Matthew Rockman et al. (2008) Development & Evolution Meeting "Genetic basis of gene expression variation among C. elegans isolates"

Matthew Rockman, Sonja Skrovanek, Leonid Kruglyak

[Development & Evolution Meeting, 2008]

Transcript abundance is a quantitative phenotype that stands between genotypes and organismal traits. To discover the genetic architecture of quantitative traits in C. elegans, we mapped genetic loci underlying variation in the abundance of 15,000 transcripts. We used Agilent microarrays to measure transcript abundances in young adult hermaphrodites from each of more than 200 recombinant inbred advanced intercross lines derived from a cross of N2 and CB4856. We identified more than 3000 significant linkages, implicating genetic variation in specific genomic locations in variation in gene expression phenotypes. The linkages are predominantly local; a gene's transcript abundance is genetically influenced by variation closely linked to the gene's own genomic location. A small number of loci influence the abundances of hundreds of transcripts. These highly pleiotropic polymorphisms may influence their transcriptional targets indirectly by altering the cell-type proportions in hermaphrodite gonads. We present experimental data implicating a chromosome I locus in shaping whole-animal transcript abundances through this cell-demographic mechanism. We also present fine-mapping results dissecting a genetically complex region of the X chromosome in which multiple linked polymorphisms influence hundreds of transcript abundances. Finally, we show that the global distribution of genetic variants with effects on transcript abundance phenotypes is biased toward chromosome arms and against chromosome centers. Cutter and Payseur (Mol. Biol. Evol. 20: 665-673) implicated linked selection (hitchhiking or background selection) in shaping neutral SNP density in such a pattern, and our results imply that linked selection also strongly shapes heritable phenotypic variation.

Matthew Rockman et al. (2007) International Worm Meeting "A high resolution recombinant inbred mapping panel implicates spat-3 in octanol avoidance behavior."

Matthew Rockman, Alison Ralph, Leonid Kruglyak

[International Worm Meeting, 2007]

The genes and mutations responsible for heritable variation in nature are largely unknown. To identify loci underlying complex trait variation in C. elegans, we generated a mapping population of 239 advanced intercross recombinant inbred lines (RILs). The lines were founded by reciprocal crosses between the N2 (Bristol) and CB4856 (Hawaii) strains. F2 males were randomly paired with F2 hermaphrodites, and random pair intercrosses were continued until the F10 generation, at which time hermaphrodites were singled and inbred by selfing for 10 additional generations. The resulting homozygous recombinant inbred line chromosomes carry random segments of DNA from N2 and CB4856, and exhibit a roughly 6-fold expansion of genetic map distances relative to an F2 population. We genotyped the strains at 1450 SNP markers to facilitate rapid high resolution mapping of the genes responsible for phenotypic variation in these strains. We have used the RIL panel to map a behavioral phenotype, avoidance of 100% octanol by well-fed worms. N2 worms reverse from octanol within a few seconds of presentation, but CB4856 worms are indifferent to it. The continuous trait distribution of time to reversal among the RILs suggests a polygenic and epistatic basis for the behavior, with the majority of lines reversing rapidly when presented with octanol. Interval mapping identified two significant quantitative trait loci (QTLs), one of which spans a small region of the X chromosome. Because F1 worms reverse rapidly and because a small minority of RILs exhibit the CB4856 phenotype, we reasoned that expression of the N2 allele of the X-linked QTL in a CB4856 background would rescue octanol avoidance. Bombardment of CB4856 with fosmids spanning the QTL interval implicated spat-3 (a suppressor of par-2 mutations); CB4856 worms carrying the N2 spat-3 DNA reverse rapidly. A spat-3 deletion strain, VC31, exhibits N2-like behavior, reversing when presented with octanol. However, F1s from a cross between VC31 and CB4856 exhibit the CB4856 behavior, failure to reverse, unlike F1s from an N2xCB4856 cross. Thus the CB4856 allele of spat-3, interacting with CB4856 alleles at other loci, confers an octanol avoidance defect. The CB4856 allele is not a null allele; it fails to suppress mutations in par-2. We introgressed spat-3 alleles from a panel of wild isolates into N2 and tested them for similar epistatic failure to complement the CB4856 allele. The results suggest that the causal polymorphism is a non-coding, regulatory insertion/deletion variant, and that the N2 allele represents the evolutionarily derived state.

Kaur, Taniya et al. (2011) International Worm Meeting "Local meiotic recombination rate variation in Caenorhabditis elegans."

Rockman, Matthew, Kaur, Taniya

[International Worm Meeting, 2011]

Meiotic recombination plays a pivotal role in shaping the genetic diversity in a population upon which natural selection may act. However, meiotic recombination rates are rarely constant across different regions of the genome in eukaryotes. A variety of factors (including DNA sequence, chromatin state, sex, environmental conditions) are postulated to explain the observed genome wide meiotic recombination rate variation. The meiotic recombination rate variation observed in Caenorhabditis elegans chromosomes is quite striking. Typically, the chromosome arms show high recombination rates whereas the chromosome centers are domains of low recombination. Pronounced boundary regions separate these distinct recombination rate domains. The causes underlying this consistent domain structure interspersed by distinctive boundary regions of transitioning recombination rates is unknown. It also remains to be determined whether the observed 'near constant' domains are in fact constant or on closer inspection of local recombination rate patterns would be irregular and punctuated with recombination hotspots. Consequently, another important unaddressed question is whether the distinctive recombination rate boundaries are discrete or diffuse. A closer inspection of the local recombination rate landscape surrounding the boundary regions is thus warranted to gain a clearer understanding of the chromosome wide recombination rate pattern. Previous work suggests that the boundary between the center and right arm of chromosome II resides at approximately position 12,020,000 base pairs (bp). To precisely map this boundary and obtain a detailed view of the local recombination landscape, recombinants between two visible markers flanking the site, unc-4 and rol-1, were selected, and 183 SNPs within this 2 Mbp interval were identified. Recombination rates were calculated after scoring crossover breakpoints in recombinant worms using the Illumina GoldenGate genotyping assay. Recombination rates from both hermaphrodite- and male-specific meiotic events were compared to investigate the effect of sex on local recombination rate patterns and the position of the recombination rate boundary. Our data for hermaphrodite specific recombination rates demonstrates at a kilo-base pair (kbp) resolution a discrete recombination rate boundary in the region of interest. Also, the high marker density spanning the recombination rate boundary region will allow us to identify potential recombination hot spots in both hermaphrodites and males at an unprecedented resolution.

Kaur, Taniya et al. (2013) International Worm Meeting "Exploring the contribution of chromosomal context in shaping the C. elegans high-resolution recombination rate landscape."

Kaur, Taniya, Rockman, Matthew

[International Worm Meeting, 2013]

Variable rates of meiotic recombination in a genome can have a profound effect on the creation of new combinations of alleles and on the efficacy of natural selection acting in different parts of a genome. A number of factors are known to shape recombination rate variability including sex, genetic background effects and specific DNA sequences. The C. elegans chromosomes (five autosomes and X chromosome) exhibit a striking pattern of a central domain of low meiotic recombination rates complemented by high recombination rate domains in the chromosome arms, separated by pronounced boundary regions. The factors underlying this consistent domain structure are unknown. The central low recombination rate domains are not physically centered on the chromosome. It is as yet unknown if the chromosomal context (arm vs. center of a chromosome) of the recombination rate boundaries encodes any positional information driving the chromosome-wide recombination rate domain structure. We generated sex-specific high-resolution recombination rate maps for the chromosome II center-right arm boundary region. In order to change the relative chromosomal position of this boundary region, we use end-to-end fusion chromosome strains derived from YA873 (IIL; XR) and YA929 (IIR; XR). Using fusion chromosome strains introgressed into the appropriate genetic background, recombinants between the two markers (unc-4 and rol-1) that flank this chromosome II boundary were selected and genotyped at 183 SNPs spanning this ~ 2Mbp region. The recombination rate maps were then generated after identifying crossover breakpoints from the Illumina GoldenGate genotyping data. We also measured the genetic map distance between unc-4 and rol-1 in these strains by scoring phenotypes in the F2 progeny. We have generated high-resolution recombination rate maps for a boundary region in two contrasting chromosomal contexts. Our data indicate that the meiotic recombination frequency in the chromosome II center-right arm boundary region is not comprehensively explained by the chromosomal position of the boundary. We also could not detect an effect of genetic background on recombination frequency in this region for the fusion chromosome strains examined.

Bernstein, Max et al. (2013) International Worm Meeting "The hunt for quantitative trait nucleotides: a near-isogenic line based approach in C. elegans."

Rockman, Matthew, Bernstein, Max

[International Worm Meeting, 2013]

The individual nucleotides responsible for phenotypic diversity have been very difficult to isolate. The majority of known causal variants that affect traits in wild populations of a species occur within protein-coding regions. These variants are typically of very large effect and may not be typical of the sorts of alleles that drive evolution. There are many confounding effects that can explain the relative lack of causal variants, or quantitative trait nucleotides (QTNs), particularly alleles of small effect, epistasis, and gene-environment interactions. One way to elucidate quantitative trait loci (QTLs) is through the creation of near-isogenic lines (NILs), where the majority of the genome (>95%) is of one genomic background. NILs have previously been created for the nematode C. elegans and identified QTLs across the genome for several phenotypes. C. elegans is a naturally inbred species, which makes it ideal for isolating QTNs. Starting with a NIL that harbors a ~1.5 Mb segment on Chromosome X originating from the Hawaiian isolate CB4856, we have created a series of more than 1000 sub-NILs that break up this segment into smaller regions by recombination. These sub-NILs will be genotyped at 278 SNPs within the 1.5 Mb segment. We are currently piloting high-throughput fitness assays similar to previous work by Elvin et al (2011) and Ramani et al (2012). Additionally, we plan to do gene expression analysis on a subset of the sub-NIL panel to characterize expression QTNs (eQTNs). QTL mapping on simulated sub-NIL datasets shows that for a single QTL model, we can accurately map small effect (h2<0.1) causal variants. Due to the linkage disequilibrium pattern in the sub-NIL panel, traditional QTL mapping methods are not applicable. We show that pairwise comparisons among groups of similar sub-NIL genotypes provide a systematic method for identifying QTLs. We have also modeled more complex QTL scenarios, including multiple QTLs and epistatic interactions. These simulations have informed us of our potential statistical power for future QTL mapping experiments.

Bernstein, Max et al. (2015) International Worm Meeting "Fine-scale recombination rate variation on the C. elegans X chromosome."

Bernstein, Max, Rockman, Matthew

[International Worm Meeting, 2015]

Meiotic recombination is a major driver for increasing genetic diversity within species. However, variation in recombination rate exists within and across chromosomes, which potentially affects how different regions of the genome evolve. In the genetic model nematode, Caenorhabditis elegans, there are broad-scale differences in recombination rate across the lengths of its six chromosomes, each of which is characterized by a gene-dense central region with a lower recombination rate flanked by the two chromosome arms with higher recombination rates. However, we do not know the extent of variation within these large domains. To address this gap in our understanding, we generated a large collection of near-isogenic lines that were screened for a single recombination event occurring within a 1.5 Mb region on the left arm of the X chromosome. From 814 genotyped recombinant lines, we found significant variation in recombination rate within this region. However, consistent with prior work in C. elegans, we do not find evidence for local recombination hotspots. Covariates that explain a significant part of the deviance in a generalized linear model are dependent on the scale of analysis. GC content is significantly negatively associated with recombination rate variation at approximately 25 kb resolution, but not at 5-10 kb resolution. This result is in contrast with what has been found in other organisms, including mammals and Apis mellifera. We also identified several small (4-6 bp) DNA motifs that are associated with recombination rate at these different resolutions. These covariates may underlie higher-order biological mechanisms, such as nucleosome positioning and chromatin conformation, that are mediating sites of crossover events.

Cattani, Victoria et al. (2013) International Worm Meeting "Evolution of ZIM proteins in Caenorhabditis."

Cattani, Victoria, Rockman, Matthew

[International Worm Meeting, 2013]

Meiosis is an essential process for sexual reproduction, allowing diploid organisms to generate haploid gametes. During this process, homolog chromosomes must recognize each other and align along their lengths to ensure accurate segregation. In Caenorhabditis elegans chromosomes, specialized regions known as pairing centers interact with four related zinc-finger proteins (HIM-8, ZIM-1, ZIM-2 and ZIM-3) to stabilize homolog interactions and initiate synapsis. Mutations in the zim genes cause meiotic defects such as broods containing inviable progeny and an elevated incidence of males. Genes involved in meiosis may diverge very quickly during evolution, despite the conservation of the meiotic process across species. In particular, certain zinc-finger proteins have undergone massive expansion during the evolution of eukaryotes. Why are zinc-finger proteins evolving rapidly? Is their function conserved in light of this rapid divergence? To investigate what molecular forces have driven the divergence of the ZIM proteins, I first identified orthologous zim genes in six species of the elegans group: C. elegans, C. briggsae, C. remanei, C. brenneri, C. sp. 11 and C. sp. 5. Consistent with previous reports, I found that the C-terminal region of these proteins is highly conserved whereas the N-terminal region is highly divergent. To evaluate the importance of this divergence in ZIM proteins I am testing whether ZIM proteins are interchangeable between closely related species. To that end, I am introducing transgenic zim alleles from C. briggsae, C. remanei, and C. sp. 11 into C. elegans zim mutants and analyzing whether they rescue the mutant phenotype.

Chang, Audrey S et al. (2011) International Worm Meeting "Removal of selection pressure leads to convergent male behavior in Caenorhabditis."

Rockman, Matthew, Chang, Audrey S

[International Worm Meeting, 2011]

Caenorhabditis males typically deposit copulatory plugs over the vulvae of their mates upon completion of sperm transfer. However, in some strains of C. elegans, males deposit copulatory plugs on other males in the absence of hermaphrodites. For example, in AB2, an Australian strain, plugs are deposited over the excretory pores of other males; in CB4856, a Hawaiian strain, plugs are frequently deposited on various parts of the male body. The cause of this anomalous plugging behavior is largely unknown. Because hermaphroditism has arisen independently multiple times in the C. elegans species group, a survey of plugging behavior in multiple strains and species of Caenorhabditis may shed light on why this trait has evolved. Our results suggest that this breakdown of male behavior is possibly a consequence of relaxed selection on male function. Additionally, we localize the genomic regions responsible for head plugging and body plugging in C. elegans. We show that two distinct loci are responsible for these aberrant male behaviors. Using these results and other techniques, we will characterize the genes underlying anomalous plugging and, furthermore, determine whether breakdown in the same genetic pathways has generated convergent behavioral phenotypes in multiple lineages.

Noble, Luke M. et al. (2013) International Worm Meeting "From locus to nucleotide to phenotype: mapping the genetic architecture of quantitative traits."

Noble, Luke M., Rockman, Matthew V.

[International Worm Meeting, 2013]

Although a decade of intensive association and mapping studies has uncovered numerous quantitative trait loci (QTL) in natural populations, these are typically large in physical extent and small in magnitude of effect. The nature of sequence variation underlying quantitative traits is still largely unknown. A powerful approach to this problem is to map the heritable effects of variation on genome-wide gene expression. Such an approach can simultaneously address the genetic basis and evolution of gene regulation, as well as the architecture of quantitative traits. More than two thousand gene expression QTL (eQTL) have been mapped with a panel of recombinant inbred lines derived from Bristol (N2) and Hawaii (CB4856) founders, which are divergent in sequence and biology. Most detected eQTL reside at or near the transcript for which expression is altered - they are local eQTL - and are amenable to experimental analysis. Strong biases are evident in the location of local eQTL across chromosomes, and in the location of sequence variation across genes. We are in the process of identifying quantitative trait nucleotides (QTN) that generate expression differences between strains, after thorough assembly, annotation and comparative analysis of the Hawaii genome, and incorporation of functional and evolutionary information. Variant effects on gene expression - regulatory, coding, or non-coding - are assayed with fluorescent reporters and confocal microscopy. The presence and penetrance of phenotypic effects will be measured for confirmed QTNs throughout ontogeny and in response to environmental perturbation. In so doing we will gain an understanding of the stability of diverse genetic networks, and of the types and consequences of natural variation within species.

Pollard, Daniel A. et al. (2011) International Worm Meeting "What eQTLs won't tell you about natural variation in gene expression: cis and trans acting variants, dominance and epistasis."

Rockman, Matthew V., Pollard, Daniel A.

[International Worm Meeting, 2011]

Variation in when, where and how much each gene is expressed potentially underlies much natural phenotypic variation. Expression QTL analysis can reveal the number, genomic location, and effect size of genetic variants affecting mRNA levels. However, it does not distinguish between cis and trans acting variants closely linked to genes. It also fails to provide dominance information, and is inefficient at capturing highly polygenic effects and the non-additive effects of epistatic interactions. To distinguish between cis and trans acting variation, we compared genome-wide expression levels in young adult C. elegans N2 and CB4856 hermaphrodites with the expression level of each strain's allele in F1 hybrids of the two strains. When the expression ratio of the two parental alleles in the F1 matches the ratio observed between the parental strains, we attribute the difference in expression level to cis acting effects. Genes whose allelic expression differs between parental strains but is equal in the F1s are considered to be governed by trans acting effects. We evaluated the degree of dominance for trans acting effects by measuring deviation in the F1 from the mean of parent strain expression levels. Finally, we investigated the role of epistatic interactions between cis and trans acting variants by modifying the genetic background for the above experiment and measuring how this affected F1 allelic expression. Our results on the proportion of cis and trans acting variants, the degree of dominance of trans acting variants, and the extent of epistatic interactions between variants, combined with previous results from eQTL studies, provide great insight into the complexity of the genetic basis for natural variation in gene expression.