BackgroundJapanese quail (Coturnix japonica), a recently domesticated poultry species, is important not only as an agricultural product, but also as a model bird species for genetic research. However, most of the biological questions concerning genomics, phylogenetics, and genetics of some important economic traits have not been answered. It is thus necessary to complete a high-quality genome sequence as well as a series of comparative genomics, evolution, and functional studies.ResultsHere, we present a quail genome assembly spanning 1.04 Gb with 86.63% of sequences anchored to 30 chromosomes (28 autosomes and 2 sex chromosomes Z/W). Our genomic data have resolved the long-term debate of phylogeny among Perdicinae (Japanese quail), Meleagridinae (turkey), and Phasianinae (chicken). Comparative genomics and functional genomic data found that four candidate genes involved in early maturation had experienced positive selection, and one of them encodes follicle stimulating hormone beta (FSHβ), which is correlated with different FSHβ levels in quail and chicken. We re-sequenced 31 quails (10 wild, 11 egg-type, and 10 meat-type) and identified 18 and 26 candidate selective sweep regions in the egg-type and meat-type lines, respectively. That only one of them is shared between egg-type and meat-type lines suggests that they were subject to an independent selection. We also detected a haplotype on chromosome Z, which was closely linked with maroon/yellow plumage in quail using population resequencing and a genome-wide association study. This haplotype block will be useful for quail breeding programs.ConclusionsThis study provided a high-quality quail reference genome, identified quail-specific genes, and resolved quail phylogeny. We have identified genes related to quail early maturation and a marker for plumage color, which is significant for quail breeding. These results will facilitate biological discovery in quails and help us elucidate the evolutionary processes within the Phasianidae family.

AbstrAct:In the present study, quantitative real-time PCR was employed to investigate expression profiling and expression difference of ESR1 and ESR2 in ovaries of Shaoxing duck and Jingjiang duck during three laying stages. Results showed the expression levels of ESR1 and ESR2 in ovaries were increased from the age of the first egg to the age of 500 days in both duck breeds. The expression of ESR1 in Shaoxing duck was lower than that in Jingjiang duck for the age of the first egg and of 180 days, and for the age of 500 days it was higher in Shaoxing duck than in Jingjiang duck. The ESR2 showed converse expression profiling in the two duck breeds. The results suggest that ESR1 and ESR2 mediate the process of egg laying in ducks, and that ESR2 may play a more important role for the ovary during egg-laying stages and may be closely related to the laying performance of the ducks.

The Hongshan chicken is a Chinese indigenous breed that has two distinctly different tail types. Some chickens have stunted tails as compared to the normal phenotype, and they are termed rumpless. Rumplessness in other chicken breeds was caused by a reduction in the number of coccygeal vertebrae. However, X-ray examination showed that rumpless Hongshan chickens possess the normal number of coccygeal vertebrae. Our analyses of the main tail feathers and tissue sections led us to speculate that their stunted tail appearance may be the result of abnormal feather development. To investigate the genetic mechanism underlying rumplessness in Hongshan chickens, we analyzed the results of various crosses. The results indicated that rumplessness is a Z-linked dominant character. In addition, we chose some normal and rumpless individuals for pool-sequencing. Nucleotide diversity and Fst were calculated, and a selective sweep was detected on the Z chromosome. These analyses allowed us to reduce the search area to 71.8–72 Mb on the Z chromosome (galGal5.0). A pseudogene LOC431648 located in this region appeared a strong candidate involving in Wnt/β-catenin signaling pathway to regulate feather development in chickens.

Follicle development is a key factor that determines the reproductive performance of poultry. The existing evidence suggests that circular RNAs (circRNAs) play an important role in a variety of biological processes, especially in posttranscriptional regulation, but the regulatory mechanism of circRNAs in duck follicle development has rarely been reported. To better explore the molecular mechanism of follicle development in ducks, we sequenced and analyzed the follicular circRNAs; 4,204 circRNAs were predicted in the duck follicles. Fourteen circRNAs were differentially expressed between the white follicles and yellow follicles. The results of our studies showed that aplacirc_013267 promoted cell apoptosis in duck GCs. Moreover, a bioinformatics prediction analysis demonstrated that aplacirc_013267 was involved in a circRNA‐miRNA‐mRNA coexpression network and was observed to sponge two follicle‐related miRNAs by a luciferase activity assay. Furthermore, we found that overexpression of aplacirc_013267 significantly increased thrombospondin‐1 (THBS1) expression and downregulated granulosa cell apoptosis. The mechanistic study showed that aplacirc_013267 directly binds to and inhibits apla‐mir‐1‐13; then, aplacirc_013267 increases the expression of THBS1 and upregulates granulosa cell apoptosis. Taken together, our findings demonstrate that circRNAs have potential effects in duck ovarian follicles and that circRNAs may represent a new avenue to understand follicular development.

In this study, the growth hormone (GH) gene was studied as a candidate gene for growth and carcass traits of three duck populations (Cherry Valley duck, Muscovy duck and Jingjiang duck). Three pairs of primers were designed to detect single nucleotide polymorphisms of introns 2, 3 and 4 of the GH gene by polymerase chain reaction-restriction fragment length polymorphism and sequencing methods. Only the products amplified from intron 2 displayed polymorphism. The results showed one novel polymorphism: a variation in intron 2 of GH gene (C172T, JN408701 and JN408702). It was associated with some growth and carcass traits in three duck populations including birth weight, 8-week weight, carcass weight, breast muscle weight, leg muscle weight, eviscerated weight, lean meat rate, dressing percentage, etc. And the TT and CT genotypes were associated with superior growth and carcass traits in carcass weight, dressing percentage and percentage of eviscerated weight. Therefore, the variation in intron 2 of GH may be a molecular marker for superior growth and carcass traits in above duck populations.

Rumplessness in Hongshan chickens has been reported as a novel sex-linked characteristic. Re-sequencing data suggest that a pseudogene on the Z chromosome, LOC431648, is affiliated with this phenotype. In this study, we chose 23 rumpless and 25 normal Hongshan chickens to localize the potential variation by means of a genome-wide association study using a high density microarray. A region on the Z chromosome was found to be closely associated with rumplessness in Hongshan chickens. The region, located in gene LINGO2, was approximately 3 Mb away from pseudogene LOC431648. The function of this gene has not yet been studied in birds. Differential expression of the candidate genes in the tail feather follicles was not detected by q-PCR, which suggests that the rumplessness trait could be attributed to other genetic mechanisms.

Follicles develop into preovulatory follicles during folliculogenesis and the majority of small yellow follicles become atretic and gets reabsorbed. In this study, based the RNA-seq results of duck ovary, epidermal growth factor receptor (EGFR) was selected as a candidate gene in follicular development and the role was explored. The results demonstrated that EGFR-P8 was the quail EGFR core promoter. It had an E2F4 binding site within EGFR core promoter. E2F4 overexpression significantly increased EGFR expression in quail granulosa cells (GCs). However, the effect was abolished when the GCs were treated with corynoxeine, an inhibitor of the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signaling pathway. Moreover, luciferase reporter assay and chromatin immunoprecipitation experiments showed that E2F4 upregulated the expression of EGFR expression, which increased E2 and P4 production. In addition, EGFR regulated GCs proliferation and affected follicular development. Taken together, our findings suggested that EGFR, which was regulated by E2F4, enhanced the expression of MAPK/ERK pathway components and follicular development. These results provided an important basis for an improved understanding of the MAPK/ERK pathway and new insight into the development of quail follicles.

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