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Short Communication · Volume 50, Issue 4, 9 July 2018 · Open Access

Development and Characterization of Twenty-Three Novel Polymorphic Microsatellite Markers for Mussel, Mytilus coruscus

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Affiliations & Notes ⌄
  1. 1National Engineering Research Center For Marine Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, No.1 Haida South Road, Changzhi Island, Zhoushan, Zhejiang, 316022, P.R. China , * Corresponding author: yeyingying5559@163.com
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
9 July 2018
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From the article

  • The characteristics of these loci were presented in Supplementary Table I. The number of alleles per locus ranging from 6 (MC18) to 21 (MC54, MC57 and MC99) with an average of 12.913, and the alleles size (bp) varied between 161 and 470 bp.
  • Five loci (MC22, MC27, MC43, MC49 and MC101) were deviated from Hardy-Weinberg equilibrium significantly (P 0.05).
  • The presence of null alleles is part of the main reason due to the five loci deviated from HWE.
  • These new 23 microsatellite loci showed a medium genetic diversity (based on the data of alleles number per locus (Na), observed and expected heterozygosity (Ho and He), HWE and PIC), and suitability for studies of potential value.

Selected from the article's own text.

Abstract

Mytilus coruscus is one of the main mussel species in China, with a widely range of aquaculture markets and importantly economic value. In this study, we used one hundred and twenty primers pairs to screen all microsatellite loci and twenty-three polymorphic loci were identified. The number of alleles per loci ranged from six to twenty-one with an average of 12.913 and the allele size varied between 161 and 470 bp. The observed and expected heterozygosity varied from 0.143 to 1.000 and from 0.351 to 0.926, with average values of 0.614 and 0.771, respectively. Eighteen loci accorded with the Hardy-Weinberg Equilibrium (HWE)(P > 0.05). The polymorphism information content (PIC) value ranged from 0.340 to 0.921 with an average of 0.751. These markers will help the further studies, provide more useful tools to choose for researching genetic diversity and population structure and provide more in-depth understanding of Mytilus coruscus.

Materials and methods

In this study, 35 specimens of M. coruscus were collected in Taishan Island, Fujian Province, China. The adductor muscle was taken out and preserved in 100% alcohol until DNA extraction using improved salting out method . The DNA was diluted to a final concentration of 50 ng/ul and stored at -20°C for further analysis.

The transcription data were used for microsatellite searching and primer designing with Illumina sequencing technology. A total of 21,723,913 paired-end clean reads (NCBI SRA database SRX 1411195) were generated from HiSeq 2000 sequencer . Online WebSat (http://wsmartins.net/websat/) was utilized to design primers based on the reads containing putative microsatellite regions.

For detection of microsatellite markers, each pair of primer was tested on 35 specimens of M. coruscus, PCR was carried out in volumes of 20µl, containing 15-40 ng of template DNA, 4 pmol of each forward and reverse primer, 10µl CW0716 2×Taq MasterMix (Cwbiotech., Peking, China). PCR amplification was performed using Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA, USA). The reaction process was as follow, after initial denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 30s, 55°C-61°C for 30s (each pair of primer was screened in gradient temperature), 72°C for 40s, and final elongation at 72°C for 10 min. In order to evaluate the amplifications, the PCR products were tested on an 8% non-denaturing polyacrylamide gel electrophoresis. Then, the genotypic of PCR products by capillary electrophoresis were performed using the BiopticQsep100 DNA Analyzer (Hangzhou Houze Biotech Company, Zhejiang, China).

For the successful primers, the number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He) and P-value of Hardy-Weinberg Equilibrium (HWE) test were obtained by the program GenAlEx 6.501 . Polymorphism information content (PIC) was calculated using the PIC_CALC 0.6. Micro-checker 2.2.3 was used to analyze the null alleles.

Results and discussion

Among 120 polynucleotide-repeat microsatellites in M. coruscus, 23 polymorphic loci were successfully amplified from the specimens of M. coruscus. The characteristics of these loci were presented in Supplementary Table I.

The number of alleles per locus ranging from 6 (MC18) to 21 (MC54, MC57 and MC99) with an average of 12.913, and the alleles size (bp) varied between 161 and 470 bp. The observed and expected heterozygosity varied from 0.143 (MC43) to 1.000 (MC31, MC63 and MC76) and from 0.351 (MC44) to 0.926 (MC101), with average values of 0.614 and 0.771, respectively. Five loci (MC22, MC27, MC43, MC49 and MC101) were deviated from Hardy-Weinberg equilibrium significantly (P<0.01). The polymorphism information content (PIC) value ranged from 0.340 (MC44) to 0.921 (MC101) and the average was 0.751.

In this study, we successfully found 18 different microsatellite markers which conformed to the HWE (P > 0.05). However, some loci tested could not attain the predicted effect (i.e., MC22, MC27, MC43, MC49 and MC101). The Wahlund effect, genotypic errors, groupings of relatives and the presences of null alleles could be the reasons for these loci deviations from HWE. It could be because these loci did not exisit in the subset of 35 individuals, or the PCR was influenced by some unknown causes or it may be because of the small sample size. So, MICRO-CHECKER 2.2.3 software was used to test these loci, and null alleles were detected from these five loci. The null alleles perhaps appear variations in SSR flanking sequence, loss of large fragments alleles or differences in the quality requirements of DNA with special loci. The presence of null alleles is part of the main reason due to the five loci deviated from HWE.

Eventually, 23 out of 120 primers pairs were selected as novel polymorphic microsatellite markers. These loci sequences were submitted to the GenBank (GenBank accession numbers: KX423595-KX423617, Supplementary Table I). These new 23 microsatellite loci showed a medium genetic diversity (based on the data of alleles number per locus (Na), observed and expected heterozygosity (Ho and He), HWE and PIC), and suitability for studies of potential value. In addition, these markers will help the further studies, serve as more useful tools for researching genetic diversity and population structure and more in-depth understanding of M. coruscus.

Acknowledgments

This study was financially supported by the Open Foundation from Fishery Sciences in the First-Class Subjects of Zhejiang (No.20160002); International Science & Technology Cooperation Program of China (No. 2015DFR30450); Public science and technology research funds projects of ocean (No. 201505025) and Public welfare project in Zhejiang Province (No. 2017C32009).

Supplementary material

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.sc3

Statement of conflict of interest

Authors have declared no conflict of interest.

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Development and Characterization of Twenty-Three Novel Polymorphic Microsatellite Markers for Mussel, Mytilus coruscus · ResearchersLinks