Recombinant Inbred Advanced Intercross Lines Research Articles

Natural variation in fecundity is correlated with species-wide levels of divergence in Caenorhabditis elegans.

Life history traits underlie the fitness of organisms and are under strong natural selection. A new mutation that positively impacts a life history trait will likely increase in frequency and become fixed in a population (e.g., a selective sweep). The identification of the beneficial alleles that underlie selective sweeps provides insights into the mechanisms that occurred during the evolution of a species. In the global population of Caenorhabditis elegans, we previously identified selective sweeps that have drastically reduced chromosomal-scale genetic diversity in the species. Here, we measured the fecundity of 121 wild C. elegans strains, including many recently isolated divergent strains from the Hawaiian islands and found that strains with larger swept genomic regions have significantly higher fecundity than strains without evidence of the recent selective sweeps. We used genome-wide association (GWA) mapping to identify three quantitative trait loci (QTL) underlying the fecundity variation. In addition, we mapped previous fecundity data from wild C. elegans strains and C. elegans recombinant inbred advanced intercross lines that were grown in various conditions and detected eight QTL using GWA and linkage mappings. These QTL show the genetic complexity of fecundity across this species. Moreover, the haplotype structure in each GWA QTL region revealed correlations with recent selective sweeps in the C. elegans population. North American and European strains had significantly higher fecundity than most strains from Hawaii, a hypothesized origin of the C. elegans species, suggesting that beneficial alleles that caused increased fecundity could underlie the selective sweeps during the worldwide expansion of C. elegans.

Genetic and genomic resources to study natural variation in Brassica rapa.

The globally important crop Brassica rapa, a close relative of Arabidopsis, is an excellent system for modeling our current knowledge of plant growth on a morphologically diverse crop. The long history of B. rapa domestication across Asia and Europe provides a unique collection of locally adapted varieties that span large climatic regions with various abiotic and biotic stress‐tolerance traits. This diverse gene pool provides a rich source of targets with the potential for manipulation toward the enhancement of productivity of crops both within and outside the Brassicaceae. To expand the genetic resources available to study natural variation in B. rapa, we constructed an Advanced Intercross Recombinant Inbred Line (AI‐RIL) population using B. rapa subsp. trilocularis (Yellow Sarson) R500 and the B. rapa subsp. parachinensis (Cai Xin) variety L58. Our current understanding of genomic structure variation across crops suggests that a single reference genome is insufficient for capturing the genetic diversity within a species. To complement this AI‐RIL population and current and future B. rapa genomic resources, we generated a de novo genome assembly of the B. rapa subsp. trilocularis (Yellow Sarson) variety R500, the maternal parent of the AI‐RIL population. The genetic map for the R500 x L58 population generated using this de novo genome was used to map Quantitative Trait Loci (QTL) for seed coat color and revealed the improved mapping resolution afforded by this new assembly.

The Gene scb-1 Underlies Variation in Caenorhabditis elegans Chemotherapeutic Responses.

Pleiotropy, the concept that a single gene controls multiple distinct traits, is prevalent in most organisms and has broad implications for medicine and agriculture. The identification of the molecular mechanisms underlying pleiotropy has the power to reveal previously unknown biological connections between seemingly unrelated traits. Additionally, the discovery of pleiotropic genes increases our understanding of both genetic and phenotypic complexity by characterizing novel gene functions. Quantitative trait locus (QTL) mapping has been used to identify several pleiotropic regions in many organisms. However, gene knockout studies are needed to eliminate the possibility of tightly linked, non-pleiotropic loci. Here, we use a panel of 296 recombinant inbred advanced intercross lines of Caenorhabditis elegans and a high-throughput fitness assay to identify a single large-effect QTL on the center of chromosome V associated with variation in responses to eight chemotherapeutics. We validate this QTL with near-isogenic lines and pair genome-wide gene expression data with drug response traits to perform mediation analysis, leading to the identification of a pleiotropic candidate gene, scb-1, for some of the eight chemotherapeutics. Using deletion strains created by genome editing, we show that scb-1, which was previously implicated in response to bleomycin, also underlies responses to other double-strand DNA break-inducing chemotherapeutics. This finding provides new evidence for the role of scb-1 in the nematode drug response and highlights the power of mediation analysis to identify causal genes.

Three Quantitative Trait Loci Explain More than 60% of Variation for Chill Coma Recovery Time in a Natural Population of Drosophila ananassae.

Ectothermic species such as insects are particularly vulnerable to climatic fluctuations. Nevertheless, many insects that evolved and diversified in the tropics have successfully colonized temperate regions all over the globe. To shed light on the genetic basis of cold tolerance in such species, we conducted a quantitative trait locus (QTL) mapping experiment for chill coma recovery time (CCRT) in Drosophila ananassae, a cosmopolitan species that has expanded its range from tropical to temperate regions. We created a mapping population of recombinant inbred advanced intercross lines (RIAILs) from two founder strains with diverging CCRT phenotypes. The RIAILs were phenotyped for their CCRT and, together with the founder strains, genotyped for polymorphic markers with double-digest restriction site-associated DNA (ddRAD) sequencing. Using a hierarchical mapping approach that combined standard interval mapping and a multiple-QTL model, we mapped three QTL which altogether explained 64% of the phenotypic variance. For two of the identified QTL, we found evidence of epistasis. To narrow down the list of cold tolerance candidate genes, we cross-referenced the QTL intervals with genes that we previously identified as differentially expressed in response to cold in D. ananassae, and with thermotolerance candidate genes of D. melanogaster. Among the 58 differentially expressed genes that were contained within the QTL, GF15058 showed a significant interaction of the CCRT phenotype and gene expression. Further, we identified the orthologs of four D. melanogaster thermotolerance candidate genes, MtnA, klarsicht, CG5246 (D.ana/GF17132) and CG10383 (D.ana/GF14829) as candidates for cold tolerance in D. ananassae.

Tightly linked antagonistic-effect loci underlie polygenic phenotypic variation in C. elegans.

Recent work has provided strong empirical support for the classic polygenic model for trait variation. Population‐based findings suggest that most regions of genome harbor variation affecting most traits. Here, we use the approach of experimental genetics to show that, indeed, most genomic regions carry variants with detectable effects on growth and reproduction in Caenorhabditis elegans populations sensitized by nickel stress. Nine of 15 adjacent intervals on the X chromosome, each encompassing ∼0.001 of the genome, have significant effects when tested individually in near‐isogenic lines (NILs). These intervals have effects that are similar in magnitude to those of genome‐wide significant loci that we mapped in a panel of recombinant inbred advanced intercross lines (RIAILs). If NIL‐like effects were randomly distributed across the genome, the RIAILs would exhibit phenotypic variance that far exceeds the observed variance. However, the NIL intervals are arranged in a pattern that significantly reduces phenotypic variance relative to a random arrangement; adjacent intervals antagonize one another, cancelling each other's effects. Contrary to the expectation of small additive effects, our findings point to large‐effect variants whose effects are masked by epistasis or linkage disequilibrium between alleles of opposing effect.

Shared Genomic Regions Underlie Natural Variation in Diverse Toxin Responses.

Phenotypic complexity is caused by the contributions of environmental factors and multiple genetic loci, interacting or acting independently. Studies of yeast and Arabidopsis often find that the majority of natural variation across phenotypes is attributable to independent additive quantitative trait loci (QTL). Detected loci in these organisms explain most of the estimated heritable variation. By contrast, many heritable components underlying phenotypic variation in metazoan models remain undetected. Before the relative impacts of additive and interactive variance components on metazoan phenotypic variation can be dissected, high replication and precise phenotypic measurements are required to obtain sufficient statistical power to detect loci contributing to this missing heritability. Here, we used a panel of 296 recombinant inbred advanced intercross lines of Caenorhabditis elegans and a high-throughput fitness assay to detect loci underlying responses to 16 different toxins, including heavy metals, chemotherapeutic drugs, pesticides, and neuropharmaceuticals. Using linkage mapping, we identified 82 QTL that underlie variation in responses to these toxins, and predicted the relative contributions of additive loci and genetic interactions across various growth parameters. Additionally, we identified three genomic regions that impact responses to multiple classes of toxins. These QTL hotspots could represent common factors impacting toxin responses. We went further to generate near-isogenic lines and chromosome substitution strains, and then experimentally validated these QTL hotspots, implicating additive and interactive loci that underlie toxin-response variation.

Collective effects of common SNPs in foraging decisions in Caenorhabditis elegans and an integrative method of identification of candidate genes.

Optimal foraging decision is a quantitative flexible behavior, which describes the time at which animals choose to abandon a depleting food supply. The total minor allele content (MAC) in an individual has been shown to correlate with quantitative variations in complex traits. We have studied the role of MAC in the decision to leave a food lawn in recombinant inbred advanced intercross lines (RIAILs) of Caenorhabditis elegans. We found a strong link between MAC and the food lawn leaving rates (Spearman r = 0.4, P = 0.005). We identified 28 genes of unknown functions whose expression levels correlated with both MAC and leaving rates. When examined by RNAi experiments, 8 of 10 tested among the 28 affected leaving rates, whereas only 2 of 9 did among genes that were only associated with leaving rates but not MAC (8/10 vs 2/9, P < 0.05). The results establish a link between MAC and the foraging behavior and identify 8 genes that may play a role in linking MAC with the quantitative nature of the trait. The method of correlations with both MAC and traits may find broad applications in high efficiency identification of target genes for other complex traits in model organisms and humans.

Collective effects of SNPs on transgenerational inheritance in Caenorhabditis elegans and budding yeast

We studied the collective effects of single nucleotide polymorphisms (SNPs) on transgenerational inheritance in Caenorhabditis elegans recombinant inbred advanced intercross lines (RIAILs) and yeast segregants. We divided the RIAILs and segregants into two groups of high and low minor allele content (MAC). RIAILs with higher MAC needed less generations of benzaldehyde training to gain a stable olfactory imprint and showed a greater change from normal after benzaldehyde training. Yeast segregants with higher MAC showed a more dramatic shortening of the lag phase length after ethanol exposure. The short lag phase as acquired by ethanol training was more dramatically lost after recovery in ethanol free medium for the high MAC group. We also found a preferential association between MAC and traits linked with higher number of additive QTLs. These results suggest a role for the collective effects of SNPs in transgenerational inheritance, and may help explain human variations in disease susceptibility.

P-D2 “HIV-1 Transgenic Nude Rat” A novel disease progressed animal model for HIV-1/AIDS

We investigated the host cell response and immune system in a new transgenic rat animal model for HIV-1. Reports of the original HIV-1 Tg rats' onset of neurological signs, learning deficits, skin lesions, and muscle wasting are consistent with end stage infection with HIV-1/AIDS in humans. We report a new animal model, the HIV-1 transgenic nude rat (HIV1TgNu+). We investigated whether we could create a more reliable animal model of HIV-1 mimicking T-cell deficiency seen in AIDS patients. This advanced model allows the investigator to utilize an immunodeficient animal, as its' precursor the HIV1Tg rat, is immunocompetent. We developed the HIV1TgNu+ rat through random pair mating for recombinant inbred advanced intercross lines of the HIV1Tg rat to the athymic nude rat. Successful breeding provides an in-vivo approach to study T-cell immunodeficiency, non AIDS cancers and opportunistic organisms in the HIV-1 setting. We phenotype the transgenic offspring based on congenital cataracts. Transgenic offspring were identified by PCR amplification of the nef gene in DNA. qPCR was performed to quantitatively measure gene transcription validating the strength of phenotypes. We identified several phenotypes that can be bred to mimic HIV-1/AIDS related pathologies. The earliest visible phenotype occurs at 2 weeks of age when cataracts are noted as the pup's eyes open. The HIV1TgNu+ rat can be selectively bred for degrees of skin disease based on fur pattern. Skin lesions appear and increase in severity starting at 4 months of age; during which a shift in the CD4/CD8 response is noted. This inflammatory response is directly proportional to outwardly observed pathologies. This transgenic rat model will provide a more consistent, reproducible model for the study of pathogenesis in HIV-1/AIDS. The HIV1TGNu+ rat has characteristics comparable to the existing HIV-1 transgenic rat model, with the additional benefit of exhibiting multiple pathologies that relates to AIDS and adds clarity on the expression of HIV-1 genes and proteins.

Natural reversal of left‐right gut/gonad asymmetry in C. elegans males is independent of embryonic chirality

Anatomical left-right (L/R) asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this "dextral" configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian ("Hw") wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes.

New Arabidopsis advanced intercross recombinant inbred lines reveal female control of nonrandom mating.

Female control of nonrandom mating has never been genetically established, despite being linked to inbreeding depression and sexual selection. In order to map the loci that control female-mediated nonrandom mating, we constructed a new advanced intercross recombinant inbred line (RIL) population derived from a cross between Arabidopsis (Arabidopsis thaliana) accessions Vancouver (Van-0) and Columbia (Col-0) and mapped quantitative trait loci (QTLs) responsible for nonrandom mating and seed yield traits. We genotyped a population of 490 RILs. A subset of these lines was used to construct an expanded map of 1,061.4 centimorgans with an average interval of 6.7±5.3 centimorgans between markers. QTLs were then mapped for female- and male-mediated nonrandom mating and seed yield traits. To map the genetic loci responsible for female-mediated nonrandom mating and seed yield, we performed mixed pollinations with genetically marked Col-0 pollen and Van-0 pollen on RIL pistils. To map the loci responsible for male-mediated nonrandom mating and seed yield, we performed mixed pollinations with genetically marked Col-0 and RIL pollen on Van-0 pistils. Composite interval mapping of these data identified four QTLs that control female-mediated nonrandom mating and five QTLs that control female-mediated seed yield. We also identified four QTLs that control male-mediated nonrandom mating and three QTLs that control male-mediated seed yield. Epistasis analysis indicates that several of these loci interact. To our knowledge, the results of these experiments represent the first time female-mediated nonrandom mating has been genetically defined.

Use of a Maize Advanced Intercross Line for Mapping of QTL for Northern Leaf Blight Resistance and Multiple Disease Resistance

Northern leaf blight [NLB; caused by Exserohilum turcicum (Pass) K.J. Leonard and E.G. Suggs] is an important fungal disease of maize (Zea mays L.) in the United States and worldwide. The IBM population, an advanced intercross recombinant inbred line population derived from a cross between the lines Mo17 and B73, was evaluated in three environments (Aurora, NY, in 2006 and 2007 and Clayton, NC in 2007) for two traits related to NLB resistance, weighted mean disease (WMD) and incubation period (IP), and for days to anthesis (DTA). Two WMD quantitative trait loci (QTL) in bins 2.00/2.01 and 4.08 were detected from the overall analysis; of these, only the QTL in bin 4.08 was detected in all three environments analyzed separately. Likewise, only one IP QTL, in bin 2.02, was detected in all three environments and from the overall analysis. Several environment‐specific QTL for each trait were also detected. Several DTA QTL were detected with the strongest effect detected in bin 8.05. Correlations between disease resistance traits and days to anthesis were uniformly low. The results from this study were compared to those of previous studies that used the IBM population to identify QTL for two other maize foliar diseases, southern leaf blight {causal agent Cochliobolus heterostrophus (Drechs.) Drechs. [anamorph = Bipolaris maydis (Nisikado and Miyake) Shoemaker; synonym = Helminthosporium maydis (Nisikado and Miyake)]} and gray leaf spot [causal agent Cercospora zeae‐maydis (Tehon and E.Y. Daniels)]. Although we did not find QTL conferring resistance to all three diseases, significant correlations between resistances to these diseases in the IBM population were identified, implying the existence of loci (and possibly genes) affecting resistance to all three diseases.

Cis-regulatory Changes atFLOWERING LOCUS TMediate Natural Variation in Flowering Responses ofArabidopsis thaliana

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (FT), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.

Recombinational Landscape and Population Genomics of Caenorhabditis elegans

Recombination rate and linkage disequilibrium, the latter a function of population genomic processes, are the critical parameters for mapping by linkage and association, and their patterns in Caenorhabditis elegans are poorly understood. We performed high-density SNP genotyping on a large panel of recombinant inbred advanced intercross lines (RIAILs) of C. elegans to characterize the landscape of recombination and, on a panel of wild strains, to characterize population genomic patterns. We confirmed that C. elegans autosomes exhibit discrete domains of nearly constant recombination rate, and we show, for the first time, that the pattern holds for the X chromosome as well. The terminal domains of each chromosome, spanning about 7% of the genome, exhibit effectively no recombination. The RIAILs exhibit a 5.3-fold expansion of the genetic map. With median marker spacing of 61 kb, they are a powerful resource for mapping quantitative trait loci in C. elegans. Among 125 wild isolates, we identified only 41 distinct haplotypes. The patterns of genotypic similarity suggest that some presumed wild strains are laboratory contaminants. The Hawaiian strain, CB4856, exhibits genetic isolation from the remainder of the global population, whose members exhibit ample evidence of intercrossing and recombining. The population effective recombination rate, estimated from the pattern of linkage disequilibrium, is correlated with the estimated meiotic recombination rate, but its magnitude implies that the effective rate of outcrossing is extremely low, corroborating reports of selection against recombinant genotypes. Despite the low population, effective recombination rate and extensive linkage disequilibrium among chromosomes, which are techniques that account for background levels of genomic similarity, permit association mapping in wild C. elegans strains.

Use of an Advanced Intercross Line Population for Precise Mapping of Quantitative Trait Loci for Gray Leaf Spot Resistance in Maize

Gray leaf spot [GLS, causal agent Cercospora zeae‐maydis (Tehon and E. Y. Daniels)] is an important fungal disease of maize in the U.S. and worldwide. The IBM population, an advanced intercross recombinant inbred line population derived from a cross between the maize lines Mo17 (resistant) and B73 (susceptible), was evaluated in three environments (Andrews, NC in 2005, 2006, and 2007) for resistance to GLS and for days from planting to anthesis (DTA). A conventional recombinant inbred line population derived from the same two parents (the “Stuber” population) was also assessed for GLS resistance in two environments (Andrews NC, 2004 and 2005). Quantitative trait loci (QTL) for GLS resistance were detected in each population. Five significant QTL were detected in the IBM population in bins 1.05, 2.04, 4.05, 9.03, and 9.05. In each case the QTL were localized to regions less than 3 centiMorgans (cM). Two QTL for GLS resistance were identified in the Stuber population in bins 2.04 and 7.05. The GLS QTL in bin 2.04 was previously identified as a QTL for southern leaf blight resistance in the IBM population. These results were compared with results from five previous GLS QTL studies and two potential GLS QTL “hotspots” were identified in bins 1.05–1.06 and 2.03–2.05. As expected, QTL were identified with much more precision in the IBM population compared to the Stuber population and to previous studies. There was no significant correlation between disease resistance and days to anthesis. Three DTA QTL were detected in bins 4.09, 8.05, and 9.02, which did not co‐localize with GLS QTL.

Precise mapping of quantitative trait loci for resistance to southern leaf blight, caused by Cochliobolus heterostrophus race O, and flowering time using advanced intercross maize lines.

The intermated B73 x Mo17 (IBM) population, an advanced intercross recombinant inbred line population derived from a cross between the maize lines B73 (susceptible) and Mo17 (resistant), was evaluated in four environments for resistance to southern leaf blight (SLB) disease caused by Cochliobolus heterostrophus race O. Two environments were artificially inoculated, while two were not inoculated and consequently had substantially lower disease pressure. Four common SLB resistance quantitative trait loci (QTL) were identified in all environments, two in bin 3.04 and one each in bins 1.10 and 8.02/3. There was no significant correlation between disease resistance and days to anthesis. A direct comparison was made between SLB QTL detected in two populations, independently derived from the same parental cross: the IBM advanced intercross population and a conventional recombinant inbred line population. Several QTL for SLB resistance were detected in both populations, with the IBM providing between 5 and, in one case, 50 times greater mapping resolution.