De novo Phased Genome Assembly, Annotation and Population Genotyping of Alectoris Chukar.

The Alectoris Chukar (chukar) is the most geographically widespread partridge species in the world, demonstrating exceptional adaptability to diverse ecological environments. However, the scarcity of genetic resources for chukar has hindered research into its adaptive evolution and molecular breeding. In this study, we have sequenced and assembled a high-quality, phased chukar genome that consists of 31 pairs of relatively complete diploid chromosomes. Our BUSCO analysis reported a high completeness score of 96.8% and 96.5%, with respect to universal single-copy orthologs and a low duplication rate (0.3% and 0.5%) for two assemblies. Through resequencing and population genomic analyses of six subspecies, we have curated invaluable genotype data that underscores the adaptive evolution of chukar in response to both arid and high-altitude environments. These data will significantly contribute to research on how chukars adaptively evolve to cope with desertification and alpine climates.

Gene Expression Profiling Reveals Potential Players of Sex Determination and Asymmetrical Development in Chicken Embryo Gonads.

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation (E4.5, E5.5, and E6.5). PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process.

One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a.

African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/µl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.

Dynamics of Small Non-coding RNA Profiles and the Intestinal Microbiome of High and Low Weight Chickens.

The host and its symbiotic bacteria form a biological entity, holobiont, in which they share a dynamic connection characterized by symbiosis, co-metabolism, and coevolution. However, how these collaborative relationships were maintained over evolutionary time remains unclear. In this research, the small non-coding RNA (sncRNA) profiles of cecum and their bacteria contents were measured from lines of chickens that have undergone long-term selection for high (HWS) or low (LWS) 56-day body weight. The results from these lines that originated from a common founder population and maintained under the same husbandry showed an association between host intestinal sncRNA expression profile (miRNA, lncRNA fragment, mRNA fragment, snoRNA, and snRNA) and intestinal microbiota. Correlation analyses suggested that some central miRNAs and mRNA fragments had interactions with the abundance of intestinal microbial species and microbiota functions. miR-6622-3p, a significantly differentially expressed (DE) miRNA was correlated with a body weight gain related bacterium, Alistipes putredinis. Our results showed that host sncRNAs may be mediators of interaction between the host and its intestinal microbiome. This provides additional clue for holobiont concepts.

Corrigendum: The Composition and Function of Pigeon Milk Microbiota Transmitted From Parent Pigeons to Squabs.

[This corrects the article DOI: 10.3389/fmicb.2020.01789.].

Heritable Gut Microbiome Associated with Salmonella enterica Serovar Pullorum Infection in Chickens.

Pullorum disease is one of the most common diarrhea-related diseases caused by Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S Pullorum); it negatively affects the poultry industry. However, limited studies have explored the association between the gut microbiota and S Pullorum infection in chickens. In the present study, we performed a microbiome comparison and a microbiome genome-wide association study (mGWAS) to investigate the association among the host genetics, the gut microbiota, and pullorum disease in chickens. We found that S Pullorum infection in chickens could alter the abundance of 39 bacterial genera (P < 0.05). The altered structure and composition of the gut microbiota were also detected in the offspring. mGWAS results revealed host genetic variants to be prominently associated with gut microbial diversity and individual microbes. The pathogens Pelomonas and Brevundimonas, which had a high abundance in positive parent chickens and their offspring, were significantly associated with several genetic mutations in immunity-related genes, such as TGIF1, TTLL12, and CCR7 This finding explained why Pelomonas and Brevundimonas were heritable in S Pullorum-infected chickens. The heritable gut microbes and identified genetic variants could provide references for the selection of resistant chickens and the elimination of pullorum disease.IMPORTANCE The present study investigated the association among the host genome, the gut microbiome, and S Pullorum infection in chickens. The results suggested that the gut microbial structure is altered in S Pullorum-infected chickens. The diversity and abundance of the gut microbiota remarkably differed between the offspring coming from S Pullorum-positive and S Pullorum-negative chickens. Heritable gut microbiota were detected in the offspring. Moreover, host genetic variants were associated with microbial diversity and individual gut microbes. The pathogens Pelomonas and Brevundimonas, which exhibited a high heritability in S Pullorum-positive parents and their offspring, were associated with several genetic mutations in immunity-related genes.

Transcriptomic Analysis of MSTN Knockout in the Early Differentiation of Chicken Fetal Myoblasts.

In mammals, Myostatin (MSTN) is a known negative regulator of muscle growth and development, but its role in birds is poorly understood. To investigate the molecular mechanism of MSTN on muscle growth and development in chickens, we knocked out MSTN in chicken fetal myoblasts (CFMs) and sequenced the mRNA transcriptomes. The amplicon sequencing results show that the editing efficiency of the cells was 76%. The transcriptomic results showed that 296 differentially expressed genes were generated after down-regulation of MSTN, including angiotensin I converting enzyme (ACE), extracellular fatty acid-binding protein (EXFABP) and troponin T1, slow skeletal type (TNNT1). These genes are closely associated with myoblast differentiation, muscle growth and energy metabolism. Subsequent enrichment analysis showed that DEGs of CFMs were related to MAPK, Pl3K/Akt, and STAT3 signaling pathways. The MAPK and Pl3K/Akt signaling pathways are two of the three known signaling pathways involved in the biological effects of MSTN in mammals, and the STAT3 pathway is also significantly enriched in MSTN knock out chicken leg muscles. The results of this study will help to understand the possible molecular mechanism of MSTN regulating the early differentiation of CFMs and lay a foundation for further research on the molecular mechanism of MSTN involvement in muscle growth and development.

Chromosome level assembly reveals a unique immune gene organization and signatures of evolution in the common pheasant.

The common pheasant Phasianus colchicus, belonging to the order Galliformes and family Phasianidae, is the most widespread species. Despite a long history of captivity, the domestication of this bird is still at a preliminary stage. Recently, the demand for accelerating its transformation to poultry for meat and egg production has been increasing. In this study, we assembled high quality, chromosome scale genome of the common pheasant by using PacBio long reads, next-generation short reads, and Hi-C technology. The primary assembly has contig N50 size of 1.33 Mb and scaffold N50 size of 59.46 Mb, with a total size of 0.99 Gb, resolving most macrochromosomes into single scaffolds. A total of 23,058 genes and 10.71 Mb interspersed repeats were identified, constituting 30.31% and 10.71% of the common pheasant genome, respectively. Our phylogenetic analysis revealed that the common pheasant shared common ancestors with turkey about 24.7-34.5 million years ago (Ma). Rapidly evolved gene families, as well as branch-specific positively selected genes, indicate that calcium-related genes are potentially related to the adaptive and evolutionary change of the common pheasant. Interestingly, we found that the common pheasant has a unique major histocompatibility complex B locus (MHC-B) structure: three major inversions occurred in the sequence compared with chicken MHC-B. Furthermore, we detected signals of selection in five breeds of domestic common pheasant, several of which are production-oriented.

Dynamic distribution of gut microbiota during embryonic development in chicken.

The gut microbiota is a complex ecological community and widely recognized in many aspects of research, but little is known on the relation between gut microbiota and embryonic development in chickens. The aim of this study was to explore the dynamic distribution of gut microbiota in chickens' embryos during stages of developments (chicken embryos that had incubated until day 3 [E3], day 12 [E12], and day 19 [E19]). Here, 16S rRNA gene sequencing was performed on the gut microbiota in chicken embryos across different developmental stages. Twenty-one phyla and 601 genera were present in chicken embryos, and 96 genera such as Ochrobactrum, Phyllobacterium, and Amycolatopsis were the core microbiota in the 3 stages of development. Second, 94 genera of microbes were found to change significantly between E3 and E12, and 143 genera significantly differed between E12 and E19 in chicken embryos (P < 0.05). Ochrobactrum and Amycolatopsis decreased with growth changes: E3 (30.4%), E12 (25.1%), and E19 (13.6%) and E3 (11.5%), E12 (7.4%), and E19 (5.6%), respectively. Contrarily, Phyllobacterium increased to 47.9% at E19, indicating the growing trend of microbial diversity among the embryos' development. Moreover, the principal component analysis showed a high level of similarities between E3 and E12 compared with E19, whereas the alpha analysis showed more diversity of gut microbiota at E19. Furthermore, the functional predictions showed that metabolic pathways such as energy metabolism and genetic information processing were significantly enriched on day 3 and day 12 in our study, suggesting their strong influence on growth, development, and immunity of chicken embryos. Our findings provide insights into the understanding of dynamic shifts of gut microbiota during chicken embryonic growth.

The Gut Microbiota of Pheasant Lineages Reflects Their Host Genetic Variation.

The host gut colonized enormous microbial community, which can be influenced by diet, diseases, behavior, age, gender, hereditary effects, and environmental factors. However, the relationship between gut microbiota and host genetic variation has not yet been elucidated. In this study, we chose five pheasant lineages-Ring-necked pheasant (RN), Manchurian pheasant (MX), Phasianus versicolor (PV), Shenhong pheasant (SP), and Melanistic mutant pheasant (MM)-to investigate the gut microbial composition of pheasants and its relationship with host genetic variation. Microbial classifications revealed 29 phyla and 241 genera presented in pheasants, with the dominant phylum of Firmicutes and the genus of Lactobacillus. Statistical analyses suggest that the relative abundance of 75 genera was significantly different among the five lineages. The most abundant genus carried by the RN and MM was Streptococcus, which was significantly lower in PV (p = 0.024). Conversely, Lactobacillus was the major genera in PV and MX. Moreover, the RN had the greatest microbial abundance, with a remarkably different microbial community than PV. The gut microbial diversity of PV was the lowest and diverged significantly from the RN and MX. Interestingly, the clustering of the MM and SP in the microbial dendrogram corresponded to their cluster in the host phylogeny. The host phylogenetic split of the RN, MX, and PV echoed their microbial distance. In conclusion, the congruence of host phylogeny and their gut microbial dendrograms implies that gut microbiota of pheasant lineages could reflect their host genetic variation.

The Composition and Function of Pigeon Milk Microbiota Transmitted From Parent Pigeons to Squabs.

Mammalian neonates obtain antibodies, nutrients, and microbiota from breast milk that help them resist the complex growth environment. Similar to mammals' lactation behavior for their offspring, parent pigeons regurgitate pigeon milk (PM) from their crops to feed the squabs. Whether pigeon milk is as valuable as mammalian milk is not clear, especially in terms of microbiota. This study adopted 16S rRNA gene sequencing to investigate the microbial composition and function in pigeon milk. We found abundant microbiota in pigeon milk. The dominant genera in parent pigeons' milk were Lactobacillus, Enterococcus, Veillonella, and Bifidobacterium. An analysis of squab milk (SM) showed that Lactobacillus also accounted for a considerable proportion, followed by Bifidobacterium. Most of the squab milk microbial genera were also detected in parent pigeons. Microbial functional analysis showed that the squab milk microbes were more involved in the pathways of carbohydrate metabolism, amino acid metabolism, and energy metabolism. These findings indicated that microbiota play an important role in squabs and can be transmitted from parent pigeons to squabs by pigeon milk. The presence of plentiful probiotics in squabs also suggests that adding probiotics in artificial pigeon milk may promote the growth and development of squabs and improve the production performance of pigeons.