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Heterosis has been extensively exploited in chicken breeding to improve laying traits in commercial hybrid stock. However, the molecular mechanisms underlying it remains elusive. This study characterizes the miRNAome in the pre-hierarchical follicles of purebred and hybrid laying hens, and investigate the functions of miRNAs with non-additive expression in the pre-hierarchical follicles as they modulate heterosis for egg number and clutch size. To achieve that aim, White Leghorn and Rhode Island Red chicken lines were reciprocally crossed to generate hybrids. The crossbreds demonstrated heterosis for egg number and clutch size, and pre-hierarchical follicles from 4 birds of each genotype were collected at 53 weeks of age. Mode of miRNA expression was characterized after miRNA sequencing. A total of 50 miRNAs including 30 novel ones, were found to exhibit non-additive expression. Dominance was the predominant mode of expression exhibited by majority of the miRNAs. Functional analysis of target genes of the known miRNAs with non-additive expression revealed Gene Ontology terms related to regulation of transcription, metabolic processes and gene expression. KEGG and REACTOME pathways including hedgehog, cellular senescence, wnt, TGF-β, progesterone-mediated oocyte maturation, oocyte meiosis, GnRH signaling, signal transduction and generic transcription, which can be linked to primordial follicle activation, growth and ovulation, were significantly enriched by target genes of miRNAs with non-additive expression. Majority of the genes enriched in these biological pathways were targeted by gga-miR-19a, gga-miR-19b, gga-miR-375, gga-miR-135a, and gga-miR-7 and 7b, thus, revealing their synergistic roles in enhancing processes that could influence heterosis for egg number and clutch size in hybrid hens.

Sexual maturation is fundamental to the reproduction and production performance, heterosis of which has been widely used in animal crossbreeding. However, the underlying mechanism have long remained elusive, despite its profound biological and agricultural significance. In the current study, the reciprocal crossing between White Leghorns and Beijing You chickens were performed to measure the sexual maturation heterosis, and the ovary lncRNAs and mRNAs of purebreds and crossbreeds were profiled to illustrate molecular mechanism of heterosis. Heterosis larger than 20% was found for pubic space and oviduct length, whereas age at first egg showed negative heterosis in both crossbreeds. We identified 1170 known lncRNAs and 1994 putative lncRNAs in chicken ovary using a stringent pipeline. Gene expression pattern showed that nonadditivity was predominant, and the proportion of nonadditive lncRNAs and genes was similar between two crossbreeds, ranging from 44.24% to 49.15%. A total of 200 lncRNAs and 682 genes were shared by two crossbreeds, respectively. GO and KEGG analysis showed that the common genes were significantly enriched in the cell cycle, animal organ development, gonad development, ECM-receptor interaction, calcium signaling pathway and GnRH signaling pathway. Weighted gene co-expression network analysis (WGCNA) identified that 7 out of 20 co-expressed lncRNA-mRNA modules significantly correlated with oviduct length and pubic space. Interestingly, genes harbored in seven modules were also enriched in the similar biological process and pathways, in which nonadditive lncRNAs, such as MSTRG.17017.1 and MSTRG.6475.20, were strongly associated with nonadditive genes, such as CACNA1C and TGFB1 to affect gonad development and GnRH signaling pathway, respectively. Moreover, the results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. Integrated with positive heterosis of serum GnRH and melatonin content detected in crossbreeds, we speculated that nonadditive genes involved in the GnRH signaling pathway elevated the gonad development, leading to the sexual maturation heterosis. We characterized a systematic landscape of ovary lncRNAs and mRNAs related to sexual maturation heterosis in chicken. The quantitative exploration of hybrid transcriptome changes lays foundation for genetic improvement of sexual maturation traits and provides insights into endocrine control of sexual maturation.

Trichomoniasis gallinae (T. gallinae) is one of the most pathogenic parasites in pigeon, particularly in squabs. Oral cavity is the main site for the host-parasite interaction. Herein, we used RNA-sequencing technology to characterize lncRNA and mRNA profiles and compared transcriptomic dynamics of squabs, including four susceptible birds (S) from infected group, four tolerant birds (T) without parasites after T. gallinae infection, and three birds from uninfected group (N), to understand molecular mechanisms underlying host resistance to this parasite. We identified 29,809 putative lncRNAs and characterized their genomic features subsequently. Differentially expressed (DE) genes, DE-lncRNAs and cis/trans target genes of DE-lncRNAs were further compared among the three groups. The KEGG analysis indicated that specific intergroup DEGs were involved in carbon metabolism (S vs. T), metabolic pathways (N vs. T) and focal adhesion pathway (N vs. S), respectively. Whereas, the cis/trans genes of DE-lncRNAs were enriched in cytokine-cytokine receptor interaction, toll-like receptor signaling pathway, p53 signaling pathway and insulin signaling pathway, which play crucial roles in immune system of the host animal. This suggests T. gallinae invasion in pigeon mouth may modulate lncRNAs expression and their target genes. Moreover, co-expression analysis identified crucial lncRNA-mRNA interaction networks. Several DE-lncRNAs including MSTRG.82272.3, MSTRG.114849.42, MSTRG.39405.36, MSTRG.3338.5, and MSTRG.105872.2 targeted methylation and immune-related genes, such as JCHAIN, IL18BP, ANGPT1, TMRT10C, SAMD9L, and SOCS3. This implied that DE-lncRNAs exert critical influence on T. gallinae infections. The quantitative exploration of host transcriptome changes induced by T. gallinae infection broaden both transcriptomic and epigenetic insights into T. gallinae resistance and its pathological mechanism.

Crossed beaks have been observed in at least 12 chicken strains around the world, which severely impairs their growth and welfare. To explore the intrinsic factor causing crossed beaks, this study measured the length of bilateral mandibular ramus of affected birds, and investigated the genome-wide DNA methylation profiles of normal and affected sides of mandibular condyle. Results showed that the trait was caused by impaired development of unilateral mandibular ramus, which is extended through calcification of mandibular condyle. The methylation levels in the CG contexts were higher than that of CHG and CHH, with the highest methylation level of gene body region, followed by transcription termination sites and downstream. Subsequently, we identified 1,568 differentially methylated regions and 1,317 differentially methylated genes in CG contexts. Functional annotation analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that these genes were involved in bone mineralization and bone morphogenesis. Furthermore, by combining the WGBS and previous RNA-Seq data, 11 overlapped genes were regulated by both long non-coding RNA and DNA methylation. Among them, FIGNL1 is an important gene in calcification of mandibular condyle. Generally, because the affected genes play key roles in maintaining mandibular calcification, these changes may be pivotal factors of crossed beaks.