Allele-specific Expression Research Articles

The conservation of allelic DNA methylation and its relationship with imprinting in maize.

Genomic imprinting refers to allele-specific expression of genes depending on parental origin, and it is regulated by epigenetic modifications. Intraspecific allelic variation for imprinting has been detected; however, the intraspecific genome-wide allelic epigenetic variation in maize and its correlation with imprinting variants remain unclear. Here, three reciprocal hybrids were generated by crossing Zea mays inbred lines CAU5, B73, and Mo17 in order to examine the intraspecific conservation of the imprinted genes in the kernel. The majority of imprinted genes exhibited intraspecific conservation, and these genes also exhibited interspecific conservation (rice, sorghum, and Arabidopsis) and were enriched in some specific pathways. By comparing intraspecific allelic DNA methylation in the endosperm, we found that nearly 15% of DNA methylation existed as allelic variants. The intraspecific whole-genome correlation between DNA methylation and imprinted genes indicated that DNA methylation variants play an important role in imprinting variants. Disruption of two conserved imprinted genes using CRISPR/Cas9 editing resulted in a smaller kernel phenotype. Our results shed light on the intraspecific correlation of DNA methylation variants and variation for imprinting in maize, and show that imprinted genes play an important role in kernel development.

Allele-dependent expression and functionality of lipid enzyme phospholipid:diacylglycerol acyltransferase affect diatom carbon storage and growth.

In the acyl-CoA-independent pathway of triacylglycerol (TAG) synthesis unique to plants, fungi, and algae, TAG formation is catalyzed by the enzyme phospholipid:diacylglycerol acyltransferase (PDAT). The unique PDAT gene of the model diatom Phaeodactylum tricornutum strain CCMP2561 boasts 47 single nucleotide variants within protein coding regions of the alleles. To deepen our understanding of TAG synthesis, we observed the allele-specific expression of PDAT by the analysis of 87 published RNA-sequencing (RNA-seq) data and experimental validation. The transcription of one of the two PDAT alleles, Allele 2, could be specifically induced by decreasing nitrogen concentrations. Overexpression of Allele 2 in P. tricornutum substantially enhanced the accumulation of TAG by 44% to 74% under nutrient stress; however, overexpression of Allele 1 resulted in little increase of TAG accumulation. Interestingly, a more serious growth inhibition was observed in the PDAT Allele 1 overexpression strains compared with Allele 2 counterparts. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that enzymes encoded by PDAT Allele 2 but not Allele 1 had TAG biosynthetic activity, and 7 N-terminal and 3 C-terminal amino acid variants between the 2 allele-encoded proteins substantially affected enzymatic activity. P. tricornutum PDAT, localized in the innermost chloroplast membrane, used monogalactosyldiacylglycerol and phosphatidylcholine as acyl donors as demonstrated by the increase of the 2 lipids in PDAT knockout lines, which indicated a common origin in evolution with green algal PDATs. Our study reveals unequal roles among allele-encoded PDATs in mediating carbon storage and growth in response to nitrogen stress and suggests an unsuspected strategy toward lipid and biomass improvement for biotechnological purposes.

Identification of Allele-Specific Expression Genes Associated with Maize Heterosis

Deciphering the molecular basis of heterosis would yield genes and markers for designing improved maize-hybrid varieties. In this study, 481 BC1F3 lines derived from Zheng58 and PH4CV were genotyped with 15,386 polymorphic SNPs markers and testcrossed with two testers (Chang7-2 and PH6WC) to generate 962 testcross lines. The yield of these testcross lines and their parental lines was evaluated across multiple environments. Genetic analysis revealed that dominance is the primary contributor to heterosis. Mapping of midparent heterosis (MPH) identified two dominant QTL, six additive-by-additive interactions, eighteen additive-by-dominance interactions, and fifty-four dominance-by-dominance interactions. These interactions encompassed 104 genetic blocks, including 24 genetic blocks that explained >1% of phenotypic variances for both MPH and hybrid performance. We compared the locations of the allele-specific expression genes (ASEGs) identified from the expression data of two hybrid lines and their parental lines with those of the 24 genetic blocks and found 15 ASEGs related to yield or biomass regulation, including two known genes BT2 and ZmNF-YC4. Fisher’s exact test analysis indicated a significant enrichment of these ASEGs in the 24 blocks, affirming the reliability of the MPH-mapping results. The co-expression network of six ASEGs, including BT2 and ZmNF-YC4, contained many genes related to yield or biomass regulation. This study unravels potential candidate genes and regulatory networks associated with maize heterosis.

Dynamic roles of small RNAs and DNA methylation associated with heterosis in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundHeterosis is a complex phenomenon wherein the hybrids outperform their parents. Understanding the underlying molecular mechanism by which hybridization leads to higher yields in allopolyploid cotton is critical for effective breeding programs. Here, we integrated DNA methylation, transcriptomes, and small RNA profiles to comprehend the genetic and molecular basis of heterosis in allopolyploid cotton at three developmental stages.ResultsTranscriptome analysis revealed that numerous DEGs responsive to phytohormones (auxin and salicylic acid) were drastically altered in F1 hybrid compared to the parental lines. DEGs involved in energy metabolism and plant growth were upregulated, whereas DEGs related to basal defense were downregulated. Differences in homoeologous gene expression in F1 hybrid were greatly reduced after hybridization, suggesting that higher levels of parental expression have a vital role in heterosis. Small RNAome and methylome studies showed that the degree of DNA methylation in hybrid is higher when compared to the parents. A substantial number of allele-specific expression genes were found to be strongly regulated by CG allele-specific methylation levels. The hybrid exhibited higher 24-nt-small RNA (siRNA) expression levels than the parents. The regions in the genome with increased levels of 24-nt-siRNA were chiefly related to genes and their flanking regulatory regions, demonstrating a possible effect of these molecules on gene expression. The transposable elements correlated with siRNA clusters in the F1 hybrid had higher methylation levels but lower expression levels, which suggest that these non-additively expressed siRNA clusters, reduced the activity of transposable elements through DNA methylation in the hybrid.ConclusionsThese multi-omics data provide insights into how changes in epigenetic mechanisms and gene expression patterns can lead to heterosis in allopolyploid cotton. This makes heterosis a viable tool in cotton breeding.

Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition.

Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.

Single-cell allele-specific expression analysis reveals dynamic and cell-type-specific regulatory effects

Differential allele-specific expression (ASE) is a powerful tool to study context-specific cis-regulation of gene expression. Such effects can reflect the interaction between genetic or epigenetic factors and a measured context or condition. Single-cell RNA sequencing (scRNA-seq) allows the measurement of ASE at individual-cell resolution, but there is a lack of statistical methods to analyze such data. We present Differential Allelic Expression using Single-Cell data (DAESC), a powerful method for differential ASE analysis using scRNA-seq from multiple individuals, with statistical behavior confirmed through simulation. DAESC accounts for non-independence between cells from the same individual and incorporates implicit haplotype phasing. Application to data from 105 induced pluripotent stem cell (iPSC) lines identifies 657 genes dynamically regulated during endoderm differentiation, with enrichment for changes in chromatin state. Application to a type-2 diabetes dataset identifies several differentially regulated genes between patients and controls in pancreatic endocrine cells. DAESC is a powerful method for single-cell ASE analysis and can uncover novel insights on gene regulation.

Transcriptome-wide identification and characterization of genes exhibit allele-specific imprinting in maize embryo and endosperm

BackgroundGenomic imprinting refers to a subset of genes that are expressed from only one parental allele during seed development in plants. Studies on genomic imprinting have revealed that intraspecific variations in genomic imprinting expression exist in naturally genetic varieties. However, there have been few studies on the functional analysis of allele-specific imprinted genes.ResultsHere, we generated three reciprocal crosses among the B73, Mo17 and CAU5 inbred lines. Based on the transcriptome-wide analysis of allele-specific expression using RNA sequencing technology, 305 allele-specific imprinting genes (ASIGs) were identified in embryos, and 655 ASIGs were identified in endosperms from three maize F1 hybrids. Of these ASIGs, most did not show consistent maternal or paternal bias between the same tissue from different hybrids or different tissues from one hybrid cross. By gene ontology (GO) analysis, five and eight categories of GO exhibited significantly higher functional enrichments for ASIGs identified in embryo and endosperm, respectively. These functional categories indicated that ASIGs are involved in intercellular nutrient transport, signaling pathways, and transcriptional regulation of kernel development. Finally, the mutation and overexpression of one ASIG (Zm305) affected the length and width of the kernel.ConclusionIn this study, our data will be helpful in gaining further knowledge of genes exhibiting allele-specific imprinting patterns in seeds. The gain- and loss-of-function phenotypes of ASIGs associated with agronomically important seed traits provide compelling evidence for ASIGs as crucial targets to optimize seed traits in crop plants.

Allele-specific expression reveals genetic drivers of tissue regeneration in mice.

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Allele specific binding of histone modifications and a transcription factor does not predict allele specific expression in correlated ChIP-seq peak-exon pairs

Allele specific expression (ASE) is widespread in many species including cows. Therefore, regulatory regions which control gene expression should show cis-regulatory variation which mirrors this differential expression within the animal. ChIP-seq peaks for histone modifications and transcription factors measure activity at functional regions and the height of some peaks have been shown to correlate across tissues with the expression of particular genes, suggesting these peaks are putative regulatory regions. In this study we identified ASE in the bovine genome in multiple tissues and investigated whether ChIP-seq peaks for four histone modifications and the transcription factor CTCF show allele specific binding (ASB) differences in the same tissues. We then investigate whether peak height and gene expression, which correlates across tissues, also correlates within the animal by investigating whether the direction of ASB in putative regulatory regions, mirrors that of the ASE in the genes they are putatively regulating. We found that ASE and ASB were widespread in the bovine genome but vary in extent between tissues. However, even when the height of a peak was positively correlated across tissues with expression of an exon, ASE of the exon and ASB of the peak were in the same direction only half the time. A likely explanation for this finding is that the correlations between peak height and exon expression do not indicate that the height of the peak causes the extent of exon expression, at least in some cases.

Genetic diversity, tissue-specific expression, and functional analysis of the ATP7A gene in sheep.

In humans, variation of the ATP7A gene may cause cranial exostosis, which is similar to "human horn," but the function of the ATP7A gene in sheep is still unknown. Tissue expression patterns and potential functional loci analysis of the ATP7A gene could help understand its function in sheep horn. In this study, we first identified tissue, sex, breed, and species-specific expression of the ATP7A gene in sheep based on the RNA-sequencing (RNA-seq) data. Second, the potential functional sites of the ATP7A gene were analyzed by using the whole genome sequencing (WGS) data of 99 sheep from 10 breeds. Last, the allele-specific expression of the ATP7A gene was explored. Our result showed the ATP7A gene has significantly higher expression in the big horn than in the small horn, and the ATP7A gene has high expression in the horn and skin, suggesting that this gene may be related to the horn. The PCA results show that the region around the ATP7A can distinguish horned and hornless groups to some extent, further indicating that the ATP7A may be related to horns. When compared with other species, we find seven ruminate specific amino acid sites of the ATP7A protein, which can be important to the ruminate horn. By analyzing WGS, we found 6 SNP sites with significant differences in frequency in horned and hornless populations, and most of these variants are present in the intron. But we still find some potential functional sites, including three missenses, three synonymous mutations, and four Indels. Finally, by combining the RNA-seq and WGS functional loci results, we find three mutations that showed allele-specific expression between big and small horns. This study shows that the ATP7A gene in sheep may be related to horn size, and several potential functional sites we identified here can be useful molecular markers for sheep horn breeding.

Environmentally robust cis-regulatory changes underlie rapid climatic adaptation

Changes in gene expression are thought to play a major role in adaptive evolution. While it is known that gene expression is highly sensitive to the environment, very few studies have determined the influence of genetic and environmental effects on adaptive gene expression differences in natural populations. Here, we utilize allele-specific expression to characterize cis and trans gene regulatory divergence in temperate and tropical house mice in two metabolic tissues under two thermal conditions. First, we show that gene expression divergence is pervasive between populations and across thermal conditions, with roughly 5 to 10% of genes exhibiting genotype-by-environment interactions. Second, we found that most expression divergence was due to cis-regulatory changes that were stable across temperatures. In contrast, patterns of expression plasticity were largely attributable to trans-effects, which showed greater sensitivity to temperature. Nonetheless, we found a small subset of temperature-dependent cis-regulatory changes, thereby identifying loci underlying expression plasticity. Finally, we performed scans for selection in wild house mice to identify genomic signatures of rapid adaptation. Genomic outliers were enriched in genes with evidence for cis-regulatory divergence. Notably, these genes were associated with phenotypes that affected body weight and metabolism, suggesting that cis-regulatory changes are a possible mechanism for adaptive body size evolution between populations. Our results show that gene expression plasticity, largely controlled in trans, may facilitate the colonization of new environments, but that evolved changes in gene expression are largely controlled in cis, illustrating the genetic and nongenetic mechanisms underlying the establishment of populations in new environments.

A targeted sequencing extension for transcript genotyping in single-cell transcriptomics.

As no existing methods within the single-cell RNA sequencing repertoire combine genotyping of specific genomic loci with high throughput, we evaluated a straightforward, targeted sequencing approach as an extension to high-throughput droplet-based single-cell RNA sequencing. Overlaying standard gene expression data with transcript level genotype information provides a strategy to study the impact of genetic variants. Here, we describe this targeted sequencing extension, explain how to process the data and evaluate how technical parameters such as amount of input cDNA, number of amplification rounds, and sequencing depth influence the number of transcripts detected. Finally, we demonstrate how targeted sequencing can be used in two contexts: (1) simultaneous investigation of the presence of a somatic variant and its potential impact on the transcriptome of affected cells and (2) evaluation of allele-specific expression of a germline variant in ad hoc cell subsets. Through these and other comparable applications, our targeted sequencing extension has the potential to improve our understanding of functional effects caused by genetic variation.

Analysis of subcellular RNA fractions demonstrates significant genetic regulation of gene expression in human brain post-transcriptionally

Gaining insight into the genetic regulation of gene expression in human brain is key to the interpretation of genome-wide association studies for major neurological and neuropsychiatric diseases. Expression quantitative trait loci (eQTL) analyses have largely been used to achieve this, providing valuable insights into the genetic regulation of steady-state RNA in human brain, but not distinguishing between molecular processes regulating transcription and stability. RNA quantification within cellular fractions can disentangle these processes in cell types and tissues which are challenging to model in vitro. We investigated the underlying molecular processes driving the genetic regulation of gene expression specific to a cellular fraction using allele-specific expression (ASE). Applying ASE analysis to genomic and transcriptomic data from paired nuclear and cytoplasmic fractions of anterior prefrontal cortex, cerebellar cortex and putamen tissues from 4 post-mortem neuropathologically-confirmed control human brains, we demonstrate that a significant proportion of genetic regulation of gene expression occurs post-transcriptionally in the cytoplasm, with genes undergoing this form of regulation more likely to be synaptic. These findings have implications for understanding the structure of gene expression regulation in human brain, and importantly the interpretation of rapidly growing single-nucleus brain RNA-sequencing and eQTL datasets, where cytoplasm-specific regulatory events could be missed.

Allele-specific quantification of human leukocyte antigen transcript isoforms by nanopore sequencing.

While tens of thousands of HLA alleles have been identified by DNA sequencing, the contribution of alternative splicing to HLA diversity is not well characterized. In this study, we sought to determine if long-read sequencing could be used to accurately quantify allele-specific HLA transcripts in primary human lymphocytes. cDNA libraries were prepared from peripheral blood lymphocytes from 12 donors and sequenced by nanopore long-read sequencing. HLA reads were aligned to donor-specific reference sequences based on the known type of each donor. Allele-specific exon utilization was calculated as the proportion of reads aligning to each allele containing known exons, and transcript isoforms were quantified based on patterns of exon utilization within individual reads. Splice variants were rare among class I HLA genes (median exon retention rate 99%-100%), except for several HLA-C alleles with exon 5 spliced out of up to 15% of reads. Splice variants were also rare among class II HLA genes (median exon retention rate 98%-100%), except for HLA-DQB1. Consistent with previous work, exon 5 of HLA-DQB1 was spliced out in alleles with a mutated splice acceptor site at rs28688207. Surprisingly, a 28% loss of exon 5 was also observed in HLA-DQB1 alleles with an intact splice acceptor site at rs28688207. We describe a simple bioinformatic workflow to quantify allele-specific expression of HLA transcript isoforms. Further studies are warranted to characterize the repertoire of HLA transcripts expressed in different cell types and tissues across diverse populations.

Chromosome-scale genome sequence of Suaeda glauca sheds light on salt stress tolerance in halophytes.

Soil salinity is a growing concern for global crop production and the sustainable development of humanity. Therefore, it is crucial to comprehend salt tolerance mechanisms and identify salt-tolerance genes to enhance crop tolerance to salt stress. Suaeda glauca, a halophyte species well adapted to the seawater environment, possesses a unique ability to absorb and retain high salt concentrations within its cells, particularly in its leaves, suggesting the presence of a distinct mechanism for salt tolerance. In this study, we performed de novo sequencing of the S. glauca genome. The genome has a size of 1.02Gb (consisting of two sets of haplotypes) and contains 54 761 annotated genes, including alleles and repeats. Comparative genomic analysis revealed a strong synteny between the genomes of S. glauca and Beta vulgaris. Of the S. glauca genome, 70.56% comprises repeat sequences, with retroelements being the most abundant. Leveraging the allele-aware assembly of the S. glauca genome, we investigated genome-wide allele-specific expression in the analyzed samples. The results indicated that the diversity in promoter sequences might contribute to consistent allele-specific expression. Moreover, a systematic analysis of the ABCE gene families shed light on the formation of S. glauca's flower morphology, suggesting that dysfunction of A-class genes is responsible for the absence of petals in S. glauca. Gene family expansion analysis demonstrated significant enrichment of Gene Ontology (GO) terms associated with DNA repair, chromosome stability, DNA demethylation, cation binding, and red/far-red light signaling pathways in the co-expanded gene families of S. glauca and S. aralocaspica, in comparison with glycophytic species within the chenopodium family. Time-course transcriptome analysis under salt treatments revealed detailed responses of S. glauca to salt tolerance, and the enrichment of the transition-upregulated genes in the leaves associated with DNA repair and chromosome stability, lipid biosynthetic process, and isoprenoid metabolic process. Additionally, genome-wide analysis of transcription factors indicated a significant expansion of FAR1 gene family. However, further investigation is needed to determine the exact role of the FAR1 gene family in salt tolerance in S. glauca.

Concerning the eXclusion in human genomics: the choice of sex chromosome representation in the human genome drastically affects the number of identified variants.

Over the past 30 years, a community of scientists has pieced together every base pair of the human reference genome from telomere to telomere. Interestingly, most human genomics studies omit more than 5% of the genome from their analyses. Under "normal" circumstances, omitting any chromosome(s) from an analysis of the human genome would be a cause for concern, with the exception being sex chromosomes. Sex chromosomes in eutherians share an evolutionary origin as an ancestral pair of autosomes. In humans, they share 3 regions of high-sequence identity (∼98-100%), which, along with the unique transmission patterns of the sex chromosomes, introduce technical artifacts in genomic analyses. However, the human X chromosome bears numerous important genes, including more "immune response" genes than any other chromosome, which makes its exclusion irresponsible when sex differences across human diseases are widespread. To better characterize the possible effect of the inclusion/exclusion of the X chromosome on variants called, we conducted a pilot study on the Terra cloud platform to replicate a subset of standard genomic practices using both the CHM13 reference genome and the sex chromosome complement-aware reference genome. We compared the quality of variant calling, expression quantification, and allele-specific expression using these 2 reference genome versions across 50 human samples from the Genotype-Tissue Expression consortium annotated as females. We found that after correction, the whole X chromosome (100%) can generate reliable variant calls, allowing for the inclusion of the whole genome in human genomics analyses as a departure from the status quo of omitting the sex chromosomes from empirical and clinical genomics studies.

Transposable elements and gene expression variation in the intraspecific hybrids of Capsella rubella

AbstractTransposable elements (TEs) are prevalent components of diverse genomes, and play an important role on the genomic stability and expression regulation of their adjacent genes. It is interesting to know the variation of TE expression and the effects of the presence/absence of TEs on gene expression after hybridization. Here we assessed the expression variation of TEs and the impacts of TEs on expression of nearby genes after hybridization based on comparisons of three pairs of reciprocal F1 hybrids and four parents in Capsella rubella. Of the 480 TE families expressed in all the four parents and six F1 hybrids, 7–23 (1.5%–4.2%) TE families were significantly differentially expressed between in silico and real F1 hybrids, indicating the expression levels of these TE families were affected during hybridization. In particular, there was a Copia TE superfamily and a non‐long terminal repeat (non‐LTR) TE differentially expressed between the reciprocal F1 hybrids of 879 and 86IT1, indicating maternal effects may have impacts on expression of TEs in these F1 hybrids. Besides the impacts on the expression of TE families of the hybridization, genes adjacent to polymorphic TEs tended to show a higher proportion (24.83%) of allele‐specific expression (ASE) in F1 hybrids. Overall, our results highlight the impacts of hybridization on the expression level variation of TEs, and the effects of TEs on ASE after hybridization.

Haplotype-Resolution Transcriptome Analysis Reveals Important Responsive Gene Modules and Allele-Specific Expression Contributions under Continuous Salt and Drought in Camellia sinensis.

The tea plant, Camellia sinensis (L.) O. Kuntze, is one of the most important beverage crops with significant economic and cultural value. Global climate change and population growth have led to increased salt and drought stress, negatively affecting tea yield and quality. The response mechanism of tea plants to these stresses remains poorly understood due to the lack of reference genome-based transcriptional descriptions. This study presents a high-quality genome-based transcriptome dynamic analysis of C. sinensis' response to salt and drought stress. A total of 2244 upregulated and 2164 downregulated genes were identified under salt and drought stress compared to the control sample. Most of the differentially expression genes (DEGs) were found to involve divergent regulation processes at different time points under stress. Some shared up- and downregulated DEGs related to secondary metabolic and photosynthetic processes, respectively. Weighted gene co-expression network analysis (WGCNA) revealed six co-expression modules significantly positively correlated with C. sinensis' response to salt or drought stress. The MEpurple module indicated crosstalk between the two stresses related to ubiquitination and the phenylpropanoid metabolic regulation process. We identified 1969 salt-responsive and 1887 drought-responsive allele-specific expression (ASE) genes in C. sinensis. Further comparison between these ASE genes and tea plant heterosis-related genes suggests that heterosis likely contributes to the adversity and stress resistance of C. sinensis. This work offers new insight into the underlying mechanisms of C. sinensis' response to salt and drought stress and supports the improved breeding of tea plants with enhanced salt and drought tolerance.

Foreign RNA spike-ins enable accurate allele-specific expression analysis at scale.

Analysis of allele-specific expression is strongly affected by the technical noise present in RNA-seq experiments. Previously, we showed that technical replicates can be used for precise estimates of this noise, and we provided a tool for correction of technical noise in allele-specific expression analysis. This approach is very accurate but costly due to the need for two or more replicates of each library. Here, we develop a spike-in approach which is highly accurate at only a small fraction of the cost. We show that a distinct RNA added as a spike-in before library preparation reflects technical noise of the whole library and can be used in large batches of samples. We experimentally demonstrate the effectiveness of this approach using combinations of RNA from species distinguishable by alignment, namely, mouse, human, and Caenorhabditis elegans. Our new approach, controlFreq, enables highly accurate and computationally efficient analysis of allele-specific expression in (and between) arbitrarily large studies at an overall cost increase of ∼5%. Analysis pipeline for this approach is available at GitHub as R package controlFreq (github.com/gimelbrantlab/controlFreq).

Design, execution, and interpretation of plant RNA-seq analyses.

Genomics has transformed our understanding of the genetic architecture of traits and the genetic variation present in plants. Here, we present a review of how RNA-seq can be performed to tackle research challenges addressed by plant sciences. We discuss the importance of experimental design in RNA-seq, including considerations for sampling and replication, to avoid pitfalls and wasted resources. Approaches for processing RNA-seq data include quality control and counting features, and we describe common approaches and variations. Though differential gene expression analysis is the most common analysis of RNA-seq data, we review multiple methods for assessing gene expression, including detecting allele-specific gene expression and building co-expression networks. With the production of more RNA-seq data, strategies for integrating these data into genetic mapping pipelines is of increased interest. Finally, special considerations for RNA-seq analysis and interpretation in plants are needed, due to the high genome complexity common across plants. By incorporating informed decisions throughout an RNA-seq experiment, we can increase the knowledge gained.