Chinese Journal of Oceanology and Limnology   2017, Vol. 35 issue(1): 198-202     PDF       
http://dx.doi.org/10.1007/s00343-016-5103-4
Institute of Oceanology, Chinese Academy of Sciences
0

Article Information

SONG Na(宋娜), CHEN Muyan(陈慕雁), GAO Tianxiang(高天翔), YANAGIMOTO Takashi
Profile of candidate microsatellite markers in Sebastiscus marmoratus using 454 pyrosequencing
Chinese Journal of Oceanology and Limnology, 35(1): 198-202
http://dx.doi.org/10.1007/s00343-016-5103-4

Article History

Received Apr. 25, 2015
accepted for publication Jul. 29, 2015
accepted in principle Jan. 5, 2016
Profile of candidate microsatellite markers in Sebastiscus marmoratus using 454 pyrosequencing
SONG Na(宋娜)1, CHEN Muyan(陈慕雁)1, GAO Tianxiang(高天翔)2, YANAGIMOTO Takashi3        
1 Fisheries College, Ocean University of China, Qingdao 266000, China;
2 Fisheries College, Zhejiang Ocean University, Zhoushan 316000, China;
3 National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Japan
ABSTRACT: Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present study using 454 pyrosequencing, and the marker profile was analyzed. A total of 2 000 000 raw sequence reads were assembled to reduce redundancy. Among them, 1 043 dinucleotide, 925 trinucleotide, 692 tetranucleotide, and 315 pentanucleotide repeats were detected. AC repeats were the most frequent motifs among the dinucleotide repeats, and AAT was the most abundant among the trinucleotide repeats. AAAT, ATAG, and ATCC were the three most common tetranucleotide motifs, and AAGAT and AATAT were the most dominant pentanucleotide motifs. The greatest numbers of loci and potentially amplifiable loci were found in dinucleotide repeats, whereas trinucleotide repeats had the fewest. In summary, a wide range of candidate microsatellite markers were identified in the present study using a rapid and efficient 454 pyrosequencing approach.
Key words: marbled rockfish     microsatellite     454 FLX pyrosequencing     genomic DNA    
1 INTRODUCTION

Microsatellites or simple sequence repeats are tandemly repeated motifs of 1-6 bases that occur throughout the genomes of organisms from viruses to eukaryotes and are characterized by a high degree of length polymorphism (Zane et al., 2002). Microsatellites have become one of the most popular molecular markers and have been widely used for constructing linkage maps and performing population genetics studies and pedigree identification due to their ubiquitous occurrence, high reproducibility, multiallelic nature, and codominant mode (Zane et al., 2002 ; Zhang and Hewitt, 2003 ; Li, 2004). Some studies have suggested that cross-species transferability of microsatellite markers is unevenly distributed across taxa (Barbará et al., 2007) and that microsatellite markers often do not transfer well from one species to another (Castoe et al., 2010). Isolating and developing microsatellite primers were timeconsuming until recently because it is necessary to create enriched microsatellite libraries (Zane et al., 2002). However, applying 454 FLX pyrosequencing reduces cost and large sequence datasets can be acquired rapidly. This recent method has been used to detect microsatellite loci in many species (Castoe et al., 2010 ; Zeng et al., 2013).

The marbled rockfish, Sebastiscus marmoratus, (Order Scorpaeniformes, Family Scorpaenidae) is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan (Kita et al., 1996). S. marmoratus has potential economic value because of its delicate meat and delicious taste. Several studies have been conducted on S. marmoratus germplasm resources due to the decline in wild populations (Dong et al., 2008 ; Su et al., 2012 ; Zhang, 2013). Various polymorphic microsatellite loci have been isolated for this species by Xu et al.(2010), Yin et al.(2012), Li et al.(2014), Liu et al.(2014), and Deng et al.(2015) using different methods. However, little is known about the distributions and characteristics of the microsatellites, and more microsatellite loci are needed for further study. In the present study, candidate S. marmoratu s microsatellite markers were identified using 454 FLX pyrosequencing and were characterized in detail. The results will provide basic data for further development of microsatellite markers in this species.

2 MATERIAL AND METHOD 2.1 Sampling and experiment

One S. marmoratus was collected from South Izu, Japan in November 2011. Muscle tissue was snapfrozen in liquid nitrogen and stored at -80℃. Total genomic DNA was extracted using a standard phenolchloroform method with some modifications for muscle tissue, and the samples were treated with RNase. Multiple DNA isolates were pooled, precipitated with ethanol-sodium acetate, washed once with 70% ethanol, and resuspended in TE buffer. A 5-μg portion of this DNA was used in the fluorophotometric pyrosequencing system (454 Life Sciences, Branford, CT, USA) to prepare a library following the manufacturer's instructions. Raw sequence reads were assembled using Newler vers. 2.3-2.6 to reduce redundancy. The assembled contigs and unassembled singletons were used as candidate targets for screening.

2.2 Data analysis

Auto-primer written in Perl ver. 5.8.8 drives the Tandem Repeats Finder ver. 4.0.4 program (Benson, 1999) for screening microsatellites whose motif length is shorter than 6 nt (MaxPeriod=5). The details of the Auto-primer work flow were described previously (Nakamura et al., 2014). Reads with microsatellite loci were screened for flanking regions using high quality polymerase chain reaction (PCR) priming sites and were regarded as potentially amplifiable loci (PAL). Auto-primer sets the minimum flanking unique sequence length to 30 bp for further screening of microsatellite-containing sequences lacking a unique flanking sequence at one or both ends.

The Primer 3 ver. 2.2.2 beta program was employed to design the primer pairs using Perl script (Rozen and Skaletsky, 2000). Low complexity and simple repeat sequences were not permitted as primer sites because they are masked by the Repbase v14.01 database (Jurka et al., 2005). The primers in the Autoprimer sets were 18-27 bp long, and the expected amplicon length was 100-400 bp, including the primers themselves. All other parameters were set to the Primer 3 default values.

The sequence accuracies of the primer-annealing sites and amplified loci, as well as the mean quality scores of the amplicons including the primerannealing sites were calculated according to the description given by Nakamura et al.(2014). Primer pairs with the highest Primer 3 assigned score were used (Rozen and Skaletsky, 2000).

3 RESULT AND DISCUSSION

A total of 2 000 000 raw sequence reads were assembled (Newbler vers. 2.3-2.6) to reduce redundancy. Microsatellites consist of tandemly repeated motifs of a particular number, and the number of repeats in the dominant position differs among species, which may be a result of natural selection or differences in microsatellite mutation rates (Katti et al., 2001). A total of 1 043 reads contained dinucleotide repeats, 925 contained trinucleotide repeats, 692 contained tetranucleotide repeats, and 315 contained pentanucleotide repeats (Fig. 1). The number of dinucleotide repeats was the highest, which was similar to previous studies on other fish, such as Megalobrama amblycephala(Zeng et al., 2013) and Pseudosciaena crocea(Li, 2014). The frequency of repeats in most eukaryotes decreases exponentially with repeat length because mutation rates are higher in longer repeats (Katti et al., 2001). Chen et al.(2010) also reported that the number of repeats is inversely correlated with repeat length, and the present results confirmed this pattern.

Figure 1 Number of identified microsatellite repeat loci (black) and the number of these loci with suitable flanking polymerase chain reaction-primer sites (potentially amplifiable loci, PAL; gray)

We screened loci to determine the suitable flanking PCR primer sites and estimated the number of promising candidate loci for PCR amplification-based scoring of variations in microsatellite length. In total, 2 937 PAL were identified (Table 1). Among the repeat classes, dinucleotides repeats had the greatest numbers of loci and the most PAL (1 043 and 1 017 respectively), and pentanucleotides had the fewest loci and PAL (315 and 315, respectively). Dinucleotide repeat loci had the lowest percentage of PAL (97.51%), and pentanucleotides had the greatest percentage of PAL within the four classes of repeats (100%, Fig. 1).

Table 1 Number of microsatellite loci identified and the potentially amplifiable (containing suitable polymerase chain reaction priming sites) subsets

The relative abundances of specific repeat motifs were highly variable among the repeats (Fig. 2). The AC repeat was the most frequent among all three types of dinucleotide repeats, whereas AT was the least frequent (Fig. 2a). Moreover, AC was the most frequent repeat among all repeat classes. Earlier studies showed that 30%-67% of microsatellites are dinucleotides, and that (AC)n is the most common dinucleotide motif in the vertebrate genome including fishes (Tóth et al., 2000 ; Chistiakov et al., 2006). The results of the present study support this conclusion. Trinucleotide repeat motifs were dominated by AAT repeats, and ACG was the least frequent (Fig. 2b). AAAT, ATAG, and ATCC were the three most frequent tetranucleotide motifs, and 76% of all tetranucleotide motifs were less than 30 repeats (Fig. 2c). AAGAT and AATAT were the most frequent pentanucleotides (Fig. 2d).

Figure 2 Observed numbers of identified microsatellite loci (black), and the subsets containing polymerase chain reactionprimer sites (potentially amplifiable loci, PAL; grey) for the dinucleotide, trinucleotide, tetranucleotide, and pentanucleotide repeat sequence motifs

Fewer long repeats and many short repeats were detected among the four classes of repeats (Fig. 3). The results showed that smaller proportions of longer loci were potentially amplifiable (Fig. 3, Table 1). Many of the potentially amplifiable regions had much longer tails.

Figure 3 Counts of the number of perfect tandem repeat units per identified (black) and per potentially amplifiable (grey) microsatellite loci Counts of perfect tandem repeat units are given for dinucleotide (a), trinucleotide (b) tetranucleotide (c), and pentanucleotide repeats (d).

Microsatellites are powerful genetic markers that can be used in population genetics, linkage analysis, and evolutionary studies. Queiros et al. suggested that reliable and accurate estimates of genetic diversity can be obtained using random microsatellites distributed throughout the genome because selecting the most polymorphic markers will generally overestimate parameters of genetic diversity, leading to misinterpretations of the actual genetic diversity, which is particularly important for managed and threatened populations (Queirós et al., 2015). In this study, we provide a long list of candidate microsatellite markers for S. marmoratus that will enhance the range of markers for this species after amplification and testing in various populations. This is the first study to analyze the characteristics of S. marmoratus microsatellites using 454 FLX pyrosequencing, and the results will improve our options to accurately develop markers for future population and conservation genetics studies.

References
Barbará T, Palma-Silva C, Paggi G M, Bered F, Fay M F, Lexer C, 2007. Cross-species transfer of nuclear microsatellite markers: potential and limitations. Molecular Ecology, 16 (18) : 3759 –3767. Doi: 10.1111/mec.2007.16.issue-18
Benson G, 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Research, 27 (2) : 573 –580. Doi: 10.1093/nar/27.2.573
Castoe T A, Poole A W, Gu W J, de Koning A P J, Daza J M, Smith E N, Pollock D D, 2010. Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence. Molecular Ecology Resources, 10 (2) : 341 –347. Doi: 10.1111/men.2010.10.issue-2
Chen M, Tan Z Y, Zeng G M, Peng J, 2010. Comprehensive analysis of simple sequence repeats in pre-miRNAs. Molecular Biology and Evolution, 27 (10) : 2227 –2232. Doi: 10.1093/molbev/msq100
Chistiakov D A, Hellemans B, Volckaert F A M, 2006. Microsatellites and their genomic distribution, evolution, function and applications: a review with special reference to fish genetics. Aquaculture, 255 (1-4) : 1 –29. Doi: 10.1016/j.aquaculture.2005.11.031
Deng H W, Li Z B, Dai G, Yuan Y, Ning Y F, Shangguan J B, Huang Y S, 2015. Isolation of new polymorphic microsatellite markers from the marbled rockfish Sebastiscus marmoratus. Genetics and Molecular Research, 14 (1) : 758 –762. Doi: 10.4238/2015.January.30.19
Dong H B, Chen G, Zhang M H, Zhang J D, Zhou H, Tang B G, Huang J S, Shi G, Jiang L, 2008. Tissue-specificities of isozymes and genetic structure of Sebastiscus marmoratus. Journal of Guangdong Ocean University, 28 (4) : 15 –20.
Jurka J, Kapitonov V V, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J, 2005. Repbase update, a database of eukaryotic repetitive elements. Cytogenetic and Genome Research, 110 (1-4) : 462 –467. Doi: 10.1159/000084979
Katti M V, Ranjekar P K, Gupta V S, 2001. Differential distribution of simple sequence repeats in eukaryotic genome sequences. Molecular Biology and Evolution, 18 (7) : 1161 –1167. Doi: 10.1093/oxfordjournals.molbev.a003903
Kita J, Tsuchida S, Setoguma T, 1996. Temperature preference and tolerance, and oxygen consumption of the marbled rockfish, Sebastiscus marmoratus. Marine Biology, 125 (3) : 467 –471.
Li H M. 2014. New Microsatellite Satellite Markers Development Based on Whole Genome Sequencing Information and Its Application in Population Genetics in Large Yellow Croaker. Zhejiang Ocean University, Zhoushan, China. 62p. (in Chinese)
Li Q H, Li Z B, Dai G, Cao Y Y, Chen X J, Chen L N, Shangguan J B, Ning Y F, 2014. Isolation and characterization of eleven microsatellite loci in the marbled rockfish,Sebastiscusmarmoratus(Scorpaenidae). Conservation Genetics Resources, 6 (1) : 53 –55. Doi: 10.1007/s12686-013-0001-y
Li Q, 2004. Advances in studies on microsatellite markers in the Pacific abalone. Periodical of Ocean University of China, 34 (3) : 365 –370.
Liu H B, Liu S F, Ye J B, Yuan Y J, Ding S X, Zhuang Z M, 2014. Polymorphic microsatellite markers in the false kelpfish Sebastiscus marmoratus: isolation, characterization, and cross-species amplification. Genetics and Molecular Research, 13 (1) : 134 –138. Doi: 10.4238/2014.January.10.4
Nakamura M, Suzuki A, Hoshida H, Akada R. 2014. Minimum GC-Rich sequences for overlap extension PCR and primer annealing. In: Valla S, Lale R eds. DNA Cloning and Assembly Methods. Humana Press, New Jersey, USA.p.165-181.
Queirós J, Godinho R, Lopes S, Gortazar C, de la Fuente J, Alves P C, 2015. Effect of microsatellite selection on individual and population genetic inferences: an empirical study using cross-specific and species-specific amplifications. Molecular Ecology Resources, 15 (4) : 747 –760. Doi: 10.1111/1755-0998.12349
Rozen S, Skaletsky H. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Misener S, Krawetz S A eds. Bioinformatics Methods and Protocols:Methods in Molecular Biology. Humana Press, New Jersey, USA. p.365-386.
Su T F, Jiang S G, Zhang D C, Zhou F L, Huang J H, Zhu C Y, Yang L S, 2012. Genetic diversity of Sebastiscus marmoratus in eastern Guangdong coastal waters and its taxonomic status in Sebastidae. Progress in Fishery Sciences, 33 (2) : 1 –8.
Tóth G, Gáspári Z, Jurka J, 2000. Microsatellites in different eukaryotic genomes: survey and analysis. Genome Research, 10 (7) : 967 –981. Doi: 10.1101/gr.10.7.967
Xu T J, Quan X Q, Sun Y N, Zhao K C, Wang R X, 2010. A first set of polymorphic microsatellite loci from the marbled rockfish, Sebastiscus marmoratus. Biochemical Genetics, 48 (7-8) : 680 –683. Doi: 10.1007/s10528-010-9349-9
Yin L N, Zhang H, Yanagimoto T, Gao T X, 2012. Isolation and characterization of nine polymorphic microsatellite markers of the marbled rockfish Sebastiscus marmoratus(Scorpaeniformes, Scorpaenidae). Russian Journal of Genetics, 48 (12) : 1264 –1266. Doi: 10.1134/S1022795412120174
Zane L, Bargelloni L, Patarnello T, 2002. Strategies for microsatellite isolation: a review. Molecular Ecology, 11 (1) : 1 –16. Doi: 10.1046/j.0962-1083.2001.01418.x
Zeng C, Gao Z X, Luo W, Liu X L, Wang W M, 2013. Characteristics of microsatellites in blunt snout bream(Megalobrama amblycephala) EST sequences using 454 FLX. Acta Hydrobiologica Sinica, 37 (5) : 982 –988.
Zhang D X, Hewitt G M, 2003. Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Molecular Ecology, 12 (3) : 563 –584. Doi: 10.1046/j.1365-294X.2003.01773.x
Zhang H. 2013. Molecular Phylogeography of Two Marine Ovoviviparous Fishes in Northwestern Pacific. Ocean University of China, Qingdao, China. 203p. (in Chinese)