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Spotlight

A showcase of research and scholarship


in selected articles from 2016
2016/17
Editorial Board ASSOCIATE EDITORS
Eduard Akhunov
EDITOR-IN-CHIEF Kansas State
Brenda Andrews University
University of Toronto Michael J. Axtell
Pennsylvania State
DEPUTY EDITOR University
Dirk Jan de Koning Danika L. Bannasch
Swedish University of University of California,
Agricultural Sciences Davis
Arash Bashirullah
EXECUTIVE EDITOR University of
Tracey DePellegrin Wisconsin-Madison
Judith Berman
MANAGING EDITOR University of Minnesota
Ruth Isaacson & Tel Aviv University
James A. Birchler
SENIOR EDITORS University of Missouri
Katrien M. Devos Charles Boone
University of Georgia University of Toronto
Susan L. Forsburg Michael Boutros
University of Southern DKFZ & University of
California Heidelberg
R. Scott Hawley Rachel Brem
Stowers Institute for Buck Institute for
Medical Research Research on Aging
Howard D. Lipshitz Patrick J. Brown
University of Toronto University of Illinois at
Lauren M. McIntyre Urbana-Champaign
Series Editor David T. Burke
University of Florida University of Michigan
Jeffrey Ross-Ibarra Medical School
University of California, Rita M. Cantor
Davis University of California,
Stephen I. Wright Los Angeles
University of Toronto Susan E. Celniker
Lawrence Berkeley
National Laboratory
Aravinda Chakravarti
Johns Hopkins
University School of
Medicine
J. Michael Cherry
Stanford University
Timothy J. Close
University of California,
Riverside
Barak A. Cohen
Washington University
School of Medicine
Josep M. Comeron
University of Iowa
Gloria M. Coruzzi Jay R. Hesselberth Chad L. Myers Kevin Thornton
New York University University of Colorado University of Minnesota University of California,
School of Medicine Irvine
William S. Davidson Corey Nislow
Simon Fraser University Charles S. Hoffman University of British David W. Threadgill
Boston College Columbia Texas A&M University
Kelly Dawe
University of Georgia James B. Holland Brian Oliver Sarah A. Tishkoff
USDA & North Carolina NIDDK, National University of
Gustavo A. de los
State University Institutes of Health Pennsylvania
Campos
Michigan State Ross Houston Andrew H. Paterson Olga Troyanskaya
University The Roslin Institute University of Georgia Princeton University
Job Dekker Emma Huang Patrick C. Phillips Mike Tyers
University of Janssen Pharma R & D University of Oregon Universit de Montral
Massachusetts
Timothy R. Hughes Craig S. Pikaard Joshua Udall
Medical School
University of Toronto Indiana University Brigham Young
Rebecca W. Doerge University
Scott A. Jackson James Prendergast
Purdue University
University of Georgia University of Veronica J. Vieland
Aime M. Dudley Edinburgh Nationwide Childrens
Mattias Jakobsson
Pacic Northwest Hospital
Uppsala University Bruce Reed
Diabetes Research
University of Waterloo Marian Walhout
Institute Jean-Luc Jannink
University of
USDA-ARS Jasper Rine
Jay C. Dunlap Massachusetts
University of California,
Dartmouth Medical Sue L. Jaspersen Medical School
Berkeley
School Stowers Institute for
Marilyn Warburton
Medical Research Antonis Rokas
Mark Estelle USDA-ARS Corn
Vanderbilt University
University of California, Stephen L. Johnson Host Plant Resistance
San Diego Washington University Matthew S. Sachs Research Unit
School of Medicine Texas A&M University
Justin D. Faris Mick Watson
USDA-ARS Cereal Nicholas Katsanis Helen K. Salz University of Edinburgh
Crops Research Unit Duke University Case Western Reserve
Jonathan F. Wendel
University
David S. Fay John K. Kim Iowa State University
University of Wyoming Johns Hopkins Michael J. Scanlon
Randall Wisser
University Cornell University
Justin C. Fay University of Delaware
Washington University Yuseob Kim David S. Schneider
Zhenbiao Yang
in St. Louis Ewha Womans Stanford University
University of California,
University
Elizabeth R. Gavis Robert A. Sclafani Riverside
Princeton University Rob J. Kulathinal University of Colorado
Dani Zamir
Temple University School of Medicine
Cayetano Gonzalez The Hebrew University
IRB Barcelona Siu Sylvia Lee Steve Scofield of Jerusalem
Cornell University USDA-ARS
Brian D. Gregory Monique Zetka
University of Jianxin Ma Tanja Slotte McGill University
Pennsylvania Purdue University University of Stockholm
David J. Gresham Christian R. Marshall Shavannor M. Smith
New York University The Hospital for Sick The University of
Children Georgia
Erich Grotewold
The Ohio State Andrew S. McCallion Marcus B. Smolka
University Johns Hopkins Cornell University
University School of
David J. Grunwald Lars M. Steinmetz
Medicine
The University of Utah European Molecular
John H. McCusker Biology Laboratory &
Kris Gunsalus
Duke University Stanford University
New York University
Medical Center
Hidenori Tachida
Kim S. McKim Kyushu University
Rutgers University
IN THEIR OWN WORDS

When we published our paper Genetic Architecture of


Resistance to Stripe Rust in a Global Winter Wheat Germplasm
Collection with G3, I was thoroughly impressed with the
quality and timeliness of the review process.
Mike Pumphrey
Washington State University

As a G3 editor, I value the opportunities to work with authors


to constantly raise the bar in providing open-access and
standardized data in a rapidly changing field.
Rob Kulathinal
Temple University
G3 Associate Editor

It has been a privilege to be part of G3 from early on (before


its first issue) as it has grown over the years. I am proud
that we have gone from a journal that primarily published
articles that did not quite have enough impact for publication
in GENETICS, to a journal that really attracts a lot of native
submissions. G3 has now clearly joined the ranks among
the established genetics journals and its reputation is
still growing. An important challenge as Deputy Editor is to
make sure we maintain and evolve our rigorous standards for
scientific quality while the areas in which we publish continue
to broaden. The editors have an ongoing dialogue about what
is useful research in their area of expertise.
Dirk Jan de Koning
Swedish University of Agricultural Sciences
G3 Deputy Editor

ON THE COVER Campbell et al. constructed a pedigree-based map of recombination in


the dog genome using a colony of Labrador Retriever and Greyhound crosses maintained
at Cornell University for over 30 years. The results suggest that even though dogs have an
inactive version of PRDM9, a gene important for directing recombination to hotspots, dogs
have similar broad-scale properties of recombination to humans. Fine-scale recombination in
2 the dog appears similar to other species lacking PRDM9. G3 6: 35173524.
G3: Genes | Genomes | Genetics
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Whats G3?
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Browse the diversity of work highlighted in this


Spotlight and see for yourself. We hope you will
consider publishing with us to ensure your work
reaches a broad audience.

Brenda J. Andrews
Editor-in-Chief,
G3: Genes | Genomes | Genetics

3
GENOME REPORTS

Whole Genome Sequence of Two Wild-Derived


Mus musculus domesticus Inbred Strains,
LEWES/EiJ and ZALENDE/EiJ, with Different
Diploid Numbers
Andrew P. Morgan, John P. Didion, Anthony G. Doran, James M. Holt,
Leonard McMillan, Thomas M. Keane, and Fernando Pardo-Manuel de Villena
G3: Genes | Genomes | Genetics December 2016 6:42114216

NEW GENOME REPORTS Many high-quality whole genome sequence


(WGS) datasets languish unpublished and undescribed because they may
notin isolationreveal substantial new biological insights. However, WGS
data are most valuable when made available to the research community in
an accessible format. To enhance the sharing of such data, G3 is pleased to
offer Genome Reports, a succinct format that follows a structured template.
Genome Reports are fast and easy for authors to submit, for reviewers to
assess, and for readers to understand.

The first Genome Report described two wild-derived mouse inbred strains
that were among the top ten inbred strains recommended for resequencing
based on their potential to increase the catalog of known mouse variants. The
work expands the number of wild-derived inbred genomes in the Mus genus
from six to eight.

ABSTRACT Wild-derived mouse inbred strains are becoming increasingly


popular for complex traits analysis, evolutionary studies, and systems
genetics. Here, we report the whole-genome sequencing of two wild-derived
mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of Mus musculus
domesticus origin. These two inbred strains were selected based on their
geographic origin, karyotype, and use in ongoing research. We generated
14 and 18 coverage sequence, respectively, and discovered over 1.1 million
novel variants, most of which are private to one of these strains. This report
expands the number of wild-derived inbred genomes in the Mus genus from
six to eight. The sequence variation can be accessed via an online query
tool; variant calls (VCF format) and alignments (BAM format) are available for
download from a dedicated ftp site. Finally, the sequencing data have also
been stored in a lossless, compressed, and indexed format using the multi-
string Burrows-Wheeler transform. All data can be used without restriction.

4
MUTANT SCREEN REPORT

A Forward Genetic Screen for Molecules


Involved in Pheromone-Induced Dauer
Formation in Caenorhabditis elegans
Scott J. Neal, JiSoo Park, Danielle DiTirro, Jason Yoon, Mayumi Shibuya, Woochan Choi,
Frank C. Schroeder, Rebecca A. Butcher, Kyuhyung Kim, and Piali Sengupta
G3: Genes | Genomes | Genetics May 2016 6:14751487

MUTANT SCREEN REPORTS This article type provides a convenient


format for succinctly describing the results of mutant screens. The Reports
fulfill one of G3s goals: to make useful data available to the community in a
timely fashion.

Animals produce and respond to complex blends of pheromones that


elicit behavioral and developmental responses. Neal et al. identified
genes required for the sensation and signaling of pheromone cues in a
Caenorhabditis elegans developmental decision. This approach led to
the identification and characterization of new molecules and neurons
involved in this process, providing insights into the mechanisms underlying
pheromone-mediated signaling.

ABSTRACT Animals must constantly assess their surroundings and integrate


sensory cues to make appropriate behavioral and developmental decisions.
Pheromones produced by conspecific individuals provide critical information
regarding environmental conditions. Ascaroside pheromone concentration
and composition are instructive in the decision of Caenorhabditis elegans
to either develop into a reproductive adult or enter into the stress-resistant
alternate dauer developmental stage. Pheromones are sensed by a small set
of sensory neurons, and integrated with additional environmental cues, to
regulate neuroendocrine signaling and dauer formation. To identify molecules
required for pheromone-induced dauer formation, we performed an unbiased
forward genetic screen and identified phd (pheromone response-defective
dauer) mutants. Here, we describe new roles in dauer formation for previously
identified neuronal molecules such as the WD40 domain protein QUI-1 and
MACO-1 Macoilin, report new roles for nociceptive neurons in modulating
pheromone-induced dauer formation, and identify tau tubulin kinases as new
genes involved in dauer formation. Thus, phd mutants define loci required
for the detection, transmission, or integration of pheromone signals in the
regulation of dauer formation.

5
INVESTIG ATIONS

Dynamics of Dark-Fly Genome Under


Environmental Selections
Minako Izutsu, Atsushi Toyoda, Asao Fujiyama, Kiyokazu Agata, and Naoyuki Fuse
G3: Genes | Genomes | Genetics February 2016 6:365376

EDITORS NOTE On November 11, 1954, a small group of fruit flies were
plunged into the dark. More than sixty years later, the descendents of those
flies have adapted to a life without light. Izutsu et al. show that these flies
outcompete the wild type in conditions of constant darkness. This competitive
difference allowed the authors to perform re-selection experiments and identify
candidate genes involved in adaptation of the Dark-fly line.

ABSTRACT Environmental adaptation is one of the most fundamental


features of organisms. Modern genome science has identified some genes
associated with adaptive traits of organisms, and has provided insights into
environmental adaptation and evolution. However, how genes contribute
to adaptive traits and how traits are selected under an environment in the
course of evolution remain mostly unclear. To approach these issues, we
utilize Dark-fly, a Drosophila melanogaster line maintained in constant
dark conditions for more than 60 years. Our previous analysis identified
220,000 single nucleotide polymorphisms (SNPs) in the Dark-fly genome,
but did not clarify which SNPs of Dark-fly are truly adaptive for living in
the dark. We found here that Dark-fly dominated over the wild-type fly in a
mixed population under dark conditions, and based on this domination we
designed an experiment for genome reselection to identify adaptive genes
of Dark-fly. For this experiment, large mixed populations of Dark-fly and the
wild-type fly were maintained in light conditions or in dark conditions, and
the frequencies of Dark-fly SNPs were compared between these populations
across the whole genome. We thereby detected condition-dependent
selections toward approximately 6% of the genome. In addition, we observed
the time-course trajectory of SNP frequency in the mixed populations
through generations 0, 22, and 49, which resulted in notable categorization
of the selected SNPs into three types with different combinations of positive
and negative selections. Our data provided a list of about 100 strong
candidate genes associated with the adaptive traits of Dark-fly.

6
An SEM image of the head of a Dark-fly adult.

7
INVESTIG ATIONS

Combinatorial Cis-regulation in
Saccharomyces Species
Aaron T. Spivak and Gary D. Stormo
G3: Genes | Genomes | Genetics March 2016 6: 653667

EDITORS NOTE Certain combinations of cis-regulatory elements (CREs)


produce nonadditive effects on gene expression, but it remains very challenging
to discover which elements interact on a genomic scale. Spivak and Stormo
developed a method to computationally identify significantly co-occurring
CREs across multiple species that are putative cooperative regulators. Previous
experimental data support most of these combinations, and the authors
also experimentally verified some of the novel predictions. They show that
these combinations can explain regulatory rewiring, in which the same set of
transcription factors control different sets of genes in different species.

ABSTRACT Transcriptional control of gene expression requires interactions


between the cis-regulatory elements (CREs) controlling gene promoters. We
developed a sensitive computational method to identify CRE combinations
with conserved spacing that does not require genome alignments. When
applied to seven sensu stricto and sensu lato Saccharomyces species,
80% of the predicted interactions displayed some evidence of combinatorial
transcriptional behavior in several existing datasets including: (1) chromatin
immunoprecipitation data for colocalization of transcription factors, (2) gene
expression data for coexpression of predicted regulatory targets, and (3)
gene ontology databases for common pathway membership of predicted
regulatory targets. We tested several predicted CRE interactions with
chromatin immunoprecipitation experiments in a wild-type strain and strains
in which a predicted cofactor was deleted. Our experiments confirmed that
transcription factor (TF) occupancy at the promoters of the CRE combination
target genes depends on the predicted cofactor while occupancy of other
promoters is independent of the predicted cofactor. Our method has the
additional advantage of identifying regulatory differences between species.
By analyzing the S. cerevisiae and S. bayanus genomes, we identified
differences in combinatorial cis-regulation between the species and showed
that the predicted changes in gene regulation explain several of the species-
specific differences seen in gene expression datasets. In some instances,
the same CRE combinations appear to regulate genes involved in distinct
biological processes in the two different species. The results of this research
demonstrate that (1) combinatorial cis-regulation can be inferred by multi-
genome analysis and (2) combinatorial cis-regulation can explain differences
in gene expression between species.

8
MOTOR PROGRAMS Drosophila larval crawling is an attractive
system to study rhythmic motor output at the level of animal behavior.
Clark et al. performed a genetic screen to identify neurons that, when
activated, could elicit distinct motor programs. The image shows
larvae of different ages during locomotion; color-coding represents
the temporal axis (blue early > white late). Each larva is a different
genotype with distinct locomotor behaviors (e.g., lower right, paralysis).
See G3 6: 20232031. Image: Matt Q. Clark.

9
INVESTIG ATIONS

Preparing for Winter: The Transcriptomic


Response Associated with Different Day
Lengths in Drosophila montana
Darren J. Parker, Michael G. Ritchie, and Maaria Kankare
G3: Genes | Genomes | Genetics May 2016 6:13731381

EDITORS NOTE Many organisms use day length to track changes in


season and adjust their physiology accordingly. Parker et al. used a northern
Finnish population of Drosophila montana to identify gene expression
changes in different light-dark cycles. Overall, many genes changed in
expression as day length decreased, including genes involved in neuron
development and metabolism. The authors also identified expression
changes in many genes associated with reproduction, suggesting that
D. montana use day length to cue reproductive changes.

ABSTRACT At northern latitudes, the most robust cue for assessing the
onset of winter is the shortening of day lengths. Many species use day length
as a cue to increase their cold tolerance and/or enter into diapause, but
little is known about changes in gene expression that occur under different
day lengths. We investigate the gene expression changes associated with
differences in light/dark cycles in Drosophila montana, a northerly distributed
species with a strong adult photoperiodic reproductive diapause. To examine
gene expression changes induced by light both prior to and during diapause,
we used both nondiapausing and diapausing flies. We found that the majority
of genes that are differentially expressed between different day lengths in
nondiapausing and diapausing flies differ. However, the biological processes
involved were broadly similar. These included neuron development and
metabolism, which are largely consistent with an increase in cold tolerance
previously observed to occur in these flies. We also found that many genes
associated with reproduction change in expression level between different
day lengths, suggesting that D. montana use changes in day length to cue
changes in reproduction both before and after entering into diapause. Finally,
we also identified several interesting candidate genes for light-induced
changes including Lsp2, para, and Ih.

10
INVESTIG ATIONS

Transcriptome Analysis Reveals that Vitamin A


Metabolism in the Liver Affects Feed Efficiency
in Pigs
Yunxia Zhao, Ye Hou, Fei Liu, An Liu, Lu Jing, Changzhi Zhao, Yu Luan, Yuanxin Miao,
Shuhong Zhao, and Xinyun Li
G3: Genes | Genomes | Genetics November 2016 6:36153624

EDITORS NOTE More than 60% of the total production costs in pig farming
go to feed, so feed efficiency (FE) is an important economic trait. Zhao et
al. compared the liver transcriptomes of pigs with extremely high and low
FE. Their results suggest that vitamin A metabolism in liver tissue plays an
important role in FE by affecting energy metabolism, which may mediate fatty
acid biosynthesis and steroid hormone metabolism.

ABSTRACT Feed efficiency (FE) is essential for pig production. In this study,
300 significantly differentially expressed (DE) transcripts, including 232
annotated genes, 28 cis-natural antisense transcripts (cis-NATs), and 40 long
noncoding RNAs (lncRNAs), were identified between the liver of Yorkshire pigs
with extremely high and low FE. Among these transcripts, 25 DE lncRNAs
were significantly correlated with 125 DE annotated genes at a transcriptional
level. These DE genes were enriched primarily in vitamin A (VA), fatty acid,
and steroid hormone metabolism. VA metabolism is regulated by energy
status, and active derivatives of VA metabolism can regulate fatty acid and
steroid hormones metabolism. The key genes of VA metabolism (CYP1A1,
ALDH1A2, and RDH16), fatty acid biosynthesis (FASN, SCD, CYP2J2, and
ANKRD23), and steroid hormone metabolism (CYP1A1, HSD17B2, and
UGT2B4) were significantly upregulated in the liver of high-FE pigs. Previous
study with the same samples indicated that the mitochondrial function and
energy expenditure were reduced in the muscle tissue of high-FE pigs.
In conclusion, VA metabolism in liver tissues plays important roles in the
regulation of FE in pigs by affecting energy metabolism, which may mediate
fatty acid biosynthesis and steroid hormone metabolism. Furthermore, our
results identified novel transcripts, such as cis-NATs and lncRNAs, which are
also involved in the regulation of FE in pigs.

11
INVESTIG ATIONS

The Mouse Universal Genotyping Array:


From Substrains to Subspecies
Andrew P. Morgan, Chen-Ping Fu, Chia-Yu Kao, Catherine E. Welsh,
John P. Didion, Liran Yadgary, Leeanna Hyacinth, Martin T. Ferris, Timothy A. Bell,
Darla R. Miller, Paola Giusti-Rodriguez, Randal J. Nonneman, Kevin D. Cook,
Jason K. Whitmire, Lisa E. Gralinski, Mark Keller, Alan D. Attie, Gary A. Churchill,
Petko Petkov, Patrick F. Sullivan, Jennifer R. Brennan, Leonard McMillan, and
Fernando Pardo-Manuel de Villena
G3: Genes | Genomes | Genetics February 2016 6:263279

EDITORS NOTE The Mouse Universal Genotyping Array (MUGA) is


designed to provide low-cost genotyping for the Collaborative Cross
and Diversity Outbred populations. Morgan et al. describe the selection
of markers for GigaMUGA, the third generation in the MUGA family, and
characterize their performance in a set of 500 reference samples spanning
classical laboratory strains, wild-derived strains, wild-caught mice, and sister
species from the Mus genus. GigaMUGA is informative across the spectrum
of relatedness, from laboratory strains and substrains to other Mus species.

ABSTRACT Genotyping microarrays are an important resource for genetic


mapping, population genetics, and monitoring of the genetic integrity of
laboratory stocks. We have developed the third generation of the Mouse
Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe
Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the
content of GigaMUGA is optimized for genetic mapping in the Collaborative
Cross and Diversity Outbred populations, and for substrain-level identification
of laboratory mice. In addition to 141,090 single nucleotide polymorphism
probes, GigaMUGA contains 2006 probes for copy number concentrated
in structurally polymorphic regions of the mouse genome. The performance
of the array is characterized in a set of 500 high-quality reference samples
spanning laboratory inbred strains, recombinant inbred lines, outbred stocks,
and wild-caught mice. GigaMUGA is highly informative across a wide range of
genetically diverse samples, from laboratory substrains to other Mus species.
In addition to describing the content and performance of the array, we provide
detailed probe-level annotation and recommendations for quality control.

12
NEW SETTLEMENTS Increasingly, researchers are interested in
estimating the heritability of traits for nonmodel organisms. Davies
et al. describe a quantitative genetic methodology for traits in highly
fecund species and illustrate their method by estimating the narrow-
sense heritability for larval settlement, a key life-history trait, in the
reef-building coral Orbicella faveolata. G3 5: 26392645. Image
shows newly settled juvenile coral recruits viewed under a fluorescent
microscope. Photo: Mikhail V. Matz.

13
INVESTIG ATIONS

Seizure Suppression by High Temperature


via cAMP Modulation in Drosophila
Arunesh Saras and Mark A. Tanouye
G3: Genes | Genomes | Genetics October 2016 6:33813387

EDITORS NOTE Drosophila bang-sensitive (BS) mutants have epilepsy-like


phenotypes and are used to study seizure disorders. In BS mutants, seizure-
like activity can be induced by either mechanical shock or high frequency
stimulation. Saras and Tanouye show seizures can be suppressed in even
the most severe mutants by heat shock. By genetic screening, they found
the suppression is dependent on intact adenylyl cyclase functionality, and by
inference, on the up-regulation of cAMP signaling.

ABSTRACT Bang-sensitive (BS) Drosophila mutants display characteristic


seizure-like activity (SLA) and paralysis after mechanical shock. After
high-frequency electrical stimulation (HFS) of the brain, they generate
robust seizures at very low threshold voltage. Here we report an important
phenomenon, which effectively suppresses SLA in BS mutants. High
temperature causes seizure suppression in all BS mutants (parabss1, eas,
sda) examined in this study. This effect is fully reversible and flies show
complete recovery from BS paralysis once the temperature effect is nullified.
High temperature induces an increase in seizure threshold after a brief pulse
of heat shock (HS). By genetic screening, we identified the involvement of
cAMP in the suppression of seizures by high temperature. We propose that
HS induces adenylyl cyclase which in turn increases cAMP concentration
which eventually suppresses seizures in mutant flies. In summary, we
describe an unusual phenomenon, where high temperature can suppress
SLA in flies by modulating cAMP concentration.

14
INVESTIG ATIONS

Novel Heterotypic Rox Sites for Combinatorial


Dre Recombination Strategies
Katherine Chuang, Eileen Nguyen, Yuri Sergeev, and Tudor C. Badea
G3: Genes | Genomes | Genetics March 2016 6:559571

EDITORS NOTE Creative genetic manipulation approaches using Site


Specific Tyrosine Recombinases, such as Cre, Flp, and Dre, allow targeting
of increasingly specific cell types and time windows. Multiple mutually
incompatible target sites are available for Cre and Flp, allowing the design
of combinatorial strategies involving inversions and excisions, cassette
replacements, and many others. Chuang et al. performed a genetic screen
to identify novel, mutually incompatible Dre recombinase target sites.
The authors demonstrate the applicability of the newly identified sites in
combinatorial inversion-excision strategies in the eukaryotic system.

ABSTRACT Site-specific recombinases (SSRs) such as Cre are widely used


in gene targeting and genetic approaches for cell labeling and manipulation.
They mediate DNA strand exchange between two DNA molecules at
dedicated recognition sites. Precise understanding of the Cre recombination
mechanism, including the role of individual base pairs in its loxP target site,
guided the generation of mutant lox sites that specifically recombine with
themselves but not with the wild type loxP. This has led to the development
of a variety of combinatorial Cre-dependent genetic strategies, such as
multicolor reporters, irreversible inversions, or recombination-mediated
cassette exchange. Dre, a Cre-related phage integrase that recognizes
roxP sites, does not cross-react with the Cre-loxP system, but has similar
recombination efficiency. We have previously described intersectional genetic
strategies combining Dre and Cre. We now report a mutagenesis screen
aimed at identifying roxP base pairs critical for self-recognition. We describe
several rox variant sites that are incompatible with roxP, but are able to
efficiently recombine with themselves in either purified systems or bacterial
and eukaryotic tissue culture systems. These newly identified rox sites are not
recognized by Cre, thus enabling potential combinatorial strategies involving
Cre, Dre, and target loci including multiple loxP and roxP variants.

15
INVESTIG ATIONS

Fine-Scale Crossover Rate Variation on the


Caenorhabditis elegans X Chromosome
Max R. Bernstein and Matthew V. Rockman
G3: Genes | Genomes | Genetics June 2016 6: 617671776

EDITORS NOTE Meiotic recombination creates genotypic diversity within


species. Crossover frequencies within species have been found to vary by
chromosome and position, with most crossovers occurring in recombination
hotspots. However, several species appear to lack hotspots despite
significant crossover location heterogeneity. C. elegans has the least fine-scale
variation in autosomal crossover distribution among organisms studied to date.
Here, an enormous new panel of recombinant near-isogenic lines extends that
finding to the X chromosome, which behaves differently from autosomes during
meiosis. Despite the absence of hotspots, subtle crossover rate heterogeneity
is associated with features of the DNA sequence and structure.

ABSTRACT Meiotic recombination creates genotypic diversity within species.


Recombination rates vary substantially across taxa, and the distribution of
crossovers can differ significantly among populations and between sexes.
Crossover locations within species have been found to vary by chromosome
and by position within chromosomes, where most crossover events occur in
small regions known as recombination hotspots. However, several species
appear to lack hotspots despite significant crossover heterogeneity. The
nematode Caenorhabditis elegans was previously found to have the least
fine-scale variation in crossover distribution among organisms studied to
date. It is unclear whether this pattern extends to the X chromosome given
its unique compaction through the pachytene stage of meiotic prophase in
hermaphrodites. We generated 798 recombinant nested near-isogenic lines
(NILs) with crossovers in a 1.41 Mb region on the left arm of the X chromosome
to determine if its recombination landscape is similar to that of the autosomes.
We find that the fine-scale variation in crossover rate is lower than that of other
model species, and is inconsistent with hotspots. The relationship of genomic
features to crossover rate is dependent on scale, with GC content, histone
modifications, and nucleosome occupancy being negatively associated
with crossovers. We also find that the abundances of 4- to 6-bp DNA motifs
significantly explain crossover density. These results are consistent with
recombination occurring at unevenly distributed sites of open chromatin.

16
INVESTIG ATIONS

Effects of DNA Methylation and


Chromatin State on Rates of Molecular
Evolution in Insects
Karl M. Glastad, Michael A. D. Goodisman, Soojin V. Yi, and Brendan G. Hunt
G3: Genes | Genomes | Genetics February 2016 6:357363

EDITORS NOTE How does epigenetic information influence genome


evolution? Glastad et al. demonstrate that although DNA methylation is
known to increase mutation, variation in synonymous substitution between
species is better explained by underlying chromatin structure, rather than
the mutational effect of DNA methylation itself. Thus, chromatin structure is
the primary epigenetic driver of gene evolution in insects with sparse DNA
methylomes.

ABSTRACT Epigenetic information is widely appreciated for its role in gene


regulation in eukaryotic organisms. However, epigenetic information can also
influence genome evolution. Here, we investigate the effects of epigenetic
information on gene sequence evolution in two disparate insects: the fly
Drosophila melanogaster, which lacks substantial DNA methylation, and the
ant Camponotus floridanus, which possesses a functional DNA methylation
system. We found that DNA methylation was positively correlated with the
synonymous substitution rate in C. floridanus, suggesting a key effect of
DNA methylation on patterns of gene evolution. However, our data suggest
the link between DNA methylation and elevated rates of synonymous
substitution was explained, in large part, by the targeting of DNA methylation
to genes with signatures of transcriptionally active chromatin, rather than
the mutational effect of DNA methylation itself. This phenomenon may be
explained by an elevated mutation rate for genes residing in transcriptionally
active chromatin, or by increased structural constraints on genes in inactive
chromatin. This result highlights the importance of chromatin structure as the
primary epigenetic driver of genome evolution in insects. Overall, our study
demonstrates how different epigenetic systems contribute to variation in the
rates of coding sequence evolution.

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INVESTIG ATIONS

The Effects of Both Recent and Long-Term


Selection and Genetic Drift Are Readily
Evident in North American Barley Breeding
Populations
Ana M. Poets, Mohsen Mohammadi, Kiran Seth, Hongyun Wang, Thomas J. Y. Kono,
Zhou Fang, Gary J. Muehlbauer, Kevin P. Smith, and Peter L. Morrell
G3: Genes | Genomes | Genetics March 2016 6:609622

EDITORS NOTE Barley is an ancient domesticated crop introduced to North


America 400 years ago; however, selection for varieties adapted to modern
farming environments is more recent. Poets et al. present a population
genetic study of a comprehensive sample of North American barley lines.
They identified gene variants involved in both recent and long-term selection,
as well as recent bouts of gene flow between breeding populations that
suggest the sharing of agronomically adaptive variation.

ABSTRACT Barley was introduced to North America ~400 yr ago but


adaptation to modern production environments is more recent. Comparisons
of allele frequencies among growth habits and spike (inflorescence) types in
North America indicate that significant genetic differentiation has accumulated
in a relatively short evolutionary time span. Allele frequency differentiation is
greatest among barley with two-row vs. six-row spikes, followed by spring
vs. winter growth habit. Large changes in allele frequency among breeding
programs suggest a major contribution of genetic drift and linked selection
on genetic variation. Despite this, comparisons of 3613 modern North
American cultivated barley breeding lines that differ for spike-type and growth
habit permit the discovery of 142 single nucleotide polymorphism (SNP)
outliers putatively linked to targets of selection. For example, SNPs within
the Cbf4, Ppd-H1, and Vrn-H1 loci, which have previously been associated
with agronomically adaptive phenotypes, are identified as outliers. Analysis
of extended haplotype sharing identifies genomic regions shared within and
among breeding populations, suggestive of a number of genomic regions
subject to recent selection. Finally, we are able to identify recent bouts of
gene flow between breeding populations that could point to the sharing of
agronomically adaptive variation. These results are supported by pedigrees
and breeders understanding of germplasm sharing.

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SEX CHROMOSOME TURNOVER Genetic control of sex determination
varies among species of African clawed frogs (Xenopus). Furman and Evans
demonstrate that yet another sex determination system exists in Xenopus borealis,
with properties that suggest some regions of vertebrate genomes might be
predisposed to develop sex determination functions. G3 6: 3625-3633.
Image shows metamorphosing froglets of Xenopus borealis. Photo: Adam Bewick.

19
INVESTIG ATIONS

Canopy Temperature and Vegetation Indices


from High-Throughput Phenotyping Improve
Accuracy of Pedigree and Genomic Selection
for Grain Yield in Wheat
Jessica Rutkoski, Jesse Poland, Suchismita Mondal, Enrique Autrique,
Lorena Gonzlez Prez, Jos Crossa, Matthew Reynolds, and Ravi Singh
G3: Genes | Genomes | Genetics September 2016 6:27992808

EDITORS NOTE Various technologies can be used to measure crop traits in


a high-throughput fashion. Rutkoski et al. show high-throughput phenotyping
of wheat crops with aerial imaging can be used to improve genomic
prediction of grain yield. They used data from aerial imagery as secondary
traits in genomic and pedigree prediction models, leading to substantial
increases in grain yield prediction accuracy.

ABSTRACT Genomic selection can be applied prior to phenotyping,


enabling shorter breeding cycles and greater rates of genetic gain relative to
phenotypic selection. Traits measured using high-throughput phenotyping
based on proximal or remote sensing could be useful for improving pedigree
and genomic prediction model accuracies for traits not yet possible to
phenotype directly. We tested if using aerial measurements of canopy
temperature, and green and red normalized difference vegetation index as
secondary traits in pedigree and genomic best linear unbiased prediction
models could increase accuracy for grain yield in wheat, Triticum aestivum L.,
using 557 lines in five environments. Secondary traits on training and test
sets, and grain yield on the training set were modeled as multivariate, and
compared to univariate models with grain yield on the training set only. Cross
validation accuracies were estimated within and across-environment, with and
without replication, and with and without correcting for days to heading. We
observed that, within environment, with unreplicated secondary trait data, and
without correcting for days to heading, secondary traits increased accuracies
for grain yield by 56% in pedigree, and 70% in genomic prediction models, on
average. Secondary traits increased accuracy slightly more when replicated,
and considerably less when models corrected for days to heading. In across-
environment prediction, trends were similar but less consistent. These results
show that secondary traits measured in high-throughput could be used in
pedigree and genomic prediction to improve accuracy. This approach could
improve selection in wheat during early stages if validated in early-generation
breeding plots.

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INVESTIG ATIONS

A Combination of CRISPR/Cas9 and


Standardized RNAi as a Versatile Platform for
the Characterization of Gene Function
Sebastian Wissel, Anja Kieser, Tetsuo Yasugi, Peter Duchek, Elisabeth Roitinger,
Joseph Gokcezade, Victoria Steinmann, Ulrike Gaul, Karl Mechtler, Klaus Frstemann,
Jrgen A. Knoblich, and Ralph A. Neumller
G3: Genes | Genomes | Genetics August 2016 6:24672478

EDITORS NOTE Minimal-in-vivo-GFP-interference (miGFPi) is a versatile


strategy for conducting loss-of-function experiments in Drosophila that avoids
the off-target effects of RNAi. miGFPi combines CRISPR/Cas9-mediated
locus tagging with an immunotag and an exogenous RNAi effector sequence
to result in a highly validated RNAi line targeting the chosen sequence.
They demonstrate the utility of miGFPi as a generalized strategy for the
characterization of gene function (including for gene isoforms) in a time
efficient and highly stringent manner.

ABSTRACT Traditional loss-of-function studies in Drosophila suffer from


a number of shortcomings, including off-target effects in the case of RNA
interference (RNAi) or the stochastic nature of mosaic clonal analysis.
Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile
strategy to characterize gene function and to conduct highly stringent, cell
type-specific loss-of-function experiments in Drosophila. miGFPi combines
CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an
immunotag and an exogenous 21 nucleotide RNAi effector sequence with the
use of a single reagent, highly validated RNAi line targeting this sequence. We
demonstrate the utility and time effectiveness of this method by characterizing
the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a,
and the previously uncharacterized gene MESR4, in the Drosophila female
germline stem cell lineage. In addition, we show that miGFPi serves as a
powerful technique to functionally characterize individual isoforms of a gene.
We exemplify this aspect of miGFPi by studying isoform-specific loss-of-
function phenotypes of the longitudinals lacking (lola) gene in neural stem
cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-
function approach that is amenable to the study of the function of all genes in
the genome in a stringent and highly time effective manner.

21
INVESTIG ATIONS

Genomic Access to Monarch Migration Using


TALEN and CRISPR/Cas9-Mediated Targeted
Mutagenesis
Matthew J. Markert, Ying Zhang, Metewo S. Enuameh, Steven M. Reppert,
Scot A. Wolfe, and Christine Merlin
G3: Genes | Genomes | Genetics April 2016 6:905915

EDITORS NOTE The general lack of functional genomic tools in emerging


model organisms has limited our understanding of the relationship between
genotype and phenotype, particularly in species with unique biology. Markert
et al. describe the development of efficient strategies using TALENs and
CRISPR/Cas9 that enable targeted mutagenesis in the migratory monarch
butterfly, with limited injection and screening efforts. By unlocking its
potential as a genetic model system, these approaches hold great promise to
accelerate our understanding of the genetic basis of migration and to facilitate
reverse genetics in many other non-traditional insect species.

ABSTRACTS The eastern North American monarch butterfly, Danaus


plexippus, is an emerging model system to study the neural, molecular,
and genetic basis of animal long-distance migration and animal clockwork
mechanisms. While genomic studies have provided new insight into
migration-associated and circadian clock genes, the general lack of simple
and versatile reverse-genetic methods has limited in vivo functional analysis
of candidate genes in this species. Here, we report the establishment of
highly efficient and heritable gene mutagenesis methods in the monarch
butterfly using transcriptional activator-like effector nucleases (TALENs)
and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9).
Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates,
we show that both TALENs and CRISPR/Cas9 generate high-frequency
nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites
(up to 100%), and that injecting fewer than 100 eggs is sufficient to recover
mutant progeny and generate monarch knockout lines in about 3 months.
Our study also genetically defines monarch CLK as an essential component
of the transcriptional activation complex of the circadian clock. The methods
presented should not only greatly accelerate functional analyses of many
aspects of monarch biology, but are also anticipated to facilitate the
development of these tools in other nontraditional insect species as well as
the development of homology-directed knock-ins.

22
INVESTIG ATIONS

Whole Genome Comparison Reveals High


Levels of Inbreeding and Strain Redundancy
Across the Spectrum of Commercial Wine
Strains of Saccharomyces cerevisiae
Anthony R. Borneman, Angus H. Forgan, Radka Kolouchova, James A. Fraser, and
Simon A. Schmidt
G3: Genes | Genomes | Genetics April 2016 6:957971

EDITORS NOTE Yeast strains for baking and brewing have been
domesticated by humans. Borneman et al. sequenced hundreds of wine
yeast strains, revealing these specialists show low genetic diversity and
high levels of inbreeding. In many cases, commercial strains from multiple
suppliers were nearly genetically identical, suggesting that there is little
potential for new strain development using existing wine yeast.

ABSTRACT Humans have been consuming wines for more than 7000 yr.
For most of this time, fermentations were presumably performed by strains of
Saccharomyces cerevisiae that naturally found their way into the fermenting
must. In contrast, most commercial wines are now produced by inoculation
with pure yeast monocultures, ensuring consistent, reliable and reproducible
fermentations, and there are now hundreds of these yeast starter cultures
commercially available. In order to thoroughly investigate the genetic diversity
that has been captured by over 50 yr of commercial wine yeast development
and domestication, whole genome sequencing has been performed on 212
strains of S. cerevisiae, including 119 commercial wine and brewing starter
strains, and wine isolates from across seven decades. Comparative genomic
analysis indicates that, despite their large numbers, commercial strains,
and wine strains in general, are extremely similar genetically, possessing all
of the hallmarks of a population bottleneck, and high levels of inbreeding.
In addition, many commercial strains from multiple suppliers are nearly
genetically identical, suggesting that the limits of effective genetic variation
within this genetically narrow group may be approaching saturation.

23
INVESTIG ATIONS

rDNA Copy Number Variants Are Frequent


Passenger Mutations in Saccharomyces
cerevisiae Deletion Collections and De Novo
Transformants
Elizabeth X. Kwan, Xiaobin S. Wang, Haley M. Amemiya, Bonita J. Brewer, and
M. K. Raghuraman
G3: Genes | Genomes | Genetics September 2016 6: 28292838

EDITORS NOTE Eukaryotic genomes contain many copies of ribosomal


DNA (rDNA), ranging from a few dozen to hundreds. Kwan et al. reveal that a
standard molecular biology procedure in yeast, lithium acetate transformation,
can result in significant changes in rDNA copy number. As a consequence,
many strains from the widely used yeast deletion collection harbor rDNA
changes that are independent of the deleted gene of interest. Such variants
can influence gene expression, telomeric silencing, and replication stress and
therefore could be unexpected and influential passenger mutations.

ABSTRACT The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus


is known to exhibit greater instability relative to the rest of the genome.
However, wild-type cells preferentially maintain a stable number of rDNA
copies, suggesting underlying genetic control of the size of this locus. We
performed a screen of a subset of the Yeast Knock-Out (YKO) single gene
deletion collection to identify genetic regulators of this locus and to determine
if rDNA copy number correlates with yeast replicative lifespan. While we found
no correlation between replicative lifespan and rDNA size, we identified 64
candidate strains with significant rDNA copy number differences. However,
in the process of validating candidate rDNA variants, we observed that
independent isolates of our de novo gene deletion strains had unsolicited
but significant changes in rDNA copy number. Moreover, we were not able
to recapitulate rDNA phenotypes from the YKO yeast deletion collection.
Instead, we found that the standard lithium acetate transformation protocol
is a significant source of rDNA copy number variation, with lithium acetate
exposure being the treatment causing variable rDNA copy number events
after transformation. As the effects of variable rDNA copy number are being
increasingly reported, our finding that rDNA is affected by lithium acetate
exposure suggested that rDNA copy number variants may be influential
passenger mutations in standard strain construction in S. cerevisiae.

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GENOME REPORTS

Many high-quality whole genome sequence (WGS) datasets


languish unpublished and undescribed because they may not
in isolationreveal substantial new biological insights. However,
WGS data are most valuable when made available to the research
community in an accessible format. To enhance the sharing of such
data, G3 is pleased to offer Genome Reports, a succinct format
that follows a structured template. Genome Reports are designed to
be fast and easy for authors to submit, for reviewers to assess, and
for readers to understand.

Learn more:
http://www.g3journal.org/content/article-types#genomereport

MUTANT SCREEN REPORTS

Dont hide away useful mutant screen results in your lab notes;
submit a Mutant Screen Report and publish your findings sooner!
The Mutant Screen Report provides a convenient format for
succinctly describing the results of mutant screens. The Reports
fulfill one of G3s goals: to make useful data available to the
community in a timely fashion.

Learn more:
http://www.g3journal.org/content/article-types#mutantreport

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