Expansion and contraction of lake basin shape the genetic structure of Sinocyclocheilus (Osteichthyes: Cypriniformes: Cyprinidae) populations in Central Yunnan, China.

Geological events can strongly affect the genetic structures and differentiation of fish populations. Especially, as an endemic fish of the genus Sinocyclocheilus in the Yunnan-Guizhou Plateau, the effects of key geological events on the distributions and genetic structures remain poorly understood. Examining the phylogeographic patterns of Sinocyclocheilus fishes can be useful for elucidating the spatio-temporal dynamics of their population size, dispersal history and extent of geographical isolation, thereby providing a theoretical basis for their protection. Here, we used single nucleotide polymorphisms (SNP) method to investigate the phylogeographic patterns of Sinocyclocheilus fishes. Our analysis supports the endemicity of Sinocyclocheilus, but the samples of different regions of Sinocyclocheilus contain multiple ancestral components, which displayed more admixed and diversified genetic components, this may be due to the polymorphism of the ancestors themselves, or gene infiltration caused by hybridization between adjacent species of Sinocyclocheilus. We estimate that the most recent common ancestor (MRCA) of Sinocyclocheilus fish in the Central Yunnan Basin at approximately 3.75~3.11 Ma, and infer that the evolution of Sinocyclocheilus in the central Yunnan Basin is closely related to the formation of plateau lakes (around 4.0~0.02 Ma), and identifies the formation of Dianchi Lake and Fuxian Lake as key geological events shaping Sinocyclocheilus population structure. It is also the first time to prove that the altitude change has a great influence on the genetic variation among the populations of Sinocyclocheilus.

Muscle transcriptome analysis provides new insights into the growth gap between fast- and slow-growing Sinocyclocheilus grahami.

Sinocyclocheilus grahami is an economically valuable and famous fish in Yunnan Province, China. However, given its slow growth (40 g/2 years) and large growth differences among individuals, its growth performance needs to be improved for sustainable future use, in which molecular breeding technology can play an important role. In the current study, we conducted muscle transcriptomic analysis to investigate the growth gaps among individuals and the mechanism underlying growth within 14 fast- and 14 slow-growth S. grahami. In total, 1,647 differentially expressed genes (DEGs) were obtained, including 947 up-regulated and 700 down-regulated DEGs in fast-growth group. Most DEGs were significantly enriched in ECM-receptor interaction, starch and sucrose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, amino acids biosynthesis and metabolism, peroxisome, and PPAR signaling pathway. Some genes related to glycogen degradation, glucose transport, and glycolysis (e.g., adipoq, prkag1, slc2a1, agl, pygm, pgm1, pfkm, gapdh, aldoa, pgk1, pgam2, bpgm, and eno3) were up-regulated, while some genes related to fatty acid degradation and transport (e.g., acox1, acaa1, fabp1b.1, slc27a1, and slc27a2) and amino acid metabolism (e.g., agxt, shmt1, glula, and cth) were down-regulated in the fast-growth group. Weighted gene co-expression network analysis identified col1a1, col1a2, col5a1, col6a2, col10a1, col26a1, bglap, and krt15 as crucial genes for S. grahami growth. Several genes related to bone and muscle growth (e.g., bmp2, bmp3, tgfb1, tgfb2, gdf10, and myog) were also up-regulated in the fast-growth group. These results suggest that fast-growth fish may uptake adequate energy (e.g., glucose, fatty acid, and amino acids) from fodder, with excess energy substances used to synthesize collagen to accelerate bone and muscle growth after normal life activities are maintained. Moreover, energy uptake may be the root cause, while collagen synthesis may be the direct reason for the growth gap between fast- and slow-growth fish. Hence, improving food intake and collagen synthesis may be crucial for accelerating S. grahami growth, and further research is required to fully understand and confirm these associations.

Identification and functional characteristics of two TLR5 subtypes in S. grahami.

TLR5, as a member of Toll-like receptors (TLRs) family in mammals, is responsible for recognizing bacterial flagellin and initiating innate immunity, but its function is still unclear in fish species. In this study, two family members of TLR5 were cloned and identified from Sinocyclocheilus grahami (S. grahami), named sgTLR5a and sgTLR5b. The length of coding sequence of sgTLR5a and sgTLR5b is 2,622 bp and 2,658 bp, encoding 873 and 885 amino acids, respectively. Molecular phylogenetic analysis indicates that sgTLR5a and sgTLR5b have the closest genetic relationship with TLR5M (membrane-type) of Cyprinus carpio and Schizothorax prenanti, respectively. sgTLR5a and sgTLR5b were widely expressed in various tested tissues, of which the expression levels were the highest in skin tissue. After stimulations of Aeromonas hydrophila (A. hydrophila) and flagellin, the expression levels of sgTLR5a and sgTLR5b in liver, spleen and head kidney tissues were strongly up-regulated, but LPS stimulation only increased the expression of sgTLR5b in these tissues. The luciferase reporter assay displayed that sgTLR5a and sgTLR5b could specifically recognize bacterial flagellin and A. hydrophila and activate the downstream NF-κB signaling pathway in HEK293T cells. Moreover, the overexpression of sgTLR5a and sgTLR5b in EPC cells up-regulated the expression levels of IL-8 and TNF. sgTLR5a and sgTLR5b were observed to locate in the intracellular region by confocal microscope. Interestingly, we found that the NF-κB signaling pathway was positively regulated by co-transfecting sgTLR5a or sgTLR5b with TLR trafficking chaperone sgUNC93B1. In conclusion, our results reveal sgTLR5a and sgTLR5b may play an important role in antibacterial response by activating the NF-κB signaling pathway.

Construction of a chromosome-level genome assembly for genome-wide identification of growth-related quantitative trait loci in Sinocyclocheilus grahami (Cypriniformes, Cyprinidae).

The Dianchi golden-line barbel, Sinocyclocheilus grahami (Regan, 1904), is one of the "Four Famous Fishes" of Yunnan Province, China. Given its economic value, this species has been artificially bred successfully since 2007, with a nationally selected breed (" S. grahami, Bayou No. 1") certified in 2018. For the future utilization of this species, its growth rate, disease resistance, and wild adaptability need to be improved, which could be achieved with the help of molecular marker-assisted selection (MAS). In the current study, we constructed the first chromosome-level genome of S. grahami, assembled 48 pseudo-chromosomes, and obtained a genome assembly of 1.49 Gb. We also performed QTL-seq analysis of S. grahami using the highest and lowest bulks (i.e., largest and smallest size) in both a sibling and random population. We screened two quantitative trait loci (QTLs) (Chr3, 14.9-39.1 Mb and Chr17, 4.1-27.4 Mb) as the major growth-related locations. Several candidate genes (e.g., map2k5, stat1, phf21a, sox6, and smad6) were also identified, with functions related to growth, such as cell differentiation, neuronal development, skeletal muscle development, chondrogenesis, and immunity. These results built a solid foundation for in-depth MAS studies on the growth traits of S. grahami.