ALBERT

Description: Mycobacterium smegmatis is a bacterium that is naturally devoid of known postreplicative DNA mismatch repair (MMR) homologs, mutS and mutL , providing an opportunity to investigate how the mutation rate and spectrum has evolved in the absence of a highly conserved primary repair pathway. Mutation accumulation experiments of M. smegmatis yielded a base-substitution mutation rate of 5.27 10 –10 per site per generation, or 0.0036 per genome per generation, which is surprisingly similar to the mutation rate in MMR-functional unicellular organisms. Transitions were found more frequently than transversions, with the A:T-〉G:C transition rate significantly higher than the G:C-〉A:T transition rate, opposite to what is observed in most studied bacteria. We also found that the transition-mutation rate of M. smegmatis is significantly lower than that of other naturally MMR-devoid or MMR-knockout organisms. Two possible candidates that could be responsible for maintaining high DNA fidelity in this MMR-deficient organism are the ancestral-like DNA polymerase DnaE1, which contains a highly efficient DNA proofreading histidinol phosphatase (PHP) domain, and/or the existence of a uracil-DNA glycosylase B (UdgB) homolog that might protect the GC-rich M. smegmatis genome against DNA damage arising from oxidation or deamination. Our results suggest that M. smegmatis has a noncanonical Dam (DNA adenine methylase) methylation system, with target motifs differing from those previously reported. The mutation features of M. smegmatis provide further evidence that genomes harbor alternative routes for improving replication fidelity, even in the absence of major repair pathways.

Description: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila . Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach.

Description: Genome-wide association studies (GWAS) have associated many single variants with complex disease, yet the better part of heritable complex disease risk remains unexplained. Analytical tools designed to work under specific population genetic models are needed. Rare variants are increasingly shown to be important in human complex disease, but most existing GWAS data do not cover rare variants. Explicit population genetic models predict that genes contributing to complex traits and experiencing recurrent, unconditionally deleterious, mutation will harbor multiple rare, causative mutations of subtle effect. It is difficult to identify genes harboring rare variants of large effect that contribute to complex disease risk via the single marker association tests typically used in GWAS. Gene/region-based association tests may have the power detect associations by combining information from multiple markers, but have yielded limited success in practice. This is partially because many methods have not been widely applied. Here, we empirically demonstrate the utility of a procedure based on the rank truncated product (RTP) method, filtered to reduce the effects of linkage disequilibrium. We apply the procedure to the Wellcome Trust Case Control Consortium (WTCCC) data set, and uncover previously unidentified associations, some of which have been replicated in much larger studies. We show that, in the absence of significant rare variant coverage, RTP based methods still have the power to detect associated genes. We recommend that RTP-based methods be applied to all existing GWAS data to maximize the usefulness of those data. For this, we provide efficient software implementing our procedure.

Description: Mutation is one of the most fundamental evolutionary forces. Studying variation in the mutation rate within and among closely-related species can help reveal mechanisms of genome divergence, but such variation is unstudied in the vast majority of organisms. Previous studies on ciliated protozoa have found extremely low mutation rates. In this study, using mutation-accumulation techniques combined with deep whole-genome sequencing, we explore the germline base-substitution mutation-rate variation of three cryptic species in the Paramecium aurelia species complex— P. biaurelia , P. sexaurelia , and P. tetraurelia . We find that there is extremely limited variation of the mutation rate and spectrum in the three species and confirm the extremely low mutation rate of ciliates.

Description: A major goal in the analysis of complex traits is to partition the observed genetic variation in a trait into components due to individual loci and perhaps variants within those loci. However, in both QTL mapping and genetic association studies, the estimated percent variation attributable to a QTL is upwardly biased conditional on it being discovered. This bias was first described in two-way QTL mapping experiments by William Beavis, and has been referred to extensively as "the Beavis effect." The Beavis effect is likely to occur in multiparent population (MPP) panels as well as collections of sequenced lines used for genome-wide association studies (GWAS). However, the strength of the Beavis effect is unknown—and often implicitly assumed to be negligible—when "hits" are obtained from an association panel consisting of hundreds of inbred lines tested across millions of SNPs, or in multiparent mapping populations where mapping involves fitting a complex statistical model with several d.f. at thousands of genetic intervals. To estimate the size of the effect in more complex panels, we performed simulations of both biallelic and multiallelic QTL in two major Drosophila melanogaster mapping panels, the GWAS-based Drosophila Genetic Reference Panel (DGRP), and the MPP the Drosophila Synthetic Population Resource (DSPR). Our results show that overestimation is determined most strongly by sample size and is only minimally impacted by the mapping design. When 〈 100, 200, 500, and 1000 lines are employed, the variance attributable to hits is inflated by factors of 6, 3, 1.5, and 1.1, respectively, for a QTL that truly contributes 5% to the variation in the trait. This overestimation indicates that QTL could be difficult to validate in follow-up replication experiments where additional individuals are examined. Further, QTL could be difficult to cross-validate between the two Drosophila resources. We provide guidelines for: (1) the sample sizes necessary to accurately estimate the percent variance to an identified QTL, (2) the conditions under which one is likely to replicate a mapped QTL in a second study using the same mapping population, and (3) the conditions under which a QTL mapped in one mapping panel is likely to replicate in the other (DGRP and DSPR).

Description: Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria.

Description: The severity of the toxic side effects of chemotherapy varies among patients, and much of this variation is likely genetically based. Here, we use the model system Drosophila melanogaster to genetically dissect the toxicity of methotrexate (MTX), a drug used primarily to treat childhood acute lymphoblastic leukemia and rheumatoid arthritis. We use the Drosophila Synthetic Population Resource, a panel of recombinant inbred lines derived from a multiparent advanced intercross, and quantify MTX toxicity as a reduction in female fecundity. We identify three quantitative trait loci (QTL) affecting MTX toxicity; two colocalize with the fly orthologs of human genes believed to mediate MTX toxicity and one is a novel MTX toxicity gene with a human ortholog. A fourth suggestive QTL spans a centromere. Local single-marker association scans of candidate gene exons fail to implicate amino acid variants as the causative single-nucleotide polymorphisms, and we therefore hypothesize the causative variation is regulatory. In addition, the effects at our mapped QTL do not conform to a simple biallelic pattern, suggesting multiple causative factors underlie the QTL mapping results. Consistent with this observation, no single single-nucleotide polymorphism located in or near a candidate gene can explain the QTL mapping signal. Overall, our results validate D. melanogaster as a model for uncovering the genetic basis of chemotoxicity and suggest the genetic basis of MTX toxicity is due to a handful of genes each harboring multiple segregating regulatory factors.

Description: The Hulunbuir short-tailed sheep ( Ovis aries ) is a breed native to China, in which the short-tail phenotype is the result of artificial and natural selection favoring a specific set of genetic mutations. Here, we analyzed the genetic differences between short-tail and normal-tail phenotypes at the genomic level. Selection signals were identified in genome-wide sequences. From 16 sheep, we identified 72,101,346 single nucleotide polymorphisms. Selection signals were detected based on the fixation index and heterozygosity. Seven genomic regions under putative selection were identified, and these regions contained nine genes. Among these genes, T was the strongest candidate as T is related to vertebral development. In T , a nonsynonymous mutation at c.G334T resulted in p.G112W substitution. We inferred that the c.G334T mutation in T leads to functional changes in Brachyury—encoded by this gene—resulting in the short-tail phenotype. Our findings provide a valuable insight into the development of the short-tail phenotype in sheep and other short-tailed animals.

Description: Chromosomal structural variations (SV) including insertions, deletions, inversions, and translocations occur within the genome and can have a significant effect on organismal phenotype. Some of these effects are caused by structural variations containing genes. Large structural variations represent a significant amount of the genetic diversity within a population. We used a global sampling of Drosophila melanogaster (Ithaca, Zimbabwe, Beijing, Tasmania, and Netherlands) to represent diverse populations within the species. We used long-read sequencing and optical mapping technologies to identify SVs in these genomes. Among the five lines examined, we found an average of 2,928 structural variants within these genomes. These structural variations varied greatly in size and location, included many exonic regions, and could impact adaptation and genomic evolution.

Description: Elucidating the mechanism of resistance to biotic and abiotic stress is of great importance in cotton. In this study, a gene containing the NAC domain, designated GbNAC1 , was identified from Gossypium barbadense L. Homologous sequence alignment indicated that GbNAC1 belongs to the TERN subgroup. GbNAC1 protein localized to the cell nucleus. GbNAC1 was expressed in roots, stems, and leaves, and was especially highly expressed in vascular bundles. Functional analysis showed that cotton resistance to Verticillium wilt was reduced when the GbNAC1 gene was silenced using the virus-induced gene silencing (VIGS) method. GbNAC1 -overexpressing Arabidopsis showed enhanced resistance to Verticillium dahliae compared to wild-type. Thus, GbNAC1 is involved in the positive regulation of resistance to Verticillium wilt. In addition, analysis of GbNAC1 -overexpressing Arabidopsis under different stress treatments indicated that it is involved in plant growth, development, and response to various abiotic stresses (ABA, mannitol, and NaCl). This suggests that GbNAC1 plays an important role in resistance to biotic and abiotic stresses in cotton. This study provides a foundation for further study of the function of NAC genes in cotton and other plants.