Introduction

Chinese sturgeon (Acipenser sinensis) is an endemic and critically endangered species in China. Chinese sturgeon was once widely distributed in the Yangtze River, Pearl River and Chinese seas1. The wild population of Chinese sturgeon has fallen drastically in the past decades due to human activities, such as the use of pesticides, industrial overfishing and environmental pollution2,3,4. Currently, the wild population of Chinese sturgeon in the Yangtze River is very small. The need for species rehabilitation is known, and efforts have been made to protect it. To prevent this species from becoming extinct, artificial propagation and tagged ranching have been performed every year since 19845,24. However, by using trinucleotide or tetranucleotide microsatellites instead of dinucleotide microsatellites, the risk of obtaining false alleles is reduced25. Studies have shown that four-base microsatellites are more stable and more accurate than other types of microsatellites26. Furthermore, PCR amplification results from tetranucleotide repeat loci are easier to interpret than in the case of dinucleotide repeat loci because only a single stutter band is typically observed, in a position four bases shorter than each allele band, and the intensity of the stutter band is generally <10% of the main band27. Currently, the human and bovine paternity test kits used worldwide consist of four-base microsatellite markers. Multiplex PCR refers to the simultaneous amplification of multiple microsatellite loci in a PCR system, resulting in multiple PCR amplification products. Multiplex PCR technology has been widely employed in many aquatic animals28,29. This technology is not only greatly improving the efficiency of genoty** but also reducing the cost of genetic analyses. Compared with the conventional simplex procedure, multiplex SSR sets and multi-loading combinations reduced the costs of the PCR related reagents by about 50% and the costs of the electrophoresis related reagents by over 85%30.

In this study we provide five duplex PCR assays performed with 10 tetranucleotide microsatellites in order to help in the traceability of individuals and in the genetic conservation management of Chinese sturgeon.

Results

A total of 412 sequences that contain tetranucleotide microsatellites were selected from the cDNA libraries. And 412 novel primer pairs were developed using Primer Premier 5.0 software. Of the 412 microsatellites that were tested with the DNA of 12 individuals, 315 resulted in no or poor amplification in Chinese sturgeon. Eighty-seven microsatellites produced ambiguous polymorphic products. The remaining 10 microsatellites were successfully amplified. Among the 31 tetranucleotide microsatellites reported by Zhu B14. that were tested with the DNA of 12 individuals, only 12 microsatellites showed polymorphism. The rest of the microsatellites showed ambiguous polymorphic products. According to the results for these microsatellites tested in the 184 individuals, we developed five novel polymorphic loci (CS57, ZHX43, ZHX69, ZHX51 and ZHX2) and selected five polymorphic loci (AS105, AS074, AS078, AS026 and AS073) previously reported in Chinese sturgeon to build five duplex PCR assays (Table 1). Sequences containing these five microsatellite markers that we developed were deposited in GenBank under accession numbers MN401754-MN401758. The MPprimer software showed no pairs of loci displayed significant linkage disequilibrium. The size of the products for the ten microsatellites in 184 Chinese sturgeon individuals has been listed in the Table 2. The observed allele number of the 10 microsatellites ranged from 7 to 16, and the total number of alleles was 106. The number of alleles per individual in CS57, ZHX43, AS105, AS074, AS078 and AS026 was 1–4. The number of alleles per individual in ZHX69, ZHX51, ZHX2 and AS073 was 2–4. The mean number of alleles per locus per individual ranged from 2.01–3.76. The expected heterozygosity (HE), observed heterozygosity (HO), polymorphic information content (PIC) and Shannon-Weiner diversity indices (H′) ranged from 0.582 to 0.899, from 0.676 to 1, from 0.518 to 0.886 and from 1.034 to 2.34, respectively (Table 2). The frequencies of null alleles ranging from 0.03 to 0.15 were reported by the program MICROCHECKER (Table 2). The 184 Chinese sturgeon individuals were accurately reconstructed in the UPGMA dendrogram using the ten microsatellites (Fig. 1).

Table 1 Characterization of 5 duplex PCR assays in Chinese sturgeon.
Full size table
Table 2 Genetic diversity in 5 duplex PCR assays in Chinese sturgeon.
Full size table
Figure 1
figure 1

UPGMA dendrogram of 184 Chinese sturgeon individuals. The dendrogram was constructed with the UPGMA clustering algorithm in PHYLIP’S NEIGHBOR version 3.69. The constructed tree file was visualized using MEGA version.

Full size image

Discussion

Microsatellites are very important for the management and conservation of fish species31. Although many microsatellites have been developed in Chinese sturgeonEstablishing the microsatellite based duplex PCR assays for population studies of Chinese sturgeon

According to the size of PCR products in 184 individuals, we obtain the range of sizes for each selected microsatellite. Duplex PCR site combinations were chosen according to the range of sizes for each selected microsatellite and the avoidance of potential hairpin structures and primer dimers. Every duplex PCR assay reaction in a total volume of 25 μl contained 0.25 U of Taq polymerase (Takara, China), 0.25 μM each primer, about 50–100 ng template DNA, 0.25 μM dNTPs, 1.5 mM MgCl2, 0.25 μM PCR buffer (Takara, China) and water. PCR was performed under the following profile: an initial step at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, and an extension at 72 °C for 10 min. The PCR products were size fractionated with 10% PAGE and visualized by silver staining. The sizes of the alleles were estimated with the pBR322 DNA/Mspl marker (Takara).

Genetic analysis

The statistics of the polymorphic parameters, including the mean expected heterozygosity (HE), observed heterozygosity (HO) and Shannon-Weiner diversity indices (H′) were calculated using ATetra1.249 software. The polymorphic information content (PIC) was calculated using the formula \(PIC=1-\mathop{\sum }\limits_{i=1}^{n}{P}_{i}^{2}-\mathop{\sum }\limits_{i=1}^{n-1}\mathop{\sum }\limits_{j=i+1}^{n}2{P}_{i}^{2}{P}_{j}^{2}\), Pi and Pj are the frequencies of I and J allele in the microsatellite loci. The presence of null alleles was assessed at a 95% confidence interval using the program MICROCHECKER50. The software MPprimers51 was used to detect whether the duplex PCR primers in this study were in linkage disequilibrium. The ten microsatellite loci were scored in a presence/absence format, so that genotypic data were transformed into allele phenotypes52. Each phenotype classified alleles as present or absent, regardless of dose. Based on the allele phenotypes, a dendrogram was constructed using the unweighted pair group arithmetic means (UPGMA) clustering algorithm in PHYLIP’S NEIGHBOR version 3.6953. The constructed tree file was visualized using MEGA version 5.154.