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As one of the keys to healthy performance, robustness of farm animals is gaining importance, and with this comes increasing interest in genetic dissection of genotype-by-environment interactions (G×E). Changes in gene expression are among the most sensitive responses conveying adaptation to environmental stimuli. Environmentally responsive regulatory variation thus likely plays a central role in G×E. In the present study, we set out to detect action of environmentally responsive cis-regulatory variation by the analysis of condition-dependent allele specific expression (cd-ASE) in porcine immune cells. For this, we harnessed mRNA-sequencing data of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with lipopolysaccharide, dexamethasone, or their combination. These treatments mimic common challenges such as bacterial infection or stress, and induce vast transcriptome changes. About two thirds of the examined loci showed significant ASE in at least one treatment, and out of those about ten percent exhibited cd-ASE. Most of the ASE variants were not yet reported in the PigGTEx Atlas. Genes showing cd-ASE were enriched in cytokine signaling in immune system and include several key candidates for animal health. In contrast, genes showing no ASE featured cell-cycle related functions. We confirmed LPS-dependent ASE for one of the top candidates, SOD2, which ranks among the major response genes in LPS-stimulated monocytes. The results of the present study demonstrate the potential of in vitro cell models coupled with cd-ASE analysis for the investigation of G×E in farm animals. The identified loci may benefit efforts to unravel the genetic basis of robustness and improvement of health and welfare in pigs.

SUMMARY: cis‐Regulatory variations contribute to trait evolution and adaptation during crop domestication and improvement. As the most important harvested organ in maize (Zea mays L.), kernel size has undergone intensive selection for size. However, the associations between maize kernel size and cis‐regulatory variations remain unclear. We chose two independent association populations to dissect the genetic architecture of maize kernel size together with transcriptomic and genotypic data. The resulting phenotypes reflected a strong influence of population structure on kernel size. Compared with genome‐wide association studies (GWASs), which accounted for population structure and relatedness, GWAS based on a naïve or simple linear model revealed additional associated single‐nucleotide polymorphisms significantly involved in the conserved pathways controlling seed size in plants. Regulation analyses through expression quantitative trait locus mapping revealed that cis‐regulatory variations likely control kernel size by fine‐tuning the expression of proximal genes, among which ZmKL1 (GRMZM2G098305) was transgenically validated. We also proved that the pyramiding of the favorable cis‐regulatory variations has contributed to the improvement of maize kernel size. Collectively, our results demonstrate that cis‐regulatory variations, together with their regulatory genes, provide excellent targets for future maize improvement. Significance Statement: GWAS associations confounded with population structure and relatedness were found to contribute to the genetic architecture of maize kernel size. Meanwhile, the favourable cis‐regulatory variations were found to involve in the improvement of maize kernel size. [ABSTRACT FROM AUTHOR]

Cotton fiber development is still an intriguing question to understand fiber commitment and development. At different fiber developmental stages, many genes change their expression pattern and have a pivotal role in fiber quality and yield. Recently, numerous studies have been conducted for transcriptional regulation of fiber, and raw data were deposited to the public repository for comprehensive integrative analysis. Here, we remapped > 380 cotton RNAseq data with uniform mapping strategies that span ∼400 fold coverage to the genome. We identified stage-specific features related to fiber cell commitment, initiation, elongation, and Secondary Cell Wall (SCW) synthesis and their putative cis-regulatory elements for the specific regulation in fiber development. We also mined Exclusively Expressed Transcripts (EETs) that were positively selected during cotton fiber evolution and domestication. Furthermore, the expression of EETs was validated in 100 cotton genotypes through the nCounter assay and correlated with different fiber-related traits. Thus, our data mining study reveals several important features related to cotton fiber development and improvement, which were consolidated in the “CottonExpress-omics” database.

Allelic imbalance (AI) occurs when alleles in a diploid individual are differentially expressed and indicates cis acting regulatory variation. What is the distribution of allelic effects in a natural population? Are all alleles the same? Are all alleles distinct? The approach described applies to any technology generating allele-specific sequence counts, for example for chromatin accessibility and can be applied generally including to comparisons between tissues or environments for the same genotype. Tests of allelic effect are generally performed by crossing individuals and comparing expression between alleles directly in the F1. However, a crossing scheme that compares alleles pairwise is a prohibitive cost for more than a handful of alleles as the number of crosses is at least (n² -n)/2 where n is the number of alleles. We show here that a testcross design followed by a hypothesis test of AI between testcrosses can be used to infer differences between nontester alleles, allowing n alleles to be compared with n crosses. Using a mouse data set where both testcrosses and direct comparisons have been performed, we show that the predicted differences between nontester alleles are validated at levels of over 90% when a parent-of-origin effect is present and of 60%-80% overall. Power considerations for a testcross, are similar to those in a reciprocal cross. In all applications, the testing for AI involves several complex bioinformatics steps. BayesASE is a complete bioinformatics pipeline that incorporates state-of-the-art error reduction techniques and a flexible Bayesian approach to estimating AI and formally comparing levels of AI between conditions. The modular structure of BayesASE has been packaged in Galaxy, made available in Nextflow and as a collection of scripts for the SLURM workload manager on github (https://github.com/McIntyre-Lab/BayesASE). [ABSTRACT FROM AUTHOR]

Accumulating evidence from small-scale studies has suggested that allele-specific expression (ASE) plays an important role in tumor initiation and progression. However, little is known about genome-wide ASE in tumors. In this study, we conducted a comprehensive analysis of ASE in individuals with colorectal cancer (CRC) on a genome-wide scale. We identified 5.4 thousand genome-wide ASEs of single nucleotide variations (SNVs) from tumor and normal tissues of 59 individuals with CRC. We observed an increased ASE level in tumor samples and the ASEs enriched as hotspots on the genome. Around 63% of the genes located there were previously reported to contain complex regulatory elements, e.g., human leukocyte antigen (HLA), or were implicated in tumor progression. Focussing on the allelic expression of somatic mutations, we found that 37.5% of them exhibited ASE, and genes harboring such somatic mutations, were enriched in important pathways implicated in cancers. In addition, by comparing the expected and observed ASE events in tumor samples, we identified 50 tumor specific ASEs which possibly contributed to the somatic events in the regulatory regions of the genes and significantly enriched known cancer driver genes. By analyzing CRC ASEs from several perspectives, we provided a systematic understanding of how ASE is implicated in both tumor and normal tissues and will be of critical value in guiding ASE studies in cancer.

Discrete colour morphs coexisting within a single population are common in nature. In a broad range of organisms, sympatric colour morphs often display major differences in other traits, including morphology, physiology or behaviour. Despite the repeated occurrence of this phenomenon, our understanding of the genetics that underlie multi-trait differences and the factors that promote the long-term maintenance of phenotypic variability within a freely interbreeding population are incomplete. Here, we investigated the genetic basis of red and black head colour in the Gouldian finch (Erythrura gouldiae), a classic polymorphic system in which naturally occurring colour morphs also display differences in aggressivity and reproductive success. We show that the candidate locus is a small (approx. 70 kb) noncoding region mapping to the Z chromosome near the Follistatin (FST) gene. Unlike recent findings in other systems where phenotypic morphs are explained by large inversions containing hundreds of genes (so-called supergenes), we did not identify any structural rearrangements between the two haplotypes using linked-read sequencing technology. Nucleotide divergence between the red and black alleles was high when compared to the remainder of the Z chromosome, consistent with their maintenance as balanced polymorphisms over several million years. Our results illustrate how pleiotropic phenotypes can arise from simple genetic variation, probably regulatory in nature. [ABSTRACT FROM AUTHOR]

Cis- and trans-regulatory mutations are important contributors to transcriptome evolution. Quantifying their relative contributions to intraspecific variation in gene expression is essential for understanding the population genetic processes that underlie evolutionary changes in gene expression. Here, we have examined this issue by quantifying genome-wide, allele-specific expression (ASE) variation using a crossing scheme that produces F1 hybrids between 18 different Drosophila melanogaster strains sampled from the Drosophila Genetic Reference Panel and a reference strain from another population. Head and body samples from F1 adult females were subjected to RNA sequencing and the subsequent ASE quantification. Cis- and trans-regulatory effects on expression variation were estimated from these data. A higher proportion of genes showed significant cis-regulatory variation (~28%) than those that showed significant trans-regulatory variation (~9%). The sizes of cis-regulatory effects on expression variation were 1.98 and 1.88 times larger than trans-regulatory effects in heads and bodies, respectively. A generalized linear model analysis revealed that both cisand trans-regulated expression variation was strongly associated with nonsynonymous nucleotide diversity and tissue specificity. Interestingly, trans-regulated variation showed a negative correlation with local recombination rate. Also, our analysis on proximal transposable element (TE) insertions suggested that they affect transcription levels of ovary-expressed genes more pronouncedly than genes not expressed in the ovary, possibly due to defense mechanisms against TE mobility in the germline. Collectively, our detailed quantification of ASE variations from a natural population has revealed a number of new relationships between genomic factors and the effects of cis- and trans-regulatory factors on expression variation. [ABSTRACT FROM AUTHOR]

The regulatory architecture of gene expression is known to differ substantially between sexes in Drosophila, but most studies performed so far used whole-body data and only single crosses, which may have limited their scope to detect patterns that are robust across tissues and biological replicates. Here, we use allele-specific gene expression of parental and reciprocal hybrid crosses between 6 Drosophila melanogaster inbred lines to quantify cis- and trans-regulatory variation in heads and gonads of both sexes separately across 3 replicate crosses. Our results suggest that female and male heads, as well as ovaries, have a similar regulatory architecture. On the other hand, testes display more and substantially different cis-regulatory effects, suggesting that sex differences in the regulatory architecture that have been previously observed may largely derive from testis-specific effects. We also examine the difference in cis-regulatory variation of genes across different levels of sex bias in gonads and heads. Consistent with the idea that intersex correlations constrain expression and can lead to sexual antagonism, we find more cis variation in unbiased and moderately biased genes in heads. In ovaries, reduced cis variation is observed for male-biased genes, suggesting that cis variants acting on these genes in males do not lead to changes in ovary expression. Finally, we examine the dominance patterns of gene expression and find that sex- and tissue-specific patterns of inheritance as well as trans-regulatory variation are highly variable across biological crosses, although these were performed in highly controlled experimental conditions. This highlights the importance of using various genetic backgrounds to infer generalizable patterns., Competing Interests: Conflicts of interest statement The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine.

As one of the keys to healthy performance, robustness of farm animals is gaining importance, and with this comes increasing interest in genetic dissection of genotype-by-environment interactions (G×E). Changes in gene expression are among the most sensitive responses conveying adaptation to environmental stimuli. Environmentally responsive regulatory variation thus likely plays a central role in G×E. In the present study, we set out to detect action of environmentally responsive cis -regulatory variation by the analysis of condition-dependent allele specific expression (cd-ASE) in porcine immune cells. For this, we harnessed mRNA-sequencing data of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with lipopolysaccharide, dexamethasone, or their combination. These treatments mimic common challenges such as bacterial infection or stress, and induce vast transcriptome changes. About two thirds of the examined loci showed significant ASE in at least one treatment, and out of those about ten percent exhibited cd-ASE. Most of the ASE variants were not yet reported in the PigGTEx Atlas. Genes showing cd-ASE were enriched in cytokine signaling in immune system and include several key candidates for animal health. In contrast, genes showing no ASE featured cell-cycle related functions. We confirmed LPS-dependent ASE for one of the top candidates, SOD2 , which ranks among the major response genes in LPS-stimulated monocytes. The results of the present study demonstrate the potential of in vitro cell models coupled with cd-ASE analysis for the investigation of G×E in farm animals. The identified loci may benefit efforts to unravel the genetic basis of robustness and improvement of health and welfare in pigs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Murani and Hadlich.)

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel ( DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis-regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele-specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis-regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis-regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis-regulatory region of tan indicated that SNPs within the region are significantly associated with allele-specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis-regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements. [ABSTRACT FROM AUTHOR]

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine. [ABSTRACT FROM AUTHOR]

Many common traits are believed to be a composite reflection of multiple genetic and environmental factors. Recent advances suggest that subtle variations in the regulation of gene expression may contribute to quantitative traits. The nature of sequence variation affecting the regulation of gene expression either in cis (that is, affecting the expression of only one of the two alleles in a heterozygous diploid) or in trans (that is, affecting the expression of both alleles in a heterozygous diploid) is a key and usually unknown feature for the breeders. If the change in expression acts entirely in cis, then the structural gene can be treated as a candidate gene and a potential target for marker-assisted selection. Therefore, gene surveys for cis-regulatory variation are a first step in identifying potential targets for marker-assisted breeding. Here, we discuss in detail the “genome-wide analysis of allele-specific expression differences” (GASED) approach. The GASED approach was developed to screen for cis-regulatory variation on a genome-wide scale. In GASED, mRNA abundance is treated as if it were a quantitative phenotypic response variable, whose genetic between-F1 hybrid variance is partitioned into additive and non-additive components. In plant breeding, this partitioning of the genetic variance is well known in the context of estimation of general and specific combining abilities for diallel crossing schemes. We demonstrate the GASED method using Arabidopsis thaliana data. The method can be used to screen for cis-regulatory variation in any crop species for which diallel crossing schemes are appropriate and genomic tools are available. [ABSTRACT FROM AUTHOR]

Cotton fiber development is still an intriguing question to understand fiber commitment and development. At different fiber developmental stages, many genes change their expression pattern and have a pivotal role in fiber quality and yield. Recently, numerous studies have been conducted for transcriptional regulation of fiber, and raw data were deposited to the public repository for comprehensive integrative analysis. Here, we remapped > 380 cotton RNAseq data with uniform mapping strategies that span ∼400 fold coverage to the genome. We identified stage-specific features related to fiber cell commitment, initiation, elongation, and Secondary Cell Wall (SCW) synthesis and their putative cis-regulatory elements for the specific regulation in fiber development. We also mined Exclusively Expressed Transcripts (EETs) that were positively selected during cotton fiber evolution and domestication. Furthermore, the expression of EETs was validated in 100 cotton genotypes through the nCounter assay and correlated with different fiber-related traits. Thus, our data mining study reveals several important features related to cotton fiber development and improvement, which were consolidated in the "CottonExpress-omics" database., Competing Interests: The CSIR-NBRI has an official collaboration with Tierra Seed Science Pvt. Ltd., for cotton research where DM and PB are employed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Prasad, Khatoon, Verma, Aalam, Kumar, Mohapatra, Bhattacharya, Bag and Sawant.)

Accumulating evidence from small-scale studies has suggested that allele-specific expression (ASE) plays an important role in tumor initiation and progression. However, little is known about genome-wide ASE in tumors. In this study, we conducted a comprehensive analysis of ASE in individuals with colorectal cancer (CRC) on a genome-wide scale. We identified 5.4 thousand genome-wide ASEs of single nucleotide variations (SNVs) from tumor and normal tissues of 59 individuals with CRC. We observed an increased ASE level in tumor samples and the ASEs enriched as hotspots on the genome. Around 63% of the genes located there were previously reported to contain complex regulatory elements, e.g., human leukocyte antigen (HLA), or were implicated in tumor progression. Focussing on the allelic expression of somatic mutations, we found that 37.5% of them exhibited ASE, and genes harboring such somatic mutations, were enriched in important pathways implicated in cancers. In addition, by comparing the expected and observed ASE events in tumor samples, we identified 50 tumor specific ASEs which possibly contributed to the somatic events in the regulatory regions of the genes and significantly enriched known cancer driver genes. By analyzing CRC ASEs from several perspectives, we provided a systematic understanding of how ASE is implicated in both tumor and normal tissues and will be of critical value in guiding ASE studies in cancer.

Cis - and trans -regulatory mutations are important contributors to transcriptome evolution. Quantifying their relative contributions to intraspecific variation in gene expression is essential for understanding the population genetic processes that underlie evolutionary changes in gene expression. Here, we have examined this issue by quantifying genome-wide, allele-specific expression (ASE) variation using a crossing scheme that produces F 1 hybrids between 18 different Drosophila melanogaster strains sampled from the Drosophila Genetic Reference Panel and a reference strain from another population. Head and body samples from F 1 adult females were subjected to RNA sequencing and the subsequent ASE quantification. Cis - and trans -regulatory effects on expression variation were estimated from these data. A higher proportion of genes showed significant cis -regulatory variation (∼28%) than those that showed significant trans -regulatory variation (∼9%). The sizes of cis -regulatory effects on expression variation were 1.98 and 1.88 times larger than trans -regulatory effects in heads and bodies, respectively. A generalized linear model analysis revealed that both cis - and trans -regulated expression variation was strongly associated with nonsynonymous nucleotide diversity and tissue specificity. Interestingly, trans -regulated variation showed a negative correlation with local recombination rate. Also, our analysis on proximal transposable element (TE) insertions suggested that they affect transcription levels of ovary-expressed genes more pronouncedly than genes not expressed in the ovary, possibly due to defense mechanisms against TE mobility in the germline. Collectively, our detailed quantification of ASE variations from a natural population has revealed a number of new relationships between genomic factors and the effects of cis - and trans -regulatory factors on expression variation., (Copyright © 2017 by the Genetics Society of America.)