In October 2021, each 1000 clams from S. constricta (average shell length 52.45 ± 3.70 mm and total body weight 11.98 ± 2.02g, one year old) and S. rivularis (average shell length 52.19 ± 3.20 mm and total body weight 9.96 ± 2.62 g, one year old) were sampled from Ninghai, Zhejiang province and Changle, Fujian province, China, respectively. Before crossing of S. constricta and S. rivularis, the parental samples have been pre-assessed to confirm pure individuals of each species by the morphology and genetic methods followed by Weng et al. (2013). When the brood stocks of two species were sexually mature, the interspecific hybridization trials were conducted. Mature clams were chosen and firstly kept dry in the shade, and then stimulated by flowing seawater to induce spawning. Sperms and eggs from different individuals of the same species were collected separately. The eggs from S. constricta were fertilized by sperms from S. rivularis to produce CR group (S. constricta♀×S. rivularis♂), while the eggs of S. rivularis were fertilized by sperms of S. constricta to generate RC group (S. rivularis♀×S. constricta♂). So two hybrids were produced: CR (S. constricta♀×S. rivularis♂) and RC (S. rivularis♀×S. constricta♂). After artificial insemination, the foot tissues of parental clams (n=30) were sampled and stored in 95% ethanol for DNA extraction.

The reciprocal hybrids were reared at tanks separately to prevent intermix, and foot tissues of live juvenile clams (n=30) of CR (shell length 12.45 ± 1.10 mm) and RC (shell length 11.47 ± 1.20 mm) were randomly sampled at 6 months of age and used for DNA extraction.

Total genomic DNA was extracted using a marine animal DNA extraction kit (Tiangen, China), and the quality and concentration of DNA were assessed by 1.2% agarose gel electrophoresis and Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA). All DNA samples were diluted to 100 ng/µL with Trise-EDTA buffer (pH 8.0) and stored at -20°C.

48 microsatellite markers from S. constricta (Liu et al., 2012b; Dong et al., 2016; Wang et al., 2016, Table 1 ) were selected and their primers were synthesized (Sangon Bioengineering, Shanghai). Cross-species amplification was carried out in S. rivularis and the polymorphic loci were identified. PCR amplifications were performed with Mastercycler Pro S thermal cycler in a 25 µL reaction system (12.5 µL 2×Taq Master Mix (Takara), 9.5 µL ddH₂O, 1.0 µL DNA and 1 µL positive and negative primers). The reaction program was as follows: first denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 s, the primer-specific annealing temperature ( Table 1 ) for 45 s, and extension at 72°C for 45 s with a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis on 8% nondenaturing polyacrylamide gel and visualized by ethidium bromide staining under UV light. Allele size was determined by a 500 bp ladder marker. Then, the expected PCR products were sequenced in Biotechnology Co., LTD, and sequence alignments were conducted by ClustalX 2.1 software.

The developed polymorphic microsatellite markers that stably amplified in S. rivularis were firstly used to analyze the genetic diversity of parental clams (S. constricta and S. rivularis), and species-specific microsatellite markers were then screened to identify two species and their hybrids (CR and RC) using 30 individuals, respectively.

The number of alleles (Na), direct count heterozygotes and homozygotes, and polymorphism information content (PIC) were calculated by CERVUS 3.0 genotype frequencies software (Kalinowski et al., 2007). The observed heterozygosity (Ho), expected heterozygosity (He) and effective number of alleles (Ne) were estimated by Popgen 32 software. The GENEPOP online software (http://genepop.curtin.edu.au) was used to calculate the Hardy-Weinberg Equilibrium (HWE), and all significant levels were adjusted by the Bonferroni correction.

48 pairs of microsatellite primers from S. constricta were assessed in S. rivularis, and the results revealed that 24 pairs of universal primers with clear bands were stably amplified in S. rivularis, with the transferability rate of 50.0% ( Table 1 ). Furthermore, 18 pairs of primers were polymorphic, accounting for 37.5% of the total primers ( Table 2 ).

The 18 polymorphic SSR markers were then used to evaluate the genetic diversity of S. constricta and S. rivularis, which displayed polymorphic differences between two species ( Table 2 ). The number of alleles in S. constricta and S. rivularis populations ranged from 2.00~6.00 (an average of 3.1667 alleles) and 2.00~5.00 (an average of 2.7222 alleles), respectively. The Ho and He changed from 0.1333 to 1.0000 (0.6958 on average) and 0.1266 to 0.7339 (0.5386 on average) in S. constricta, respectively, while Ho and He of S. rivularis varied from 0.0333 to 1.0000 (0.5574 on average) and 0.0333 to 0.7006 (0.4376 on average), respectively. The polymorphic information content (PIC) ranged from 0.117 to 0.677 (0.4556 on average) in S. constricta, while it changed from 0.141 to 0.677 (0.4101 on average) in S. rivularis. After Bonferroni correction, 8 and 5 loci deviated from Hardy-Weinberg equilibrium in S. constricta and S. rivularis, respectively. In general, S. constricta showed higher genetic diversity than S. rivularis.

Of all 24 universal microsatellites, a total of two species-specific markers (comp147145_c0 and comp149438_c0) were screened between two species S. constricta and S. rivularis ( Table 3 ; Supplementary Figure S1 ). Furthermore, the PCR products of these species-specific markers were sequenced, and the results showed that the repeat numbers of core repeating units were different between two species, which were consistent with above genotyping results ( Table 3 ; Supplementary Figure S1 ). Overall, there is a significant difference in the number and length of fragments amplified and no allele overlap between two species by two species-specific microsatellites, which is useful for distinguishing S. constricta and S. rivularis.

According to the principle that hybrid bands are parent-based complementary bands, the purity of hybrid can be identified quickly, accurately and efficiently. These two species-specific microsatellites were then used to verify the hybrids nature of the reciprocal hybrids ( Figure 1 ). For microsatellite marker comp147145_c0, two bands (about 290 and 310bp) were amplified in S. constricta, only one band about 250bp was observed in S. rivularis, while the hybrids CR and RC crosses possessed two bands (about 290bp and 250bp) that inherited one band from each parent, respectively ( Figure 1A ). For microsatellite marker comp149438_c0, only one band about 165bp was amplified in S. constricta, only one band about 195bp appeared in S. rivularis, while these two bands (about 165bp and 195bp) were both appeared in the hybrids CR and RC that come from their parents ( Figure 1B ). Consequently, the hybrids CR and RC possessed bands derived from both parents amplified by microsatellites comp147145_c0 and comp149438_c0, which could be used for quick genetic identification of S. constricta, S. rivularis and their reciprocal hybrids.

Owing to relatively conservatism, microsatellite markers show a considerable degree of transferability to related species, and can be cross-species amplified in closely related species, which is especially important for some species lacking of research of interspecific evolutionary relationships, interspecific comparison mapping, molecular marker assisted breeding and so on (Li, 2006). Especially for some species lacking of microsatellite markers, the transferability is of great significance for the evaluation of genetic diversity and germplasm identification.

Previous studies have reported the cross-species amplification of microsatellite markers in mollusks. In two scallops, 21 microsatellite markers of P. yessensis could amplify specific bands in C. farreri, of which 17 markers were polymorphic, with a polymorphism proportion of 28.33% (Zhan et al., 2008). In another example, 15 microsatellite markers from Argopecten irradians irradians were used to cross-species amplified in C. farreri, P. yesoensis and Amusium pleuronectes, and the transferability rates were 60%, 60% and 20%, respectively (Li et al., 2008). In hard clams, the interspecific transferability of 53 polymorphic microsatellite markers from M. meretrix revealed that 19 polymorphic markers in M. lamarckii and 10 in M. lyrata were developed, resulting in the transferability rates of 35.8% and 18.9%, respectively (Dong et al., 2013b). In this study, 48 microsatellite markers of S. constricta were tested in S. rivularis, of which 24 markers were successfully amplified and 18 were polymorphic, with the transferability rate of 50.0%. Meanwhile, the polymorphic microsatellite markers of S. constricta were also cross-species amplified in S. linearis, and 20 markers could be amplified and displayed polymorphisms, resulting in a transferability rate of 33.3% (Dong et al., 2016), which was lower than that of S. rivularis in the present study. Previous studies have reported that the cross-species transferability rate of microsatellites depends upon the evolutionary distance between the source and target species, and the higher the genomic homology, the greater conservation of flanking regions, the higher transferability rate of microsatellite markers (FitzSimmons et al., 1995). One possible explanation was that S. constricta was more closely related to S. rivularis than S. linearis, and S. constricta and S. rivularis had a relatively closer affinity. Therefore, the microsatellite markers developed by cross-species amplification could enrich some species with fewer markers available, and especially could be utilized for genetic diversity, comparative mapping and evolutionary biology among different species.

Microsatellite markers are highly polymorphic and widely distributed in eukaryotic genomes (Schlötterer, 2000), which has been widely used in genetic diversity analysis, construction of genetic linkage maps, variety identification and so on (Li, 2006). In mollusks, microsatellite markers have also been widely used in genetic diversity analysis in Japanese scallop P. yesoensis (Li et al., 2014), Manila clam Ruditapes philippinarum (Tan et al., 2020), Eastern oyster Crassostrea virginica (Wang and Guo, 2007), etc. In the present study, 18 polymorphic microsatellite markers were developed by cross-species amplification, and were used to evaluate the genetic diversity of S. constricta and S. rivularis. From the genetic parameters of Na, Ho, He and PIC, S. constricta showed higher genetic diversity than S. rivularis. Genetic diversity of a population or species could be evaluated by PIC value, and PIC < 0.25 represents low polymorphism, 0.25 ≤ PIC ≤ 0.5 represents moderate polymorphism, and PIC ≥ 0.5 represents high polymorphism (Botstein et al., 1980). The average PIC of S. constricta and S. rivularis were 0.4556 and 0.4101, respectively, suggesting that the genetic diversity of two species both above the middle level, with a higher genetic diversity.

S. rivularis is similar in shape to S. constricta, including similar size, elongate quadrate, shell surface covered with a yellow-green periostracum, and an oblique furrow running from the umbo to ventral margin on the center of the shell, which is the unique character of S. constricta. Therefore, S. rivularis has always been considered to be S. constricta until it was recently identified as a new species in 2007. To identify two razor clams, shell phenotype (shell length/shell height ratio) and sperm morphology were used to distinguish them by Huang and Zhang (2007). Afterwards, the ultrastructure of mature sperms by electron microscope and transmission electron microscopy was applied to differentiate two species (Huang et al., 2011). With the advance of molecular genetics in recent decades, mitochondrial COІ and 16S rRNA fragments were also used to identify S. rivularis and S. constricta (Weng et al., 2013). Generally, the external morphological measurement method may have great instability and imprecision, because their shell morphology may change under the habitat condition influence. Moreover, this method is difficult to identify juvenile clams and their hybrids. The observation of mature sperm ultrastructure has to rely on expensive instruments (electron microscope and transmission electron microscopy), and can only be used in breeding season due to collecting mature sperms, while female clams and hybrids cannot be distinguished. The COІ and 16S rRNA, belonging to mitochondrial DNA, are maternally inherited, so that they cannot be used for hybrid identification. Therefore, it is of great significance to explore a simple, accurate and reliable method for identification of S. constricta, S. rivularis and their hybrids.

Hybridization has been used for the genetic improvement and played an important role in the development shellfish aquaculture, and the Pacific oyster is a good example (Guo, 2009). Meanwhile, hybridization identification is essential for breeding, conservation, and understanding of their biological and genetic differences. Hybrids of closely related species often possess intermediated morphological traits, including body shape and other meristic traits (Konopiński and Amirowicz, 2018; Elliott et al., 2020). In many cases, minor deviations away from one or the other parental species can lead to difficulty in correctly diagnosing pure from F1 individuals, particularly when relying on morphology alone (Lessios, 2007). So the identification of pure and hybrid lineages in fundamental in developing policies for the conservation and management of native species (Allendorf et al., 2001), and DNA molecular marker techniques have been widely applied for the identification of aquaculture species and their hybrids in mollusks. For example, nuclear and mitochondrial primers were used to identify hybrids of Mytilus coruscus × Mytilus galloprovincialis in mussel hatcheries of China (Zhang et al., 2020), and two-step PCR method using three primers was conducted to identify abalones H. discus hannai, H. fulgens and their hybrids (You et al., 2015).

In comparison with other molecular markers, microsatellite markers are highly abundant in various eukaryotic genomes, and provide more polymorphism information for distinguishing different species or identify hybridization in aquatic animals (Li, 2006; Luo et al., 2010). For example, abalones H. fulgens, H. rufescens and their hybrids could be distinguished by species-specific microsatellite markers (Luo et al., 2010). Cruz et al. (2005) also developed a quick diagnosis for genetic identification of hybrids between red and Pacific abalone using microsatellites, and two species-specific microsatellite markers of H. discus hannai and H. fulgens could be used to distinguish the hybrids and their parents. In the present study, two species-specific microsatellite markers (comp147145_c0 and comp149438_c0) were both amplified in two closely related species, and there is a significant difference in the number and length of fragments amplified without allele overlap in two razor clams, which could be used as species-specific markers to clearly distinguish two razor clams. Furthermore, these two microsatellite loci confirmed that the hybrids CR and RC consisted of two haploid sets, one originating from each parental species, which could be used for quick genetic identification of S. constricta, S. rivularis and their reciprocal hybrids.

Consequently, these results provide a valuable and convenient method for distinguishing among two species and crossbred offspring.