Development and characterization of a duplex PCR assay in Chinese sturgeon (Acipenser sinensis) for genetic analysis

Yacheng Hu,Xueqing Liu,Hejun Du,Kan Xiao,Binzhong Wang,Jing Yang

doi:10.1038/s41598-020-60401-y

Yacheng Hu, Xueqing Liu + Show 4 more

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https://doi.org/10.1038/s41598-020-60401-y

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Abstract

Chinese sturgeon (Acipenser sinensis) has been listed as a critically endangered species on the IUCN Red List and is an endemic fish of China. Five sets of duplex polymerase chain reactions (PCR) assays were developed with 10 tetranucleotide microsatellites for Chinese sturgeon. The size of CS57, ZHX43, ZHX69, AS105, ZHX51, AS074, ZHX2, AS078, AS026 and AS073 products in 184 Chinese sturgeon individuals ranged from 257–305, 191–241, 251–285, 172–244, 236–260, 169–209, 194–234, 92–176, 165–257 and 120–164, respectively. 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 index (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. Despite many advantages, the use of microsatellites as genetic analysis tools can be limited by the cost of the associated experiment. To solve this problem, this set of five duplex PCRs will provide tools that are more helpful, less expensive and less time consuming than others used for genetic analyses in Chinese sturgeon.

Highlights

Chinese sturgeon (Acipenser sinensis) has been listed as a critically endangered species on the IUCN Red List and is an endemic fish of China
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 polymerase chain reactions (PCR) assays (Table 1)
Many microsatellites have been developed in Chinese sturgeon[13,14], their associated PCR assays are all single assays

Summary

Introduction

Chinese sturgeon (Acipenser sinensis) has been listed as a critically endangered species on the IUCN Red List and is an endemic fish of China. The use of microsatellites as genetic analysis tools can be limited by the cost of the associated experiment To solve this problem, this set of five duplex PCRs will provide tools that are more helpful, less expensive and less time consuming than others used for genetic analyses in Chinese sturgeon. The Chinese sturgeon is listed as a Critically Endangered species by the International Union for Conservation of Nature and Natural Resources Red List (IUCN Red List)[7] and is a first class protected animal in China[8]. Multiplex PCR technology has been widely employed in many aquatic animals[28,29] This technology is greatly improving the efficiency of genotyping and 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

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