The aim of this study was to analyze the cellular heterogeneity of duck testes before and after maturation using single-cell sequencing technology, to construct a developmental map of duck testicular cells, and to further reveal the process and regulatory mechanisms of testicular maturation.

Each three testes samples from 10 week (immature testis group, IMT) and 23 week old (mature testis group, MT) ducks were collected, and single-cell suspensions were prepared for single-cell sequencing. The quality control, standardization, dimensionality reduction, and cluster analysis on the obtained sequencing data were performed to construct testicular maps of ducks at different developmental stages. Cell subpopulations were annotated, intergroup differences were analyzed, and key regulatory genes and signaling pathways of testicular development and maturation were screened.

More than 30 million high-quality sequencing data were obtained from the each six samples. After quality control, 54702 valid cells were obtained (27756 in the IMT group and 26946 in the MT group). Through cell clustering and identification of marker genes, 21 cell sub-populations were successfully identified, including 10 germ cell sub-populations and 11 somatic cell sub-populations. During the development and maturation of duck testes, spermatogonial stem cells, spermatogonia, primary spermatocytes, and Sertoli cells type 1 and type 3 decreased significantly, while intermediate spermatocytes, secondary spermatocytes, round spermatids, elongated spermatids, and Sertoli cells type 2 and type 4 increased significantly. Differential gene analysis identified 4495 differential genes (1737 up-regulated and 2758 down-regulated). Among them, genes such as Gm614, SPATC1, TSSK6, ZIC1, PHF7, Armc12 and FAM166C were significantly up-regulated in germ cells, and genes such as PKIA, SNX22, Wnt6, LAMA1, SCN4B, CDH6 and FAM110C were significantly down-regulated in Sertoli cells. Functional enrichment analysis showed that the top 20 GO terms all belonged to the cell component category. KEGG analysis indicated that signaling pathways such as protein processing in the endoplasmic reticulum and cellular senescence played key regulatory roles. Furthermore, protein-protein interaction network analysis of differentially expressed genes of these two signaling pathways showed that SEC63 regulated the protein processing process in the endoplasmic reticulum through interactions with ERLEC1, AMFR and RAD23B, etc., while CDK6 might regulate the cellular senescence signaling pathway through interactions with CHEK2, CCNA2, CCNA1 and CDC25, etc.

This study constructed the single-cell transcription map of duck testes before and after maturity for the first time, revealed the dynamic change rules of cell sub-populations during testicular development, discovered a large number of differentially expressed genes, and suggested that SEC63 and CDK6 might play important roles in the testicular maturation mechanism by regulating the endoplasmic reticulum stress response and cell cycle progression, respectively.

This study aimed to screen and identify single nucleotide polymorphisms (SNPs) and candidate genes associated with egg production traits in Jinding ducks using genome-wide association study (GWAS) technology, providing the reference information for molecular breeding of Jinding ducks.

A group of 441 healthy female Jinding ducks from the same batch was used as the research subjects, and the number of eggs laid by each duck from the first egg until 43, 55, and 72 weeks of age (referred to W43, W55 and W72, respectively) was collected. Blood samples were collected from the wing veins of all 441 individuals for whole-genome resequencing. The sequencing data was aligned with the duck reference genome (ZJU1.0) to identify SNPs across the genome. After quality control using plink software to obtain high-quality SNPs, the heritability of egg production at 43, 55 and 72 weeks of age as well as the genetic and phenotypic correlations between each pair of traits were estimated using multi-trait animal model of the ASReml-R 4.2 software. Gemma software was used to separately conduct genome-wide association analyses for the three egg production traits, identifying the associated marker loci shared by them. Then, Bedtools software was employed to annotate these loci and mine for candidate genes near the markers.

The heritability of egg production at 43, 55, and 72 weeks of age in Jinding ducks was relatively low, ranging from 0.17 to 0.30, and decreased gradually with increasing age. There was a high genetic positive correlation between egg production at different ages, with higher genetic correlation coefficients between adjacent time points than between non-adjacent time points. The genetic correlation coefficient between 43 and 55 weeks of age was 0.75, and between 55 and 72 weeks of age was 0.89. The GWAS results showed that 174 suggestively significant SNPs (P<9.43×10^-7) were identified for the egg production trait at 43 weeks of age, distributed on chromosomes 3, 13, and 21; 297 suggestively significant SNPs (P<9.43×10^-7) were identified for the egg production trait at 55 weeks of age, distributed on chromosomes 3 and 13; 36 suggestively significant SNPs (P<9.43×10^-7) were identified for the egg production trait at 72 weeks of age, all located on chromosome 3.20 overlapping suggestively significant SNPs (P<9.43×10^-7) were shared among the three egg production traits, all located on chromosome 3. These loci formed two large haplotype blocks and involved 5 candidate genes, including the VSNL1 gene involved in calcium ion action, the MSGN1 gene involved in mesoderm formation, the KCNS3 gene with potassium ion channel function, and the SMC6 and GEN1 genes involved in DNA repair.

The genetic parameters of egg production traits at 43, 55 and 72 weeks of age in Jinding ducks were estimated, and 20 suggestively significant SNPs and 5 candidate genes affecting egg production traits at 3 different ages were identified through GWAS, providing the reference information for molecular breeding of egg production traits in Jinding ducks.