Utilizing Next-Generation Sequencing to Develop and Characterize Microsatellite Loci in Cuttlefish (Sepia pharaonis) and Cross-Amplification in Other Sepiidaes

A total of 32 specimens of S. pharaonis were collected from Cangnan sea area (Fujian Province, China). Muscle tissues of S. pharaonis were obtained from each individual, preserved in 95% ethanol and stored at -20°C before DNA extraction. Total DNA was extracted from muscles using standard phenol–chloroform procedures (Sambrook et al., 1989).

An illumina-based RNA-Seq approach was used to characterize the novel microsatellite loci for S. pharaonis collected from the Cangnan Sea area in the The Beijing Genomics Institute (BGI, Shenzhen, China). Transcriptome contigs were obtained and screened for microsatellites using MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html). Primers were designed for microsatellite loci using the program Primer3.0 (http://www.onlinedown.net/soft/51549.htm).

For SSR marker validation and population genetic analysis sixty primer pairs were arbitrarily chosen, synthesized and used to test for polymorphisms in 10 individuals. The PCR amplification was performed in a 2720 PCR machine (ABI, USA) and in a reaction mixture (10 µL) containing 2-10 ng DNA (0.5 µL), 0.5 µL of each forward and reverse primers, 5 µL 2×Es Taq MasterMix and 3.5 µL of double distilled water. PCR was performed as follows: 5 min at 95°C, 30-35 cycles of 30 s at 95 °C, 30 s at 55–61 °C, 40 s at 72 °C, and 10 min at 72 °C. PCR products were detected using capillary electrophoresis (BIOptic’s Qsep100 dna-CE, Taiwan), and allele size was estimated using Q-Analyzer Software.Primers that amplified reproducible and score-able peaks of the expected size were further characterized using 32 wild-caught S. pharaonis individuals. The PCR products were genotyped using the method mentioned above.

To verify the transferability of the developed microsatellite loci from S. pharaonis three closely species of the sepiidae, we tested cross-amplification on the following three sepiidaes: Sepia lycidas, Sepia esculenta, and Sepiella japonica, with 10 individuals from each species.

The number of alleles (NA), observed (HO) heterozygosity and expected (HE) heterozygosity were calculated using ARLEQUIN ver. 3.5.1.3 (Excoffier and Lischer, 2010). The polymorphic information content (PIC) was calculatedaccording to Botstein (1980). GENEPOP ver. 4.0.10 was used to examine conformation to Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium between all pairs of loci (Raymond and Rousset, 1995). Significance values were adjusted for multiple comparisons using Bonferroni corrections where necessary (Rice, 1989). Finally, all loci were assessed using MICRO-CHECKER to check for null alleles andscoring errors (Van-Oosterhout et al., 2004).

In this study, we obtained approximately 98.12 nt bases from S.pharaonis the Illumina Hiseq 2000 platform. Morethan 56228 microsatellite loci with at least 4 repeats of mono-nucleotide to hexa-nucleotide motifs were detected (Table I). Among these microsatellites, mono-nucleotide motifs were the most frequent (51.65%), followed by di- (30.51%) and tri-nucleotides (15.56%). Quad-, penta- and hexa-nucleotide SSRs had a much lower frequency (1.98%, 0.22% and 0.08%, respectively) (Table I).

Of 56228 microsatellite loci, we randomly selected 60 microsatellite loci with polynucleotide-repeat types to test primer pairs. Out of 60 primers pairs examined, 21 microsatellite loci appeared to be polymorphic in the population of S. pharaonis. The characteristics of these loci are shown in Table II. The number of alleles per loci ranged from 2 to 9, with an average of 4.81. Observed heterozygosity ranged from 0.000 to 0.531 and expected heterozygosity from 0.031 to 0.751, with an average of 0.200 and 0.422, respectively. Five loci (CL1142, CL1770, CL2683, CL3494, CL3674) significantly departed from Hardy-Weinberg equilibrium after Bonferroni correction (P<0.05), perhaps because of population stratification, genotyping errors, or other confounding factors (Zintzaras and Lau, 2008). The polymorphic information content (PIC) values ranged from 0.030 to 0.717. Of these 21 loci, six loci were highly informative (PIC > 0.5), nine showed as mildly informative (0.25 < PIC < 0.5), and six were lowly informative (PIC < 0.25) (Table II). No significant evidence for null alleles or linkage disequilibrium was detected (P>0.05). The polymorphism rate of polynucleotide SSRs (35%) developed for S. pharaonis in this study was similar to that in P. bengalensis (34.3%; Eo et al., 2016), Vriesea simplex (33.3%; Neri et al., 2015) but higher than that in Labeo rohita (12.2%; Chhotaray et al., 2015) and Artemia parthenogenetica (11.5%; Nougué et al., 2015). This finding indicates that the polynucleotide-repeat microsatellites may also be powerful tools to study population structure and genetic diversity of S.pharaonis.

To examine these polymorphic microsatellite markers developed in S.pharaonis for utility with other species, cross-amplification of these microsatellite loci was tested on three other species (S .lycidas, S. esculenta and S. japonica) (Table III). The results showed that all 21 loci except CL9851 and UN11117 were effectively amplified, and 10 of 19 loci showed high polymorphisms in S. lycidas, indicating a higher transferability of these microsatellite markers in S. lycidas (Table III). Fifteen loci amplified and 4 of 15 loci showed polymorphisms in S. esculenta. Thirteen of 21 loci were cross-amplified in S. japonica, but all were monomorphic. As expected, cross-amplification levers were higher in S. lycidas and S. esculenta than

in S. japonica due to a closer phylogenetic relationship between these three species.

To the best of our knowledge, our study is the first to report the isolation of microsatellite markers in S. pharaonis using high-throughput sequencing technology and to test the cross-amplification in related species: S. lycidas, S. esculenta and S. japonica. These microsatellite loci will be powerful tools to study population structure and genetic diversity, which may provide new information to guide its conservation and management strategies for S. pharaonis. More importantly, most of them showed good applicability in three closely related species. The results indicated that the five loci had good transferability at the genus level.

This work was financially supported by Chinese National Natural Science Foundation (41406138), Natural Science Foundation of Zhejiang Province ( LY130190001), the International Science and Technology Cooperation Program of China (No. 2014DFT30120).

+, amplified; –, no amplification; Numbers of alleles are indicated in brackets.

Authors have declared no conflict of interest.