Pseudobagrus vachellii (Richardson, 1846) is a commercially important freshwater fish species in China. To understand the effects of artificial breeding on the genetic diversity of three consecutive P. vachellii breeding populations (F1, F2, and F3) since 2012, a genetic analysis was conducted using polymorphic microsatellite markers. The mean allele number, expected heterozygosity, observed heterozygosity, and the polymorphic information content from generation F1 to F3 decreased from 7.75 to 5.63, from 0.77 to 0.63, from 0.83 to 0.77, and from 0.72 to 0.58, respectively. Analysis of molecular variance showed greater genetic divergence within the three generations (93.67%) than that among the generations (6.33%), and the overall differentiation level was moderate. Additionally, the lowest genetic differentiation was between F2 and F3 (F_st = 0.0484), and the highest was between F1 and F3 (F_st = 0.12864). Inbreeding occurred in each generation and was the highest in generation F3. Structural analysis showed that the three P. vachellii generations were most likely divided into two different genetic clusters. Although genetic diversity declined slightly in the mass selection lines after three breeding generations, overall genetic diversity was maintained at a relatively high level. To minimize the loss of genetic diversity and inbreeding in the subsequent breeding process, a moderate number of parents can be used for each generation. Information regarding the genetic diversity and structure of the selective P. vachellii breeding generations obtained in this study will be useful for future broodstock management and selective breeding programs.

genetic diversity, genetic structure, microsatellites, Pseudobagrus vachellii, selective breeding

Pseudobagrus vachellii (Richardson, 1846) represents the family Bagridae and is an endemic freshwater fish species widely distributed in China. However, wild populations have rapidly declined owing to habitat destruction, water pollution, and overfishing. As an edible fish, it has the largest body size and is the fastest-growing group in the genus Pseudobagrus. Moreover, it is the male parent of the hybrid yellow catfish “Huangyou 1” (GS-02-001-2018). Pseudobagrus vachellii possesses high nutritional value and has a great taste, with low bone amounts in the muscle (Zheng et al. 2021). However, long farming periods can result in problems such as slow growth rate, differences in morphological traits, mottling of body color, short and thick body shapes (Fig. 1), and decreased stress resistance in the retained population. These problems greatly affect the market value of P. vachellii.

Figure 1. 

Comparison between Pseudobagrus vachellii populations. (A) Poor germplasm population and (B) mass-selected population by our project team.

Artificial selective breeding is an effective way to improve performance traits, breed excellent aquatic varieties, and increase productivity; however, it also decreases genetic diversity in breeding populations (Gjedrem et al. 2012; Chen et al. 2017; Phuc et al. 2021; Swain et al. 2022). As a raw material, for artificial selection in captive populations, maintaining the genetic diversity of the breeding population is a prerequisite for the continuous utilization of aquatic germplasm resources, and genetic assessments should be conducted periodically (Diyie et al. 2021). For many fish species, usually in the late stages of artificial selection breeding, the number of breeding individuals gradually decreases, which leads to increased inbreeding, decreased population genetic diversity, and loss of genetic variability in artificially bred seed populations. This eventually leads to the loss of product development and utilization (Ortega-Villaizan et al. 2011; Liu et al. 2018).

Because of the long-term artificial breeding of Pseudobagrus vachellii, nonstandard conservation, and germplasm degradation have occurred in nursery farms. The mass selection program of P. vachellii derived from the wild Huai River and Yangtze River populations for growth traits and morphological characteristics has been conducted since 2012 for three generations. Compared with that of the unselected P. vachellii, the growth rate increased by 15 percentage points, which showed obvious advantages (Duan et al. 2023). However, the changes in the genetic variation and diversity among these three breeding generations are unknown.

The microsatellite marker technique is a sensitive, simple, and efficient method for studying the genetic diversity of aquatic animals (Fu et al. 2017). Some microsatellite markers have been successfully developed for the genetic analysis of P. vachellii (see Wang et al. 2021; Zheng et al. 2020). Eight polymorphic microsatellite loci were used to monitor changes in genetic diversity and structure during the selection process in this study. This useful information can be used to evaluate whether the levels of variation are appropriate for a long-term selective breeding program. Moreover, it provides guidelines for avoiding genetic variation loss and inbreeding for future P. vachellii selective breeding programs and promotes the sustainable and healthy development of the P. vachellii industry.

Mass selection process and pond culture management. In our selective breeding program, three generations of Pseudobagrus vachellii were produced between 2012 and 2021 at the Fishery Research Institute of the Anhui Academy of Agricultural Sciences in Hefei, Anhui, China (Fig. 2). From the founder group comprising more than 800 P. vachellii individuals, 80 individuals with large sizes and similar morphological traits were screened as breeding parents. The female-to-male ratio was approximately 1:1, and they were artificially bred in May 2014. Approximately 300 000 F1-generation hatchlings, which were cultivated using natural bait in the ponds (rotifers, branchiostomatids, copepods, etc.) for one week, were fed compound feed powder (with a protein content of 45%) for cultivation and then changed to compound feed granules (with a protein content of 40%–45%). When the fish were half a month old, they were fed compound feed pellets (protein content of 40%–45%), and selection was carried out at 1 month, 12 months, and 32 months of age, respectively, using growth rate and morphological traits as the main selection indexes. Healthy and disease-free individuals with fast growth rates and similar morphological traits were selected and retained for cultivation. In June 2017, 60 breeding parents with a good degree of sexual maturity and morphology were selected from the F1 generation at a male-to-female ratio of approximately 1:1. They were then artificially inseminated to obtain approximately 220 000 F2 generation first hatchery fry. The fry were bred in the same way as the F1 generation and were selected at one, 12, and 32 months of age, using growth rate and morphological traits as the selection indices. In June 2021, 60 breeding parents with a good degree of sexual maturity and morphology were selected from the F2 generation at a female-to-male ratio of approximately 1:1. Artificial insemination was then performed to obtain approximately 220 000 fry in the F3 generation. The fry cultivation method was the same as that of the F1 generation, and selection was carried out at 1 month and 12 months of age, respectively, using growth rate, morphological traits, and hypoxia tolerance as selection indices. The number of individuals in the selection group was then approximately 7500. The female breeding parents of the F1, F2, and F3 generation all weighed over 150 g and the males weighed not less than 250 g. During breeding, oxytocin was injected artificially, and after the effect time was reached, insemination was performed by squeezing the eggs artificially, and the fertilized eggs were incubated on a mesh. Each female fish produced 6000~9000 eggs.

Figure 2. 

Mass selection of Pseudobagrus vachellii and sample collection.

Fish materials and sample collection. The care and use of experimental animals in this study complied with the guidelines and policies approved by the Experimental Animal Welfare and Ethical Committee of the Anhui Academy of Agricultural Sciences. Twenty-six to thirty individuals from each generation were sampled randomly, and 85 unrelated individuals were randomly selected from each generation (Fig. 2). The fish body length was 31.77 ± 9.99 cm and the body weight was 434.24 ± 291.48 g. Muscle tissue samples were quickly removed, cleaned with 0.70% physiological saline, and stored at 4°C in 95% ethanol for subsequent experiments.

DNA extraction and genotyping. Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. A panel of eight microsatellite markers previously developed for Pseudobagrus vachellii were amplified using polymerase chain reaction (PCR) at annealing temperatures (Zheng et al. 2020; Wang et al. 2021) (Table 1). The PCR reactions were conducted using a Peltier thermal cycler using a 30 μL reaction mixture. Each reaction mixture contained 3 μL of 10× PCR buffer, and final concentrations of 2.5 μL (2.5 mmol · L^–1) deoxynucleotide triphosphates, 1 μL (10 μmol · L^–1) of each forward and reverse primer, 0.3 μL (5 U · μL^–1) of Taq DNA polymerase (Transgen, Beijing, China), and 1–2 μL (50 ng · μL^–1) of template DNA that was added to 30 μL double-distilled H₂O. Temperature profiles for the PCR consisted of an initial denaturation at 94°C for 5 min, 31–34 cycles of 94°C for 30 s, annealing at primer-specific temperatures (53–60°C) for 40 s, extension at 72°C for 50 s, and a final extension at 72°C for 10 min (Wang et al. 2022). The PCR products were separated and sized on an ABI 3730xl automated sequencer with a ROX 500 size standard, and the resulting genotype traces were scored in GeneMapper 3.7 (all Applied Biosystems). The presence of null alleles, large allele dropouts, scoring of stutter peaks, and typographic errors were assessed using a micro-checker (Van Oosterhout et al. 2004).

Data analysis. The microsatellite data were analyzed using web-based Genepop software (http://genepop.curtin.edu.au/), with Markov chain parameters of 10 000 dememorizations, 500 batches, and 5000 iterations per batch to determine whether each locus deviated from the Hardy–Weinberg equilibrium and to test the linkage equilibria. The number of alleles (N_a), number of effective alleles (N_e), observed heterozygosity (H_o), expected heterozygosity (H_e), Shannon diversity index (I), and Nei’s genetic distance (D_a) values were calculated using Popgene 1.32, respectively (Nei 1972; Yeh et al. 1997). The genetic differentiation coefficient (F_st) is one of the most widely used descriptive statistics for evaluating genetic differentiation between and among populations and can provide important insights into the evolutionary processes that influence genetic variation among populations (Holsinger and Weir 2009). According to the rule by Wright (1965), an F_st value of 0.000–0.049 represents low differentiation, values of 0.05–0.25 indicate moderate differentiation and values higher than 0.25 indicate high differentiation among populations. The F_st and genetic variation were analyzed using analysis of molecular variance (AMOVA) with Arlequin 3.5 software (Excoffier and Lischer 2010). An unweighted pair group method with arithmetic mean (UPGMA) phylogenetic tree based on Nei’s genetic distance was constructed using MEGA software (version 7.0) (Kumar et al. 2016). The polymorphism information content (PIC) of each locus and population was calculated using Cervus 3.0 (Kalinowski et al. 2007). The genotypes determined using COANCESTRY (V1.0.1.1; Wang 2011) were used to measure relatedness estimates (R) between generations and within-generation genotypes as described by Wang (2002), and the inbreeding coefficient (F) was determined as described by Ritland (1996). Microsatellite data were also analyzed using the STRUCTURE 2.3.3 program and the admixture model was used to estimate population genetic structure among and within species (Evanno et al. 2005). We conducted an analysis with ten iterations for each population size (K) of one–eight, and with the Markov chain Monte Carlo running for 500 000 iterations and an initial burn-in of 100 000 iterations. The K values described by Evanno et al. (2005) were calculated to identify the most reasonable K using the Structure Harvester program (Earl and vonHoldt 2012). The runs were averaged using CLUMPP version 1.1.2 (Jakobsson and Rosenberg 2007), and the results were visualized using DISTRUCT version 1.1 (Rosenberg 2004).

Summary statistics. No evidence of allelic stutter or large allele dropouts was found in the dataset, and no null alleles were detected at any of the eight loci. Almost all eight loci were highly polymorphic (PIC > 0.5) (Botstein et al. 1980). In this study, microsatellite markers were used for the genetic analysis of three consecutive P. vachellii selective breeding generations. The genetic indicators of the eight microsatellite loci are listed in Table 2. The mean observed H_o was 0.80, the mean expected H_e was 0.7506, the mean N_a per locus was 8.625, the effective N_e was 4.448, and I was 1.6396. A comparison of the genetic information of three consecutive selective breeding generations of P. vachellii is shown in Table 3. A relatively high level of overall genetic diversity was observed (H_e = 0.6323–0.7663, PIC = 0.5754–0.7183), whereas the number of microsatellite alleles (N_a, N_e), heterozygosity (H_o, H_e), and PIC decreased slightly in the mass selection lines.

Genetic variation and differentiation among generations. AMOVA revealed that the variation among populations was only 6.33%, whereas the variation within populations was 93.67%. Wright (1965) proposed that F_st < 0.05 indicated low differentiation, 0.05 < F_st < 0.15 indicated moderate differentiation and F_st > 0.15 indicated high differentiation. The overall F_st value was 0.06329, which indicated a moderately differentiated degree (0.05 < F_st < 0.15) (Table 4). The F_st values for the three generations ranged from 0.0484–0.1286. The lowest genetic differentiation was observed between F2 and F3 (F_st = 0.0484) with the smallest genetic distance (D_a = 0.142), whereas the highest genetic differentiation was observed between F1 and F3 (F_st = 0.1286) with the largest genetic distance (D_a = 0.4373) (Table 5). The UPGMA phylogenetic tree based on Nei’s genetic distance (Nei 1972) indicated that the three generations could divide the population into two clades. The analysis revealed that F2 and F3 formed sister relations and were clustered with F1 (Fig. 3). Structural analysis showed that K = 2 was the ideal number of subtypes; that is, three consecutive Pseudobagrus vachellii selective breeding generations were most likely to be divided into two different genetic clusters (Fig. 4). Different colors represent different genetic clusters in the figure, and the degree of gene purification increased with the development of breeding.

Figure 3. 

Nei’s unweighted pair group method with arithmetic mean (UPGMA) tree of three consecutive Pseudobagrus vachellii selective breeding generations of based on microsatellites. Note: Scale bar denotes genetic distance.

Figure 4. 

Population genetic structure of the three consecutive Pseudobagrus vachellii generations. The assignment results show that K = 2 (parameter introduced by Evanno et al. 2005). The two colors represent two different genetic clusters. The Y-axis denotes the proportion of ancestral components in an individual relative to other populations.

Partner relatedness and inbreeding coefficient analysis. The results showed that the relation (R) and inbreeding coefficient (F) within each generation had positive values and were the largest in generation F3; the relatedness increased in succeeding generations (Table 6). The R value between generations was negative, except for in F2 and F3, which were positive. Although the F value among all three generations was negative compared to the relatedness between F1 and F2 (−0.08417), the relatedness between F1 and F3 decreased (−0.13296) (Table 7).

Selection quickly improves certain traits, but the genetic diversity is usually lower than that of founder populations (Zhang et al. 2010). Many fish species have high fecundity and require relatively few parents to produce offspring; therefore, maintaining genetic diversity over successive generations is a recognized challenge for aquaculture breeding programs (Zhang et al. 2010; Ortega-Villaizan et al. 2011; Liu et al. 2018; Varney and Wilbur 2020). The presently reported study revealed that the genetic diversity of Pseudobagrus vachellii declined slightly after three generations of breeding. These results are similar to those obtained from other artificial breeding of aquatic animals. For example, a report showed that even when using a relatively large number of banana shrimp (Penaeus merguiensis De Man, 1888) broodstocks, a substantial loss of allelic diversity within lines over 14 generations is still observed (Knibb et al. 2014). Li et al. (2018) found a slight decrease in genetic diversity over three successive generations of early- and late-maturing strains of the Chinese mitten crab (Eriocheir sinensis Milne Edwards, 1853). In cultured silver-lipped pearl oysters, Pinctada maxima (Jameson, 1901), genetic diversity decreased, and the effective population size was reduced (Lind et al. 2009). High genetic diversity was observed among generations of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758), in Ghana (Diyie et al. 2021), and Wang et al. (2022) reported that the genetic diversity of cultured Procambarus clarkii (Girard, 1852) tends to decline. These studies are important for ensuring the sustainability of the aquaculture industry.

Generally, 0.25 < PIC < 0.50 meant that the single sequence repeat (SSR) loci were moderately polymorphic, and PIC > 0.50 meant that the SSR loci were highly polymorphic (Botstein et al. 1980). In the presently reported study, with an increase in breeding generations, the genetic diversity of the three artificially selected populations gradually decreased, and the PIC values were 0.7183, 0.6728, and 0.5754, respectively, indicating that the SSR loci were highly polymorphic. The overall number of alleles declined from 7.75 to 5.63, and a similar decline in the number of alleles was reported in previous studies (Zhang et al. 2018; Varney and Wilbur 2020). A study of three successive selection lines of Pacific abalone showed that the mean H_o and H_e values decreased from 0.679 to 0.622 and 0.756 to 0.649, respectively (Chen et al. 2017). These results were similar to those of the presently reported study in that after three consecutive P. vachellii selective breeding generations, the mean H_o and H_e values from the F1 generation to the F3 generation decreased from 0.8250 to 0.7672 and from 0.7663 to 0.6323, respectively. These results revealed a high level of genetic diversity in the three successive generations of breeding populations.

In the presently reported study, the F_st among the various generations of P. vachellii was 0.06329, indicating a moderate degree of differentiation. Additionally, the lowest genetic differentiation was observed between F2 and F3 (F_st = 0.0484), whereas the highest genetic differentiation was observed between F1 and F3 (F_st = 0.12864), indicating that the genetic similarity of the selected offspring increased gradually. However, the genetic structure of F3 changed significantly compared to that of F1.

In terms of successive generations of mass selection, strategies to avoid inbreeding are of critical concern (Fu et al. 2017). Inbreeding depression, through the loss of genetic variation, can ultimately limit long-term genetic progress through selective breeding (Evans et al. 2004; Varney and Wilbur 2020). The presently reported study showed that there was no inbreeding among the three generations; however, inbreeding occurred in each generation, and the largest inbreeding occurred in generation F3. These results are consistent with those of previous studies on long-term artificial selection, as genotypes become more homogeneous, leading to inbreeding depression (Zhang et al. 2010; Chen et al. 2017). Therefore, in the subsequent breeding process, pooling of fertilized eggs from multiple crosses to create cohorts and moderate numbers of parents for each generation (at least 30 pairs) can be used to minimize the loss of genetic diversity and inbreeding depression.

In conclusion, the presently reported study revealed that the genetic similarity of the offspring increased gradually by artificial selection, and the genetic diversity of P. vachellii declined slightly after three generations of breeding. However, the level of genetic diversity was still high, which has the potential for further breeding. To minimize the negative influence of inbreeding, new strains can be bred by appropriately increasing the number of breeding parents in the subsequent breeding process, thereby reducing the probability of inbreeding and adopting high selection pressure.

This work was financially supported by the Young Talent Program of Anhui Academy of Agricultural Sciences (2023–2027) and the Wuhu City Science and Technology Planning Project (No. 2022ly15).