2 Fisheries College, Jimei University, Xiamen 361021, China
The Manila clam, Ruditapes philippinarum (Adams et Reeve, 1850), is an economically important bivalve mollusk. Except for the vitally edible value, the species also plays the role as a model organism to coastal pollution (Hégaret et al., 2007; Cong et al., 2018). The traits of eurythermic and euryhaline make the species widely distributed in the west coast of the Pacific from Russia to the Philippines, especially along the estuaries and near coastal waters in China (Wu et al., 2011; FAO, 2014). However, in recent years, with the rapid development of the economy, the clam resources and germplasm are declining resulted from beach reutilization, overfishing, environment pollution, and destruction, etc. Additionally, the two to three weeks of the planktonic larval stage prompts its larvae to disperse to new habitats, even settle down, and breed (Yasuda et al., 2007). If so, the population structure may make a difference over time.
Highly polymorphic genetic markers can provide an effective way to reveal the population structure and promote heredity development (Yasuda et al., 2007). Up to now, microsatellite marker, i.e. simple sequence repeat (SSR), is one more popular molecular marker technology for genetic research, because of its abundance, co-dominance, highly allelic variability and wide dispersal throughout the genome (Chistiakov et al., 2006). Furtherly, there were generally two types of SSRs—genomic SSR and genic SSR, in terms of their correspondingly original sequences (Ellis and Burke, 2007). Genomic SSRs are not only obtained from genomic high-throughput sequencing but also from SSRs library construction by magnetic beads enrichment, which perhaps had certain inefficiency and limitations with different biotin probes. Nevertheless, it is reported that genic SSRs are more conservative and have a relatively higher transferability in related or cryptic species than genomic SSRs (Varshney et al., 2005a; Yamini et al., 2013). In other words, genomic SSRs are more polymorphic than genic SSRs (Ellis and Burke, 2007; Kong et al., 2014). In aquaculture, some reports about SSRs in the field of genetic research are reported, for example, pearl mussel Hyriopsis cumingii (Bai et al., 2013), ridge-tail white prawn Exopalaemon carinicauda (Li et al., 2015), schilbid catfish Silonia silondia (Mandal et al., 2016) and pen shell Atrina pectinata (Sun et al., 2017), etc. For R. philippinarum, there have been many SSR loci obtained from expressed sequence tag (EST) sequences (Nie et al., 2014, 2015; Yan et al., 2015; Zhu et al., 2015). In any event, the high-throughput sequencing has made enormous contributions to important organisms, for example, non-model or rare species in terms of population genetic information.
In this study, high-throughput sequencing was carried out on the Illumina platform to obtain the transcriptome of R. philippinarum, and then a de novo assembly was conducted to assemble the short clean reads. The characterization of all genic SSRs was analyzed from all perspectives including SSR types and distribution percentages. Finally, we screened randomly 34 polymorphic and 3 monomorphic SSR loci. The results will promote conservation and breeding studies of the clam species.2 MATERIAL AND METHOD 2.1 Specimen preparation
Healthy and adult samples used for cDNA library construction were purchased from the aquatic wholesale market (Gaoqi, Xiamen, China) in April 2017. The gill tissues for RNA extraction were dissected and quick-frozen in liquid nitrogen. In addition, 32 wild and living individuals from coastal waters (Jimei, Xiamen, China) in November 2017 were used to detect subsequent population genetics and polymorphism. All the fresh samples were then stored at -80℃.2.2 cDNA library construction and SSR-enriched sequences obtainment
Total RNA was extracted using TRIzol reagent (Life Technologies, CA, USA) according to the Product Manual. The cDNA library construction and RNA-Seq were finished by Gene Denovo Biotechnology Co. (Guangzhou, China) with the platform Illumina HiSeqTM 4000. Clean reads were achieved by removing low-quality sequences from raw data: (1) reads containing adapters; (2) reads containing more than 10% of unknown nucleotides (N); and (3) reads containing more than 50% of low quality (Q value ≤10) bases. Next, all unigenes were obtained by de novo assembling clean reads with the Trinity program (Grabherr et al., 2011). Microsatellites were searched and located among all the unigenes of transcriptome with the software microsatellite identification tool (MISA, http://pgrc.ipk-gatersleben.de/misa/), and the parameter settings were as follows: (1) definition (unit size, min repeats): 2-6 3-5 4-4 5-4 6-4; (2) interruptions (max difference between 2 SSRs): 100 bp. Meanwhile, the statistical analysis of SSR category and corresponding characterization were performed.2.3 Specific primers of SSR loci screening and polymorphism detection
Two hundred and thirty-four SSR sequences were randomly selected and 100 primers designed with Primer Premier 5.0.32. Genomic DNA of 32 wild individuals was extracted using the cell/tissue genomic DNA extraction kit (GENErayTM Biotechnology, Shanghai, China). The DNA pool with 10 high-quality samples were applied to test the primers' specificity and the optimum annealing temperature (Ta) with the gradient polymerase chain reaction (PCR) process: 1 cycle (95℃ predegeneration for 5 min); 30 cycles (45 s at 94℃ degeneration, 30 s at 60–40℃ gradient annealing, 45 s at 72℃ extending); 1 cycle (72℃ extending for 10 min). Moreover, the PCR amplification was realized by a volume of 10-μL reaction system with 2*Taq Master Mix (Novoprotein Scientific, Shanghai, China).
Next, the polymorphism of the above specific primers were examined using a set of 32 DNA samples in touchdown-PCR method as follows: 1 cycle (95℃ for 5 min); 15 cycles (94℃ for 45 s; from Ta+15℃, -1℃/cycle for 30 s; 72℃ for 45 s); 20 cycles (94℃ for 45 s; 30 s at Ta; 72℃ for 45 s); 1 cycle (72℃ for 10 min). Finally, the 32 PCR amplifications of every specific primer were separated and detected by 6% polyacrylamide gel electrophoresis on the Sequi-Gen Sequencing Cell (Bio-Rad, USA), followed by silver staining.2.4 Genetic date statistics
The Micro-Checker was used to inspect null alleles (van Oosterhout et al., 2004; Wen et al., 2013). Some population genetic information and index (PopGene 32, Version 1.32) (Yeh et al., 2000) were used to calculate the Hardy-Weinberg equilibrium (P-HWE), linkage disequilibrium, the number of alleles per locus (Na), observed heterozygosities (Ho) and expected heterozygosities (He). Cervus 3.0.3 (Marshall et al., 1998) was used to estimate the polymorphism information content (PIC).3 RESULT 3.1 Characterization of SSR-enriched sequences from transcriptome
After sequencing and de novo assembly, 106 831 unigenes and 58 273 004 base-pairs were obtained. Among them 2 428 sequences containing 2 664 SSRs identified as shown in Table 1. The results show that there is 1 microsatellite locus in every ~40 sequences or every ~21 874 bp. According to the different repeat units, these 2 664 SSRs can be categorized into dinucleotides (864, 32.43%), tri-nucleotides (1 236, 46.40%), tetra-nucleotides (521, 19.56%), pentanucleotides (40, 1.50%), and hexa-nucleotides (3, 0.11%) (Fig. 1). Apparently, the repeat units (≤ tetranucleotides) were the main body, and tri-nucleotides were top, followed by di-nucleotides and tetranucleotides. Oppositely, only 1.61% was the pentaand hexa-nucleotides. Additionally, AT/AT (19.14%) was the most common in di-nucleotides, then AC/GT (11.26%), and the least CG/CG (0.04%). Moreover, among all the repeat units, AAC/GTT was dominant for taking 19.82%. All the frequency of classified repeat types was listed in the order in Appendix 1. The repeat numbers of different types of SSRs also present some interesting differences, for example, the repeat times of di-nucleotides were mainly 6–11 (Table 2), but penta-nucleotides were only 4–6. Obviously, among the number 6–10, the SSRs percentages progressively decreased with the increasing base number of the repeat unit (e.g. di-, tri-, tetra-, penta-, hexa-) at the same of repeat times. Meanwhile, every type of SSR possessing a minimum of repeat times frequently had the highest proportion at the same repeat unit, such as (di-)6, 495; (tri-)5, 859; (tetra-)4, 363.3.2 Isolation and analysis of polymorphic SSR loci
After the specific detection of 100 primers using the gradient PCR, 60 primers with target genes were first obtained and then tested for polymorphism of the wild 32 R. philippinarum genomic DNA. Of them, 34 polymorphic SSR loci had 112 alleles (average 3.29/ locus) and ranged from 2 to 7 (Na). In addition, three monomorphic SSR loci were also amplified (e.g. MG871457, MG871458, MG871459). All the genetic information was listed in Table 3, and the above 37 SSR loci had GenBank accession No. assigned (MG871423–MG871459). The Ho and He were 0.100 0–1.000 0 (mean=0.348 8) and 0.191 3–0.723 6 (mean=0.429 0), respectively. The PIC value was from 0.183 to 0.720, including four low polymorphic loci (PIC < 0.25), 14 moderate polymorphic loci (0.25 < PIC < 0.5), and 16 high polymorphic loci (PIC > 0.5). In other words, the polymorphism of 34 SSR loci was at a higher level. At the same time, apart from five SSR polymorphic loci (e.g. MG871423, MG871428, MG871429, MG871434, MG871435), all the other 29 polymorphic loci conformed to the Hardy-Weinberg equilibrium after the Bonferroni correction (adjusted P=0.001 471). In the meantime, no genotypic linkage disequilibrium or genotyping error (null allele) was observed.4 DISCUSSION
As shown in this study, the partition ratio of SSRs (1 SSR locus / ~40 sequences) was far below those of Sepiella japonica (1 SSR locus / 2 sequences) (Lü et al., 2017) and Atrina pectinata (1 SSR locus / 5 sequences) (Sun et al., 2017), which might be resulted from the ignorance of the mononucleotide repeats in the study. However, the percentages for the other SSRs types were similar to the corresponding ones. In other words, the abundance of SSR loci distribution is not only concerned with SSR search criteria but also affected by the database size and SSR loci development approaches (Varshney et al., 2005b; Parchman et al., 2010). In this research, tri-nucleotides (1 236, 46.40%) was the dominant part of all SSR repeat categories, followed by di-nucleotides (864, 32.43%). The results are consistent with the reports that trinucleotides primarily occur in exons, while di-, tetra-, and penta-nucleotide repeats occur mainly within the untranslated regions (UTRs) (Qiu et al., 2010). And, the database in the study is also mRNA transcriptome with the main exons. It may be that the cytosine (C) is methylated easily and turned into thymine (T) via deamination (Schorderet and Gartler, 1992) so that the AT part was top in di-nucleotides. Sun et al. (2017) reported that AC/GT (29.35%) is the highest, followed by AT/AT (25.41%). Therefore, different animals employ different SSRs patterns of mutability in genomic evolution (Chistiakov et al., 2006). Slightly differences were found in the genomes of Lottia gigantean and C. gigas, in which di-nucleotides or hexa-nucleotides present the majorities (Jiang et al., 2014). Altogether, we can conclude that the number of SSRs, representing genetic diversity and variability, may be in a downtrend with the increasing repeat unit or times of repetition.
Of the 34 polymorphic SSR loci, the average alleles (3.29/locus) were lower than that of R. philippinarumof 5.97 (Zhu et al., 2015), or 7.76 (Nie et al., 2014). Although in the same species, the different proportion of SSR types and repeat times may affect the polymorphism (Bouck and Vision, 2007). Zhu et al. (2015) reported that di-nucleotide repeats were the most abundant type (71.05%) of 38 EST-SSRs, but in the present 34 SSRs, tri-nucleotide repeats 19 (55.88%) were top, followed by di- 7 (23.53%) and tetra- 7 (23.53%). In the population diversity detection, Ho (mean=0.348 8) was lower than He (mean=0.429 0). We infer that as inbreeding has become more common, the homozygote is higher than heterozygote in the Xiamen's population. However, this view needs sufficient wild samples for further verification. Additionally, the PIC value of 30 (88.24%) SSR loci was at the medium-to-high level (PIC > 0.25), showing that the development efficiency of EST-derived SSR is optimistic and feasible and can avoid the triviality in traditional methods.5 CONCLUSION
This study can prove that the method of SSRs derived from transcriptome is not only much more effective than the traditional ones but also able to create a general and comprehensive landscape for R. philippinarum SSRs. Perhaps, those SSRs that usually from a traditional approach represent some aspects only owing to the differences in probe type or the efficiency of SSRs enrichment, and so on. In addition, the 37 SSR loci, particularly 34 polymorphic loci, will contribute to the genetic and phylogenetic analysis of R. philippinarum, even to the shellfish and mollusks, for example, cross-species amplification, genetic breeding, and population conservation.6 DATA AVAILABILITY STATEMENT
The datasets and materials supporting the conclusions of this article are included in the article.
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