Analysis of genetic characteristics of D. magna population by SSR markers
D. magna is a small planktonic cladoceran. D. magna has been used as a model species for ecotoxicology, as it is sensitive to environmental stressors and environmental changes [29] (Lee et al. 2019). At present, research on D. magna mainly focuses on reproduction ecology, toxic and toxicological effects [30], and water pollution purification [31–32]. Lee et al. [29] assembled and characterized the genome of D. magna, which has greatly helped the molecular research of D. magna. Robinson et al. [33] used statistics calculated from multilocus microsatellite datasets to estimate population ages in data generated through coalescent simulations and in samples from populations of known age in a metapopulation of D. magna in Finland. Our research uses SSR markers to identificate of wild and artificially domesticated species.
In our study, 26 polymorphic SSR loci were developed using RAD-seq technology, and genetic diversity analysis was performed on 12 wild D. magna populations and one artificially selected D. magna population in China. The average number of alleles for the 26 pairs of SSR primers is 4.654. With an average of more than four alleles, one can better assess the genetic diversity of a population [34], so the developed microsatellite markers show good polymorphism and can provide help in further molecular research on D. magna. Diversity analysis of D. magna populations in the 13 regions shows that the mean values of Nei’s genetic identity and Shannon’s Information index are 0.2233 and 0.3073, respectively, indicating that the genetic marker diversity of D. magna is relatively high. The genetic diversity of D. magna is relatively high in Shanxi and Guangdong and is the lowest in Jiangsu. In addition, it also showed good genetic diversity in artificially domesticated D. magna (DZ). Genetic distance and Nei’s genetic identity analysis, NJ cluster analysis, PCoA analysis, and STRUCTURE genetic structure analysis were conducted on germplasm samples from D. magna in different regions; the results were basically consistent and complementary. The research results showed that the genetic diversity of D. magna from different sources was quite different. The genetic relationship between Anhui and Hunan’s D. magna germplasm is the closest, and the genetic relationship between Anhui and Jiangsu is the furthest. NJ cluster analysis and genetic distance analysis gave the same results. PCoA analysis results showed that D. magna in Anhui, Hunan, Jining, Liaoning, Taiheshui, Yantai, and Yunnan can be clustered into one group. The differences in D. magna in Beijing and Hainan are also small, while D. magna in Guangdong, Sichuan, and Jiangsu are quite different from other regions, and the clustering results have no correlation with geographic origin. The STRUCTURE genetic structure plot divides D. magna from the 13 regions into two groups. The genetic backgrounds of D. magna in Anhui, Hunan, Jining, Liaoning, Shanxi, Yunnan, Yantai, and Taihe Water are relatively simple, and the genetic structure of D. magna in Beijing, Hainan, and Jiangsu is also very simple but significantly different from the previous group, while the genes of D. magna in Guangdong and Sichuan are shared with the two groups.
Genetic differentiation of D. magna population
From the sampling site distribution and clustering results, there is no absolute correlation between the gene distribution of D. magna and its geographic origin, but on the whole, there are still large differences in the genetic structure of D. magna in the north and south of China, which may be a result of differences in temperature, climate, precipitation, and water environment. However, there are exceptions; the genetic distance between D. magna in Beijing and Hainan is relatively small, which may be due to similarities in the water environment causing D. magna in the two places to evolve in a similar direction. Human interference is an extremely significant activity affecting gene exchange in species, with the high similarity of D. magna genes in different regions likely to be the result of artificial introduction. Meanwhile, zooplankton have weak swimming ability and are highly susceptible to factors such as water level fluctuations, tidal currents, and water erosion [35]. Ingestion by large aquatic animals such as fish, shrimps, and crabs will also accelerate gene exchange between D. magna from different regions. However, based on our research, the genetic structure of D. magna in different regions is quite different. Human construction activities destroy the habitats of D. magna, which fragment the habitat and spread populations, resulting in low genetic similarity between D. magna in different regions. In addition to its weak swimming ability, D. magna mainly reproduces asexually, which inevitably affects gene exchange between populations due to long-term geographic isolation, which may lead to obvious genetic differentiation between populations, increase in genetic distance, and decrease in population size, leading to population selfing and genetic drift, which ultimately leads to a decrease in genetic diversity. Therefore, we should raise awareness of environmental protection, protect the safety and integrity of the water environment, and also cultivate and introduce D. magna into different regions and rivers in order to increase gene flow between populations, prevent the aggravation of genetic differentiation, and increase genetic diversity. A high level of genetic diversity can provide a source of genes to optimize the germplasm resources of D. magna. As a natural open bait for high-level aquatic animals such as fish and shrimps, high-quality D. magna germplasm resources can improve the quality of aquatic products, shorten the breeding cycle of aquatic products, and improve the economic benefits of aquaculture [36]. In addition, artificial domestication of D. magna (Taihe Water sampling site) can quickly remove algae and other organic particles, increasing the transparency of the water body by 1.0-1.5 m in a short time, so that submerged plants can be transplanted and survive. Submerged plants quickly reduce the content of nutrients such as nitrogen and phosphorus in the water body through nutrient competition, and inhibit the growth and reproduction of blooms and algae[4], playing a major role in the restoration of water ecosystems. Therefore, studying the genetic structure of D. magna in different regions can not only protect its genetic diversity at a molecular level, but it can also enrich the D. magna gene bank and increase our understanding of its evolutionary potential.
The life history of D. magna is unique; it consists of parthenogenetic generation and bisexual reproduction, and these two reproductive methods can alternate with changes in the external environment [37]. When the external environment is suitable, such as suitable temperature, sufficient food, and sufficient biological space, D. magna reproduces mainly by parthenogenesis, and the egg cells produced by females can directly develop into female offspring without fertilization. This is the most important way for it to reproduce. Once external conditions deteriorate, such as lack of food, high or low pH, the amount of dissolved oxygen in the water drops sharply, metabolic wastes accumulate, population density increases, or the light cycle shortens, the reproductive mode of D. magna will change from asexual to bisexual. When environmental conditions improve again, the reproductive mode of D. magna will switch from asexual reproduction to bisexual reproduction [38]. All D. magna samples used in this experiment must be the offspring of asexual reproduction, so it is necessary to ensure a suitable culture environment. In wild waters environment, D. magna reproduces asexually under most conditions, but in some harsh environments, some individuals will reproduce with both sexes and produce fertilized eggs covered by a saddle to resist the poor external environment. Therefore, there will still be differences in the genetic structure of D. magna in the same water area, even if the difference is very small. In addition, the sampling sites in this study are evenly distributed, but only a few sampling sites were chosen, China has a vast territory and a large north-south span. The sampling sites of a certain river cannot fully reflect the genetic diversity of all D. magna in a province or city. Although the polymorphic SSR markers developed in this experiment are abundant in quantity, provided reliable results, and co-dominant markers, the SSR markers still have co-migration problems. Today, the rapid development of new molecular markers such as EST, SNP, and gene chips have brought genetic diversity research more efficient and rich results and will be the focus of our next research.