3.1 Polymorphism of the microsatellite loci
As codominant markers, microsatellite markers have a high mutation rate among tandem repeat sequences and can form many alleles. Therefore, microsatellite markers have been known with highly polymorphic (Weber 1990; Gur-Arie et al. 2000). According to Botstein et al. (1980), PIC is divided into three grades: PIC < 0.25 (indicating a low polymorphism level), 0.25 < PIC < 0.5 (a moderate polymorphism level), and PIC > 0.5 (a high polymorphism level). Eleven microsatellite markers were developed in this study. The mean PIC of the 11 markers was 0.459, with the values for SSR01, SSR02, SSR 029 and SSR40 above 0.5 and only the value for SSR48 lower than 0.25, indicating that the developed microsatellite markers were highly polymorphic.
Analysis of microsatellite DNA characteristics found that most of them being dinucleotide and trinucleotide repeats. This result was consistent with the distribution characteristics of microsatellite loci in most plant (Sonah et al. 2011; Shan et al. 2018; Zhang et al. 2018). Besides, CG-rich repeats types are the most populous. The CG-rich SSRs identified here is mostly owing to the high GC content of N. yezoensis genome (Xu et al. 2006; Wang et al. 2007; Kim et al. 2021). SSR01 and SSR02 had the longest sequences and were highly polymorphic. Generally speaking, the longer the microsatellite DNA sequence or the more nucleotide repeats, the higher polymorphism of this microsatellite locus (Gou et al. 2007). In addition, the genetic parameters of SSR markers screened in this study were all higher than those of Wei (2012) when both cultivated and wild N. yezoensis populations were used as samples. And the number of allele per SSR were higher than the SSRs developed by Kim et al (2021). The Ho of 11 microsatellite markers were all lower than the He, and the microsatellite markers all deviated significantly from the Hardy-Weinberg Equilibrium. Purifying selection, population substructure, copy number variation, genotyping error and sampling effect are all potential causes of departure from Hardy-Weinberg Equilibrium (Chen et al. 2017; Kang et al. 2013; Qi et al. 2017). These markers would be valuable tools for N. yezoensis germplasm resource evaluation, genetic diversity protection and breeding.
3.2 Genetic diversity of wild N. yezoensis resource in China
The wild populations of N. yezoensis collected in this study were distributed in 6 cities along the coast of the Yellow Sea and Bohai Sea, which covered the major natural distribution of this species in China (Zhang and Zheng 1962; Cao et al. 2018). The collecting sites were almost geographically separated from each other. Fujio et al. (1985) found that the natural population structure of N. yezoensis has more geographical genetic differentiation than many other diploid organisms. It has been reported that the degree of genetic differentiation among laver populations is related to geographical location and growth environment (Xu et al. 2001; Yang et al. 2002). It has been tested in the other aquatic organisms as well (Niu et al. 2007; Guo et al. 2011). The present AMOVA analysis of 13 wild populations showed that genetic variation mainly existed within the population. The phylogenetic tree, structure and PCoA analysis consistently showed that the wild populations were divided into two distinct groups with Miaodao Strait as the boundary (Fig. 2). However, no obvious correlation between the genetic variation and geographical distance was found among the wild populations within each group in this study. The previous study revealed the similar phenomenon that the genetic distance between laver species is not always related to geography or growing environment (Cui et al. 2006). The genetic differentiation coefficients (Fst) among the 9 wild populations from south of Miaodao Strait were 0.018–0.117, indicating a high level of gene flow and low level of genetic differentiation (Wright 1978). Yu (2010) also analyzed the genetic diversity of N. yezoensis collected from Qingdao, Jiangsu and Japan using microsatellite markers, and got the similar conclusion. During the long evolutionary process of wild N. yezoensis, ocean drift may result in high gene exchanges and similar genetic background between wild populations in different geographical locations in the same sea area.
3.3 Genetic diversity of cultivated N. yezoensis resource in China
The cultivation area of N. yezoensis in China is distributed in Jiangsu, Shandong, and Liaoning province, with more than 99% of the production and processing enterprises are distributed along the coast of Jiangsu province (An et al. 2018; Lu et al. 2018). The semi-floating raft or standing-pole culture systems are generally used for laver cultivation, which leads to the limitation of laver aquaculture in shallow water (Wang et al. 2016). Intensive cultivation and long-term selective breeding of cultivated N. yezoensis germplasm leads to reduced genetic diversity of stock, fragility to climate change, susceptibility to disease, and reduced product quality (Li et al. 2018). New strains of N. yezoensis with varying good agronomic characters such as adaptation to non-drying culture system (Wang et al. 2016; Li et al. 2018), resistance to high temperature (Zhang et al. 2011) and disease (Park and Hwang 2014) were cultured to expand culture space, improve product quality, and adapt to different sea conditions and market demands.
The cultivated populations of N. yezoensis were collected from 5 cities along the coast of Jiangsu, Shandong and Liaoning province. Rizhao cultivated populations had the highest diversity (I = 0.698, P = 90.91%), and Nantong had a low diversity level (I = 0.208, P = 36.16%). AMOVA analysis of 9 cultivated populations showed that the genetic variation mainly existed within the population, which was consistent with the previous study (Yang et al. 2016). The average polymorphism of 9 cultured N. yezoensis populations in this study was higher than that of the wild populations, and the percentage of polymorphic loci of the most cultivated populations was at a high level (P = 90.91%). The previous studies based on different molecular markers found that there were great genetic differences among cultivated populations of different N. yezoensis cultivars (Mei et al. 2000; Chen et al. 2012). This suggested that the germplasm of cultivated N. yezoensis in China are diversified.
3.4 Comparison of diversity between cultivated and wild populations
Generally, the genetic diversity is higher in wild populations than in cultured ones, owing to multi-generational selection and purification of the cultivars. Wei (2012) used SSR markers to analyze the genetic diversity of some cultivated populations from Rizhao and Lianyungang (average I = 0.3537) and some wild populations from Qingdao, Yantai and Weihai (average I = 0.3856). They found that the genetic diversity of the wild populations was slightly higher than that of the cultivated populations. The genetic variation was largely present within the populations (Wei 2012). In this study, a higher genetic diversity was identified in the cultivated populations (average I = 0.469, P = 78.79%) than in the wild populations (average I = 0.352, P = 63.64%). In recent years, wild resources of seaweeds were deteriorated and biodiversity was declining due to global climate and coastal environmental change (Sun et al. 2010; Tanaka et al. 2012; Koh and Kim 2020). New germplasm resources from wild populations and new strains obtained from mutation or cross breeding have been recruited for aquaculture (Zhang et al. 2011; Park and Hwang 2014; Li et al. 2018). Usually, a mixture of seedlings from several sources is used in a nursery farm, which may increase the genetic diversity within the cultured populations.
The genetic diversity of wild N. yezoensis populations was lower than the cultivated ones. Whether years of N. yezoensis cultivation resulted in the lower genetic diversity of wild populations? From the structure analysis, it can be seen that significant differences were present between the cultured populations and the wild ones. On one hand, it has been reported that some of the N. yezoensis cultivars in China were introduced from abroad (Qiao et al. 2007). On the other hand, the distinct genetic background of some cultured populations may also result from mutation or cross breeding.
According to Wright (1978) classification, there was a high genetic differentiation between the cultivated populations and the wild populations (Fst=0.529–0.683) in the Changshan Islands. Along Qingdao Peninsula, gene exchange was present between some of the cultivated populations and the wild ones (Fst=0.218–0.641). In Haizhou Bay, there were a high gene exchange and a low genetic variation between the cultured populations and the wild one, especially between RZ-C and RZ-W (Fst=0.095). RZ-W, RZ-C and LYG-C1 were aggregated into a group. As mentioned above, there is the longest N. yezoensis culture history along Haizhou Bay (40–50 years). Industrial aquaculture of N. yezoensis in Qingdao and Dalian started just 5–7 years ago. The gene exchange between the cultivated populations and the wild populations seemed to be related to the length of culture time. However, it can be seen from the structure analysis that the gene flow between the cultivated populations and the wild populations was asymmetric, and the gene flow from the cultivated populations to the wild populations was limited. In other words, even if there is an impact of N. yezoensis cultivation on wild populations, it is a long-term and slow process.
N. yezoensis is a monoecious and self-fertilized seaweed, and the thallus is capable of repeating itself by means of asexual reproduction through archeospores (Fujio et al. 1985). The spreading of archeospores may cause genetic exchange and the decrease of genetic variation between the geographically adjacent populations (Cao et al. 2018). Nevertheless, the survival and germinating of archeospores depends on suitable environmental conditions. Although there is the longest N. yezoensis culture history of about 50 years in Jiangsu province, no flourishing of spontaneous populations have been identified along the coast. It may be due to the sandy substrate. It has been found that the cultivated population of U. pinnatifida (Shan et al. 2018; Li et al. 2019) and S. japonica (Shan et al. 2019) had higher gene exchange with the spontaneous populations on the cultivation rafts, but less gene exchange with the spontaneous populations in the adjacent subtidal zone. In practice, there are very few individuals of N. yezoensis growing on the cultivation stem ropes and rafts. In this study, the QD-C4 was genetically distinct from the other 3 populations (QD-C1, C2, C3) that were cultured at the same time and next to each other. It indicated that the genetic impact between the adjacent populations was limited.
In conclusion, the 11 SSRs markers efficiently distinguished the wild and cultured populations of N. yezoensis. The relationship between some populations can be well-traced. e.g., QD-C4 and LYG-C1 seemed to come from the same parents or germplasm, and the germplasm for DL-C and LYG-C2 had very close genetic relationship. It was firstly identified that there are at least two distinct genetic resources of wild N. yezoensis in China. The genetic exchange between the cultivated N. yezoensis populations and the wild populations seemed to be related to the length of culture time. Even if there is a genetic impact of N. yezoensis aquaculture on wild resource, it is a long-term and slow process.