Comparative genome and transcriptome integration studies reveal the mechanism of pectoral muscle development and function in pigeons

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Pigeon breed resources provide a genetic model for the study of phenomics. The pectoral muscles are play a key role for the meat production performance of the meat pigeon and the athletic ability of the High flyers. Euro-pigeons and Silver King are commercial varieties that exhibit good meat production performance. In contrast to the domestication direction of meat pigeons, the traditional Chinese ornamental pigeon breed, High flyers, has a small and light body. Here, we investigate the molecular mechanism of the pectoral muscle development and function of pigeons using whole-genome and RNA sequencing data Result: The selective sweep analysis (FST and log2 (θπ ratio)) revealed 293 and 403 positive selection genes in Euro-pigeons and Silver King, respectively, of which 65 genes were shared. With the Silver King and Euro-pigeon as the control group, the High flyers were selected for 427 and 566 genes respectively. There were 673 differentially expressed genes in the breast muscle transcriptome between the commercial meat pigeons and ornamental pigeons. Pigeon genome selection signal combined with the breast muscle transcriptome revealed that 6 genes from commercial varieties of pigeons and 5 genes from Chinese traditional ornamental pigeons were positively selected. These genes were involved in pathways related to muscle development and function. Conclusion: Integrated selection signal, transcriptome analysis, and functional annotation identified SYNE1 as a key gene enriched in the actin binding and muscle cell differentiation pathways. Moreover, SYNE1 gene mutations have been associated with human muscular dystrophy. The differential expression of this gene reveals that it has a negative regulatory role in the development and function of pigeon breast muscle. Pigeons Pectoral muscle development Selective sweep Transcriptome SYNE1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Archaeological evidence indicates that pigeons were historically consumed as food for thousands of years[ 1 ] and represented an important protein source for humans. In Europe, North America, and Asia, squab is considered a delicacy and is very popular among consumers[ 2 ]. The pigeon industry gradually emerged in China during the early 1970s. After nearly 40 years of development, the number of pigeons in stock, out of stock, and the total production of pigeons ranked in China first in the world. According to the 2018 China Poultry Industry Development Report, there were 256,000 pairs of grandparent breeding pigeons, 41.2 million pairs of parent breeding pigeons, an increase of 5% over 2017, and an annual production of 643 million squabs [ 3 ]. Similar to other types of livestock and poultry, breeders tend to cultivate large sized, full-breasted pigeons with high fecundity. There are substantial differences in body shape among different pigeon breeds. The largest pigeon can reach 1,000 g and small pigeons can reach to 250 g, which is nearly a four-fold difference in body mass[ 4 ]. For example, Euro-pigeon and Silver King weigh over 600 g at 4 weeks of age[ 5 ], whereas some local varieties only weigh 250 g[ 6 ]. In contrast to meat pigeons, some performance and carrier pigeons are selected for athletic capability, and these pigeons are generally highly adept at flying. Carrier pigeons have the capacity for long-distance exploration and are known as messengers[ 7 ], whereas flipping pigeons are recognized as air dancers. High flying pigeons are more famous for their high altitude flying and also have important ornamental traits. Compared with chickens, ducks, and geese, pigeons are the only altricial poultry in China, and are monogamous. Molecular breeding can accelerate the breeding process of meat pigeons. Most studies investigating quality traits using pigeon genomics have focused on the feather crown[ 8 ], feather color[ 9 ], and foot feathers[ 10 , 11 ]. Moreover, analyses of complex traits have mainly focused on competition ability[ 12 ]. Using genomics and transcriptomics, researchers have revealed that the CASK gene is the key gene involved in the homing ability of carrier pigeons[ 13 ]. The relationship between candidate gene polymorphisms and meat quality traits has also been studied[ 14 – 16 ], and the physical and chemical characteristics of pigeon meat were previously reported[ 2 ]. Many studies have found that the MSTN gene is related to muscle development in cattle[ 17 , 18 ], pigs[ 19 , 20 ], chickens[ 21 , 22 ], and sheep[ 23 ]. In addition, MSTN gene expression in the breast muscle of pigeons was found to be significantly higher than that in other tissues, and to increase with age[ 24 ]. The genetic mechanism of body weight and size traits in pigs[ 25 ], horses[ 26 ], and ducks[ 27 ] was revealed using multi-omics; however, few genome-wide studies have been conducted on gene mapping of pigeon growth traits, particularly the development of pectoral muscles. Pigeon breast muscle accounted for approximately 30% of the slaughter weight[ 2 ]. Moreover, the growth and development of breast muscle is extremely important for meat performance and the flying ability of pigeons. In this study, we selected three breeds and sequenced a total of 23 individuals, including eight Euro-pigeons (EU), eight Silver King pigeons (SK), and seven High flyers. European meat pigeons and SK pigeons are larger and have better meat performance. HF are good at flying at high altitudes, with the characteristics of a small size and light posture. The distinct purposes of the pigeons diverged these breeds via human selection. The selection signal revealed genes that were positively selected among each of the breeds. Next, 28-day-old pigeon breast muscle transcriptome differences combined with the selection signal revealed the key genes required for pigeon muscle growth. This study is of great significance for the discovery of candidate genes that affect pigeon growth and development traits, and provides new insight into muscle development and function of altricial birds using functional omics research. Results A total of 23 samples were sequenced from different pigeons, with high quality data volume of 199.3 Gb, high sequencing quality (Q20 ≥ 96.0%, Q30 ≥ 90.2%), and normal GC distribution. None of the 23 samples were contaminated (Table S1). The library was successfully constructed and sequenced. The average map rate of the population samples was 97.6%, the average sequencing depth of the genome (excluding gap regions) was 7.30 (only reads with a comparison quality > 0 are considered), and the average coverage was 98.8% (at least one base is covered) (Table S2). A total of 5,673,290 SNPs were detected (Table S3). Population structure Based on the degree of SNP differences among individuals of different pigeon breeds, PCA analysis revealed that European meat pigeon and Silver King Pigeon had a closer genetic distance, while Chinese traditional ornamental pigeon and commercial meat pigeon had a longer genetic distance (Figure 1 A). The phylogenetic tree analysis revealed that commercial meat pigeon breeds and HF pigeons formed two independent branches, indicating that there was a large genetic distance between them. Although there are obvious differences in feather color between EU pigeons and SK pigeons, the genetic distance between them was relatively close, as well as a cross between them, which may be related to the cross utilization of meat pigeons in the production process (Figure 1 B). Positive selection of meat pigeon and High flyers By analyzing selection signals, 293 genes were positively selected in the EU population and 403 genes were positively selected in the SK population. There were 65 overlapping genes (Table S4; Figure 2A and B). When EU was used as a control group, 566 genes were selected by HF pigeons. When SK was used as the control group, 427 genes were selected by HF pigeons. There were 262 overlapping genes (Table S4; Figure 2C and D). Comparative transcriptome of the pectoral muscle A total of 12 breast muscle samples (4 EUs, 4 SKs and 4 HFs) of three pigeon breeds were sequenced according to the standard operation, and a total of 94.73 G of raw data was obtained. After performing quality control, 92.48 G of clean data were obtained, 97.62% of which were effective reads. The proportion of bases with quality values ≥ 20 (sequencing error rate less than 0.01) was 99.97%, the proportion of bases with quality values ≥ 30 (sequencing error rate less than 0.001) was 98.11%, and the proportion of GC content was 48.42% (Table S5). Hisat was used to compare the reference genome of valid data after preprocessing, revealing a comparison rate of 90.86% (Table S6). The percentage of exon annotated sequences was 91.63%, whereas the percentage of intron and intergenic reads was 4.78% and 3.59%, respectively (Figure S1). Analysis of differentially expressed genes (DEGs) The expression level of genes mainly uses FPKM (Fragments Per Kilobase of exon model per Million mapped reads) to measure the abundance value of gene expression. R package DESeq2 was used to analyze significant differences between samples. The expression profiles of 26,640 genes were obtained using Ballgown package to provide file input for FPKM quantification. The genes with FC > 2 times or FC < 0.5 times and P value < 0.05 were defined as differentially expressed genes. A total of 1,016 differentially expressed genes were obtained by comparing the transcriptome of breast muscle samples from EU and HF pigeons, including 408 up-regulated genes and 608 down-regulated genes (Table S7; Figure 3A). There were 1,294 differentially expressed genes between SK and HF pigeons, including 465 up-regulated genes and 829 down-regulated genes (Table S8; Figure 3B). A total of 322 genes were identified in EU vs SK group, of which 195 were up-regulated and 127 were down regulated (Table S9). The results showed that there were more differences in breast muscle gene expression between meat pigeons and HF; however, there were fewer differences between meat pigeons, which was consistent with the phenotypic analysis results. Further analysis with a Wayne diagram showed that there were 673 overlapping genes in the EU vs HF and SK vs HF groups (Figure 3). Pigeon genome selection signal combined with the breast muscle transcriptome revealed that six genes from commercial varieties of pigeons (Figure 3C) and five genes from Chinese traditional ornamental pigeons (Figure 3D) were positively selected. GO and KEGG enrichment analysis A total of 305 significant GO terms were identified in the EU vs HF group, the top 20 of which were related to muscle function, including actin binding (GO:0003779, P=0.0003) and myosin filament (GO:0032982, P=0.0003) (Table S10; Figure 4A). Moreover, the insulin-like growth factor binding (GO:0005520, P=0.00003) pathway, which is related to growth and development, was also enriched. For SK and HF, the differentially expressed genes were enriched in 358 significant pathways (Table S11; Figure 4B). Similarly, the first 20 pathways included actin binding (GO:0003779, P=0.0000000000000) and myosin filament (GO:0032982, P=0.00009). In addition, muscle contraction (GO:0006936) was also related to muscle function. Although 516 GO terms were enriched in the pigeon breeds, the top 20 GO terms were not related to muscle development or function. This finding indicated that there were few differences in the pectoral muscle between the two meat-type pigeon breeds (Table S12). A KEGG pathway enrichment analysis of the differentially expressed genes in the breast muscle tissue of commercial meat pigeons and HF was carried out. The differentially expressed genes in the breast muscle of EU and HF were significantly enriched in 217 pathways, 21 of which were extremely significantly enriched (P<0.05) (Table S13; Figure 4C). The differentially expressed genes in the breast muscle of SK and HF were significantly enriched in 178 pathways, of which 31 were extremely significantly enriched (P < 0.05) (Table S14; Figure 4D). The significantly enriched pathways exhibited by the two meat breeds included participation in cell proliferation, differentiation, metabolism, and synthesis. These pathways included cell adhesion molecules (CAMs), ECM-receptor interaction, glycolysis/gluconeogenesis, focal adhesion, insulin signaling pathway, mucin type O-glycan biosynthesis, biotin metabolism, and the adipocytokine signaling pathway. Differential gene expression in the breast muscle of meat pigeon breeds was enriched in 111 pathways, 19 of which were significantly enriched (P < 0.05) (Table S15). Verification of key gene expression Four genes, including SYNE1 , INSC , IGFBP1 and MAFF exhibited significant different expression patterns between the meat breeds and the HF breed. The expression patterns of these genes in pigeon breast muscle were consistent with the RNA sequencing results, which verified the accuracy of the comparative transcriptome sequencing results (Figure 5; Table S16). Among these genes, SYNE1 was selected in the HF population, which was related to actin binding function and flight ability. The HF breed has a lighter body that is suitable for gliding in the air, but has a slower flight speed and is not suitable for long-distance flight. It is speculated that mutations in the SYNE1 gene leads to weakening of its chest muscle function. The INSC gene also plays an important role in the growth and development of pigeon breast muscle. IGFBP1 play a role in the growth and metabolism of pigeons and regulate the development of the pectoral muscle. Discussion In this study, a genomics perspective was used to analyze the genes of two meat pigeon breeds (EU and SK) and an ornamental breed. To further study the genetic mechanism associated with the phenotype differences in the pectoral muscle between the two different breeds, a comparative transcriptome was used to reveal the key genes related to growth and development and more importantly, to discover genes that regulate muscle function. The selection signal combined with the comparative transcriptome revealed INSC , CALCB , ZBTB21 , B2M , and LOC110356506 as the genes that affect breast muscle development in meat pigeons. INSC can regulate mesoderm differentiation of mouse embryonic stem (ES) cells[ 28 ]. In addition, the human INSC gene, which is closely associated with the CALCB gene by an interval of about 30 kb, was assigned to human chromosome 11p15.2-p15.1[ 29 ]. While a short-term RNA interference-mediated CALCB knockdown had no effect on the proliferation and clonogenic growth of EwS cells in vitro , its long-term knockdown decreased EwS growth both in vitro and in vivo . In the differential expression analysis, 673 genes were identified, among which the genes related to growth and development included IGFBP1 , IGFBP4 , FOXO3 , HMGA1 , and FAM184B . Studies have shown that the IGFBP1 gene has an important regulatory effect on fetal growth and development. Both hypoxia and leucine deprivation can increase the level of the IGFBP1 gene expression and phosphorylation, inhibit the effect of IGF , and lead to impaired embryonic development[ 30 , 31 ]. IGF-1 and IGFBP1 have anabolic effects on skeletal muscle and are related to the preservation of lean meat[ 32 ]. Compared with commercial meat pigeons, HFs are selected for light weight individuals. At the same time, the flying ability of HFs is inferior to that of carrier pigeons, characterized by a short hovering distance and slow flying speed. Genomic and transcriptomic analyses revealed that SLC16A10 , S100B , SYNE1 , HECW2 , and CASQ2 genes were positively selected. Studies in mice have shown that the SLC16A10 gene is involved in promoting the cellular transport of thyroid hormone[ 33 ]. Studies in pigs have demonstrated that a low protein diet induces higher expression of the SLC16A10 gene, resulting in limited protein synthesis and growth of the longissimus dorsi[ 34 ]. Mutations in the HECW2 gene can cause neurodevelopmental delay, and the clinical features shared by patients include severe developmental delay and hypotonia[ 35 ]. In addition, this gene mutation has also been linked to epilepsy-associated developmental delay[ 36 ]. CASQ2 plays an important role in regulating Ca 2+ release in the sarcoplasmic reticulum, buffering of Ca 2+ in the sarcoplasmic reticulum, and promoting the closure of cardiac ryanodine receptors during diastole[ 37 ]. CASQ2 is expressed in slow muscle throughout the lifespan of mice, but only in fast muscle during the newborn stage and early development. Indeed, CASQ2 −/− mice display ultrastructural changes only in the rapid twitch muscle [ 38 ]. A defect in nesprin-1 encoded by the SYNE1 gene can cause Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by joint contracture, myasthenia, and cardiac abnormalities[ 39 – 42 ]. The protein encoded by SYNE1 is widely expressed in a variety of tissues and connects the outer membrane of the nuclear membrane with the cytoskeleton by interacting with F-actin[ 43 ] and is highly expressed in striated muscle[ 44 ]. At the same time, several studies throughout the world have reported that SYNE1 gene mutations can cause autosomal receptive cerebellar ataxia type 8 (SCAR8)[ 45 – 49 ]. The SYNE1 gene was selected in HF pigeons and found to be significantly up-regulated ( P < 0.01) compared with that in meat pigeons. The body weight and breast muscle weight of HF pigeons are significantly lower ( P < 0.01) than those of commercial meat pigeons (table S1), indicating that this gene has a negative regulatory effect on the growth and development of pigeons. With the enrichment analysis, SYNE1 was found to be involved in actin binding (GO: 0003779) and muscle cell differentiation (GO: 0042692). It is speculated that mutations in the SYNE1 gene will affect the development and function of HF breast muscle. Conclusion This study revealed the selection of genome regions between commercial meat pigeons and Chinese traditional ornamental pigeons using genomics analyses. The results of transcriptome sequencing and differential expression analysis revealed the molecular mechanism of breast muscle phenotypic differences between commercial meat pigeons and Chinese traditional ornamental pigeons. Multi-omics further revealed multiple genes related to cell differentiation, muscle development, and skeletal muscle function. Among these genes, INSC and CALCB were related to cell differentiation and were positively selected and up-regulated in both EU pigeons and SK pigeons. Thus, these genes may be involved in promoting the growth and development of the pigeon breast muscle. More importantly, we found that the SYNE1 gene was related to muscular dystrophy in the ornamental pigeon population, and also played an important role in pigeon skeletal muscle function and muscle cell differentiation. Therefore, mutations in the SYNE1 gene may affect the function of breast muscle cells by promoting a lighter body and reduced flight speed in high flying pigeons. Materials And Methods Pigeons and ethics approval The whole genome of 23 pigeons, including eight European meat pigeons, eight SK pigeons, and seven HF pigeons were sequenced. All pigeons are from Shanghai Jinhuang Pigeon Industry Co., Ltd. After blood collection from the pterygoid vein, DNA was extracted using a Tiangen kit, and the DNA concentration was detected with a Nanodrop Spectrophotometer 2000. An Illumina Hiseq PE150 sequencing platform was used for sequencing. Pigeons used in this study were approved by the Ethics and Animal Welfare Committee of Shanghai Academy of Agricultural Sciences (No. SAASPZ0521012). Comparison of reference genomes The effective high quality sequencing data were compared to the reference genome (reference genome download link: https://www.ncbi.nlm.nih.gov/genome/10719?genome_ assembly_ id=39619) using BWA[50] software (parameter: mem-t 4-K 32-m), and comparison results were removed with SAMTOOLS[51] (parameter: rmdup). SNP detection and annotation We used SAMTOOLS [51] and other software to detect population SNPs. The Bayesian model was used to detect polymorphic loci in the population, and quality control SNPs were obtained through the following filtering and screening method: 1) Q20 quality control (SNPs with a quality value of Q20 (i.e., the sequencing error rate was greater than 1% were filtered out); 2) the SNPs were at least 5 bp apart from each other (Since the probability of two SNPs being so close is extremely low, it is considered to be due to errors in sequencing, experimental factors, or analysis, and the two SNPs are removed); 3) the support number (coverage depth) of the SNP was between [1/3, 5] times of the average depth. Evolutionary tree of pigeon populations We used neighbor joining methods to construct the evolutionary tree. After SNP detection, the individual SNPs can be used to calculate the distance between populations. TreeBeST-1.9.2 software (https://mybiosoftware.com/treebest-1-9-2-softwares-phylogenetic-trees.html) was used to calculate the distance matrix. Using the distance matrix, a phylogenetic tree was constructed using the neighbor joining method. The bootstrap values were calculated 1000 times. We used EIGENSOFT (v5.0; https://www.hsph.harvard.edu/alkes-price/software/) for principal component analysis (PCA) on an individual scale for the 23 pigeons. Analysis of selection signal based on F ST & θπ We calculated the genome-wide distribution of F ST values [52] and θπ ratios among the seven pigeon breeds using a sliding-window approach (40-kb windows with 20-kb increments). The θπ ratios were log2( θπ ratio) transformed. We considered the windows with the top 5% values for the F ST and log2( θπ ratio) simultaneously as candidate outliers under strong selective sweeps. Furthermore, the overlap information of a selected signal was obtained using the “vennDiagram” package in R (https://www.omicstudio.cn/tool/6). Comparative transcriptome analysis of the pectoral muscle The breast muscles of four EU meat pigeons, four SK pigeons, and four HF pigeons were collected for transcriptome analysis, and the slaughter traits were recorded (Table 1). Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's procedure. The amount and purity of the RNA in each sample was quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent, CA, USA) with an RIN number > 7.0, and confirmed by electrophoresis with denaturing agarose gel. Finally, we performed 2 × 150 bp paired-end sequencing (PE150) with an Illumina Novaseq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) following the vendor's recommended protocol. Table 1. Slaughter traits of three pigeon breeds Breeds Live weight （ g ） Slaughter weight (g) eviscerated weight (g) pectorales weight (g) Euro-pigeon 599.25 ± 64.20 a 523.00 ± 65.56 a 404.50 ± 50.49 a 119.5 ± 19.21 a Silver King 482.75 ± 34.54 b 423.50 ± 34.38 b 331.50 ± 21.27 b 86.00 ± 5.89 b High flyer 311.00 ± 32.76 c 262.50 ± 32.80 c 177.00 ± 25.53 c 64.95 ± 9.16 c Sequence and primary analysis Cutadapt software ( https://cutadapt.readthedocs.io/en/stable/ , version: cutadapt-1.9) was used to remove the reads that contained adaptor contamination (command line: ~cutadapt -a ADAPT1 -A ADAPT2 -o out1.fastq -p out2.fastq in1.fastq in2.fastq -O 5 -m 100). After removing the low quality and undetermined bases, HISAT2 software ( https://daehwankimlab.github.io/hisat2/ , version: hisat2-2.0.4) was used to map reads to the Cliv_1.0 rock pigeon reference genome, (command line: ~hisat2 -1 R1.fastq.gz -2 R1.fastq.gz -S sample_mapped.sam). The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/, version: stringtie-1.3.4d. Linux_x86_64) with default parameters (command line: ~stringtie -p 4 -G genome.gtf -o output.gtf -l sample input.bam). The transcriptomes from all of the samples were merged to reconstruct a comprehensive transcriptome using GffCompare software (http://ccb.jhu.edu/software/stringtie/gffcompare.shtml, version: gffcompare-0.9.8. Linux_x86_64). After the final transcriptome was generated, StringTie and Ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the levels of transcript expression and determine the mRNA expression level by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads (millions) × exon_length (kB)]), (command line: ~stringtie -e -B -p 4 -G merged.gtf -o samples.gtf samples.bam). The differentially expressed mRNAs with a fold-change > 2 or fold-change < 0.5, and p-value < 0.05 were selected using DESeq2[53] (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html). Gene ontology enrichment and pathway analysis The function of the differentially expressed genes in different types of pigeon breast muscle was investigated by comparing the following groups: EU vs SK, EU vs HF, and SK vs HF. Goseq was used to perform the enrichment analysis of the GO term [54]. Based on the KEGG database, KOBAS 2.0 software was used to analyze the pathways associated with the differentially expressed genes [55]. Ggplot 2 was used to analyze the enrichment of the GO and KEGG databases, and the results were displayed as a scatter plot (bubble chart). Real-time quantitative PCR analysis Using 12 RNA samples isolated from pigeons (four EU pigeons, four SK pigeons, and four HF pigeons), the RNA OD value was detected with an ultra-micro nucleic acid protein analyzer (scandrop100). SYBR Green I was used to detect the expression of target genes in the samples using the A260/A280 ratio, β-actin as an internal reference gene, and four target genes (primers are listed in Table 2). The fluorescence quantitative PCR program and system was performed as follows: Step 1: 95℃ for 3 min; Step 2: 95℃ for 10 s; Step 3: 60℃ for 30 s + plate read; repeat step 2 for 39 cycles. Melt curve analysis: (60℃ – 95℃, +1℃/cycle, holding time of 4 s). Declarations Acknowledgments The authors are grateful to The Shanghai Jinhuang Pigeon Co., Ltd., provided support in the process of collecting research materials on High Flyers and commercial pigeon breeds. The authors would like to thank Mr. Liu for providing technical support in bioinformatics analysis. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Authors’ contributions Experimental design: J.Y. and W.D.; Project leader: C.Y.; Article writer: H.H.; Data analysis and mining: H.H. and X.W.; Sample collection: X.C., W.L., Y.T., and A.B.; Research materials: Q.W. and W.Z. Funding This research has been supported by the Key Projects of Developing Agriculture by Science and Technology in Shanghai (2018-02-08-00-12-F01546) and the Shanghai Grants from the SAAS Program for Excellent Research Team (SPERT). Ethics approval and consent to participate The process of collecting pigeon samples strictly follows the animal ethics procedures and guidelines of the People’s Republic of China. Pigeons used in this study were approved by the Ethics and Animal Welfare Committee of Shanghai Academy of Agricultural Sciences (No. SAASPZ0521012). Consent for publication Not applicable. 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Hamza W, Ali Pacha L, Hamadouche T, Muller J, Drouot N, Ferrat F, Makri S, Chaouch M, Tazir M, Koenig M et al : Molecular and clinical study of a cohort of 110 Algerian patients with autosomal recessive ataxia . BMC medical genetics 2015, 16 :36. Synofzik M, Smets K, Mallaret M, Di Bella D, Gallenmüller C, Baets J, Schulze M, Magri S, Sarto E, Mustafa M et al : SYNE1 ataxia is a common recessive ataxia with major non-cerebellar features: a large multi-centre study . Brain: a journal of neurology 2016, 139 (Pt 5):1378–1393. Mademan I, Harmuth F, Giordano I, Timmann D, Magri S, Deconinck T, Claaßen J, Jokisch D, Genc G, Di Bella D et al : Multisystemic SYNE1 ataxia: confirming the high frequency and extending the mutational and phenotypic spectrum . Brain: a journal of neurology 2016, 139 (Pt 8):e46. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform . Bioinformatics 2009, 25 (14):1754–1760. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S: The Sequence Alignment/Map format and SAMtools . Bioinformatics 2009, 25 (16):2078–2079. Weir BS, Cockerham CC: ESTIMATING F-STATISTICS FOR THE ANALYSIS OF POPULATION STRUCTURE . Evolution 1984, 38 (6). Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL: StringTie enables improved reconstruction of a transcriptome from RNA-seq reads . Nature biotechnology 2015, 33 (3):290–295. Young MD, Wakefield MJ, Smyth GK, Oshlack A: Gene ontology analysis for RNA-seq: accounting for selection bias . Genome Biol 2010, 11 (2):R14. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T et al : KEGG for linking genomes to life and the environment . Nucleic Acids Res 2008, 36 (Database issue):D480-484. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-468678","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":23720307,"identity":"8fd25a9c-2799-477b-aa2f-afce8180950f","order_by":0,"name":"Haobin Hou","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haobin","middleName":"","lastName":"Hou","suffix":""},{"id":23720308,"identity":"17be8a11-c500-4780-97a0-8ad61c8c8912","order_by":1,"name":"Xiaoliang Wang","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoliang","middleName":"","lastName":"Wang","suffix":""},{"id":23720309,"identity":"42dc5c9f-f211-4b0f-ba46-43b443d33092","order_by":2,"name":"Changsuo Yang","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changsuo","middleName":"","lastName":"Yang","suffix":""},{"id":23720310,"identity":"a6a16e7a-26d1-4dd8-b5a9-0a97f4038989","order_by":3,"name":"Xia Cai","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Cai","suffix":""},{"id":23720311,"identity":"10db8059-5fd9-49f2-9ccd-d95603e28db3","order_by":4,"name":"Wenwei Lv","email":"","orcid":"","institution":"Shanghai Academy of Agricultural 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Company","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Quanli","middleName":"","lastName":"Wu","suffix":""},{"id":23720315,"identity":"d86f475f-0109-4d4e-a292-7a1ad85646ef","order_by":8,"name":"Weimin Zhao","email":"","orcid":"","institution":"Shanghai Jinhuang Pigeon Company","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Zhao","suffix":""},{"id":23720316,"identity":"7353e47b-3558-4d34-b4ec-21e32279ca9a","order_by":9,"name":"Junfeng Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHACNoYEEMnefPjBBwMbO+K18PEcSzOcUZCWTJwWEJCTyDGQ5vlwiLGBkHqD44ePPXjwxyaPTSItwdjG4AAzA/vhoxvwajmTlm6Q2JZWzMbz+MDjHIM7fAw8aWk38Go5kGMmkdhwOLGNHWhLjsEzZgYJHjP8Ws6/MZNI+APUwgD0i4XBYcYGglpuAG1JYANq4QBqYSBGi+SNZ2C/JLaBArnHIC2ZjZBf+M4nH3v4449N4vx2YFQCGXb87IeP4dWicABdhA2fchCQbyCkYhSMglEwCkYBAAoIT3Hqx2GUAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Junfeng","middleName":"","lastName":"Yao","suffix":""},{"id":23720317,"identity":"a63d5ad9-b3a8-46ae-afda-136d17e52459","order_by":10,"name":"Weixing Ding","email":"","orcid":"","institution":"Shanghai Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weixing","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2021-04-27 13:44:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-468678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-468678/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3389/fgene.2021.735795","type":"published","date":"2021-12-21T08:26:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":8561441,"identity":"381d4968-3b3a-4651-927d-72e96d0d7f7b","added_by":"auto","created_at":"2021-04-28 18:24:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":513668,"visible":true,"origin":"","legend":"A) The 3D PCA plot of pigeon population. EU (Euro-pigeon), SK (Silver King), HF (High flyers). B) Phylogenetic tree of three pigeon breeds. ","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/210fd535e6df97007f12a44b.png"},{"id":8561444,"identity":"b79b9be1-d2ed-4d3d-b953-248c61e017c6","added_by":"auto","created_at":"2021-04-28 18:24:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1619428,"visible":true,"origin":"","legend":"Distribution of log2 values (θπ•control/θπ•selected) and the top 5% highest FST values calculated in 40-kb sliding windows with 20-kb increments. A) Compared with HF, EU are subject to positive selection of genomic regions. B) Compared with HF, SK are subject to positive selection of genomic regions. C) Compared with EU, HF are subject to positive selection of genomic regions. D) Compared with SK, HF are subject to positive selection of genomic regions.","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/c18134e43ba5d2fb836e1191.png"},{"id":8560676,"identity":"2da76730-799e-486a-9c2d-651639bd8c37","added_by":"auto","created_at":"2021-04-28 18:21:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1353721,"visible":true,"origin":"","legend":"A) Volcano plot of EU. VS. HF breast muscle differential gene expression level. B) Volcano plot of SK. VS. HF breast muscle differential gene expression level. C) Venn diagram of the commercial pigeon positive selection signal combined with DEGs analysis of the breast muscle transcriptome. D) Venn diagram of the HF positive selection signal combined with DEGs analysis of the breast muscle transcriptome.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/f178b963b5b75b56915b9467.png"},{"id":8561443,"identity":"5096bcea-3c09-436f-b36a-0af86344b2dc","added_by":"auto","created_at":"2021-04-28 18:24:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":959670,"visible":true,"origin":"","legend":"Top 20 significantly enriched GO and KEGG pathways of the differentially expressed genes.","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/039f72ca622c500bf7c339b3.png"},{"id":8561837,"identity":"e2167600-0865-4139-95cc-4bb3c3d4402e","added_by":"auto","created_at":"2021-04-28 18:27:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38940,"visible":true,"origin":"","legend":"A) Gene expression of pigeon breast muscle transcriptome sequencing. FPKM (Fragments Per Kilobase of exon model per Million mapped reads). B) Expression of five significant differentially expressed genes validated by qRT-PCR.","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/78e0622d7dc53d3cdd4c9c62.png"},{"id":16604062,"identity":"f0271bfe-3f39-4909-bca6-49818ddb0aa3","added_by":"auto","created_at":"2021-12-20 08:26:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2710886,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/a020b9ea-75b3-4384-997e-bde9f5524ac3.pdf"},{"id":8561440,"identity":"df66c915-6c1a-4fb0-a01a-262fd96811d5","added_by":"auto","created_at":"2021-04-28 18:24:47","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-468678/v1/9f8f0aac5caacdd75b1548f6.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative genome and transcriptome integration studies reveal the mechanism of pectoral muscle development and function in pigeons","fulltext":[{"header":"Background","content":" \u003cp\u003eArchaeological evidence indicates that pigeons were historically consumed as food for thousands of years[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and represented an important protein source for humans. In Europe, North America, and Asia, squab is considered a delicacy and is very popular among consumers[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pigeon industry gradually emerged in China during the early 1970s. After nearly 40 years of development, the number of pigeons in stock, out of stock, and the total production of pigeons ranked in China first in the world. According to the 2018 China Poultry Industry Development Report, there were 256,000 pairs of grandparent breeding pigeons, 41.2\u0026nbsp;million pairs of parent breeding pigeons, an increase of 5% over 2017, and an annual production of 643\u0026nbsp;million squabs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Similar to other types of livestock and poultry, breeders tend to cultivate large sized, full-breasted pigeons with high fecundity. There are substantial differences in body shape among different pigeon breeds. The largest pigeon can reach 1,000 g and small pigeons can reach to 250 g, which is nearly a four-fold difference in body mass[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, Euro-pigeon and Silver King weigh over 600 g at 4 weeks of age[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], whereas some local varieties only weigh 250 g[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In contrast to meat pigeons, some performance and carrier pigeons are selected for athletic capability, and these pigeons are generally highly adept at flying. Carrier pigeons have the capacity for long-distance exploration and are known as messengers[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], whereas flipping pigeons are recognized as air dancers. High flying pigeons are more famous for their high altitude flying and also have important ornamental traits.\u003c/p\u003e \u003cp\u003eCompared with chickens, ducks, and geese, pigeons are the only altricial poultry in China, and are monogamous. Molecular breeding can accelerate the breeding process of meat pigeons. Most studies investigating quality traits using pigeon genomics have focused on the feather crown[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], feather color[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and foot feathers[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, analyses of complex traits have mainly focused on competition ability[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Using genomics and transcriptomics, researchers have revealed that the \u003cem\u003eCASK\u003c/em\u003e gene is the key gene involved in the homing ability of carrier pigeons[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The relationship between candidate gene polymorphisms and meat quality traits has also been studied[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the physical and chemical characteristics of pigeon meat were previously reported[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Many studies have found that the \u003cem\u003eMSTN\u003c/em\u003e gene is related to muscle development in cattle[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], pigs[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], chickens[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and sheep[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, \u003cem\u003eMSTN\u003c/em\u003e gene expression in the breast muscle of pigeons was found to be significantly higher than that in other tissues, and to increase with age[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genetic mechanism of body weight and size traits in pigs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], horses[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and ducks[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was revealed using multi-omics; however, few genome-wide studies have been conducted on gene mapping of pigeon growth traits, particularly the development of pectoral muscles. Pigeon breast muscle accounted for approximately 30% of the slaughter weight[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, the growth and development of breast muscle is extremely important for meat performance and the flying ability of pigeons. In this study, we selected three breeds and sequenced a total of 23 individuals, including eight Euro-pigeons (EU), eight Silver King pigeons (SK), and seven High flyers. European meat pigeons and SK pigeons are larger and have better meat performance. HF are good at flying at high altitudes, with the characteristics of a small size and light posture. The distinct purposes of the pigeons diverged these breeds via human selection. The selection signal revealed genes that were positively selected among each of the breeds. Next, 28-day-old pigeon breast muscle transcriptome differences combined with the selection signal revealed the key genes required for pigeon muscle growth. This study is of great significance for the discovery of candidate genes that affect pigeon growth and development traits, and provides new insight into muscle development and function of altricial birds using functional omics research.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003eA total of 23 samples were sequenced from different pigeons, with high quality data volume of 199.3 Gb, high sequencing quality (Q20 \u0026ge; 96.0%, Q30 \u0026ge; 90.2%), and normal GC distribution. None of the 23 samples were contaminated (Table S1). The library was successfully constructed and sequenced. The average map rate of the population samples was 97.6%, the average sequencing depth of the genome (excluding gap regions) was 7.30 (only reads with a comparison quality \u0026gt; 0 are considered), and the average coverage was 98.8% (at least one base is covered) (Table S2). A total of 5,673,290 SNPs were detected (Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePopulation structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the degree of SNP differences among individuals of different pigeon breeds, PCA analysis revealed that European meat pigeon and Silver King Pigeon had a closer genetic distance, while Chinese traditional ornamental pigeon and commercial meat pigeon had a longer genetic distance (Figure 1 A). The phylogenetic tree analysis revealed that commercial meat pigeon breeds and HF pigeons formed two independent branches, indicating that there was a large genetic distance between them. Although there are obvious differences in feather color between EU pigeons and SK pigeons, the genetic distance between them was relatively close, as well as a cross between them, which may be related to the cross utilization of meat pigeons in the production process (Figure 1 B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePositive selection of meat pigeon and High flyers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy analyzing selection signals, 293 genes were positively selected in the EU population and 403 genes were positively selected in the SK population. There were 65 overlapping genes (Table S4; Figure 2A and B). When EU was used as a control group, 566 genes were selected by HF pigeons. When SK was used as the control group, 427 genes were selected by HF pigeons. There were 262 overlapping genes (Table S4; Figure 2C and D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative transcriptome of the pectoral muscle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 12 breast muscle samples (4 EUs, 4 SKs and 4 HFs) of three pigeon breeds were sequenced according to the standard operation, and a total of 94.73 G of raw data was obtained. After performing quality control, 92.48 G of clean data were obtained, 97.62% of which were effective reads. The proportion of bases with quality values \u0026ge; 20 (sequencing error rate less than 0.01) was 99.97%, the proportion of bases with quality values \u0026ge; 30 (sequencing error rate less than 0.001) was 98.11%, and the proportion of GC content was 48.42% (Table S5). Hisat was used to compare the reference genome of valid data after preprocessing, revealing a comparison rate of 90.86% (Table S6). The percentage of exon annotated sequences was 91.63%, whereas the percentage of intron and intergenic reads was 4.78% and 3.59%, respectively (Figure S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of differentially expressed genes (DEGs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression level of genes mainly uses FPKM (Fragments Per Kilobase of exon model per Million mapped reads) to measure the abundance value of gene expression. R package DESeq2 was used to analyze significant differences between samples. The expression profiles of 26,640 genes were obtained using Ballgown package to provide file input for FPKM quantification. The genes with FC \u0026gt; 2 times or FC \u0026lt; 0.5 times and P value \u0026lt; 0.05 were defined as differentially expressed genes. A total of 1,016 differentially expressed genes were obtained by comparing the transcriptome of breast muscle samples from EU and HF pigeons, including 408 up-regulated genes and 608 down-regulated genes (Table S7; Figure 3A). There were 1,294 differentially expressed genes between SK and HF pigeons, including 465 up-regulated genes and 829 down-regulated genes (Table S8; Figure 3B). A total of 322 genes were identified in EU vs SK group, of which 195 were up-regulated and 127 were down regulated (Table S9).\u003c/p\u003e\n\u003cp\u003eThe results showed that there were more differences in breast muscle gene expression between meat pigeons and HF; however, there were fewer differences between meat pigeons, which was consistent with the phenotypic analysis results. Further analysis with a Wayne diagram showed that there were 673 overlapping genes in the EU vs HF and SK vs HF groups (Figure 3). Pigeon genome selection signal combined with the breast muscle transcriptome revealed that six genes from commercial varieties of pigeons (Figure 3C) and five genes from Chinese traditional ornamental pigeons (Figure 3D) were positively selected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO and KEGG enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 305 significant GO terms were identified in the EU vs HF group, the top 20 of which were related to muscle function, including actin binding (GO:0003779, P=0.0003) and myosin filament (GO:0032982, P=0.0003) (Table S10; Figure 4A). Moreover, the insulin-like growth factor binding (GO:0005520, P=0.00003) pathway, which is related to growth and development, was also enriched. For SK and HF, the differentially expressed genes were enriched in 358 significant pathways (Table S11; Figure 4B). Similarly, the first 20 pathways included actin binding (GO:0003779, P=0.0000000000000) and myosin filament (GO:0032982, P=0.00009). In addition, muscle contraction (GO:0006936) was also related to muscle function. Although 516 GO terms were enriched in the pigeon breeds, the top 20 GO terms were not related to muscle development or function. This finding indicated that there were few differences in the pectoral muscle between the two meat-type pigeon breeds (Table S12).\u003c/p\u003e\n\u003cp\u003eA KEGG pathway enrichment analysis of the differentially expressed genes in the breast muscle tissue of commercial meat pigeons and HF was carried out. The differentially expressed genes in the breast muscle of EU and HF were significantly enriched in 217 pathways, 21 of which were extremely significantly enriched (P\u0026lt;0.05) (Table S13; Figure 4C). The differentially expressed genes in the breast muscle of SK and HF were significantly enriched in 178 pathways, of which 31 were extremely significantly enriched (P \u0026lt; 0.05) (Table S14; Figure 4D). The significantly enriched pathways exhibited by the two meat breeds included participation in cell proliferation, differentiation, metabolism, and synthesis. These pathways included cell adhesion molecules (CAMs), ECM-receptor interaction, glycolysis/gluconeogenesis, focal adhesion, insulin signaling pathway, mucin type O-glycan biosynthesis, biotin metabolism, and the adipocytokine signaling pathway. Differential gene expression in the breast muscle of meat pigeon breeds was enriched in 111 pathways, 19 of which were significantly enriched (P \u0026lt; 0.05) (Table S15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification of key gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour genes, including \u003cem\u003eSYNE1\u003c/em\u003e, \u003cem\u003eINSC\u003c/em\u003e, \u003cem\u003eIGFBP1\u003c/em\u003e and \u003cem\u003eMAFF\u003c/em\u003e exhibited significant different expression patterns between the meat breeds and the HF breed. The expression patterns of these genes in pigeon breast muscle were consistent with the RNA sequencing results, which verified the accuracy of the comparative transcriptome sequencing results (Figure 5; Table S16). Among these genes, \u003cem\u003eSYNE1\u003c/em\u003e was selected in the HF population, which was related to actin binding function and flight ability. The HF breed has a lighter body that is suitable for gliding in the air, but has a slower flight speed and is not suitable for long-distance flight. It is speculated that mutations in the \u003cem\u003eSYNE1\u003c/em\u003e gene leads to weakening of its chest muscle function. The\u003cem\u003e INSC\u003c/em\u003e gene also plays an important role in the growth and development of pigeon breast muscle. \u003cem\u003eIGFBP1\u003c/em\u003e play a role in the growth and metabolism of pigeons and regulate the development of the pectoral muscle.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eIn this study, a genomics perspective was used to analyze the genes of two meat pigeon breeds (EU and SK) and an ornamental breed. To further study the genetic mechanism associated with the phenotype differences in the pectoral muscle between the two different breeds, a comparative transcriptome was used to reveal the key genes related to growth and development and more importantly, to discover genes that regulate muscle function. The selection signal combined with the comparative transcriptome revealed \u003cem\u003eINSC\u003c/em\u003e, \u003cem\u003eCALCB\u003c/em\u003e, \u003cem\u003eZBTB21\u003c/em\u003e, \u003cem\u003eB2M\u003c/em\u003e, and \u003cem\u003eLOC110356506\u003c/em\u003e as the genes that affect breast muscle development in meat pigeons. \u003cem\u003eINSC\u003c/em\u003e can regulate mesoderm differentiation of mouse embryonic stem (ES) cells[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, the human \u003cem\u003eINSC\u003c/em\u003e gene, which is closely associated with the \u003cem\u003eCALCB\u003c/em\u003e gene by an interval of about 30 kb, was assigned to human chromosome 11p15.2-p15.1[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While a short-term RNA interference-mediated \u003cem\u003eCALCB\u003c/em\u003e knockdown had no effect on the proliferation and clonogenic growth of EwS cells \u003cem\u003ein vitro\u003c/em\u003e, its long-term knockdown decreased EwS growth both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. In the differential expression analysis, 673 genes were identified, among which the genes related to growth and development included \u003cem\u003eIGFBP1\u003c/em\u003e, \u003cem\u003eIGFBP4\u003c/em\u003e, \u003cem\u003eFOXO3\u003c/em\u003e, \u003cem\u003eHMGA1\u003c/em\u003e, and \u003cem\u003eFAM184B\u003c/em\u003e. Studies have shown that the \u003cem\u003eIGFBP1\u003c/em\u003e gene has an important regulatory effect on fetal growth and development. Both hypoxia and leucine deprivation can increase the level of the \u003cem\u003eIGFBP1\u003c/em\u003e gene expression and phosphorylation, inhibit the effect of \u003cem\u003eIGF\u003c/em\u003e, and lead to impaired embryonic development[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eIGF-1\u003c/em\u003e and \u003cem\u003eIGFBP1\u003c/em\u003e have anabolic effects on skeletal muscle and are related to the preservation of lean meat[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCompared with commercial meat pigeons, HFs are selected for light weight individuals. At the same time, the flying ability of HFs is inferior to that of carrier pigeons, characterized by a short hovering distance and slow flying speed. Genomic and transcriptomic analyses revealed that \u003cem\u003eSLC16A10\u003c/em\u003e, \u003cem\u003eS100B\u003c/em\u003e, \u003cem\u003eSYNE1\u003c/em\u003e, \u003cem\u003eHECW2\u003c/em\u003e, and \u003cem\u003eCASQ2\u003c/em\u003e genes were positively selected. Studies in mice have shown that the \u003cem\u003eSLC16A10\u003c/em\u003e gene is involved in promoting the cellular transport of thyroid hormone[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Studies in pigs have demonstrated that a low protein diet induces higher expression of the \u003cem\u003eSLC16A10\u003c/em\u003e gene, resulting in limited protein synthesis and growth of the longissimus dorsi[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Mutations in the \u003cem\u003eHECW2\u003c/em\u003e gene can cause neurodevelopmental delay, and the clinical features shared by patients include severe developmental delay and hypotonia[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, this gene mutation has also been linked to epilepsy-associated developmental delay[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. \u003cem\u003eCASQ2\u003c/em\u003e plays an important role in regulating Ca\u003csup\u003e2+\u003c/sup\u003e release in the sarcoplasmic reticulum, buffering of Ca\u003csup\u003e2+\u003c/sup\u003e in the sarcoplasmic reticulum, and promoting the closure of cardiac ryanodine receptors during diastole[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. \u003cem\u003eCASQ2\u003c/em\u003e is expressed in slow muscle throughout the lifespan of mice, but only in fast muscle during the newborn stage and early development. Indeed, \u003cem\u003eCASQ2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice display ultrastructural changes only in the rapid twitch muscle [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA defect in nesprin-1 encoded by the \u003cem\u003eSYNE1\u003c/em\u003e gene can cause Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by joint contracture, myasthenia, and cardiac abnormalities[\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The protein encoded by \u003cem\u003eSYNE1\u003c/em\u003e is widely expressed in a variety of tissues and connects the outer membrane of the nuclear membrane with the cytoskeleton by interacting with F-actin[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and is highly expressed in striated muscle[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. At the same time, several studies throughout the world have reported that \u003cem\u003eSYNE1\u003c/em\u003e gene mutations can cause autosomal receptive cerebellar ataxia type 8 (SCAR8)[\u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The \u003cem\u003eSYNE1\u003c/em\u003e gene was selected in HF pigeons and found to be significantly up-regulated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with that in meat pigeons. The body weight and breast muscle weight of HF pigeons are significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than those of commercial meat pigeons (table S1), indicating that this gene has a negative regulatory effect on the growth and development of pigeons. With the enrichment analysis, \u003cem\u003eSYNE1\u003c/em\u003e was found to be involved in actin binding (GO: 0003779) and muscle cell differentiation (GO: 0042692). It is speculated that mutations in the \u003cem\u003eSYNE1\u003c/em\u003e gene will affect the development and function of HF breast muscle.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThis study revealed the selection of genome regions between commercial meat pigeons and Chinese traditional ornamental pigeons using genomics analyses. The results of transcriptome sequencing and differential expression analysis revealed the molecular mechanism of breast muscle phenotypic differences between commercial meat pigeons and Chinese traditional ornamental pigeons. Multi-omics further revealed multiple genes related to cell differentiation, muscle development, and skeletal muscle function. Among these genes, \u003cem\u003eINSC\u003c/em\u003e and \u003cem\u003eCALCB\u003c/em\u003e were related to cell differentiation and were positively selected and up-regulated in both EU pigeons and SK pigeons. Thus, these genes may be involved in promoting the growth and development of the pigeon breast muscle. More importantly, we found that the \u003cem\u003eSYNE1\u003c/em\u003e gene was related to muscular dystrophy in the ornamental pigeon population, and also played an important role in pigeon skeletal muscle function and muscle cell differentiation. Therefore, mutations in the \u003cem\u003eSYNE1\u003c/em\u003e gene may affect the function of breast muscle cells by promoting a lighter body and reduced flight speed in high flying pigeons.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003ePigeons and ethics approval\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; \u0026nbsp;The whole genome of 23 pigeons, including eight European meat pigeons, eight SK pigeons, and seven HF pigeons were sequenced. All pigeons are from Shanghai Jinhuang Pigeon Industry Co., Ltd. After blood collection from the pterygoid vein, DNA was extracted using a Tiangen kit, and the DNA concentration was detected with a Nanodrop Spectrophotometer 2000. An Illumina Hiseq PE150 sequencing platform was used for sequencing. Pigeons used in this study were approved by the Ethics and Animal Welfare Committee of Shanghai Academy of Agricultural Sciences (No. SAASPZ0521012).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eComparison of reference genomes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eThe effective high quality sequencing data were compared to the reference genome (reference genome download link: https://www.ncbi.nlm.nih.gov/genome/10719?genome_ assembly_ id=39619) using BWA[50] software (parameter: mem-t 4-K 32-m), and comparison results were removed with SAMTOOLS[51] (parameter: rmdup).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eSNP detection and annotation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWe used SAMTOOLS [51] and other software to detect population SNPs. The Bayesian model was used to detect polymorphic loci in the population, and\u0026nbsp;quality control\u0026nbsp;SNPs were obtained through the following filtering and screening method: 1) Q20 quality control (SNPs with a quality value of Q20 (i.e., the sequencing error rate was greater than 1% were filtered out); 2) the SNPs were at least 5 bp apart from each other (Since the probability of two SNPs being so close is extremely low, it is considered to be due to errors in sequencing, experimental factors, or analysis, and the two SNPs are removed); 3) the support number (coverage depth) of the SNP was between [1/3, 5] times of the average depth.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eEvolutionary tree of pigeon populations\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWe used neighbor joining methods to construct the evolutionary tree. After SNP detection, the individual SNPs can be used to calculate the distance between populations. TreeBeST-1.9.2 software (https://mybiosoftware.com/treebest-1-9-2-softwares-phylogenetic-trees.html) was used to calculate the distance matrix. Using the distance matrix, a phylogenetic tree was constructed using the neighbor joining method. The bootstrap values were calculated 1000 times.\u0026nbsp;We used EIGENSOFT (v5.0; https://www.hsph.harvard.edu/alkes-price/software/) for principal component analysis (PCA) on an individual scale for the 23 pigeons.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eAnalysis of selection signal based on \u003cem\u003eF\u003csub\u003eST\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u0026amp; \u003cem\u003e\u0026theta;\u0026pi;\u003c/em\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eWe calculated the genome-wide distribution of\u003cem\u003e\u0026nbsp;F\u003csub\u003eST\u003c/sub\u003e\u003c/em\u003e values [52] and \u003cem\u003e\u0026theta;\u0026pi;\u003c/em\u003e ratios among the seven pigeon breeds using a sliding-window approach (40-kb windows with 20-kb increments). The \u003cem\u003e\u0026theta;\u0026pi;\u003c/em\u003e ratios were log2(\u003cem\u003e\u0026theta;\u0026pi;\u003c/em\u003e ratio) transformed. We considered the windows with the top 5% values for the \u003cem\u003eF\u003csub\u003eST\u003c/sub\u003e\u003c/em\u003e and log2(\u003cem\u003e\u0026theta;\u0026pi;\u003c/em\u003e ratio) simultaneously as candidate outliers under strong selective sweeps. Furthermore, the overlap information of a selected signal was obtained using the \u0026ldquo;vennDiagram\u0026rdquo; package in R (https://www.omicstudio.cn/tool/6).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eComparative transcriptome analysis of the pectoral muscle\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eThe breast muscles of four EU meat pigeons, four SK pigeons, and four HF pigeons were collected for transcriptome analysis, and the slaughter traits were recorded (Table 1).\u003cspan style=\"color:#333333;\"\u003e\u0026nbsp;\u003c/span\u003eTotal RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer\u0026apos;s procedure. The amount and purity of the RNA in each sample was quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent, CA, USA) with an RIN number \u0026gt; 7.0, and confirmed by electrophoresis with denaturing agarose gel.\u003cspan style=\"color:#333333;\"\u003e\u0026nbsp;\u003c/span\u003eFinally, we performed 2 \u0026times; 150 bp paired-end sequencing (PE150) with an Illumina Novaseq\u0026trade; 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) following the vendor\u0026apos;s recommended protocol.\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:left;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:left;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eTable 1. Slaughter traits of three pigeon breeds\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:6.1in;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.55pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eBreeds\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLive weight\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e（\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eg\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e）\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.25pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSlaughter weight (g)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eeviscerated weight (g)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003epectorales weight (g)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.55pt;border: medium none;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eEuro-pigeon\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border: medium none;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e599.25\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e64.20\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.25pt;border: medium none;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e523.00\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e65.56\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: medium none;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e404.50\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e50.49\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border: medium none;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e119.5\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e19.21\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.55pt;border: medium none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSilver King\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border: medium none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e482.75\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e34.54\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.25pt;border: medium none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e423.50\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e34.38\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: medium none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e331.50\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e21.27\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border: medium none;padding: 0in 5.4pt;height: 23.2pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e86.00\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e5.89\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63.55pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eHigh flyer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e311.00\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e32.76\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.25pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e262.50\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e32.80\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e177.00\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e25.53\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 23.65pt;vertical-align: top;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e64.95\u003c/span\u003e\u003cspan style=\"font-size:13px;\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e9.16\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height: 150%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003eSequence and primary\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='line-height: 150%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003eanalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCutadapt software (\u003c/span\u003e\u003ca href=\"https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9\" target=\"_blank\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ehttps://cutadapt.readthedocs.io/en/stable/\u003c/span\u003e\u003c/a\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e, version: cutadapt-1.9) was used to remove the reads that contained adaptor contamination (command line: ~cutadapt -a ADAPT1 -A ADAPT2 -o out1.fastq -p out2.fastq in1.fastq in2.fastq -O 5 -m 100). After removing the low quality and undetermined bases, HISAT2 software (\u003c/span\u003e\u003ca href=\"file:///D%3A/%E8%BD%AC%E5%BD%95%E7%BB%84%E6%B5%8B%E5%BA%8F/%E8%BD%AC%E5%BD%95%E7%BB%84%E6%B5%8B%E5%BA%8F%E7%BB%93%E6%9E%9C1/Report/Report/2020K02sxjA001-B34_houhaobin_report/%3Ca%20href=\" target=\"_blank\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ehttps://daehwankimlab.github.io/hisat2/\u003c/span\u003e\u003c/a\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e, version: hisat2-2.0.4) was used to map reads to the Cliv_1.0 rock pigeon reference genome, (command line: ~hisat2 -1 R1.fastq.gz -2 R1.fastq.gz -S sample_mapped.sam). The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/, version: stringtie-1.3.4d. Linux_x86_64) with default parameters (command line: ~stringtie -p 4 -G genome.gtf -o output.gtf -l sample input.bam). The transcriptomes from all of the samples were merged to reconstruct a comprehensive transcriptome using GffCompare software (http://ccb.jhu.edu/software/stringtie/gffcompare.shtml, version: gffcompare-0.9.8. Linux_x86_64). After the final transcriptome was generated, StringTie and Ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the levels of transcript expression and determine the mRNA expression level by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads (millions) \u0026times; exon_length (kB)]), (command line: ~stringtie -e -B -p 4 -G merged.gtf -o samples.gtf samples.bam). The differentially expressed mRNAs with a fold-change \u0026gt; 2 or fold-change \u0026lt; 0.5, and p-value \u0026lt; 0.05 were selected using DESeq2[53] (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eGene ontology enrichment and pathway analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eThe function of the differentially expressed genes in different types of pigeon breast muscle was investigated by comparing the following groups: EU vs SK, EU vs HF, and SK vs HF. Goseq was used to perform the enrichment analysis of the GO term [54]. Based on the KEGG database, KOBAS 2.0 software was used to analyze the pathways associated with the differentially expressed genes [55]. Ggplot 2 was used to analyze the enrichment of the GO and KEGG databases, and the results were displayed as a scatter plot (bubble chart).\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;line-height:150%;\"\u003e\u003cstrong\u003e\u003cspan style='line-height:150%;font-family:\"Times New Roman\",serif;'\u003eReal-time quantitative PCR analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;text-indent:21.0pt;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eUsing 12 RNA samples isolated from pigeons (four EU pigeons, four SK pigeons, and four HF pigeons),\u0026nbsp;the RNA OD value was detected with an ultra-micro nucleic acid protein analyzer (scandrop100).\u0026nbsp;SYBR Green I was used to detect the expression of target genes in the samples\u0026nbsp;using the A260/A280\u0026nbsp;ratio,\u0026nbsp;\u0026beta;-actin as an internal reference gene, and four target genes (primers are listed in Table 2).\u0026nbsp;The fluorescence quantitative PCR program and system was performed as follows:\u0026nbsp;Step 1: 95℃ for 3 min; Step 2: 95℃ for 10 s; Step 3: 60℃ for 30 s + plate read; repeat step 2 for 39 cycles. Melt curve analysis: (60℃ \u0026ndash; 95℃, +1℃/cycle, holding time of 4 s).\u003c/span\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to The Shanghai Jinhuang Pigeon Co., Ltd., provided support in the process of collecting research materials on High Flyers and commercial pigeon breeds. The authors would like to thank Mr. Liu for providing technical support in bioinformatics analysis. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental design: J.Y. and W.D.; Project leader: C.Y.; Article writer: H.H.; Data analysis and mining: H.H. and X.W.; Sample collection: X.C., W.L., Y.T., and A.B.; Research materials: Q.W. and W.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has been supported by the Key Projects of Developing Agriculture by Science and Technology in Shanghai (2018-02-08-00-12-F01546) and the Shanghai Grants from the SAAS Program for Excellent Research Team (SPERT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe process of collecting pigeon samples strictly follows the animal ethics procedures and guidelines of the People\u0026rsquo;s Republic of China. Pigeons used in this study were approved by the Ethics and Animal Welfare Committee of Shanghai Academy of Agricultural Sciences (No. SAASPZ0521012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBlasco R, Finlayson C, Rosell J, Marco AS, Finlayson S, Finlayson G, Negro JJ, Pacheco FG, Vidal JR: \u003cb\u003eThe earliest pigeon fanciers\u003c/b\u003e. \u003cem\u003eSci Rep\u003c/em\u003e 2014, \u003cb\u003e4\u003c/b\u003e:5971.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKokoszyński D, Stęczny K, Żochowska-Kujawska J, Sobczak M, Kotowicz M, Saleh M, Fik M, Arp\u0026aacute;šov\u0026aacute; H, Hrnč\u0026aacute;r C, Włodarczyk K: \u003cb\u003eCarcass Characteristics, Physicochemical Properties, and Texture and Microstructure of the Meat and Internal Organs of Carrier and King Pigeons\u003c/b\u003e. \u003cem\u003eAnimals: an open access journal from MDPI\u003c/em\u003e 2020, \u003cb\u003e10\u003c/b\u003e(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAssociation CAA: \u003cb\u003eChina Poultry Industry Development Report in 2018\u003c/b\u003e. 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issue):D480-484.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pigeons, Pectoral muscle development, Selective sweep, Transcriptome, SYNE1","lastPublishedDoi":"10.21203/rs.3.rs-468678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-468678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: Various morphological breeds of rock pigeons have emerged as a result of human domestication. Pigeon breed resources provide a genetic model for the study of phenomics. The pectoral muscles are play a key role for the meat production performance of the meat pigeon and the athletic ability of the High flyers. Euro-pigeons and Silver King are commercial varieties that exhibit good meat production performance. In contrast to the domestication direction of meat pigeons, the traditional Chinese ornamental pigeon breed, High flyers, has a small and light body. Here, we investigate the molecular mechanism of the pectoral muscle development and function of pigeons using whole-genome and RNA sequencing data\nResult: The selective sweep analysis (FST and log2 (θπ ratio)) revealed 293 and 403 positive selection genes in Euro-pigeons and Silver King, respectively, of which 65 genes were shared. With the Silver King and Euro-pigeon as the control group, the High flyers were selected for 427 and 566 genes respectively. There were 673 differentially expressed genes in the breast muscle transcriptome between the commercial meat pigeons and ornamental pigeons. Pigeon genome selection signal combined with the breast muscle transcriptome revealed that 6 genes from commercial varieties of pigeons and 5 genes from Chinese traditional ornamental pigeons were positively selected. These genes were involved in pathways related to muscle development and function.\nConclusion: Integrated selection signal, transcriptome analysis, and functional annotation identified SYNE1 as a key gene enriched in the actin binding and muscle cell differentiation pathways. Moreover, SYNE1 gene mutations have been associated with human muscular dystrophy. The differential expression of this gene reveals that it has a negative regulatory role in the development and function of pigeon breast muscle.","manuscriptTitle":"Comparative genome and transcriptome integration studies reveal the mechanism of pectoral muscle development and function in pigeons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-28 18:21:45","doi":"10.21203/rs.3.rs-468678/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c36046b3-ec4b-4579-b31a-0e330b451f97","owner":[],"postedDate":"April 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2021-12-20T08:26:25+00:00","versionOfRecord":{"articleIdentity":"rs-468678","link":"https://doi.org/10.3389/fgene.2021.735795","journal":{"identity":"frontiers-in-genetics","isVorOnly":true,"title":"Frontiers in Genetics"},"publishedOn":"2021-12-21 08:26:25","publishedOnDateReadable":"December 21st, 2021"},"versionCreatedAt":"2021-04-28 18:21:45","video":"","vorDoi":"10.3389/fgene.2021.735795","vorDoiUrl":"https://doi.org/10.3389/fgene.2021.735795","workflowStages":[]},"version":"v1","identity":"rs-468678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-468678","identity":"rs-468678","version":["v1"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}