2College of Marine Life Science, Ocean University of China, Qindao, China
*J-Y.X. and G-B.X. contributed equally to this work.
Single nucleotide polymorphisms (SNPs) are high-density natural sequence variations in genomes. They are considered to be the major genetic source of phenotypic variability within a given species and serve as excellent genetic markers. SNPs are useful in identifying candidate genes that contribute to disease and phenotypic traits. In non-model organisms, the application of SNPs has been limited, because of the expense and technical difficulties entailed in currently available SNP isolation techniques. In the present study, we have developed a rapid and effective method to isolate SNPs throughout the genome randomly. The DNA fragments containing SNPs could be isolated efficiently from background DNA. We analyzed ten isolated DNA fragments with this method in half-smooth tongue sole (Cynoglossus semilaevis)—a newly exploited and commercially important cultured marine flatfish in China—and found that nine of the fragments contained SNPs. The findings were confirmed successfully in different individuals. The method presented here is cost-effective and applicable to essentially any organism.
The identification of genes affecting complex traits is a very difficult and challenging task. For many complex traits, the observable variation is quantitative, and loci affecting such traits are generally termed quantitative trait loci (QTLs). In contrast with monogenic traits, it is impossible to identify all the genomic regions responsible for complex trait variation without a genetic map constructed with molecular markers. Single nucleotide polymorphisms (SNPs) can be used as genetic markers for constructing high-density genetic maps and to carry out association studies related to diseases (1). As a result of their abundance, heredity stability, and the availability of high-throughput analysis technologies, SNP markers have begun to replace traditional markers such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeat markers (SSRs or microsatellites) for fine mapping and association studies. SNPs have become an important application in the development and research of genetic markers.
Studies utilizing SNPs have become feasible with the availability of a variety of methods of SNP detection and genotyping. As the demand for genetic analysis increases, SNP detection technologies are being developed at an accelerated pace. SNP detection encompasses two broad areas: (i) scanning DNA sequences for previously unknown polymorphisms, and (ii) screening (genotyping) individuals for known polymorphisms. Scanning for new SNPs can be further divided into the global (or random) approach, and the regional (or targeted) approach (2).
There are several strategies that can be applied to new SNP discovery. The most straightforward method is direct sequence comparison using public or other sequence databases (3,4) and locus-specific amplification of target genome regions followed by sequence comparison (5,6). For SNP discovery in candidate genomic regions, a prescreening of SNPs prior to sequence determination is needed. There are a number of methods to prescreen SNPs (7,8,9), such as single-strand conformational polymorphism (10), denaturation kinetics (11), chemical cleavage (12,13,14), enzyme cleavage (15,16,17,18), array hybridization (19), mismatch repair detection (20), and bacteriophage Mu DNA transposition (21,22).
The main drawback of these methods is the requirement for prior sequence information (i.e., at the very least, the region for primer design should be clear). Such sequences are usually the product of whole-genome shotgun sequencing applications and have been mostly limited to model organisms or humans. In non-model organisms, there have been certain research efforts focusing on SNP discovery in candidate genes or genomic regions (23). However, these efforts are limited to highly homologous regions in which the sequence can be obtained by homologous cloning.
There are certain methods which have been developed for exploiting SNPs randomly in the genome, such as representation shotgun sequencing (24), primer-ligation–mediated PCR (25) and degenerate oligonucleotide–primed PCR (26,27). Despite the advantage of reducing the number of clones required in the analysis, these methods still require the sequencing of tens of thousands of clones to obtain data suitable for SNP discovery, and the efficiency of SNP discovery has been much lower than expected. Therefore, there is still room for considerable improvement in the specificity, sensitivity, and cost-effectiveness of SNP detection methods. Here we describe a novel and effective method to develop SNPs randomly throughout the entire genome in essentially any organism. We were able to specifically isolate the sequences containing SNPs using the half-smooth tongue sole (Cynoglossus semilaevis) as the experiment model. The half-smooth tongue sole is a newly exploited and commercially important cultured marine flatfish in China. Moreover, the females grow 2–4 times faster than males, which is useful for studying both sex determination and development (28,29). A high-density genetic map is necessary for this purpose, and SNPs represent an excellent choice for fulfilling this requirement.Materials and methods Purification and preparation of CEL I nuclease
CEL I nuclease was purified as described (18). Briefly, celery stalks (500 g) were juiced at 4°C, adjusted to 0.1 M Tris-HCl, pH 7.7, 100 µM phenylmethanesulphonylfluoride (PMSF), and spun for 20 min at 2600× g to pellet debris. The supernatant was brought to 25% saturation in (NH4)2SO4, mixed for 30 min at 4°C and spun at 16,000× g at 4°C for 40 min. The resulting supernatant was adjusted to 80% (NH4)2SO4, mixed for 30 min at 4°C and spun at 16,000× g for 1.5 h. The pellet was suspended in 0.1 M Tris-HCl, 0.5 M KCl pH 7.7, 100 µM PMSF (1/10 starting volume). The suspension was transferred to a dialysis tube and dialyzed against a total of 2 L of the same buffer, with four changes over 4 h. Aliquots of extract were stored at -20°C. Different dilutions of CEL I were tested and an optimal concentration of 1 U/µL was determined so that the CEL I cleaved SNPs efficiently but did not nonspecifically cleave dsDNA.