Comparative study of PGCs cultivation systems HiS and FAcs: a transcriptomic and cellular biology perspective.

In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs.

Role of PI3K/AKT signaling pathway involved in self-renewing and maintaining biological properties of chicken primordial germ cells.

Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

Comparison of primordial germ cell differences at different developmental time points in chickens.

Objective: Recently, the application in the field of germplasm resource conservation has become an important application of primordial germ cells (PGCs). However, due to the lack of deep understanding of the biological characteristics of PGCs at different time points, there is no systematic scheme for the selection of PGCs at which time points in practical application, which affects the practical application effect of PGCs. This study aims to clarify the differences in PGCs during development. Methods: Here, migration experiment, EdU proliferation assay and cell apoptosis assay were conducted to compare the differences in the migration ability, the proliferation ability and the recovery efficiency among female and male PGCs at E3.5, E4.5 and E5.5, which were explained by the following transcriptome sequencing analysis. Results: We found that there were larger differences between female and male PGCs at different embryonic ages, while smaller differences between female and male PGCs at the same embryonic age. Further comparison showed that the cell migration ability of female and male PGCs decreased gradually during development, so female and male PGCs at E3.5 are more suitable for in vitro allotransplantation. At the same time, the proliferation ability of PGCs gradually decreased during development, and cell adhesion and extracellular matrix communication were weakened, indicating that female and male PGCs of E3.5 are more suitable for in vitro long-term culture cell line establishment. Interestingly, female and male PGCs at E5.5 showed strong DNA damage repair ability, thus more suitable for in vitro long-term cryopreservation. Conclusion: This study provides a theoretical basis for systematically selecting PGCs at suitable developmental time points as cell materials for efficient utilization by analyzing the characteristics of female and male PGCs at different developmental time points based on transcriptome.

Ubiquitination plays an important role during the formation of chicken primordial germ cells.

As an important post-translational modification, ubiquitination plays an important role in regulating protein homeostasis in eukaryotic cells. In our previous studies, both the transcriptome and proteome suggested that ubiquitination is involved in the formation of chicken primordial germ cells (PGCs). Here, affinity enrichment combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) was used to analyze the ubiquitome during the differentiation from embryonic stem cells (ESCs) to PGCs, and we identify that 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins. Furthermore, GO and KEGG enrichment analysis showed that ubiquitination regulates key proteins to participate in the progression of key events related to PGC formation and the transduction of key signals such as Wnt, MAPK and Insulin signals, followed by the detailed explanation of the specific regulatory mechanism of ubiquitination through the combined proteome and ubiquitome analysis. Moreover, both the activation and inhibition of neddylation were detrimental to the maintenance of the biological characteristics of PGCs, which also verified the importance of ubiquitination. In conclusion, this study provides a global view of the ubiquitome during the formation of PGCs by label-free quantitative ubiquitomics, which lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs.

Identification and functional analysis of circular RNA expression profiles associated with ammonia exposure in chicken lungs.

Ammonia acts as a detrimental atmospheric pollutant, posing a sever threat to respiratory tract health and causing lung injury in humans and animals. Circular RNAs (circRNAs) are a distinctive class of non-coding RNA generated by back-splicing of linear RNA, implicated in various biological processes. However, their role in the immune response of chicken lungs to ammonia exposure remains unclear. In this study, we examined the expression profiles of circRNAs in chicken lungs under ammonia stimulation. In total, 61 differentially expressed (DE) circRNAs were identified between the ammonia exposure and control groups, including 17 up-regulated and 44 down-regulated circRNAs. The source genes of these DE circRNAs were predominantly enriched in Influenza A, SNARE interactions in vesicular transport, and Notch signaling pathway. Notably, nine DE circRNAs (circNBAS, circMTIF2, circXPO1, circSNX24, circRAB11A, circARID3B, circUSP54, circPPARA, and circERG) were selected for validation the reliability and authenticity of RNA-seq data. Results showed the back-splicing circular structure, as well as the reliability and accuracy of RNA-seq data in quantifying circRNA expression, as the RT-qPCR results were in agreement with the RNA-seq data. Moreover, we constructed the circRNA-miRNA-mRNA regulatory networks and identified several regulatory networks in chicken lungs under ammonia stimulation, including circRAB11A-gga-miR-191b-3p-BRD2 and circARID3B-gga-miR-1696-CKS2. Taken together, our study delineates the circRNA expression profile and their potential roles in the immune response of chicken lungs to ammonia exposure. These findings offer insights into molecular mechanisms that may mitigate diseases associated with ammonia induced respiratory tract pollution in humans and animals.

DDX5 Can Act as a Transcription Factor Participating in the Formation of Chicken PGCs by Targeting BMP4.

As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4-a key gene for PGC formation-and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro.

Identification of Two Potential Gene Insertion Sites for Gene Editing on the Chicken Z/W Chromosomes.

The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.

Mechanisms of Embryonic Stem Cell Pluripotency Maintenance and Their Application in Livestock and Poultry Breeding.

Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock.

The Effect of Inhibiting the Wingless/Integrated (WNT) Signaling Pathway on the Early Embryonic Disc Cell Culture in Chickens.

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/ß-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal-Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.

lncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-ß signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

The biogenesis, identification, and functionality of circWWP2 in lipopolysaccharide stimulated macrophages.

CircRNA, a non-coding RNA, is an ideal biomarker and a suitable potential therapeutic target for various disease due to its high stability, species conservation and cell/tissue specificity. Our previous study has found a circular RNA WWP2 (circWWP2) was significantly decreased in chicken macrophages during bacterial infection. However, the function of circWWP2 in chicken macrophages remains unclear. In this study, it was demonstrated that circWWP2 was a stable circular RNA created by back-splicing of exons 2 to 4 of WWP2 via PCR amplification, Sanger sequencing, RNase R exonuclease digestion, and RT-qPCR. Moreover, bioinformatics analysis showed circWWP2 could interact with 13 miRNAs and target 3,264 genes, which were significantly enriched in lysosomes, IgA-producing intestinal immune networks for IgA production, and Notch signaling pathway. Furthermore, CCK8 and RT-qPCR indicated that overexpression of circWWP2 could promote lipopolysaccharide (LPS)-induced cellular injury by decreasing cell viability and increasing the expression levels of pro-inflammatory cytokines and pro-apoptosis genes, and NO production. CircWWP2 may exert a potential target for the treatment of bacterial infection. Further experiments are necessary to validate the specific mechanism that circWWP2 regulates LPS induced cellular immune responses.

Autophagy activation alleviates the LPS-induced inflammatory response in endometrial epithelial cells in dairy cows.

PROBLEM: Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. METHODS OF STUDY: In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. RESULTS: Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. CONCLUSIONS: These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.

The Establishment and Optimization of a Chicken Primordial Germ Cell Induction Model Using Small-Molecule Compounds.

In recent years, inducing pluripotent stem cells to differentiate into functional primordial germ cells (PGCs) in vitro has become an important method of obtaining a large number of PGCs. However, the instability and low induction efficiency of the in vitro PGC induction system restrict the application of PGCs in transgenic animal production, germplasm resource conservation and other fields. In this study, we successfully established a two-step induction model of chicken PGCs in vitro, which significantly improved the formation efficiency of PGC-like cells (PGCLCs). To further improve the PGC formation efficiency in vitro, 5025 differentially expressed genes (DEGs) were obtained between embryonic stem cells (ESCs) and PGCs through RNA-seq. GO and KEGG enrichment analysis revealed that signaling pathways such as BMP4, Wnt and Notch were significantly activated during PGC formation, similar to other species. In addition, we noted that cAMP was activated during PGC formation, while MAPK was suppressed. Based on the results of our analysis, we found that the PGC formation efficiency was significantly improved after activating Wnt and inhibiting MAPK, and was lower than after activating cAMP. To sum up, in this study, we successfully established a two-step induction model of chicken PGCs in vitro with high PGC formation efficiency, which lays a theoretical foundation for further demonstrating the regulatory mechanism of PGCs and realizing their specific applications.

Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture.

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 µg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 µg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 µg/mL and 100 µg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 µg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

Analysis of the Promoter Regions of gga-miR-31 and Its Regulation by RA and C-jun in Chicken.

The role of gga-miR-31 in chicken germ cell differentiation and spermatogenesis is of significant importance. The transcriptional properties of gga-miR-31 are crucial in establishing the foundation for the formation of chicken spermatogonia stem cells and spermatogenesis. In this study, a series of recombinant vectors including varying lengths of the gga-miR-31 promoter were predicted and constructed. Through the utilization of the dual luciferase reporting system, the upstream -2180~0 bp region of gga-miR-31 was identified as its promoter region. Furthermore, it was predicted and confirmed that the activity of the gga-miR-31 promoter is increased by retinoic acid (RA). The binding of RA to the gga-miR-31 and Stra8 promoter regions was found to be competitive. Through the deletion of C-jun binding sites and the manipulation of C-jun expression levels, it was determined that C-jun inhibits the activity of the gga-miR-31 promoter. Furthermore, the combined treatment of C-jun and RA demonstrated that the positive regulatory effect of RA on the gga-miR-31 promoter is attenuated in the presence of high levels of C-jun. Overall, this study establishes a foundation for further investigation into the regulatory mechanisms of gga-miR-31 action, and provides a new avenue for inducing chicken embryonic stem cells (ESC) to differentiate into spermatogonial stem cells (SSC), and sperm formation.

The First Crested Duck Genome Reveals Clues to Genetic Compensation and Crest Cushion Formation.

The Chinese crested (CC) duck is a unique indigenous waterfowl breed, which has a crest cushion that affects its survival rate. Therefore, the CC duck is an ideal model to investigate the genetic compensation response to maintain genetic stability. In the present study, we first generated a chromosome-level genome of CC ducks. Comparative genomics revealed that genes related to tissue repair, immune function, and tumors were under strong positive selection, indicating that these adaptive changes might enhance cancer resistance and immune response to maintain the genetic stability of CC ducks. We also assembled a Chinese spot-billed (Csp-b) duck genome, and detected the structural variations (SVs) in the genome assemblies of three ducks (i.e., CC duck, Csp-b duck, and Peking duck). Functional analysis revealed that several SVs were related to the immune system of CC ducks, further strongly suggesting that genetic compensation in the anti-tumor and immune systems supports the survival of CC ducks. Moreover, we confirmed that the CC duck originated from the mallard ducks. Finally, we revealed the physiological and genetic basis of crest traits and identified a causative mutation in TAS2R40 that leads to crest formation. Overall, the findings of this study provide new insights into the role of genetic compensation in adaptive evolution.

Genetic diversity and breed identification of Chinese and Vietnamese local chicken breeds based on microsatellite analysis.

South Asia and Southeast Asia are the origins of domestic chickens and are rich in poultry genetic resources, resulting in many unique local chicken breeds. However, with the rapid intensification of poultry farming worldwide, many local chicken breeds are threatened with extinction. In response to China's "One Belt, One Road" policy, it is imperative to strengthen the conservation and breeding of local chicken breeds in China and Vietnam. This study characterized 18 microsatellite molecular genetic markers to analyze the genetic diversity of 21 local chicken populations in southern China (Yunnan and Guangxi Provinces) and Vietnam, breed identification tags for microsatellite loci were constructed. The results showed that a total of 377 alleles were detected in all breeds, and the most alleles (44) and the highest polymorphic information content (0.7820) were detected at the LEI0094 locus. The average polymorphic information content (PIC) content of the whole population was 0.65, indicating moderate polymorphism. The genetic diversity of the whole population was rich, except for two loci MCW0111 and MCW0016, that showed heterozygote excess at microsatellite loci, and the population had high genetic differentiation. The Vietnamese breeds showed low pairwise fixation coefficient (FST) and Nei's standard genetic distance (DS) between them. According to the neighbor-joining dendrogram constructed by DS and the analysis of population genetic structure using the structure program, Longshengfeng chicken, Yunlong dwarf chicken, Tengchong white chicken, Xiayan chicken, and Daweishan mini chicken are similar, and Xishuangbanna game fowl, Wuding chicken, and Lanping silky chicken are similar to Yanjin black-bone chicken. In addition, excluding Dongtao chicken, other Vietnamese breeds are clustered together, indicating that the southern chicken breeds are closely related and have experienced better breeding. Overall, the whole population is rich in genetic resources, and the chicken breeds in the three regions are genetically close because of geographical factors and human activities. Dongtao chicken in Vietnamese, Chinese Yunnan local chicken breeds (Gallus gallus spadiceus), and red jungle fowl chickens (Gallus gallus) may have the same origin. We also constructed unique microsatellite molecular markers for 20 cultivars using 15 microsatellite loci. This study provides valuable insights to facilitate breed identification, improve cultivar protection, and new germplasm construction.

China's Yunnan, Guangxi, and Vietnam are rich in biodiversity, and a wide variety of local chicken breeds exist. Due to the rapid development of intensive farming, the biodiversity of chicken breeds in these two regions has gradually decreased. To protect the diversity of local breeds, and to promote the exchange of germplasm resources and the creation of new germplasm between China and Vietnam, this study analyzed the genetic diversity of some chicken breeds in Yunnan, Guangxi, and Vietnam by using microsatellite molecular markers. Studies have shown that the entire population has rich genetic resources, and Vietnamese chickens have a strong kinship with some local chicken breeds in Yunnan Province, China, and there is also a high degree of hybridization between Vietnamese local chickens. In addition, since some chicken breeds have more similarities in appearance, this study constructed unique molecular tags for different breed-specific alleles as one of the methods to accurately distinguish different breeds for breed protection and subsequent breeding.

Inhibition of Autophagy Maintains ESC Pluripotency and Inhibits Primordial Germ Cell Formation in Chickens.

Autophagy plays an important role in the pluripotency and differentiation of stem cells. Transcriptome data showed that the autophagy genes MAP1LC3A and MAP1LC3B were significantly upregulated in primordial germ cells (PGCs). The Kyoto Encyclopedia of Genes and Genome (KEGG) results showed that the lysosome signaling pathway, which is related to autophagy, was significantly enriched in PGCs. Quantitative RT-PCR, western blotting, and transmission electron microscopy (TEM) results showed that autophagy was expressed in both embryonic stem cells (ESCs) and PGCs but was significantly activated in PGCs. To explore the role of autophagy in the differentiation of chicken ESCs into PGCs, autophagy was activated and inhibited using rapamycin and bafilomycin A1, respectively. Results of qRT-PCR, flow cytometry, and indirect immunofluorescence showed that the efficiency of PGC formation significantly decreased after autophagy inhibition. Our results showed, for the first time, that autophagy plays an indispensable role in the formation of chicken PGCs, which lays the foundation for studying the mechanism of autophagy in chicken PGCs and in bird gene editing and the rescue of endangered birds.