Sinocyclocheilus grahami (Cypriniformes, Cyprinidae) is an endemic fish species in China, with restricted distribution in Dianchi Lake and surrounding streams on the Yunnan Plateau (Zhao and Zhang, 2009). As one of the “Four Famous Fishes” in Yunnan, S. grahami is an economically valuable species known for its excellent quality and higher crude protein (∼20%), essential amino acids (∼18%) and polyunsaturated fatty acids (∼0.34%) content than Ctenopharyngodon idellus, Hypophthalmichthys nobilis, or Cyprinus carpio (Zhao et al., 2013), and possesses huge breeding potential, especially in freshwater aquaculture. From the 1960s, the species became highly endangered due to habitat destruction, water pollution, and alien species invasion (Yang et al., 2007). As such, over the past 2 decades, our team has successfully established an artificial breeding program to ensure the survival of the species and lay a foundation for its production, resulting in the creation of a new national breed (“S. grahami, Bayou No. 1”, hereafter S. grahami) with accelerated growth and weakened intermuscular bones via four generations of artificial selection (Yang et al., 2007; Pan et al., 2009; Yin et al., 2021). However, slow growth (40 g/2 years, 2 years = mature age) and growth gaps among individuals remain problematic, and further selective breeding is required to obtain a faster and more stable growing strain.

With the rapid development of molecular biology and sequencing technology, modern breeding techniques attempt to target the regulatory genes underpinning desired phenotypes and achieve superior varieties via the selection or manipulation of these genes, i.e., molecular-assisted breeding (Eze, 2019; Liu et al., 2022). Therefore, it has become increasingly important to understand the regulatory mechanisms and major genes behind desired phenotypes.

Although growth is a vital characteristic of farmed species, it is a complex trait influenced by many genes with minor effects. As such, the genetic mechanisms underlying growth remain unclear, although various relevant genes have been identified, including growth axis-related genes [growth hormone (gh), growth hormone receptor (ghr), insulin-like growth factor I (igf1), insulin-like growth factor II (igf2), somatostatin (sst)] and muscle growth regulating genes [myostatin (mstn), myogenic regulatory factors (mrfs)] and appetite, food intake regulate genes [melanocortin receptor-4 (mc4r), ghrelin (ghrl)] (De-Santis and Jerry, 2007; Blanco et al., 2017; Baldini and Phelan, 2019). Nevertheless, many genes related to growth remain unresolved, and the mechanisms for growth differ in different species (Laghari et al., 2014; Yu et al., 2016; Chen et al., 2022). Hence, mechanistic analysis of S. grahami is required to better guide breeding.

Fish growth can be achieved via skeletal muscle growth, primarily determined by hyperplasia and hypertrophy of muscle fibers (muscle cells) (Stoiber et al., 2002; Johnston, 2006; Fuentes et al., 2013). Muscle constitutes 50%–70% of body weight of most commercially important fish species and is the main consumed product (Li et al., 2019). Therefore, muscle growth plays a critical role in fish growth. Transcriptomic analysis plays a significant role in all fields of biological research and is widely used to study gene expression profiles and functional mechanisms of genotypes (Klopfleisch and Gruber, 2012; Papatheodorou et al., 2015). For instance, muscle transcriptomic studies have identified several genes correlated with growth in a variety of fish species, including Micropterus salmoides (Li et al., 2017), Ctenopharyngodon idella (Lu et al., 2020), Schizothorax prenanti (Li et al., 2019), and Mylopharyngodon piceus (Zhang et al., 2020).

Here, we focused on exploring the molecular mechanisms and major genes underlying growth of S. grahami, and aimed to verify differences in gene expression profiles between fast- and slow-growth fish and identify key genes involved in body length/weight based on muscle transcriptomic analysis. We hope that our study can provide valuable information for further studying of growth mechanisms and breeding strategies in S. grahami and other farmed species.

As a critical economic trait, growth is important for the development of aquaculture. A growing number of transcriptomic studies have identified growth mechanism diversity in species, tissues, and environments (Fu et al., 2019; Prieto et al., 2019; Lu et al., 2020; Wang et al., 2021). In our study, 1,647 DEGs (947 up-regulated and 700 down-regulated DEGs in the fast-growth group) were obtained from muscle tissue between the fast- and slow-growth groups. Most DEGs were significantly enriched in metabolic pathways, such as starch and sucrose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, glycine, serine and threonine metabolism, arginine and proline metabolism, biosynthesis of amino acids, peroxisome, and PPAR signaling pathway (Figure 2C). These findings are similar to those in previous studies on C. idella (Lu et al., 2020), Eriocheir sinensis (Wang et al., 2021), and Paramisgurnus dabryanus (Zhao et al., 2021), suggesting that metabolism plays a crucial role in growth, and the fast-growth group exhibit higher expression of some metabolic genes than slow-growth group.

Achieving somatic growth requires depletion of available nutrients and energy acquired from the environment, which are converted into cellular and tissue components through metabolic cellular reactions (Sousa et al., 2010; Lukas et al., 2011; Canosa and Bertucci, 2020). Glucose and fatty acids are the most important sources of energy for animal growth (Judge and Dodd, 2020). Animals preferentially use dietary glucose and fatty acids for energy supply. When the body enters a long period of fasting or starvation, stored glycogen, fatty acids, and protein will be successively decomposed and utilized to maintain normal life activities (Judge and Dodd, 2020). In our study, adiponectin (adipoq), 5′-AMP-activated protein kinase subunit gamma-1 (prkag1), and solute carrier family 2, facilitated glucose transporter member 1 (slc2a1) were up-regulated and enriched in the adipocytokine signaling pathway. Studies have shown that adiponectin (encoded by adipoq) can activate AMP-activated protein kinase (AMPK) subunits (encoded by prkag1), thereby directly regulating glucose metabolism and insulin sensitivity (Yamauchi et al., 2002; Schönke et al., 2015). Adiponectin is also an important appetite regulator, modulating energy homeostasis by increasing appetite, boosting substrate storage, and decreasing energy expenditure (Wolf, 2003; Jeon et al., 2021). Glucose transporter 1, a uniporter protein encoded by slc2a1, facilitates glucose diffusion across the cell membrane, regulating the first limiting step (glucose transport into cells) for glucose homeostasis (Yan, 2017; Coudert et al., 2018). Moreover, several genes involved in glycogen degradation and glycolysis were up-regulated in the fast-growth group (Figure 5), including glycogen debranching enzyme (agl), glycogen phosphorylase, muscle form (pygm), phosphoglucomutase-1 (pgm1), ATP-dependent 6-phosphofructokinase, muscle type (pfkm), fructose-bisphosphate aldolase A (aldoa), glyceraldehyde-3-phosphate dehydrogenase (gapdh), phosphoglycerate kinase 1 (pgk1), phosphoglycerate mutase 2 (pgam2), 2,3-bisphosphoglycerate mutase (bpgm), beta-enolase (eno3), pyruvate kinase PKM (pkm), L-lactate dehydrogenase A chain (ldha), and acetyl-coenzyme A synthetase, cytoplasmic (acss2). High expression of these genes indicates that glucose utilization ability is higher in the fast-growth S. grahami fish.

In addition, several genes related to glycolysis were down-regulated in fast-growth S. grahami (Figure 5), including ATP-dependent 6-phosphofructokinase, platelet type (pfkp), aldolase b, fructose-bisphosphate (aldob), fructose-bisphosphate aldolase C (aldoc), phosphoglycerate mutase 1 (pgam1), alpha-enolase (eno1), pyruvate kinase L/R (pklr), and lactate dehydrogenase Bb (ldhbb). Despite similar functions as pfkm, aldoa, pgam2, eno3, and pkm, which are mainly expressed in the muscle, pfkp, aldob, aldoc, pgam1, eno1, and pklr are mainly expressed in non-muscle tissues (e.g., brain, liver, blood) (Verma and Dutta, 1994; Zhang et al., 2001; Caspi et al., 2014; Ausina et al., 2018; Tarnopolsky, 2018; Ždralević et al., 2018). Their downregulation in the fast-growth group and upregulation in the slow-growth group may be a sign of energy homeostasis, whereby energy was supplied to the brain and liver to maintain normal vital activities in the slow-growth group, but excess energy was supplied to the muscles for growth in the fast-growth group.

Several down-regulated DEGs in the fast-growth group were also enriched in the peroxisome and PPAR signaling pathways, which mainly regulate fatty acid transport and β oxidation to degradation (Ordovás et al., 2006; Morais et al., 2007; Watkins et al., 2007; Fidaleo et al., 2011; Shinoda et al., 2020), including peroxisomal acyl-coenzyme A oxidase 1 (acox1), 3-ketoacyl-CoA thiolase B, peroxisomal (acaa1), very long-chain acyl-CoA synthetase (slc27a2), fatty acid binding protein 1-B.1 (fabp1b.1), and long-chain fatty acid transport protein 1 (slc27a1) (Figure 5). These results indicate that fatty acid utilization is lower in the faster growing fish. In addition, some DEGs involved in the regulation of cholesterol and polyunsaturated fatty acid synthesis were also down-regulated in the fast-growth group. Cholesterol and polyunsaturated fatty acids are extremely important biological molecules that play essential roles in membrane structure and are precursors for the synthesis of other biological molecules (Simons and Ikonen, 2000; Christie and Harwood, 2020). Endogenous cholesterol and polyunsaturated fatty acid biosynthesis are affected by existing intracellular levels, i.e., higher food intake leads to lower endogenous biosynthesis in the body, while lower food intake has the opposite effect (Simons and Ikonen, 2000; Xu et al., 2020). This suggests that the fast-growth group had a higher dietary intake of cholesterol and polyunsaturated fatty acids, and thus could not mobilize endogenous synthesis to meet body needs.

Amino acids can directly promote muscle growth in fish, both by stimulating rates of protein synthesis and reducing rates of protein degradation (Seiliez et al., 2008; Cleveland and Radler, 2019). In the current study, we identified several DEGs correlated with glycine, serine, arginine, and proline metabolism (Figure 5), e.g., serine-pyruvate aminotransferase, mitochondrial (agxt), serine hydroxymethyltransferase 1 (shmt1), glutamate-ammonia ligase (glutamine synthase) (glula), cystathionine gamma-lyase (cth), and prolyl 4-hydroxylase subunit alpha-1 (p4ha1). In the fast-growth group, agxt, shmt1, cth, and glula were down-regulated. Agxt encodes serine-pyruvate aminotransferase, which catalyzes the conversion of alanine and glyoxylate into pyruvate and glycine, respectively (Cellini et al., 2007; Montioli et al., 2015), shmt1 catalyzes the transfer of serine to glycine (Wang et al., 2013; Pinthong et al., 2014), cth catalyzes the conversion of cystathionine into cysteine (Krück et al., 2009), and glula regulates de novo glutamine production from glutamate (Eelen et al., 2018). P4ha1, which catalyzes the formation of 4-hydroxyproline (Rappu et al., 2019; Tolonen et al., 2022), was up-regulated in the fast-growth group. These results indicate that the fast-growth group may exhibit stronger 4-hydroxyproline synthesis, lower glycine, cysteine, and glutamine synthesis, and somewhat lower amino acid synthesis ability than the slow-growth group.

Based on the expression of genes related to glucose, fatty acid, and amino acid metabolism, the fast-growth group exhibited higher glucose and lower fatty acid utilization and lower amino acid synthesis activity compared to the slow-growth group. These results suggest that the fast-growth group consumed adequate energy (glucose, fatty acid, and amino acid) from fodder, with excess energy substances used for growth after maintenance of normal life activities. Therefore, energy intake and metabolism are crucial for S. grahami growth. Energy intake may be the root cause for the gap in growth between fast- and slow-growing S. grahami fish. Thus, the genes that regulate appetite and food intake (e.g., adipoq) require further analysis.

The synthesis of macromolecular substances (e.g., protein) is the foundation for growth and is based on the uptake and metabolism of energy substances. Here, we identified several collagen synthesis genes with a high degree of node connection (degree ≥ 18) based on WGCNA, which allows exploration of the correlations among large-scale gene expression data and phenotypes (Langfelder and Horvath, 2008). These genes, including col1a1, col1a2, col5a1, col6a2, col10a1, and col26a1, were up-regulated in the fast-growth group and significantly positively correlated with body length/weight. Genes with a high degree of node connection are significantly correlated with proximity to the center of the network (Zhou et al., 2022). Thus, col1a1, col1a2, col5a1, col6a2, col10a1, and col26a1 may be crucial genes for S. grahami growth. The upregulation of these genes implies that collagen synthesis ability is higher in faster growing fish (Tolonen et al., 2022). Collagen accounts for one-third of total protein in postnatal animals. It is the main component of connective tissue and plays an important role in force transmission and tissue structure maintenance, especially tendons, ligaments, bone, and muscle, as well as in growth, development, and health (KJÆR, 2004; Zhang et al., 2005; Li and Wu, 2018). Glycine, proline, and hydroxyproline are major amino acids, accounting for 57% of total amino acids in collagen (Li and Wu, 2018). In our study, p4ha1 was up-regulated in the fast-growth group. This may promote collagen synthesis as p4ha1 catalyzes 4-hydroxyproline formation, which is essential for proper three-dimensional folding of newly synthesized procollagen chains (Rappu et al., 2019; Tolonen et al., 2022). However, some genes related to glycine and proline synthesis were not markedly different or down-regulated in the fast-growth group. These results suggest that the fast-growth group consumes sufficient amino acids for direct utilization, thereby reducing the endogenous synthesis of amino acids, which is energy efficient and will promote body growth. The increased synthesis of 4-hydroxyproline but not glycine and proline may be because 4-hydroxyproline is produced from proline-containing collagen rather than from free amino acids (Gorres and Raines, 2010).

WGCNA also identified bglap and krt15 as crucial genes with the largest expression differences in S. grahami growth (Figure 1E; Figure 3D). Studies have shown that bglap and krt15 play critical roles in bone formation and mineralization and in structural integrity (Lee et al., 2007; Bose et al., 2013; Komori, 2020). We also identified several growth factors and muscle-growth related genes that were up-regulated in the fast-growth group, such as bone morphogenetic protein 2 (bmp2), bone morphogenetic protein 3 (bmp3), transforming growth factor beta-1 (tgfb1), transforming growth factor beta-2 (tgfb2), growth/differentiation factor 10 (gdf10), myogenin (myog), myosin-binding protein C, fast-type (mybpc2), myosin heavy chain, fast skeletal muscle (myh), myosin light chain, skeletal muscle (myl1, myl3), troponin I, fast skeletal muscle (tnni2), troponin T, fast skeletal muscle (tnnt3), and troponin C, skeletal muscle (tnnc2). Growth/differentiation factors (GDFs), BMPs, and TGF-β are multi-functional growth factors belonging to the TGF-β superfamily and play important roles in development and tissue homeostasis via regulation of cell proliferation, migration, and differentiation, ECM production, multiple cellular signal transduction, cardiogenesis, somite formation, neurogenesis, and musculoskeletal development (Cunningham et al., 1995; Macias et al., 1997; Hino et al., 2004; Maatouk et al., 2009; Heldin and Moustakas, 2016; Zhou et al., 2016). Studies have also found that TGF-β stimulates collagen synthesis and mediates metabolic pathways by regulating the expression of glucose transporter 1 (slc2a1) (Johnston and Gillis, 2017; Yu et al., 2019; Zhou et al., 2021). Myog belongs to the Myogenic Regulatory Factors (MRFs) family, plays a crucial role in myogenesis (De-Santis and Jerry, 2007). Myosin heavy chain and myosin light chain are major component for skeletal muscle myosins (Schiaffino and Reggiani, 1996). Troponin is the key calcium-dependent regulator of striated muscles, and composed of troponin C (TnC), troponin I (TnI), and troponin T (TnT) (Rasmussen and Jin, 2021). Myosin, troponin and myosin-binding protein C are crucial components of skeletal muscle, which essential for myogenesis, muscle contraction (Das et al., 2019; Song et al., 2021; Rasmussen and Jin, 2021). The high expression of these genes would strengthen the muscle growth and contraction.

The WGCNA results also indicated that the differences in collagen synthesis may be the direct cause of the growth gap between the fast- and slow-growth groups of S. grahami. Glycine, proline and hydroxyproline are the main amino acids for collagen synthesis. Endogenous amino acid synthesis (e.g., proline and hydroxyproline) consumes a large amount of adenosine triphosphate (ATP) but is inadequate to meet optimal growth and connective tissue repair (Li and Wu, 2018). Hence, adequate amounts of dietary proline and hydroxyproline are essential for maximizing growth performance and feed efficiency in farmed S. grahami.

Based on transcriptomic analysis of S. grahami muscle, we identified various genes related to glucose, fatty acid, and amino acid uptake and metabolism, and collagen synthesis, which play crucial roles in promoting bone and muscle growth. Energy uptake and collagen synthesis may be the key factors for the growth gap between fast- and slow-growth S. grahami, and energy uptake may be the root cause, while collagen synthesis may be the direct reason. The reasons for differences in uptake and how to improve intake and collagen synthesis require further research. Our findings provide new insights into the mechanism underlying the growth gap between fast- and slow-growth S. grahami and provide an important theoretical basis for guiding S. grahami breeding. Furthermore, these results may provide valuable information for further studying of growth mechanisms and breeding strategies in other species.