Development of 30 SNP Markers for the Daphnia Magna based on Restriction Site-associated DNA Sequencing (RAD-seq)

: Daphnia magna belongs to the Cladocera and plays an important role in the water ecosystem. With the intensification of water pollution, the wild population of D. magna has declined rapidly in recent years, and insufficient molecular markers have limited effective research and conservation of this species. In this research, 30 novel single nucleotide polymorphism (SNP) markers were developed in a cultivar of Daphnia magna and 12 wild Daphnia magna using restriction site - associated DNA sequencing (RAD - seq). The minor allele frequency, observed heterozygosity, and expected heterozygosity ranged from 0.115 to 0.721, 0.073 to 0.800, and 0.077 to 0.520, respectively. The PIC ranged from 0.071 to 0.403. Six loci showed significant deviations from the Hardy - Weinberg equilibrium after Bonferroni correction (p < 0.05).These newly developed polymorphic SNP markers for D. magna are of great significance in terms of the genetic breeding of D. magna , identification of wild and artificially domesticated species and conservation genetics research.

Daphnia magna belongs to the Cladocera and is an important species of zooplankton (Jeong and Simpson 2019). It mainly lives in freshwater. Daphnia magna helps to increase the diversity of phytoplankton in the water body and build a stable phytoplankton community structure, as well preventing the occurrence of cyanobacteria blooms (Sarnelle 2007). It is the key to balance in the water ecosystem.
In China, the artificially domesticated Daphnia magna has been used to inhibit Cyanobacteria blooms and restore submerged vegetation, which has been used in more than 400 ecological restoration projects (Huo et al. 2010;Peng et al. 2011). In addition, it is widely used in water pollution monitoring and aquatic organism toxicological research (Dietrich et al. 2010;Fan et al. 2011). However, in recent years, with the rapid expansion of the population and increases in the discharge of domestic sewage, the aquatic ecosystem has been severely damaged; relevant research shows that 54% of Asian, 53% of European, 48% of North American, 41% of South American, and 28% of African rivers are eutrophic water bodies, and eutrophication of water bodies is currently the most serious problem facing rivers and lakes (Bagousse et al. 2012), as a result, the habitat of D. magna has been severely damaged, and its genetic diversity is also declining sharply. To date, a comprehensive study of D.
magna population genetic variations is still lacking, and very little information is available on the genome and effective molecular markers of D. magna. This hinders correlational studies on this species. Thus, it is very important to investigate the genetic diversity of this species for scientific management and sustainable utilization in the future.
Genetic methods based on DNA markers are extremely efficient at revealing population distributions and the laws of evolution (Hao et al. 2017;Chen et al. 2018; technologies, the differences and dynamics of populations can be understood. The disclosure of genetic diversity and genetic structure is also the basis of species genetic management (Manel et al. 2003;Schwartz et al. 2007). Because single nucleotide polymorphism (SNPs) are codominant, dimorphic, allelic, and widely distributed (Vignal et al. 2002;Céline et al. 2018;Honorio et al. 2019), they have become among the most advanced DNA markers. Nowadays, SNP have been widely used in genetic structure analysis, cultivar identification, genetic diversity evaluation, construction of high-density genetic linkage maps, and molecular-assisted breeding (Gupta et al. 2008;Pujolar et al. 2013;Vasemagi and Primmer 2005).
Restriction site-associated DNA sequencing (RAD-seq) is an important sequencing technology for simplifying the genome. It has a high number of markers and high density, and can be applied in many fields. For research subjects without a reference genome, RAD-seq technology is the best choice for simplifying genome establishment (Miller et al. 2007). This technology has been widely used in research in the fields of population genetics, genetic map construction, and systematic evolution ). Here, we adopted restriction site-associated DNA (RAD-seq) technology (Miller et al. 2007)     To our knowledge, this is the first time SNP markers were developed for D.
magna, thus, filling the gap in the field. The application of RAD-seq technology is continuously expanding, and it has been widely used for gene mapping (Guo et al. 2015), high-precision genetic map construction ) and assessments of genetic diversity (Pootakham et al. 2011). Our study identified 30 polymorphic SNP markers using RAD-seq, which can efficiently and accurately identify the D.  Figure 1 The map of the sampling locations of the 13 populations of Daphnia magna in the present study. Note:

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