticus (N F and N), we analyzed the same six microsatellite loci within the growth related-genes IGF-II, PRL I, GH and insulin. The polymorphism and genotypic proportions in the N and N F strains are shown in Table 3. IGF-II-MS03 was highly variable. A total of 19 genotypes were found in the two strains. We found eleven genotypes for PRL I-MS02, nine genotypes for GH-MS01, seven genotypes for PRL I-MS01, seven genotypes for ISP-MS01 and a six-allele locus for IGF II-MS01. Each genotype consists of two alleles at each locus, one smaller and one larger. Among these microsatellite markers, only PRL I-MS01 and PRL I-MS02 were able to discriminate between the N and N  Figure 3A). For PRL I-MS02, a genotype consisting of 206-bp and 222-bp alleles was observed in the N F strain, while a genotype consist of 240-bp and 260-bp alleles was observed in the N strain ( Figure 3B). These results demonstrate that PRL I microsatellite markers can efficiently discriminate between O. niloticus strains (N and N F ) based on electrophoretic genotype.

Tracing the parentage of hybrid tilapia
High proportions of commercial tilapia from Taiwan consist of hybrids of O. aureus and O. niloticus. To assess the practical ability of PRL I-MS01 and PRL I-MS02 to trace the parentage of hybrid tilapia, the genotypes of hybrid tilapia derived from mating of N and N F mating with A were analyzed to confirm whether the alleles at each locus matched those of the parental strains. In hybrid tilapia derived from female N and male A parents, the PRL I-MS01 locus exhibited 260-bp and 312-bp alleles. In hybrid tilapia derived from female N F and male A parents, the PRL-MS01 locus exhibited 248-bp and 312bp alleles. PRL I-MS02 was not able to distinguish between the A and N strains. Therefore, this marker can be used to trace hybrid tilapia progeny derived only from N F and A. In the latter population, PRL nitrite, 0.01-0.02 mg/l. Aquaria were cleaned twice daily by suction to remove faeces. Water that was removed during aquarium cleaning was replaced with clean water pre-cooled to the same temperature with ice cubes.

Growth performance of O. niloticus strains
The N F strain, which exhibited rapid growth compare to the original N strain, was developed through generational selective breeding. To demonstrate that the N F strain possessed the rapid-growth phenotype, we measured the growth curve of N F and N strain over one month. Most commercial tilapia is derived from hybrids of O. niloticus and O. aureus. Therefore, we also evaluated the growth performance of the hybrid progeny of N F xA and NxA. The N F xN F progeny population exhibited better growth performance in body length and body weight than NxN population. Among hybrid populations, the N F xA population showed better growth performance in body length and body weight than the NxA population. Growth performance traits increased in the following order: N F x N F >N F xA>NxN>NxA>AxA ( Figure 2). The order of SGR value based on body length or weight for each population was consistent with the growth performance. As shown in Table 2   a Measured at the beginning of the growth period (one month). b Measured at the end of the growth period (six month). c Calculated over the growth period from one to six months. I-MS02 exhibited 206-bp and 246-bp alleles derived from the maternal N F strain and a 246-bp allele derived from the paternal A strain ( Figure  3B). These results demonstrate that the PRL I-MS01 microsatellite marker can be used to trace the origin of hybrid tilapia products.

Characterization of tandem repeats within PRL I microsatellite loci
Based on sequences obtained from Gene Bank database (NCBI Accession no. X92380), GT and CA tandem repeats are known to exist in the PRL I-MS01 and PRL I-MS02 microsatellite loci. However, the copy numbers of GT and CA tandem repeats in the N and N F strains are unclear. Most microsatellite genotypes in the N strain consisted of 260-bp and 288-bp alleles (PRL I-MS01) and 240-bp and 260-bp alleles (PRL I-MS02). In the N F strain, the PRL I-MS01 and PRL I-MS02 microsatellite genotypes were 248/274 bp and 206/222 bp, respectively.
To clarify differences in tandem repeats between N and N F , small alleles of PRL I-MS01 and PRL I-MS02 were cloned and sequenced to identify the copy numbers of tandem repeats. As shown in Table 4, the numbers of GT tandem repeats in N and N F were 35 and 27, respectively, while the numbers of CA tandem repeats in N and N F were 33 and 16, respectively. These results further demonstrate that PRL I-MS01 and PRL I-MS02 are useful genetic markers to characterize O. niloticus tilapia strains.

Discussion
Tilapia has become the second most important aquaculture fish in the world. Therefore, researchers worldwide have endeavored to improve its growth characteristics. From an economic benefits perspective, farm-raised tilapia selected for large body size are almost exclusively male because of the xenogenic hybridization technology used. Taiwan is one of the major tilapia-producing countries and is an important supplier to the global market. In Taiwan   to supplement conventional approaches is required to strengthen the tilapia aquaculture industry and facilitate optimal breeding. Microsatellite markers have been used previously to identify tilapia species, trace parentage, measure genetic diversity and facilitate genetic mapping [4,[21][22][23][24][25].
We used six microsatellite regions located within four growthrelated genes, including IGF-II, PRL-I, GH and insulin, to discriminate between five tilapia species. Only two microsatellite markers located in the promoter region of the PRL I gene could be used to identify species of tilapia. We found that the genotypes of A, M, H and N could be differentiated using PRL I-MS01. However, the genotypes of these four strains could not be differentiated using PRL I-MS02. The genotype of S could be uniquely distinguished from the other four species using PRL I-MS01. These results demonstrate that the combination of the PRL I-MS01 and PRL I-MS02 microsatellite markers can characterize the five tilapia species examined.
Prolactin (PRL) is a hormone released by the pituitary gland. It is similar in structure and function to growth hormone (GH) and somatolactin (SL). In fish, PRL is involved in the regulation of electrolyte homeostasis and immune function [26,27]. Furthermore, knockdown of the PRL gene in zebrafish embryos leads to phenotypes of shorter body length and smaller head and eye size, suggesting that PRL is also involved in growth [18]. The N and N F strains of O. niloticus exhibit significant differences in body size, body weight and specific growth rate. Farmed hybrid tilapia produced by crossing A with N or N F also show differences in body size, body weight and specific growth rate. To assess whether the difference in growth performance between N and N F can be predicted by PRL I microsatellites, the genotypes of N and N F were analyzed using PRL I-MS01 and PRL I-MS02. Our findings reveal that both microsatellite regions are efficient genetic markers to distinguish between N and N F tilapia strains. Traceability of farmed hybrid tilapia is important to ensure the overall quality and safety of farm-raised tilapia. PRL I microsatellites were also used to verify whether the genotypes of hybrid tilapia match those of their parents. Our findings reveal that the progeny of hybrid tilapia inherit PRL I alleles from the paternal and maternal genotypes at this locus. Thus, the origin of hybrid tilapia can be traced based on PRL I microsatellite genotypes.
Recent studies have demonstrated that functional microsatellites located in protein-coding regions, untranslated regions (UTRs) and introns can regulate gene expression and function by affecting transcription and translation [10,11,28]. For example, the microsatellite in the 5' flanking region of the GH gene of teleosts is associated with growth [29]. Furthermore, CA tandem repeats within intron 1 of the IGF-I gene affect circulating concentrations of IGF-I in swine [30]. Sequence data confirms that the copy numbers of GT tandem repeats in PRL I-MS01 and CA tandem repeats in PRL I-MS02 vary significantly between the N and N F tilapia strains. Streelman and Kocher [24] have demonstrated that different lengths of PRL I microsatellite alleles with CA repeats can affect gene expression [31,32]. The difference in copy number of PRL I microsatellites may affect the diversity of PRL I gene expression, further influencing the observed growth performance differences between N and N F . This hypothesis requires further study.
Nevertheless, it is not clear whether selection for low cortisol response is always recommendable. Further analysis will be carried out on the consistency of stress/cortisol response on this and the next generation to see whether the selection for this characteristic may help to decrease husbandry problems and improve the profitability of aqua cultured fish.
We found that PRL I microsatellites, including PRL I-MS01 and PRL I-MS02, are useful genetic markers to characterize Oreochromis tilapia species and to distinguish between the N and N F strains of O. niloticus. Microsatellite markers will provide an efficient tool to strengthen the management of the tilapia aquaculture industry and facilitate the conventional breeding of superior strains.