Natural Genetic Variation Research Articles

Chromosome-level assembly of the Clinopodium gracile genome

Clinopodium gracile is an important medicinal herb in the Lamiaceae family. This species lacks corresponding genomic resources, which significantly limits the study of its active compound synthesis pathways, breeding practices, and assessment of natural genetic variations. We assembled the chromosomal-level genome of C. gracile using Oxford Nanopore (ONT) technology and Hi-C sequence. The assembled genome is 307.3 Mb in size and consists of 9 chromosomes. The scaffold N50 was 36.3 Mb. The BUSCO completeness (Embryophyta_db10) of the genome was 97.2%. The genome annotates 40,083 protein coding genes. C. gracile and S. miltiorrhiza diverged approximately 30.615 million years ago. C. gracile has not undergone recent species-specific WGD events. A high proportion of young LTRs indicates a recent transposable element (TE) transposition burst in C. gracile.

Drosophila Toxicogenomics: genetic variation and sexual dimorphism in susceptibility to 4-Methylimidazole

Background4-methylimidazole is a ubiquitous and potentially carcinogenic environmental toxicant. Genetic factors that contribute to variation in susceptibility to its toxic effects are challenging to assess in human populations. We used the Drosophila melanogaster Genetic Reference Panel (DGRP), a living library of natural genetic variation, to identify genes with human orthologs associated with variation in susceptibility to 4-methylimidazole.ResultsWe screened 204 DGRP lines for survival following 24-hour exposure to 4-methylimidazole. We found extensive genetic variation for survival, with a broad sense heritability of 0.82; as well as genetic variation in sexual dimorphism, with a cross-sex genetic correlation of 0.59. Genome-wide association analyses identified a total of 241 candidate molecular polymorphisms in or near 273 unique genes associated with survival. These polymorphisms had either sex-specific or sex-antagonistic effects, and most had putative regulatory effects. We generated interaction networks using these candidate genes as inputs and computationally recruited genes with known physical or genetic interactions. The network genes were significantly over-represented for gene ontology terms involving all aspects of development (including nervous system development) and cellular and organismal functions as well as canonical signaling pathways, and most had human orthologs.ConclusionsThe genetic basis of variation in sensitivity to acute exposure to 4-methylimidazole in Drosophila is attributable to variation in genes and networks of genes known for their effects on multiple developmental and cellular processes, including possible neurotoxicity. Given evolutionary conservation of the underlying genes and pathways, these insights may be applicable to humans.

Systems biology approaches identify metabolic signatures of dietary lifespan and healthspan across species

Dietary restriction (DR) is a potent method to enhance lifespan and healthspan, but individual responses are influenced by genetic variations. Understanding how metabolism-related genetic differences impact longevity and healthspan are unclear. To investigate this, we used metabolites as markers to reveal how different genotypes respond to diet to influence longevity and healthspan traits. We analyzed data from Drosophila Genetic Reference Panel (DGRP) strains raised under AL and DR conditions, combining metabolomic, phenotypic, and genome-wide information. We employed two computational and complementary methods across species—random forest modeling within the DGRP as our primary analysis and Mendelian randomization in human cohorts as a secondary analysis. We pinpointed key traits with cross-species relevance as well as underlying heterogeneity and pleiotropy that influence lifespan and healthspan. Notably, orotate was linked to parental age at death in humans and blocked the DR lifespan extension in flies, while threonine supplementation extended lifespan, in a strain- and sex-specific manner. Thus, utilizing natural genetic variation data from flies and humans, we employed a systems biology approach to elucidate potential therapeutic pathways and metabolomic targets for diet-dependent changes in lifespan and healthspan.

Revolutionizing livestock sustainability: Pioneering breeding strategies for superior forage biomass and quality

Livestock primarily rely on forage crops as a source of feed and nutrition. The milk productivity of a cow or meat production in goat/sheep could directly be associated with the availability of a sufficient quantity of quality green fodders with essential nutrients in a balanced ratio. Feeding the cereal/grass: legume fodders in the required proportion will not only improve productivity but also the reproductive capacity of animals. However, many countries of the world experience a huge gap between demand and availability of green fodder. In this context, emphasis should be placed on developing efficient forage genotypes with increased biomass and quality as per the requirements of animals, duly considering their digestibility. Breeding approaches encompassing required classical approaches, including wide hybridization to exploit natural genetic variability, biotechnological tools such as transgenic technology, marker-assisted selection, genomic selection, and various omics techniques alongside high-throughput phenotyping using multispectral cameras, would help to sustain livestock productivity by meeting out the present and future fodder requirements coupled with enhanced nutrients

EQTL mapping in transgenic alpha-synuclein carrying Caenorhabditis elegans recombinant inbred lines.

Protein aggregation of α-synuclein (αS) is a genetic and neuropathological hallmark of Parkinson's disease (PD). Studies in the model nematode Caenorhabditis elegans suggested that variation of αS aggregation depends on the genetic background. However, which genes and genetic modifiers underlie individual differences in αS pathology remains unknown. To study the genotypic-phenotypic relationship of αS aggregation, we constructed a Recombinant Inbred Line (RIL) panel derived from a cross between genetically divergent strains C. elegans NL5901 and SCH4856, both harboring the human αS gene. As a first step to discover genetic modifiers 70 αS-RILs were measured for whole-genome gene expression and expression quantitative locus analysis (eQTL) were mapped. We detected multiple eQTL hot-spots, many of which were located on Chromosome V. To confirm a causal locus, we developed Introgression Lines (ILs) that contain SCH4856 introgressions on Chromosome V in an NL5901 background. We detected 74 genes with an interactive effect between αS and the genetic background, including the human p38 MAPK homologue pmk-1 that has previously been associated with PD. Together, we present a unique αS-RIL panel for defining effects of natural genetic variation on αS pathology, which contributes to finding genetic modifiers of PD.

Gene regulatory network inference from CRISPR perturbations in primary CD4+ T cells elucidates the genomic basis of immune disease

The effects of genetic variation on complex traits act mainly through changes in gene regulation. Although many genetic variants have been linked to target genes in cis, the trans-regulatory cascade mediating their effects remains largely uncharacterized. Mapping trans-regulators based on natural genetic variation has been challenging due to small effects, but experimental perturbations offer a complementary approach. Using CRISPR, we knocked out 84 genes in primary CD4+ Tcells, targeting inborn error of immunity (IEI) disease transcription factors (TFs) and TFs without immune disease association. We developed a novel gene network inference method called linear latent causal Bayes (LLCB) to estimate the network from perturbation data and observed 211 regulatory connections between genes. We characterized programs affected by the TFs, which we associated with immune genome-wide association study (GWAS) genes, finding that JAK-STAT family members are regulated by KMT2A, an epigenetic regulator. These analyses reveal the trans-regulatory cascades linking GWAS genes to signaling pathways.

Genome-wide Association Studies of Photosynthetic and Agronomic Traits in Cowpea Collection.

Exploring genomic regions linked with drought tolerance and photosynthesis in cowpea could accelerate breeding of climate-resilient cowpea varieties A Genome-wide association study (GWAS) was conducted to identify marker-trait associations for agronomic and photosynthetic traits measured under well-watered and water-stressed conditions. One hundred and twelve cowpea accessions from IITA were phenotyped for agronomic and photosynthetic traits across three locations in two years: Ibadan, Ikenne (2020 and 2021) and Kano (2021 and 2022). The accessions were genotyped using 19,000 DArT-Seq SNP markers from which 9,210 markers were utilized for GWAS analysis using BLINK and mixed linear model (MLM) in GAPIT. Results revealed significant accession × environment interactions for measured traits while ΦPSII, ΦNO and ΦNPQ had significant and consistent correlations with grain yield across conditions. GWAS identified five SNP markers having consistent associations with grain yield under well-watered and water-stressed conditions and three markers associated with ΦNPQ and ΦNO. Gene annotations revealed Vigun04g169000 and Vigun08g168900 genes linked with grain yield and highly expressed under water-stressed conditions have functional roles in regulating plant development and adaptive response to environmental stress. Vigun07g133400, Vigun07g132700 and Vigun07g258000 genes linked with ΦNPQ and ΦNO are involved in activities controlling photoprotection and stress-induced damage in plants. This study identified natural genetic variation in cowpea and correlations between photosynthetic traits and grain yield under real-field drought conditions. The identified SNP markers upon validation would be valuable in marker-assisted selection and useful for cowpea breeders to harness the role of photosynthesis in genetic enhancement of cowpea tolerance to drought.

Genomic variation of European beech reveals signals of local adaptation despite high levels of phenotypic plasticity

Local adaptation is key for ecotypic differentiation and species evolution. Understanding underlying genomic patterns can allow the prediction of future maladaptation and ecosystem stability. Here, we report the whole-genome resequencing of 874 individuals from 100 range-wide populations of European beech (Fagus sylvatica L.), an important forest tree species in Europe. We show that genetic variation closely mirrors geography with a clear pattern of isolation-by-distance. Genome-wide analyses for genotype-environment associations (GEAs) identify relatively few potentially adaptive variants after correcting for an overwhelming signal of statistically significant but non-causal GEAs. We characterize the single high confidence genomic region and pinpoint a candidate gene possibly involved in winter temperature adaptation via modulation of spring phenology. Surprisingly, allelic variation at this locus does not result in any apparent fitness differences in a common garden. More generally, reciprocal transplant experiments across large climate distances suggest extensive phenotypic plasticity. Nevertheless, we find indications of polygenic adaptation which may be essential in natural ecosystems. This polygenic signal exhibits broad- and fine-scale variation across the landscape, highlighting the relevance of spatial resolution. In summary, our results emphasize the importance, but also exemplify the complexity, of employing natural genetic variation for forest conservation under climate change.

Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

The Genetic Architecture of Arabidopsis thaliana in Response to Native Non-Pathogenic Bacterial Species Revealed by Genome-Wide Association Mapping in Field Conditions

Non-pathogenic bacteria can substantially contribute to plant health by mobilizing and supplying nutrients, providing protection against pathogens, and alleviating abiotic stresses. However, the number of genome-wide association studies reporting the genetic architecture of the response to individual members of the beneficial microbiota remains limited. In this study, we established a genome-wide association study under field conditions to estimate the level of genetic variation and the underlying genetic architecture among 162 accessions of Arabidopsis thaliana originating from 54 natural populations in the southwest of France in response to 13 strains of seven of the most abundant and prevalent non-pathogenic bacterial species isolated from the leaf compartment of A. thaliana in the same geographical region. Using a high-throughput phenotyping methodology to score vegetative growth-related traits, extensive genetic variation was detected among A. thaliana accessions in response to these leaf bacteria, at both the species and strain levels. The presence of crossing reaction norms between each strain and the mock treatment indicates that declaring a strain as a plant growth-promoting bacterium is highly dependent on the host genotype tested. In line with the strong genotype-by-genotype interactions, we detected a complex and highly flexible genetic architecture between the 13 strains. Finally, the candidate genes underlying the quantitative trait loci revealed significant enrichment in several biological pathways, including cell, secondary metabolism, signaling, and transport. Altogether, plant innate immunity appears as a significant source of natural genetic variation in plant–microbiota interactions and opens new avenues for better understanding the ecologically relevant molecular dialog during plant–microbiota interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genetic variation in phenology of wild Arabidopsis thaliana plants.

Phenology and the timing of development are often under selection, but at the same time influence selection on other traits by controlling how traits are expressed across seasons. Plants often exhibit high natural genetic variation in phenology when grown in controlled environments, and many genetic and molecular mechanisms underlying phenology have been dissected. There remains considerable diversity of germination and flowering time within populations in the wild and the contribution of genetics to phenological variation of wild plants is largely unknown. We obtained collection dates of naturally inbred Arabidopsis thaliana accessions from nature and compared them to experimental data on the descendant inbred lines that we synthesized from two new and 155 published controlled experiments. We tested whether the genetic variation in flowering and germination timing from experiments predicted the phenology of the same inbred lines in nature. We found that genetic variation in phenology from controlled experiments significantly, but weakly, predicts day of collection from the wild, even when measuring collection date with accumulated photothermal units. We found that experimental flowering time breeding values were correlated to wild flowering time at location of origin estimated from herbarium collections. However, local variation in collection dates within a region was not explained by genetic variation in experiments, suggesting high plasticity across small-scale environmental gradients. This apparent low heritability in natural populations may suggest strong selection or many generations are required for phenological adaptation and the emergence of genetic clines in phenology.

Detection and analysis of complex structural variation in human genomes across populations and in brains of donors with psychiatric disorders.

Complex structural variations (cxSVs) are often overlooked in genome analyses due to detection challenges. We developed ARC-SV, a probabilistic and machine-learning-based method that enables accurate detection and reconstruction of cxSVs from standard datasets. By applying ARC-SV across 4,262 genomes representing all continental populations, we identified cxSVs as a significant source of natural human genetic variation. Rare cxSVs have a propensity to occur in neural genes and loci that underwent rapid human-specific evolution, including those regulating corticogenesis. By performing single-nucleus multiomics in postmortem brains, we discovered cxSVs associated with differential gene expression and chromatin accessibility across various brain regions and cell types. Additionally, cxSVs detected in brains of psychiatric cases are enriched for linkage with psychiatric GWAS risk alleles detected in the same brains. Furthermore, our analysis revealed significantly decreased brain-region- and cell-type-specific expression of cxSV genes, specifically for psychiatric cases, implicating cxSVs in the molecular etiology of major neuropsychiatric disorders.

Identifying Grapevine Rootstocks Tolerant to Copper Excess

The aim of the current study is to identify grapevine rootstocks with the potential to tolerate excessive Cu concentrations. Four grapevine rootstock genotypes were tested: Paulsen 1103, IAC 572, SO4 and Isabel. The plants were cultivated in nutrition solution added to the following treatments: 0.3 µM Cu and 80 µM Cu. Growth, nutrient concentration in tissue and the physiological and biochemical parameters were assessed. Rootstocks showed different growth responses to Cu excess in the solution. SO4, IAC 572 and Isabel markedly reduced growth under Cu excess compared to plants in the control solution, whereas genotype Paulsen 1103 showed a less pronounced effect. The root system of all genotypes presented a Cu increase under a high Cu concentration, as well as higher POD activity and H2O2 concentration than the control. Isabel presented the greatest sensitivity to Cu excess, as shown by leaf wilting and yellowing. Paulsen 1103 rootstock presented smaller changes in the observed parameters in the high Cu concentration solution than in the control solution. Our results indicate that Paulsen 1103 is the most tolerant to Cu excess, whereas Isabel is the most sensitive. There are natural genetic variations in tolerance to this abiotic stress that typically affect grapevine plants.

Wide-ranging genetic variation in sensitivity to rapamycin in Drosophila melanogaster.

The progress made in aging research using laboratory organisms is undeniable. Yet, with few exceptions, these studies are conducted in a limited number of isogenic strains. The path from laboratory discoveries to treatment in human populations is complicated by the reality of genetic variation in nature. To model the effect of genetic variation on the action of the drug rapamycin, here we use the growth of Drosophila melanogaster larvae. We screened 140 lines from the Drosophila Genetic References Panel for the extent of developmental delay and found wide-ranging variation in their response, from lines whose development time is nearly doubled by rapamycin, to those that appear to be completely resistant. Sensitivity did not associate with any single genetic marker, nor with any gene. However, variation at the level of genetic pathways was associated with rapamycin sensitivity and might provide insight into sensitivity. In contrast to the genetic analysis, metabolomic analysis showed a strong response of the metabolome to rapamycin, but only among the sensitive larvae. In particular, we found that rapamycin altered levels of amino acids in sensitive larvae, and in a direction strikingly similar to the metabolome response to nutrient deprivation. This work demonstrates the need to evaluate interventions across genetic backgrounds and highlights the potential of omic approaches to reveal biomarkers of drug efficacy and to shed light on mechanisms underlying sensitivity to interventions aimed at increasing lifespan.

Understanding natural genetic variation for nutritional quality in grain and identification of superior haplotypes in deepwater rice genotypes of Assam, India

Rice grown under deepwater ecosystem is considered to be natural farming and hence they are considered to be input efficient. Thus, to identify gene responsible for nutritional content under natural conditions, a genome-wide association study (GWAS)was performed. GWAS identified single nucleotide polymorphisms (SNPs) significantly associated with various nutritional quality traits such as Zn (mg/kg), Fe (mg/kg), Protein (%), Oil (%), Amylose (%), Starch (%), Phytic acid (%), Phenol (%) and TDF (%) in 184 deepwater rice accessions evaluated over 2 consecutive years. A total of 278 SNPs distributed across 12 chromosomes were found to be significantly associated with Zn, Oil and Phenol content. Among them, eight high confidence SNPs were significant and identified on chr1 (AX-95933712), chr7 (AX-95957036), and chr8 (AX-95965181) for Zn content. Similarly, on chr2 (AX-95945186), chr8 (AX-95964718), and chr11 (AX-95961099) have been found to be associated with Oil content and on chr3 (AX-95922121) and chr4 (AX-95963889) for Phenol content. Genomic regions of ± 220 kb flanking the three consistent lowest p value containing SNPs for each trait were considered for finding superior haplotypes. These SNPs showed significant phenotypic variations with different identified haplotype blocks. The allelic variations with phenotypes were considered to be superior haplotypes i.e., Block 1: Hap 1 (ACCC) for high Zn content, Block 2: Hap 1 (CT) for high Oil content, and Block 2: Hap 1(CGGG) for low Phenol content. The discovered superior haplotype with high nutritional content could be important for understanding the mechanisms involving nutrient use efficiency. Thus, the present study demonstrated that developing rice varieties with appropriate nutritional quality traits will be possible through the incorporation of such superior haplotypes in breeding programs.

Position-dependent function of human sequence-specific transcription factors

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1–3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF’s distinct functional profiles—many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

Mitochondrial genetics through the lens of single-cell multi-omics.

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease.

A PLURIPOTENT STEM CELL PLATFORM FOR IN VITRO SYSTEMS GENETICS STUDIES OF MOUSE DEVELOPMENT.

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.

Evaluation of Genetic Variability for Yield Improvement in Bitter Gourd (Momordica charantia L.) Genotypes

The degree of genetic variability found in any particular germplasm is a major factor in determining the best breeding program or technique to be used. Exploitation of the natural genetic variability present within a crop species can aid in meeting the rising demand through the identification and modification of the adaptive and productive genes present. Breeders thus identify natural genetic variability as the key to crop improvement. The present investigation was undertaken at the Experimental field of Urban Technological Park Habbak, Srinagar, Jammu and Kashmir during kharif-2022. To investigate several aspects of genetic variability, including mean, range, PV, GV, PCV, GCV, heritability, genetic gain, and genetic advance among the genotypes, the experiment was set up in a Randomized Block Design with three replications and a plant spacing of 2×1 m for thirty genotypes. For every character under study, it was discovered that the estimates of the phenotypic coefficient of variation were marginally greater than the respective genotypic coefficient of variation, indicating a possible involvement of environment in the expression of these traits. Fruit yield hectare-1 (q) exhibited the largest genotypic and phenotypic coefficient of variation (39.788 and 38.970). For yield hectare-1 (q), high heritability and high genetic gain (0.95 and 78.62) were observed. This suggests that additive gene effects are most likely the cause of the heritability and increases the likelihood that this characteristic would be fixed by selection. For every characteristic, the estimates of broad sense heritability were high. All these factors help in selection of better parents for the development of commercial varieties/hybrids. Considering the potential nutritional and economic benefits of bitter gourd (Momordica charantia L.), there is an imperative necessity to isolate such breeding lines having desirable traits, high yield potential along with better quality.

Molecular Mechanisms of Genotype-Dependent Lifespan Variation Mediated by Caloric Restriction: Insight from Wild Yeast Isolates.

Caloric restriction (CR) is known to extend lifespan across different species and holds great promise for preventing human age-onset pathologies. However, two major challenges exist. First, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. Second, genetic differences causing variations in response to CR and genetic factors contributing to variability of CR response on lifespan are largely unknown. Here, we took advantage of natural genetic variation across 46 diploid wild yeast isolates of Saccharomyces species and the lifespan variation under CR conditions to uncover the molecular factors associated with CR response types. We identified genes and metabolic pathways differentially regulated in CR-responsive versus non-responsive strains. Our analysis revealed that altered mitochondrial function and activation of GCN4-mediated environmental stress response are inevitably linked to lifespan variation in response to CR and a unique mitochondrial metabolite might be utilized as a predictive marker for CR response rate. In sum, our data suggests that the effects of CR on longevity may not be universal, even among the closely related species or strains of a single species. Since mitochondrial-mediated signaling pathways are evolutionarily conserved, the dissection of related genetic pathways will be relevant to understanding the mechanism by which CR elicits its longevity effect.