Genetic structure analysis of Megalobrama terminalis, Culter alburnus, Chanodichthys mongolicus and their hybrids based on genotyping by sequencing

Distant hybridization is an essential means of breeding among aquatic animal species, genera, subfamilies, and even families, combining parents’ dominant traits to obtain excellent new hybrid varieties. Previous studies on Megalobrama and Culter’s distant hybridization show that Megalobrama and Culter’s hybrid offspring have significant heterosis. However, the analysis of the genetic structure of the fish hybridized from Megalobrama and Culter is still relatively limited, and there is little research on single nucleotide polymorphism markers (SNPs). This study aims to analyze the genetic structure of Megalobrama terminalis, Culter alburnus, Chanodichthys mongolicus, and their hybrids by high-quality SNPs obtained by genotyping by sequencing (GBS) technology. In the study, the total DNA extracted from the fin strips of 6 individuals that included M. terminalis, C. alburnus, C. mongolicus, and their hybrids (F₁), was double digested. Simplified genome sequencing was performed using Stacks software to construct a reference genome for SNP comparison. As a result, six individuals produced 7.01 GB of clean data, with an average of 1.17 GB per sample. All samples were used as a group to detect SNP mutations. A total of 399 145 SNP sites were detected. After quality control filtering, 97 911 SNP sites were obtained. SNP analysis indicated that, of parents and their hybrids, the average observed heterozygosity was 0.339 4. The average expected heterozygosity was 0.285 3. The average polymorphic information content was 0.273 7, average nucleotide diversity was 0.372 7, the average minimum allele frequency was 0.247 2, and the average genotyping rate was 93.94%. The principal component analysis (PCA) showed that M. terminalis, C. alburnus, C. mongolicus, and their hybrids clustered at different points, but the clustering points of the two hybrids of M. terminalis and C. alburnus were close. The PCA’s result indicated apparent genetic differentiation among M. terminalis, C. alburnus, C. mongolicus, and their hybrids. Based on the genetic differentiation index and genetic distance between different populations, the results show that the differentiation indexes between M. terminalis, C. alburnus, C. mongolicus, and their hybrids are 0.309 6-0.894 0, and the genetic distance is 0.370 5-2.244 3. The phylogenetic tree constructed using the maximum likelihood (ML) method shows that M. terminalis, C. alburnus, C. mongolicus, and their hybrids are distinguished. C. mongolicus firstly formed a new branch. M. terminalis (♀) × C. mongolicus (♂) was separated as a single branch. However, M. terminalis, C. alburnus, and their hybrids formed a broad branch. In this big branch, M. terminalis first differentiated and formed a separate branch. M. terminalis (♀) × C. alburnus (♂) differentiated again. Finally, M. terminalis (♂) × C. alburnus (♀) differentiated. The genetic structure diagram further supports the analysis results of the phylogenetic tree. The three parents are divided into three subgroups. The hybrids of M. terminalis and C. alburnus are obtained by crossing M. terminalis and C. alburnus. In comparison, the hybrids of M. terminalis (♀) × C. mongolicus (♂) are obtained by crossing M. terminalis (♀) and C. mongolicus (♂). This study will provide more genetic data for the hybrid breeding of M. terminalis, C. alburnus, and C. mongolicus, such as providing SNPs for constructing a high-density genetic map and screening SNP s associated with traits.