Scientific Papers

Construction of a high-density genetic linkage map and QTL mapping of growth and cold tolerance traits in Takifugu fasciatus | BMC Genomics

High density genetic linkage mapping is an important prerequisite for genetic profiling of the location of genes or QTLs associated with target traits [18, 19]. Linkage mapping has been applied to many aquatic species, and growth and cold tolerance are economically important traits for T. fasciatus. To date, no QTL analysis has been reported for growth or cold tolerance traits in T. fasciatus. Therefore, the linkage mapping and QTL analysis in this study will provide a powerful tool for further research and breeding.

High-resolution genetic map

With technological development, genetic mapping has been widely applied to aquatic animal breeding [20]. To date, genetic linkage maps have been constructed for many aquatic animals, such as tiger puffer (Takifugu rubripes) [21] and Chinese giant salamander (Andrias davidianus) [22]. The selection of a mapping population is a prerequisite for constructing a genetic linkage map. Different mapping family types, including recombined inbred lines (RIL), haploid (HAP) and doubled haploid (DH), subgeneration (F1), subgeneration (F2) and back crossing (BC), have advantages and disadvantages [23]. At present, the F1 mapping family is used to construct genetic linkage maps for most aquatic species. In this study, 1,462,637 high-quality SNP markers were screened, and 4,891 SNP markers were obtained for the construction of the F1 T. fasciatus genetic linkage map. The total length was 2281.35 cM, with an average length of 108.243 cM. The average genetic distance of the markers was 0.535 cM, indicating that the marker genetic map constructed in this study was of high density, good quality and uniform marker distribution, and the map contained 22 LGs, which was consistent with the chromosome number of T. fasciatus, indicating that the map had high credibility. As far as we know.This map is the first one to be produced for T. fasciatus, and it is the highest linkage genetic map among all genetic maps for the Takifugu genus. Contrary to the previous genetic map established by Shi [4], which used a full-sib offspring of Takifugu bimaculatus, the total length of their map was 2039.74 cM, and the number of markers was much 1079. The genetic linkage map we constructed is superior to their, which reduced the average genetic distance between neighboring markers from 1.13 cM to 0.535 cM. In the present study, genetic maps of high density and high resolution were constructed, providing an important tool for future fine mapping of MAS.

QTL localization and candidate gene identification of growth-related traits

Growth traits are key to the genetic improvement of economically farmed fish, and many fish species, such as Ctenopharyngodon idellus [24] and Pelteobagrus vachelli [25], have been targeted for growth-related QTL based on the construction of genetic linkage maps. In this study, QTLs for the growth traits of T. fasciatus were analysed for the first time based on a constructed genetic linkage map. QTL analysis is helpful for identifying trait-related linkage markers and predicting candidate genes. Current studies have shown that fish growth traits are mainly regulated by microefficient polygenes. Growth phenotypic traits were not previously isolated but were interlinked. To facilitate subsequent analysis of QTL localization results, 11 growth phenotypic traits (BW, TL, BL, BT, HL, SL, CPL, CPH, IW, ED and BH) were subjected to correlation coefficient analysis in this study, and the results showed strong correlations among BW, TL, and BL. A total of 19 QTLs related to BW, TL and BL were detected in 11 LGs, which indicated that growth traits were influenced by multiple QTLs and genes. For example, Wang et al. [26] identified QTLs for BW in two LGs of Lates calcarife. Liu et al. [27] identified QTLs for BW in Larimichthys polyactis on 3 different LGs. Moreover, BW and BL in this study had the same confidence interval on LG2, 10, 15, 16, 18, 19 and 20, which confirms their relatively high correlation coefficients. This high correlation coefficient has been found in other aquaculture species (e.g.) [21, 27, 28].

The study of candidate genes related to T. fasciatus growth traits can help to improve breeding efficiency of the species. In this study, three candidate genes, IGF1 (insulin-like growth Factor 1), IGF2 (insulin-like growth Factor 2) and ADGRB2 (adhesion g protein-coupled receptor B2), were screened from growth-related QTLs and qRT-PCR experiments were performed. The results showed that the three genes showed different levels of expression in fast-growing fish, indicating that these genes play a potential regulatory function in the rapid growth of T. fasciatus. IGF1 and IGF2 are both considered to be important receptor synthesis mediators that exert biological effects in growth hormone receptor synthesis and belong to a class of growth hormone receptor mediators where insulin plays a decisive role in the growth of tissue cells in the immediate postnatal period in mammals [29]. IGF1 has a specific growth hormone-binding receptor protein (IGFBP) and a growth hormone-binding receptor protein with a specific IGF receptor, an endocrine growth hormone and an endocrine and paracrine growth regulator of its own [30, 31]. The role of IGF1 in growth has been relatively well studied, with the main focus on livestock [32], and relatively little research and reporting in aquaculture. Davis et al. studied polymorphisms in the IGF1 gene and found that this gene polymorphism had a significant effect on weight gain and milk yield growth in cows [33]. In the present study, the expression of IGF1 and IGF2 was elevated in fast-growing fish, and we speculate that they play a role in promoting the growth of T. fasciatus. ADGRB is an evolutionarily ancient subgroup of the GPCR (adhesion G protein-coupled receptors) superfamily, and plays a key molecular switch role in many important physiological processes in organisms [34], such as brain development, neurodevelopment, angiogenesis, water and salt regulation, inflammation and cells. ADGRB2 was significantly expressed in fast growing T. fasciatus, and we speculate that ADGRB2 promotes the fast growth of T. fasciatus. However, research on this gene is still relatively scarce and further in-depth studies can be conducted in the future.

QTL localization and candidate gene identification of cold tolerance traits

T. fasciatus is a warm water migratory economic fish, and breeding cold-tolerant species can promote healthy development of the fish farming industry. In addition, cold tolerance is considered to be an important trait in cultured fish, and is considered to be a qualitative trait controlled by multiple genes [1, 35]. In this study, a total of 11 QTLs associated with cold tolerance were identified, each scattered on different LGs, namely LG2, 3, 5, 6, 11, 12, 14, 15, 17, 19 and 20, suggesting that cold tolerance traits may be controlled by multiple genes on multiple chromosomes. Liu et al. [21] constructed a high-density genetic linkage map for Takifugu rubripes (a close relative of T. fasciatus) and a QTL map of cold resistance traits, and a total of eight QTLs related to cold resistance were detected in LGs 5, 7, 10, 15, 16, and 22. This result could also suggest that the cold tolerance trait may be controlled by multiple genes on multiple chromosomes. Although there are common chromosomes between growth-related traits and cold tolerance traits, they are not in the same location, suggesting that growth and cold tolerance traits are not well correlated and that finding a gene that is correlated with both growth and cold tolerance may be difficult. In the future, we will develop synergistic gene for the growth and low-temperature tolerance of T. fasciatus.

In this study, three genes associated with low-temperature tolerance, HSP90 (Heat shock protein 90), HSP70 (Heat shock protein 70) and HMGB1 (High mobility group Box 1), were screened from the low-temperature tolerance-related QTL for expression analysis under low-temperature stress. All three genes showed different levels of expression in the tissues under low temperature stress, indicating that these genes play a potential regulatory function in the low temperature adaptation of T. fasciatus. HSP90 plays a crucial role in protein folding, cell signalling and protein degradation. HSP90 can be regulated by a variety of environmental stressors, such as heat shock, heavy metals and pathogenic infections [36, 37]. Peng et al. found that kaluga (Huso dauricus) showed significant changes in HSP90 expression in its muscle, gill and liver at low temperatures [38]. The results of Li et al. [39] also showed that HSP90 is significantly expressed at low temperatures in Oryzias melastigma larvae. Similarly, oue results show that HSP90 is significantly expressed in the muscles of T. fasciatus at low temperatures. HSP70 is a kind of highly conservative protein that is rapidly synthesized under stress stress [40]. Such stress response can provide protection, tolerance, and cross tolerance; can mitigate stress caused by abnormal or denatured proteins; can have the effect of activating other cell genes; and can inhibit cell apoptosis caused by ATP loss [41, 42]. Liu et al. [43] found that low-temperature stress increased HSP70 levels in Puntius tetrazona, suggesting that the expression of the molecular chaperone HSP70 in P. tetrazona may play a key role in the response to acute cold stress. In this study, the qRT-PCR results showed that the expression of HSP70 in muscle showed an increasing trend in the low temperature group. HMGB1 is an abundant and charge-rich nuclear protein that plays an important function in organisms [44, 45]. In this study, the expression of HMGB1 in the muscle of T. fasciatus showed an increasing trend in the low temperature group. Therefore, the increased HMGB 1 expression in the muscle of T. fasciatus during cold stress may be a response of the body to protect the fish from low-temperature damage.

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