Evolution of cis- and trans-regulatory divergence in the chicken genome between two contrasting breeds analyzed using three tissue types at one-day-old

- Evolution of cis- and trans-regulatory divergence in the chicken genome.
- To understand the role of cis- and trans-regulatory variation on population divergence in chicken, we developed reciprocal crosses of two chicken breeds, White Leghorn and Cornish Game, which exhibit major differences in body size and reproductive traits, and used them to determine the degree of cis versus trans variation in the brain, liver, and muscle tissue of male and female 1-day-old specimens..
- Compared with cis-regulatory divergence, trans-acting genes were more extensive in the chicken genome.
- In addition, considerable compensatory cis- and trans-regulatory changes exist in the chicken genome.
- Our research is the first study to describe the regulatory divergence between two contrasting breeds.
- The results suggest that artificial selection associated with domestication in chicken could have acted more on trans-regulatory divergence than on cis-regulatory divergence..
- Numerous transcriptional regulatory factors, which can be classified into cis-regulatory elements and trans-regulatory factors, regulate gene expression [1].
- In contrast, trans-regulatory factors regulate (or modify) the expression of distant genes by combining with their target sequences [1, 2].
- Cis- and trans-regulatory elements are thought to vary based on key genetic and evolutionary properties [5, 6]..
- By comparison, trans- regulatory factors interact with target sequences to regulate both alleles [1].
- Trans-regulatory divergence is enriched for dominant effect, while the effects of cis-regulatory variants are additivity [6, 7].
- Beneficial cis-regulatory variants are more likely to be enriched to fixation in the course of.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- Both cis- and trans-regulatory variation are play key roles in phenotypic variation [1, 8–10].
- Therefore, it is critical to investigate gene regulatory divergence in birds..
- The rapid change under domestication offers a unique model for revealing the relative importance of the cis- and trans-regulatory variation underlying phenotypic change.
- We used reciprocal crosses of White Leghorn (WL), a key layer breed selected for its high egg output, and Cornish Game breeds (CG), a cornerstone broiler breed selected for its rapid growth and muscle development [30], to assess the role of different forms of regulatory variation in the brain, liver, and muscle tissue of 1-day-old males and females..
- The profile of the parental genomes and gene expression in different tissues, sexes of progenies.
- To identify breed- specific variants, we sequenced the genes of four parents of the two reciprocal crosses, recovering on average 100.73 million pair-end reads per sample after quality control.
- We picked SNPs that were homozygous in each parental bird but different from each other in the same cross (heterozygous in the hybrid progenies), resulting in two heterozygous SNP lists with 1.4 million heterozy- gous SNPs on average for the two reciprocal crosses, in- dividually, to identify the allele-specific RNA-Seq reads of the offspring in the following steps..
- To eliminate the effect of the sex chromosomes, we removed all Z and W genes from our analysis and focused entirely on autosomal loci.
- Tissue was the most significant factor influen- cing gene expression, sex played a leading role in the brain, strain influenced gene expression of liver the most, while in the muscle, the parent-of-origin seemed the most powerful because samples were divided into.
- Table 1 Studies that have classified gene regulatory divergence in genomes Species Tissue Sex Cis Trans Cis and.
- Consequently, we retained all three variables in our subsequent analyses, resulting in 12 treatment groups, comprised of three tis- sues, two sexes, and two reciprocal crosses in the present study..
- To identify the parental origin of the mRNA of the off- spring, we explored a novel pipeline using the ‘asSeq’.
- Briefly, a set of R scripts was available for genotype phasing based on the 1.4 million heterozy- gous SNPs identified in the preceding step.
- Approxi- mately 2% of the SNPs mentioned above were located in the exon region.
- We also concatenated two female liver samples in the same manner.
- Genes were classified into different categories based on the type of regulatory divergence.
- Approximately a fifth of the genes contained het- erozygous SNPs and were expressed in our progeny samples (Additional file 1: Table S1).
- cross 4), in 14.71% in the brain, 36.45% in the liver, and 38.38% in muscle (consider the heterozygous SNP list of cross 2, for example).
- In males, 17.64% of the genes in the brain, 41.87% of the genes in the liver, and 37.84% of the genes in muscle were expressed significantly differentially (Additional file 1: Table S1)..
- Expressed genes were classified into different categories based on the type of gene regulatory divergence .
- expected, considering the relatively recent divergence time of the two breeds investigated.
- More than 70, 40%, and ap- proximately 50% of the genes in the brain, liver, and muscle, respectively, were classified as conserved.
- Nonethe- less, we observed substantial cis- and trans-variation in the hybrid crosses.
- Genes regulated by both cis- and trans-regulatory varia- tions were divided into four categories, including “cis + trans (same.
- Unexpectedly, we observed only few loci with consistent cis- or trans-regulatory divergence across dif- ferent groups (Additional file 1: Figure S6).
- The stable cis- or trans-regulatory divergence genes seem to play key roles in phenotypic divergence.
- We counted the number of variants located 1 kb upstream of transcription start sites of each gene using the genome data of the four parents.
- The results showed greater varia- tions upstream of cis-regulatory divergence genes than upstream of trans-acted genes in all samples (Fig.
- The ratio of the number of non-synonymous SNPs to the number of synonymous SNPs (pN/pS) in the coding sequences of each gene was calculated in the present study..
- Previous studies on regulatory divergence genes did not select identical time points from the embryo to adult.
- therefore, different results would be obtained from the regulatory divergence genes across different development stages..
- First, the SNP list we used to identify the parental origin was fil- tered strictly from the re-sequencing data of the four parents.
- The coordinate position shows the average log2 expression fold-change between the alleles in the hybrids (y-axis) and between the two purebreeds (x-axis).
- The proportion of each category is summarized in the bar graph c, where we removed the conserved and ambiguous genes, and further subdivided the cis + trans category genes into two categories, based on whether the cis and trans variants acted in the same direction or in opposite directions.
- The number above the bar represents the proportion of genes in the regulatory category, and the number on the bar represents the gene count of the category.
- The SNPs were used to mark the parental origins of the alleles of each gene, which in- creased the accuracy of classification.
- 4 Sequence conservation analysis of the cis- and trans-regulatory divergence genes.
- The number following the regulatory category name in the legend refers to the mean value of variation count of all genes in this category.
- b The pN/pS values in cis- and trans-regulatory divergence genes.
- The y-axis refers to the mean value of all genes in the category.
- Significance of the difference between the two regulatory categories is labeled above the bar.
- most genes exhibited conserved or ambiguous expression, and more trans-regulatory variants compared to cis- regulatory variants, which could be attributed to the rela- tively short differentiation time between WL and CG.
- The large proportion of trans-regulatory mutations observed in the present study suggest that artificial selection has pri- marily acted on trans-regulatory mutations, but the neutral cis-regulatory mutations have not accumulated substan- tially over the relatively short period since the breeds were established..
- Genes regulated by both cis- and trans-variations act in opposite directions more often than not, and most genes were classified as “compensatory” in the present study.
- Despite the lack of a complete dosage compensation mechanism on the sex chromosome [24–28], an extensive compensatory trend persists in the chicken genome..
- There were few loci with consistent cis- or trans- regulatory variation among different tissues and between different sexes.
- However, the cis- and trans-regulatory diver- gence classification is much more complex than the ASE analysis.
- It is always controlled by the interaction of cis-regulatory DNA sequences and trans-regulatory factors, which could complicate the identification of regulatory divergence.
- Our results are consistent with the findings of a recent study in Drosophila [7], which de- tected greater variants 1 kb upstream of transcription start sites of cis-regulatory divergence genes than upstream of transcription start sites trans-acted genes, suggesting that our classification results were reliable.
- Our results suggest that trans-regulatory divergence genes were subjected to high selective con- straint in the course of chicken domestication and could.
- In the present study, we present a pipeline for exploring ASE in the hybrid progenies of inbred lines without a spe- cific reference genome.
- Using the genome sequences of par- ents and RNA-seq data of offspring, we classified the genes expressed in the chicken genome into different categories based on the type of regulatory divergence involved.
- More instances of trans-regulatory divergence than instances of cis-regulatory divergence were observed due to the relatively short history of divergence in the two parental breeds.
- Con- siderable compensatory cis- and trans-regulatory changes exist in the chicken genome.
- The sequence conservation analysis results suggested that artificial selection associated with domestication could have potentially acted on genes regulated by trans-variations in the course of the establish- ment of commercial chicken breeds..
- The inbred chickens used in our study were obtained from the National Engineering Laboratory for Animal Breeding of the China Agricultural University.
- The 4 parental chickens of the two reciprocal crosses were released after collected brachial vein blood, and the 23 1-day-old chickens were beheaded before we collected tissues..
- The re-sequencing data of the four parents were mapped to the chicken reference genome (Gallus_gallus-5.0, http://hgdownload..
- The four simulated parental ge- nomes were used to replace the reference genome in the RNA-Seq data alignment of the hybrid crosses.
- In the case of one read containing more than one SNPs, we set the par- ameter of prop.cut to 0.9, that is, we assigned a read to one of the two parental alleles if the proportion of those heterozygous SNPs suggested the read that was from that allele was greater than 0.9.
- We filtered the expressed genes using the following criterion: for each sex and each tissue, the total reads of the three pure- bred offspring and the three hybrid offspring have to be between 6 and 1000.
- To categorize regulatory variations, we referenced the methods applied in the study of regulatory divergence in Drosophila [7] and house mouse [36].
- Re-sequencing data from four parents were used to study the sequence conservation of cis- and trans- regulatory divergence genes.
- The pN/pS ratio of the coding sequence and the number of variants in 1 kb up- stream from the transcription start site were calculated using the results of SNP annotation performed using SnpEff [53].
- Synonymous muta- tion refers to the variant in the coding region causing a codon that produces the same amino acid..
- Intersection of different groups of cis- and trans- regulatory genes.
- The ratio of the numbers of non-synonymous SNPs to the numbers of.
- The ratio of the numbers of non-synonymous SNPs to the numbers of synonymous SNPs (pN/pS) in different groups of cross 3.
- YJ participated in the reciprocal cross experiment, the collection of samples, and revised the manuscript.
- ZJ and XZ provided some suggestions for the improvement of the study and substantively revised the manuscript.
- JL provided experimental animals and assisted in the carrying out of the reciprocal cross experiment.
- NY participated in the design of the study.
- This work was supported by the Beijing Innovation Team of the Modern Agro-industry Technology Research System (BAIC04 – 2016, BAIC04 – 2017).
- The funding body did not exert influence on the design of the study, on data collection, analysis, and interpretation, or on the writing of the manuscript..
- The datasets generated and/or analyzed during the current study are available in the NCBI BioProject (https://submit.ncbi.nlm.nih.gov/subs/.
- The author NY is a member of the editorial board (Associate Editor) of this journal..
- Evolutionary changes in cis and trans gene regulation.
- The roles of cis- and trans- regulation in the evolution of regulatory incompatibilities and sexually dimorphic gene expression.
- Regulatory divergence in Drosophila revealed by mRNA-seq.
- Extensive compensatory cis-trans regulation in the evolution of mouse gene expression.
- Regulatory divergence between parental alleles determines gene expression patterns in hybrids.
- Parental imprinting of the mouse H19 gene.
- Parental imprinting of the mouse insulin-like growth factor II gene.
- Dosage compensation of the active X chromosome in mammals.
- evolutionary accumulation of cis- and trans-effects on gene expression.
- A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118

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