Nonadditive and Asymmetric Allelic Expression of Growth Hormone in Hybrid Tilapia

Abstract

Hybridization is a common breeding technique that can improve germplasm through heterosis in aquaculture. However, the regulation of key gene expression, including the details of transcriptional level changes at the beginning of hybridization events, remains largely undefined, especially in teleosts. In this study, by interspecies crossing between two pure lines of Nile tilapia and blue tilapia, we obtained a hybrid tilapia population as a model to elucidate heterosis, and we traced the molecular outcomes of growth hormone (GH) expression and allele-specific expression (ASE) in hybrids. The hybrids display growth vigor compared to their parents in the 120-day growth trial. GH mRNA expression was uniquely expressed in the pituitary. Higher GH expression was found in the hybrid than the midparent value, in both males and females, showing a nonadditive pattern. We identified four single-nucleotide polymorphism sites between Nile tilapia and blue tilapia. Subsequently, by pyrosequencing, we found asymmetric allelic expression in hybrids with higher maternal allelic transcript ratios in both males and females. Fasting significantly increased GH expression in hybrids, but asymmetric allelic expression was not affected by feeding or fasting conditions. Finally, we identified cis and trans effects via overall expression and ASE values in the hybrid, which showed that the cis and trans effects promoted the expression of maternal subgenome in the hybrid, contributing to the expression superiority of GH in hybrid tilapia. Taken together, the results of our study first illustrated the concept of GH expression superiority and its formation mechanism in hybrid fish with growth vigor.

Keywords: allele specific expression; growth hormone; heterosis; hybrid; nonadditive expression; tilapia.

Figure 1

Generation of hybrid tilapia and growth performance. (A) The parents of the hybrid (Nile tilapia and blue tilapia) were obtained nine times after self-mating to guarantee a pure genetic background. Then, hybrid tilapia was obtained by Nile tilapia and blue tilapia crossing. The genotypes of Nile tilapia, blue tilapia, and hybrid were represented as NN, BB and NB, respectively. (B) Body weight and SGR comparison among Nile tilapia, blue tilapia, and hybrid. Data are presented as the mean ± SEM (n = 20). Asterisks indicate significant differences between groups (P < 0.05).

Figure 2

Overall expression of GH in hybrid tilapia. (A) Tissue distribution analysis of GH mRNA expression by RT-PCR. (B) Comparison of GH mRNA expression in pituitary from 5-month-old Nile tilapia, blue tilapia, and hybrid by qPCR. (C) Comparison of GH mRNA expression in pituitary from 2-year-old Nile tilapia, blue tilapia, and hybrid by qPCR. Data are presented as the mean ± SEM (n = 5). Asterisks indicate significant differences between groups (P < 0.05). Dashed lines indicate MPV calculated from the average value of Nile tilapia and blue tilapia.

Figure 3

Identification of SNP between Nile tilapia and blue tilapia. (A) Four SNPs were identified from the cDNA sequences of Nile tilapia and blue tilapia by alignment using ClustalW. Bent arrow represents the transcription start site. Empty boxes represent the 5′ and 3′ UTRs. Red boxes represent exons. Broken lines indicate introns. Red arrows and asterisks indicate the SNP sites. (B) Validation of the third SNP site (nucleotide 501) from the DNA sequences by Sanger sequencing in Nile tilapia (N1–N6), blue tilapia (B1–B6), and hybrid (NB1–NB6). Red arrows indicate the SNP sites.

Figure 4

ASE analyses of GH in hybrid. (A) Asymmetric allelic expression of GH in male hybrids and female hybrids by pyrosequencing. Six individuals of male hybrids and six female hybrids were included based on the C/G SNP site (nucleotide 501). (B) Allelic transcript ratios of C/G in hybrid calculated from males (n = 6), females (n = 6) and total studied individuals (n = 12). Data are presented as the mean ± SEM. Asterisks indicate significant differences between groups (P < 0.05). Mat, maternal; Pat, parental.

Figure 5

Expression of GH in hybrid tilapia in feeding and fasting conditions. (A) Increasing overall expression of GH in hybrid tilapia by RT-PCR after fasting in hybrid tilapia. Data are presented as the mean ± SEM (n = 4). Asterisks indicate significant differences between groups (P < 0.05). (B) ASE of GH in feeding and fasting hybrids (n = 5) by pyrosequencing based on the C/G SNP site (nucleotide 501). (C) Allelic transcript ratios of C/G in feeding and fasting hybrids. Data are presented as the mean ± SEM (n = 6). Asterisks indicate significant differences between groups (P < 0.05). Mat, maternal; Pat, parental.

Figure 6

GH cis and trans regulation in hybrid tilapia. (A) Identification of enhancing cis and trans effects was found in the hybrid by calculating the value of overall expression (data from Figure 2 ) and ASE (data from Figure 4 ). (B) Possible mode of N and B allele interactions contributes to gene expression changes in the hybrid. In hybrids, both cis and trans effects tend to increase the transcriptional activities of GH genes in the same direction. These regulations were more sensitive for the subgenome of Nile tilapia. As a result, GH in the hybrid is higher than that in its parents, while the expressions of B alleles were not changed. Nile tilapia, blue tilapia, and hybrid were represented as NN, BB, and NB, respectively. Red and blue boxes indicate genes of Nile tilapia and blue tilapia, respectively. Lines attached to the genes indicate cis elements. Asterisks indicate trans regulators (Nile tilapia in red; blue tilapia in blue). +: low expression level; ++: medium expression level; +++: high expression level.