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Development and Validation of Single Nucleotide Polymorphism (SNP) Markers from an Expressed Sequence Tag (EST) Database in Olive Flounder (Paralichthys olivaceus)

ABSTRACT To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29, respectively. In conclusion, we identified SNP and polymorphism in olive flounder using newly designed marker, it supports that developed markers are suitable for SNP detection and diversity analysis in olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.


INTRODUCTION
The olive flounder (Paralichthys olivaceus) is an economically important fish in Korea and Japan, and total yields of aquacultured olive flounder increased and reached to 40,805 tons in 2011 (FAO) in Korea. Recently, breeding research is focused only culture techniques and develop-ment of research also insufficient to further increase production. Higher breeding research including the development of molecular markers is essential to produce of good breeds and high value fisheries and to enhance our competitiveness in fisheries industry. Identification and functional characterization of economic traits (or breeding traits) related genes, the development of molecular breeding techniques using DNA markers to molecular breeding is meaningful (Hubert et al., 2009;Yoon, 2006). Moreover, application of these genes in the aquaculture (molecular selective breeding) has attracted worldwide attention.
Recently, large-scale DNA sequencing such as BAC library projects and EST projects have provided usable data for a great variety of species (Hayes et al., 2005). In particular, the sequence analysis was significantly increased in economic worth of fish. This information can be used to increase the accuracy of selection breeding research for economical traits, thereby increasing the rate of genetic gain and production efficiency.
The molecular marker is an important element for breeding research, and there are various methods to used for the research. The kind of molecular techniques are RAPD (random-amplified polymorphic DNA), RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), MS (microsatellites) and SNP (single nucleotide polymorphisms) (Liu & Cordes, 2004). Among possible molecular markers, microsatellites have been most useful for population genotyping technology, but is not suitable to high-quality data about influence gene expression and the functions. Unlike MS, single nucleotide polymerphisms (SNPs) are the most abundant type of variation in DNA sequence among individuals in a population (Fahrenkrug et al., 2002). Moreover, these markers appear to have low mutation rates (a mutation occurs every 2 × 10 8 nucleotides) and hence are extremely stable than other genetic markers (The International SNP Map Working Group, 2001). Although, SNPs have been recently proposed as an alternative to microsatellites for genotyping individuals (Vignal et al., 2002), have a disadvantage of time and cost-consuming when a certain selection mechanism does not exist.
Whereas, if developed SNPs from of expressed sequence tags (ESTs) can be particularly valuable, also it is possible to reduce the time and cost for developing a useful SNP marker. In aquaculture species, SNPs are especially important if they cause differences in economic traits, or are linked to the mutations that do so.
Wild and cultured olive flounder have different growth rate and resistance to disease because of differences in the growth environment. For example, wild olive flounder has generally small size and slow growth but strong immunity, whereas cultured have generally rapid growth and heavy weight but weak immunity. Each feature is associated with a SNP, and therefore SNP analysis using wild and culture samples can be a help to find regions involved superior traits. In this study, we develop useful marker to detect the single nucleotide polymorphism (SNP) from EST in olive flounder and then apply to sample of wild and cultured.
Moreover, we were identified substitution of SNP and population diversity analysis using developed marker.

EST database and candidate SNP detection 1) ESTs database assemble
In this study used polymorphic sites were identified from expressed sequence tags (ESTs) derived from P.
olivaceus. Using to the previous studied EST data, bioinformatic analysis was conducted to determine gene identities using Gene Master Software (Ensoltek). ESTs were assembled in clusters of contiguous sequences (contig) using ICAtools program (Parsons, 1995). Gene annotation procedures and homology searches of the sequenced ESTs have been locally done by BLASTX for amino acid similarity comparisons. Matches with the Expect value (E) less than 10xe-4 were considered to be significant. All ESTs that were not identified as orthologs of known genes were designated as unknown EST clones.

2) Candidate SNP detection
Total of 755 SNP genes were selected from chromosome 24. The selection of SNP gene criteria is as below; the frequency of minor allele is 30 percent, each gene is related with immune and growth, and evenly exists on chromosome 24. As a result, 144 markers were selected, developed the primer for detecting of specific gene. Primers of SNPs were designed using Primer 3 software (Rozen and Skaletsky, 2000) adjusting length products to 100-300 bp (Table 1).

1) Sampling, amplification and analysis
Wild and cultured each of eight olive flounder were randomly obtained from East Sea and Jeju Island in Korea, respectively. Genomic DNA was extracted from pectoral fin with the standard method of phenol-chloroform (He et al., 2008) and a total of 144 primers were designed.

Identification of SNPs from EST database
The olive flounder EST-derived database contains 4,327 ESTs and identified 693 contigs and 514 SNP were detected.
The   polymorphic SNPs (Fig. 2). With C/T being the commonest (12) and C/G the least common (1) alteration observed.
Transition/transversion ratio (ts/tv) was 1.910, SNP substitutions result of development marker shows transitions rate higher than transversions rate (Table 3). These results correspond with the EST resource SNP's ones.

SNP genotype and allele frequency
The

DISCUSSIONS
Genomic regions of immune and protein synthesisrelated genes is directly related to life of organisms so that   its importance is well known (He et al., 2011;He et al., 2012). In addition, single base substitution of specific genomic region can be a positive or negative a factor for survival, growth and adaptation to the environment. Because these usefulness, the SNP research has been actively proceeding in various fish species; but hasn't yet been nephrosin, elastases factors, MHC II alpha beta antigens and apolipoprotein involved in innate immunity (Dijkstra et al., 2013;Tsai ea al., 2004;Vera et al., 2011). In addition, ferritin has an important role for iron storage and detoxification, the storage of iron is closely related to the immune response to bacterial infection protected. Finally, transferrin could be related to the inflammation and enhanced resistance to oxidative stress by parasite infections (Paramá et al., 2007). Also, SNP detected genes in olive flounder show a The diversity analysis using developed SNP marker in our research shows the heterozygosity value is higher in the wild sample of 0.34 than the cultured sample of 0.29, meaning higher diversity of the wild than the cultured. The cultured sample has got smaller diversity due to continuous inbreeding in the group without any inflow of new species (new mother group), resulting in lower diversity than the wild (Xing et al., 2014). Result of our heterozygosity is a correlation with diversity study outcome of olive flounder using microsatellite (Jung et al., 2009). It supports the appropriateness and usefulness of newly developed SNP marker to apply not only to SNP detection but also to other diversity analysis.
In conclusion, we was identified the substitution using the new marker from sample, it supports that development marker on this study is suitable for SNP detection in olive flounder. In addition, the marker is an appropriate tool for diversity analysis. The outcome of this study will be used in the selection of breeding with economic traits and will facilitate the molecular breeding programs of olive flounder.