The full-length transcriptional of the multiple spatiotemporal embryo-gonad tissues in chicken (Gallus gallus)

Abstract

Objectives

Chicken (Gallus gallus), as the most economically important poultry, is a classical and ideal model for studying the mechanism of vertebrate developmental biology and embryology. However, the sex determination and differentiation in chicken is still elusive, which limited the application and slowed down many basic studies in chicken.

Data description

We applied PacBio Iso-seq to multiple spatiotemporal embryo-gonad tissues in the male and female chicken, which contain the blastoderm (E0, un-differentiation stage), genital ridge (E3.5–6.5, sex-differentiation stage) and gonads (E18.5, full-sex-differentiation stage). We obtained 51,479 and 48,356 full-length transcripts in male and female chicken embryo, respectively. The comprehensive annotated and evaluated these transcripts. The 1,293 and 1,556 candidate lncRNAs, 5,766 and 4,211 AS events in male and female. Collectively, our data constitutes a grand increase in the known number of lncRNA, AS (Alternative splicing) and Poly(A) during chicken embryo sex-differentiation and plays an important role in improving current genome annotation. In the meantime, the data will be enriched the functional studies in other birds.

Objective

The Chicken (Gallus gallus) is widely used in developmental biology and embryology due to its economic value in the poultry industry [1,2,3,4]. Understanding sex determination and differentiation is crucial as it impacts traits like growth and reproduction [5,6,7]. While female chickens are preferred in layer breeding, males are favored for meat production [8]. Despite the clear Z and W sex chromosomes, the mechanisms behind sex determination in chickens is still elusive [9, 10]. The embryonic gonad originates from the genital ridge at day 3.5 (E3.5) and undergoes sex-specific changes by E6.5, developing into either ZZ testis or ZW ovary [11, 12]. Although the chicken genome was sequenced in 2004 and RNA-seq has advanced, identifying genes involved in sex differentiation is still challenging [13,14,15,16]. CA Smith identified a series of homologous genes in the Z/W chromosome and described the discrepancy via RNA-seq [17]. However, the Short-read sequencing is insufficient for accurately identifying long non-coding RNAs (lncRNAs) and alternatively spliced (AS) genes, which may critical for this process [18,19,20]. The full-length (FL) RNA-seq provides a more accurate representation of lncRNA and gene isoforms, improving genome annotation related to sex differentiation.

In this study, we performed the PacBio Iso-seq for multiple spatiotemporal embryo-gonad tissues of chicken at different times of the developmental stages including; the blastoderm (E0, un-differentiated stage), genital ridge (E3.5–6.5, sex-differentiation stage) and gonads (E18.5, full-sex differentiation stage). Collectively, we obtained 51,479 and 48,356 full-length transcripts from male and female chicken embryonic reproductive organs, respectively. The subsequent systemic functional annotation of these full-length transcripts detected 1,293 and 1,556 candidate LncRNA as well as 5,766 and 4,211 AS events in male and female sex-determining tissues, respectively. This comprehensive dataset provides valuable insights into the roles of lncRNAs and AS events during sex differentiation in chickens and is a critical resource for future studies on sex determination in birds. These data also provide a valuable resource for genomic annotation at different specific chicken embryological developmental stages.

Data description

The Fertilized eggs from Rugao Yellow Chicken were obtained from the Poultry Institute, Chinese Academy of Agricultural Sciences. Eggs were incubated at 37 °C with 75% humidity using a Brinsea Incubator Ova-Easy 100. Embryonic tissues from both male and female chickens were collected at three developmental stages: blastoderm (E0), genital ridge (E3.5–6.5), and gonads (E18.5) (Fig. 1). Tissues were flash-frozen in liquid nitrogen and stored at -80 °C for RNA extraction and sex identification. The RNA was extracted using TRIzol reagent, following the manufacturer’s protocol. RNA integrity was assessed using a NanoDrop2000 spectrophotometer and an Agilent 2100 Bioanalyzer (Data file 6). Sex identification was conducted via PCR amplification of the Chd1 gene, with males showing a single 580 bp band and females showing two bands (580 bp and 423 bp). The two iso-seq libraries were created by pooling RNA from male and female tissues separately. Equal amounts of RNA from each developmental stage (10 μg per tissue) were combined. cDNA was synthesized using the SMARTer PCR cDNA Synthesis Kit, and SMRT bell libraries were constructed using the Pacific Biosciences DNA Template Prep Kit. Sequencing was performed using the PacBio Sequel System(Pac Bio’s Iso-seq™). The PacBio data were processed and evaluated following the figured standard pipeline of the Iso-seq analysis (Fig. 1). Briefly, the raw data were processed using the SMRTlink software to generate circular consensus sequences (CCS). Sequences were refined using IsoSeq3, generating high-quality (HQ) non-chimeric sequences[21, 22]. HQ isoforms were mapped to the Galgal6 reference genome using minimap2 [23], and redundant sequences were collapsed using Cupcake-ToFU. The resulting non-redundant transcripts were analyzed with SQANTI2 for quality control [24]. High-quality transcripts were further annotated using the OrthoDB database, and sequences were aligned with NR, SwissProt, and COG/KOG databases for functional annotation (Fig. 2 and Data file 9). Gene Ontology (GO) classification and KEGG pathway analysis were conducted for deeper insights [25]. TheFunctional annotation was performed using databases like NR, SwissProt, and Pfam [26, 27]. GO terms were assigned to each isoform, identifying key biological processes, cellular components, and molecular functions. KEGG pathway analysis classified transcripts into cellular processes, metabolism, and organismal systems, among others (Fig. 3). A total of 1,293 male and 1,556 female candidate lncRNAs were identified using a customized pipeline based on CPC2, CNCI, Pfam, and PLEK databases [27,28,29].As a final outcome, the interesting isoforms without coding potentials are considered as our candidate lncRNA(Fig. 4) [30, 31].The Alternative splicing (AS) events were identified using Astalavista, with exon skipping being the most common type (Data file 10, 11 and 12 ) [32]. Male tissues showed more AS events than female, suggesting a role in sex differentiation. Poly(A) site analysis revealed differences in distribution between male and female tissues, which exhibit the different pattern of Poly(A) site like UBP1, indicating that alternative polyadenylation may play a critical role during sex differentiation (Fig. 5). All the results indicating that the complexity and diversity of transcription is enhanced by AS and other post-transcription regulation during chicken sex differentiation. Finally, the transcripts were clustered in to total 26,089 male and 23,889 female non-redundant transcripts which were used for further analysis. All data obtained from the Iso-Seq3 analysis were listed in Data file 7. The BUSCO orthologs software was used to assess the transcript completion. As we concerned, the percentage of complete and single-copy BUSCO genes (vertebrata_odb9 dataset, 65 species, 2586 sequences) is 59.1% and 52.6% in male and female full-length transcripts, respectively (Data file 8).

Fig. 1

Experimental design and standard Iso-Seq pipeline for raw data processing

Table 1 Overview of data files/data sets

Fig. 2

The annotation statistics of male and female

Fig. 3

KEGG pathway and GO functional annotations of the male and female full-length transcripts

Fig. 4

Characterization of identified novel lncRNAs

Fig. 5

The total number of AS events and Poly(A) Sites

Collectively, our data represent the first comprehensive full-length transcriptomic resource for chicken embryo sex differentiation, including predicted lncRNA, alternative splicing (AS) events, and Poly(A) signals identified through Iso-seq technology. These findings significantly expand the known repertoire of lncRNAs, AS events, and Poly(A) signals involved in chicken embryo sex differentiation, contributing to improved genome annotation. Importantly, this data may also inform breeding strategies by providing molecular insights into sex determination, offering potential applications to address challenges in the poultry industry related to sex differentiation. Furthermore, the findings enrich functional studies in other bird species and provide a valuable resource for broader vertebrate developmental biology research.

Limitations

The long-read sequencing technology can accurately identify the full length of transcripts, but it still has a significant disadvantage of a high error rate. This issue can be mitigated by combining it with RNA-seq. Therefore, in this study, the results obtained from analyzing samples of both sexes independently still need to be verified through molecular biology techniques (such as qPCR, Northern Bolting, Western Blotting, and in situ Hybridization) on these transcripts in the future.

Code availability

The version and parameters of main software tools are described below:

• (1) SMARTLink: version (v6.0), parameters: pbccs.task options.max.length = 20,000 pbccs.task options.min_length = 300.

• (2) Cupcake-ToFU: version (v4.1), parameters: -i 0.85.

• (3) BUSCO: version (v3.0.1), parameters: default.

• (4) diamond: version (v0.9.7), parameters: --more-sensitive -e 1e-5.

• (5) kobas: version (v3.0), parameters: default.

• (6) blast+: version (v2.6.0), parameters: -evalue 1e-10.

• (7) CPC2: version (v2), parameters: default.

• (8) CNCI: version (v2.0), parameters: -m ve.

• (9) Pfam: version (v2015-06-02), parameters: -e_seq 0.001.

• (10)   Astalavista (v4.0), parameters: default.

• (11)  TAPIS (v1.2.1), parameters: default.