Animal Genomes Research Articles

Haplogenome assembly reveals structural variation in Eucalyptus interspecific hybrids.

De novo phased (haplo)genome assembly using long-read DNA sequencing data has improved the detection and characterization of structural variants (SVs) in plant and animal genomes. Able to span across haplotypes, long reads allow phased, haplogenome assembly in highly outbred organisms such as forest trees. Eucalyptus tree species and interspecific hybrids are the most widely planted hardwood trees with F1 hybrids of Eucalyptus grandis and E. urophylla forming the bulk of fast-growing pulpwood plantations in subtropical regions. The extent of structural variation and its effect on interspecific hybridization is unknown in these trees. As a first step towards elucidating the extent of structural variation between the genomes of E. grandis and E. urophylla, we sequenced and assembled the haplogenomes contained in an F1 hybrid of the two species. Using Nanopore sequencing and a trio-binning approach, we assembled the separate haplogenomes (566.7 Mb and 544.5 Mb) to 98.0% BUSCO completion. High-density SNP genetic linkage maps of both parents allowed scaffolding of 88.0% of the haplogenome contigs into 11 pseudo-chromosomes (scaffold N50 of 43.8 Mb and 42.5 Mb for the E. grandis and E. urophylla haplogenomes, respectively). We identify 48,729 SVs between the two haplogenomes providing the first detailed insight into genome structural rearrangement in these species. The two haplogenomes have similar gene content, 35,572 and 33,915 functionally annotated genes, of which 34.7% are contained in genome rearrangements. Knowledge of SV and haplotype diversity in the two species will form the basis for understanding the genetic basis of hybrid superiority in these trees.

Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence.

Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites' regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

EndHiC: assemble large contigs into chromosome-level scaffolds using the Hi-C links from contig ends.

The application of PacBio HiFi and ultra-long ONT reads have enabled huge progress in the contig-level assembly, but it is still challenging to assemble large contigs into chromosomes with available Hi-C scaffolding tools, which count Hi-C links between contigs using the whole or a large part of contig regions. As the Hi-C links of two adjacent contigs concentrate only at the neighbor ends of the contigs, larger contig size will reduce the power to differentiate adjacent (signal) and non-adjacent (noise) contig linkages, leading to a higher rate of mis-assembly. We design and develop a novel Hi-C based scaffolding tool EndHiC, which is suitable to assemble large contigs into chromosomal-level scaffolds. The core idea behind EndHiC, which distinguishes it from other Hi-C scaffolding tools, is using Hi-C links only from the most effective regions of contig ends. By this way, the signal neighbor contig linkages and noise non-neighbor contig linkages are separated more clearly. Benefiting from the increased signal to noise ratio, the reciprocal best requirement, as well as the robustness evaluation, EndHiC achieves higher accuracy for scaffolding large contigs compared to existing tools. EndHiC has been successfully applied in the Hi-C scaffolding of simulated data from human, rice and Arabidopsis, and real data from human, great burdock, water spinach, chicory, endive, yacon, and Ipomoea cairica, suggesting that EndHiC can be applied to a broad range of plant and animal genomes. EndHiC is a novel Hi-C scaffolding tool, which is suitable for scaffolding of contig assemblies with contig N50 size near or over 10Mb and N90 size near or over 1Mb. EndHiC is efficient both in time and memory, and it is interface-friendly to the users. As more genome projects have been launched and the contig continuity constantly improved, we believe EndHiC has the potential to make a great contribution to the genomics field and liberate the scientists from labor-intensive manual curation works.

CAULIFINDER: a pipeline for the automated detection and annotation of caulimovirid endogenous viral elements in plant genomes

Plant, animal and protist genomes often contain endogenous viral elements (EVEs), which correspond to partial and sometimes entire viral genomes that have been captured in the genome of their host organism through a variety of integration mechanisms. While the number of sequenced eukaryotic genomes is rapidly increasing, the annotation and characterization of EVEs remains largely overlooked. EVEs that derive from members of the family Caulimoviridae are widespread across tracheophyte plants, and sometimes they occur in very high copy numbers. However, existing programs for annotating repetitive DNA elements in plant genomes are poor at identifying and then classifying these EVEs. Other than accurately annotating plant genomes, there is intrinsic value in a tool that could identify caulimovirid EVEs as they testify to recent or ancient host-virus interactions and provide valuable insights into virus evolution. In response to this research need, we have developed CAULIFINDER, an automated and sensitive annotation software package. CAULIFINDER consists of two complementary workflows, one to reconstruct, annotate and group caulimovirid EVEs in a given plant genome and the second to classify these genetic elements into officially recognized or tentative genera in the Caulimoviridae. We have benchmarked the CAULIFINDER package using the Vitis vinifera reference genome, which contains a rich assortment of caulimovirid EVEs that have previously been characterized using manual methods. The CAULIFINDER package is distributed in the form of a Docker image.

Investigating the Structural Organization and Expression of the Animal Genome Report of the 2021 Laureate of the Lomonosov Grand Gold Medal of the Russian Academy of Sciences

Investigating the Structural Organization and Expression of the Animal Genome Report of the 2021 Laureate of the Lomonosov Grand Gold Medal of the Russian Academy of Sciences

Comparative analysis reveals the long-term coevolutionary history of parvoviruses and vertebrates.

Parvoviruses (family Parvoviridae) are small DNA viruses that cause numerous diseases of medical, veterinary, and agricultural significance and have important applications in gene and anticancer therapy. DNA sequences derived from ancient parvoviruses are common in animal genomes and analysis of these endogenous parvoviral elements (EPVs) has demonstrated that the family, which includes twelve vertebrate-specific genera, arose in the distant evolutionary past. So far, however, such "paleovirological" analysis has only provided glimpses into the biology of ancient parvoviruses and their long-term evolutionary interactions with hosts. Here, we comprehensively map EPV diversity in 752 published vertebrate genomes, revealing defining aspects of ecology and evolution within individual parvovirus genera. We identify 364 distinct EPV sequences and show these represent approximately 200 unique germline incorporation events, involving at least five distinct parvovirus genera, which took place at points throughout the Cenozoic Era. We use the spatiotemporal and host range calibrations provided by these sequences to infer defining aspects of long-term evolution within individual parvovirus genera, including mammalian vicariance for genus Protoparvovirus, and interclass transmission for genus Dependoparvovirus. Moreover, our findings support a model of virus evolution in which the long-term cocirculation of multiple parvovirus genera in vertebrates reflects the adaptation of each viral genus to fill a distinct ecological niche. Our findings show that efforts to develop parvoviruses as therapeutic tools can be approached from a rational foundation based on comparative evolutionary analysis. To support this, we published our data in the form of an open, extensible, and cross-platform database designed to facilitate the wider utilisation of evolution-related domain knowledge in parvovirus research.

The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus

BackgroundThe first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation.ResultsIn the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica.ConclusionsThe compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

Transcriptome annotation of 17 porcine tissues using nanopore sequencing technology.

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long-read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue-specific gene expression analyses showed that 9749 transcripts were highly tissue-specific, and cerebellum contained the most tissue-specific transcripts. As the same samples were used for the annotation of cis-regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome.

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

High-quality genome assembles from key Hawaiian coral species.

Coral reefs house about 25% of marine biodiversity and are critical for the livelihood of many communities by providing food, tourism revenue, and protection from wave surge. These magnificent ecosystems are under existential threat from anthropogenic climate change. Whereas extensive ecological and physiological studies have addressed coral response to environmental stress, high-quality reference genome data are lacking for many of these species. The latter issue hinders efforts to understand the genetic basis of stress resistance and to design informed coral conservation strategies. We report genome assemblies from 4 key Hawaiian coral species, Montipora capitata, Pocillopora acuta, Pocillopora meandrina, and Porites compressa. These species, or members of these genera, are distributed worldwide and therefore of broad scientific and ecological importance. For M. capitata, an initial assembly was generated from short-read Illumina and long-read PacBio data, which was then scaffolded into 14 putative chromosomes using Omni-C sequencing. For P. acuta, P. meandrina, and P. compressa, high-quality assemblies were generated using short-read Illumina and long-read PacBio data. The P. acuta assembly is from a triploid individual, making it the first reference genome of a nondiploid coral animal. These assemblies are significant improvements over available data and provide invaluable resources for supporting multiomics studies into coral biology, not just in Hawai'i but also in other regions, where related species exist. The P. acuta assembly provides a platform for studying polyploidy in corals and its role in genome evolution and stress adaptation in these organisms.

The effects of transpositions of functional I retrotransposons depend on the conditions and dose of parental exposure

Purpose Transposable elements (TEs) cause destabilization of animal genomes. I retrotransposons of Drosophila melanogaster, as well as human LINE1 retrotransposons, are sources of intra- and interindividual diversity and responses to the action of internal and external factors. The aim of this study was to investigate the response to irradiation for the offspring of Drosophila melanogaster with the increased activity of inherited functional I elements. Materials and methods The material used was dysgenic Drosophila females with active I retrotransposons obtained as a result of crossing irradiated/non-irradiated parents of a certain genotype. Non-dysgenic females (without functional I elements) were used as controls. The effects of different conditions (irradiation of both parents simultaneously or separately) and doses (1–100 Gy) of parental irradiation have been assessed by analyzing SF-sterility, DNA damage and lifespan. The presence of full-size I retrotransposons was determined by PCR analysis. Results The maternal exposure and exposure of both parents are efficient in contrast with paternal exposure. Irradiation of mothers reduces the reproductive potential and viability of their female offspring which undergo high activity of functional I retrotransposons. Though I retrotranspositions negatively affect the female gonads, irradiation of the paternal line can increase the lifespan of SF-sterile females. Radiation stress in the range of 1–100 Gy increases DNA fragmentation in both somatic and germ cells of the ovaries with high I-retrotransposition. Conclusions These results allow for the specificity of the radiation-induced behavior of I retrotransposons and their role in survival under conditions of strong radiation stress.

Contrasting modes of macro and microsynteny evolution in a eukaryotic subphylum

Examination of the changes in order and arrangement of homologous genes is key for understanding the mechanisms of genome evolution in eukaryotes. Previous comparisons between eukaryotic genomes have revealed considerable conservation across species that diverged hundreds of millions of years ago (e.g., vertebrates,1,2,3 bilaterian animals,4,5 and filamentous fungi6). However, understanding how genome organization evolves within and between eukaryotic major lineages remains underexplored. We analyzed high-quality genomes of 120 representative budding yeast species (subphylum Saccharomycotina) spanning ∼400 million years of eukaryotic evolution to examine how their genome organization evolved and to compare it with the evolution of animal and plant genome organization.7 We found that the decay of both macrosynteny (the conservation of homologous chromosomes) and microsynteny (the conservation of local gene content and order) was strongly associated with evolutionary divergence across budding yeast major clades. However, although macrosynteny decayed very fast, within ∼100 million years, the microsynteny of many genes-especially genes in metabolic clusters (e.g., in the GAL gene cluster8)-was much more deeply conserved both within major clades and across the subphylum. We further found that when genomes with similar evolutionary divergence times were compared, budding yeasts had lower macrosynteny conservation than animals and filamentous fungi but higher conservation than angiosperms. In contrast, budding yeasts had levels of microsynteny conservation on par with mammals, whereas angiosperms exhibited very low conservation. Our results provide new insight into the tempo and mode of the evolution of gene and genome organization across an entire eukaryotic subphylum.

Transposable element expansion and low-level piRNA silencing in grasshoppers may cause genome gigantism

BackgroundTransposable elements (TEs) have been likened to parasites in the genome that reproduce and move ceaselessly in the host, continuously enlarging the host genome. However, the Piwi-interacting RNA (piRNA) pathway defends animal genomes against the harmful consequences of TE invasion by imposing small-RNA-mediated silencing. Here we compare the TE activity of two grasshopper species with different genome sizes in Acrididae (Locusta migratoria manilensis♀1C = 6.60 pg, Angaracris rhodopa♀1C = 16.36 pg) to ascertain the influence of piRNAs.ResultsWe discovered that repetitive sequences accounted for 74.56% of the genome in A. rhodopa, more than 56.83% in L. migratoria, and the large-genome grasshopper contained a higher TEs proportions. The comparative analysis revealed that 41 TEs (copy number > 500) were shared in both species. The two species exhibited distinct “landscapes” of TE divergence. The TEs outbreaks in the small-genome grasshopper occurred at more ancient times, while the large-genome grasshopper maintains active transposition events in the recent past. Evolutionary history studies on TEs suggest that TEs may be subject to different dynamics and resistances in these two species. We found that TE transcript abundance was higher in the large-genome grasshopper and the TE-derived piRNAs abundance was lower than in the small-genome grasshopper. In addition, we found that the piRNA methylase HENMT, which is underexpressed in the large-genome grasshopper, impedes the piRNA silencing to a lower level.ConclusionsOur study revealed that the abundance of piRNAs is lower in the gigantic genome grasshopper than in the small genome grasshopper. In addition, the key gene HENMT in the piRNA biogenesis pathway (Ping-Pong cycle) in the gigantic genome grasshopper is underexpressed. We hypothesize that low-level piRNA silencing unbalances the original positive correlation between TEs and piRNAs, and triggers TEs to proliferate out of control, which may be one of the reasons for the gigantism of grasshopper genomes.

Animal-SNPAtlas: a comprehensive SNP database for multiple animals.

Single-nucleotide polymorphisms (SNPs) as the most important type of genetic variation are widely used in describing population characteristics and play vital roles in animal genetics and breeding. Large amounts of population genetic variation resources and tools have been developed in human, which provided solid support for human genetic studies. However, compared with human, the development of animal genetic variation databases was relatively slow, which limits the genetic researches in these animals. To fill this gap, we systematically identified ∼ 499 million high-quality SNPs from 4784 samples of 20 types of animals. On that basis, we annotated the functions of SNPs, constructed high-density reference panels and calculated genome-wide linkage disequilibrium (LD) matrixes. We further developed Animal-SNPAtlas, a user-friendly database (http://gong_lab.hzau.edu.cn/Animal_SNPAtlas/) which includes high-quality SNP datasets and several support tools for multiple animals. In Animal-SNPAtlas, users can search the functional annotation of SNPs, perform online genotype imputation, explore and visualize LD information, browse variant information using the genome browser and download SNP datasets for each species. With the massive SNP datasets and useful tools, Animal-SNPAtlas will be an important fundamental resource for the animal genomics, genetics and breeding community.

SYNPHONI: scale-free and phylogeny-aware reconstruction of synteny conservation and transformation across animal genomes.

Current approaches detect conserved genomic order either at chromosomal (macrosynteny) or at subchromosomal scales (microsynteny). The latter generally requires collinearity and hard thresholds on syntenic region size, thus excluding a major proportion of syntenies with recent expansions or minor rearrangements. 'SYNPHONI' bridges the gap between micro- and macrosynteny detection, providing detailed information on both synteny conservation and transformation throughout the evolutionary history of animal genomes. Source code is freely available at https://github.com/nsmro/SYNPHONI, implemented in Python 3.9. Supplementary data are available at Bioinformatics online.

AnimalTFDB 4.0: a comprehensive animal transcription factor database updated with variation and expression annotations.

Transcription factors (TFs) are proteins that interact with specific DNA sequences to regulate gene expression and play crucial roles in all kinds of biological processes. To keep up with new data and provide a more comprehensive resource for TF research, we updated the Animal Transcription Factor Database (AnimalTFDB) to version 4.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB4/) with up-to-date data and functions. We refined the TF family rules and prediction pipeline to predict TFs in genome-wide protein sequences from Ensembl. As a result, we predicted 274 633 TF genes and 150 726 transcription cofactor genes in AnimalTFDB 4.0 in 183 animal genomes, which are 86 more species than AnimalTFDB 3.0. Besides double data volume, we also added the following new annotations and functions to the database: (i) variations (including mutations) on TF genes in various human cancers and other diseases; (ii) predicted post-translational modification sites (including phosphorylation, acetylation, methylation and ubiquitination sites) on TFs in 8 species; (iii) TF regulation in autophagy; (iv) comprehensive TF expression annotation for 38 species; (v) exact and batch search functions allow users to search AnimalTFDB flexibly. AnimalTFDB 4.0 is a useful resource for studying TF and transcription regulation, which contains comprehensive annotation and classification of TFs and transcription cofactors.

Allele-specific expression reveals functional SNPs affecting muscle-related genes in bovine

Single nucleotide polymorphisms showing allele-specific expression (ASE SNPs) are useful for cis-regulatory variants discovery. Despite this potential, there are expensive costs involved in genome-level ASE analysis for large sample sizes. If different data resolutions are available, genotype imputation can be used to mitigate this limitation. Aiming to increase the power to detect regulatory variants, we used a large dataset (>4 million) of imputed SNP genotypes and RNA-Seq data from 190 Nelore steers. Differences between major and minor allele expressions in muscle were tested with a Binomial Test. We identified 38,177 ASE SNPs (FDR ≤ 0.05) within 7304 linkage disequilibrium blocks. After that, we searched for aseQTLs (i.e., neighboring SNPs potentially regulating the ASE SNPs' allelic expression) by comparing the ASE of heterozygous to homozygous sample groups under a Wilcoxon Rank Sum test. We identified 21,543 aseQTLs potentially regulating 430 ASE SNPs (FDR ≤ 0.05). A total of 3333 cis-eQTLs (being 2098 ASE SNPs and 1075 aseQTLs) were associated with the expression of 758 transcripts (FDR ≤ 0.05), demonstrating the cis-regulatory effect of these ASE SNPs and aseQTLs. Data integration showed reproducibility with previous studies in bovine ASE and genomic imprinting. Furthermore, we identified 36,756 novel ASE regions due to the imputation approach. Comparisons with epigenetics data from Functional Annotation of Animal Genomes (FAANG) suggest a regulatory potential of the ASE-related SNPs. The affected genes were enriched in metabolic pathways essential for muscle homeostasis. These findings reinforce the potential of using ASE for discovering cis-regulatory SNPs that may affect muscle-related traits.

Toxicity by descent: A comparative approach for chemical hazard assessment.

Toxicology is traditionally divided between human and eco-toxicology. In the shared pursuit of environmental health, this separation does not account for discoveries made in the comparative studies of animal genomes. Here, we provide evidence on the feasibility of understanding the health impact of chemicals on all animals, including ecological keystone species and humans, based on a significant number of conserved genes and their functional associations to health-related outcomes across much of animal diversity. We test four conditions to understand the value of comparative genomics data to inform mechanism-based human and environmental hazard assessment: (1) genes that are most fundamental for health evolved early during animal evolution; (2) the molecular functions of pathways are better conserved among distantly related species than the individual genes that are members of these pathways; (3) the most conserved pathways among animals are those that cause adverse health outcomes when disrupted; (4) gene sets that serve as molecular signatures of biological processes or disease-states are largely enriched by evolutionarily conserved genes across the animal phylogeny. The concept of homology is applied in a comparative analysis of gene families and pathways among invertebrate and vertebrate species compared with humans. Results show that over 70% of gene families associated with disease are shared among the greatest variety of animal species through evolution. Pathway conservation between invertebrates and humans is based on the degree of conservation within vertebrates and the number of interacting genes within the human network. Human gene sets that already serve as biomarkers are enriched by evolutionarily conserved genes across the animal phylogeny. By implementing a comparative method for chemical hazard assessment, human and eco-toxicology converge towards a more holistic and mechanistic understanding of toxicity disrupting biological processes that are important for health and shared among animals (including humans).

Regulatory frameworks can facilitate or hinder the potential for genome editing to contribute to sustainable agricultural development.

The advent of new breeding techniques (NBTs), in particular genome editing (GEd), has provided more accurate and precise ways to introduce targeted changes in the genome of both plants and animals. This has resulted in the use of the technology by a wider variety of stakeholders for different applications in comparison to transgenesis. Regulators in different parts of the world are now examining their current frameworks to assess their applicability to these NBTs and their products. We looked at how countries selected from a sample of geographical regions globally are currently handling applications involving GEd organisms and what they foresee as opportunities and potential challenges to acceptance of the technology in their jurisdictions. In addition to regulatory frameworks that create an enabling environment for these NBTs, acceptance of the products by the public is vitally important. We, therefore, suggest that early stakeholder engagement and communication to the public be emphasized to foster public acceptance even before products are ready for market. Furthermore, global cooperation and consensus on issues cutting across regions will be crucial in avoiding regulatory-related bottlenecks that affect global trade and agriculture.

237 Using Metabolomic Approaches to Understand and Reduce Animal Variation in Meat Quality Traits

Abstract Failure to meet consumer expectations for meat quality attributes such as color, tenderness, and flavor leads to loss of customer satisfaction and market share. Thus, much effort has been dedicated to understanding the biological variation in these traits. However, traditional approaches explained only a limited amount of biological variation. Investigations of the genome, transcriptome, proteome, and metabolome of meat animals has greatly increased the understanding of biochemical processes affecting meat quality attributes. Gene expression in response to environmental factors results in protein production. These proteins, which vary in functionality, are involved in cellular processes which produce metabolites. Thus, the proteome comprises the machinery of cellular functions and provides a great deal of information about genomic expression resulting in the phenotype. However, quantifying the metabolome provides additional information about the functionality of the machinery, and thus, even greater information about the phenotype. Untargeted metabolomic investigations have suggested novel mechanisms influencing meat tenderness, lean color stability, and flavor. Future work with targeted metabolomic approaches should focus on validating these mechanisms and studying these mechanisms under variable production systems. Moreover, future work should integrate metabolomic data with genomic, transcriptomic, and proteomic data to fully understand the biological basis of meat quality phenotypes.