bluuebunny/crossref_metadata_2025 huggingface dataset:
RAG4 Gene Encodes a Glucose Sensor in Kluyveromyces lactis
Authors: Betina Svätopluk , Goffrini Paola , Ferrero Iliana , Wésolowski-Louvel Micheline
Published: April 2021

The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished.
A Role for the Swe1 Checkpoint Kinase During Filamentous Growth of Saccharomyces cerevisiae
Authors: La Valle Roberto , Wittenberg Curt
Published: April 2021

In this study we show that inactivation of Hsl1 or Hsl7, negative regulators of the Swe1 kinase, enhances the invasive behavior of haploid and diploid cells. The enhancement of filamentous growth caused by inactivation of both genes is mediated via the Swe1 protein kinase. Whereas Swe1 contributes noticeably to the effectiveness of haploid invasive growth under all conditions tested, its contribution to pseudohyphal growth is limited to the morphological response under standard assay conditions. However, Swe1 is essential for pseudohyphal differentiation under a number of nonstandard assay conditions including altered temperature and increased nitrogen. Swe1 is also required for pseudohyphal growth in the absence of Tec1 and for the induction of filamentation by butanol, a related phenomenon. Although inactivation of Hsl1 is sufficient to suppress the defect in filamentous growth caused by inactivation of Tec1 or Flo8, it is insufficient to promote filamentous growth in the absence of both factors. Moreover, inactivation of Hsl1 will not bypass the requirement for nitrogen starvation or growth on solid medium for pseudohyphal differentiation. We conclude that the Swe1 kinase modulates filamentous development under a broad spectrum of conditions and that its role is partially redundant with the Tec1 and Flo8 transcription factors.
Genetic Evidence Supports a Role for the Yeast CCR4-NOT Complex in Transcriptional Elongation
Authors: Denis Clyde L , Chiang Yueh-Chin , Cui Yajun , Chen Junji
Published: April 2021

The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively. The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins. We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation. First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects. A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs. Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16. Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation. Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele. The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation. These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process.
Regions of Lower Crossing Over Harbor More Rare Variants in African Populations of Drosophila melanogaster
Authors: Andolfatto Peter , Przeworski Molly
Published: April 2021

A correlation between diversity levels and rates of recombination is predicted both by models of positive selection, such as hitchhiking associated with the rapid fixation of advantageous mutations, and by models of purifying selection against strongly deleterious mutations (commonly referred to as “background selection”). With parameter values appropriate for Drosophila populations, only the first class of models predicts a marked skew in the frequency spectrum of linked neutral variants, relative to a neutral model. Here, we consider 29 loci scattered throughout the Drosophila melanogaster genome. We show that, in African populations, a summary of the frequency spectrum of polymorphic mutations is positively correlated with the meiotic rate of crossing over. This pattern is demonstrated to be unlikely under a model of background selection. Models of weakly deleterious selection are not expected to produce both the observed correlation and the extent to which nucleotide diversity is reduced in regions of low (but nonzero) recombination. Thus, of existing models, hitchhiking due to the recurrent fixation of advantageous variants is the most plausible explanation for the data.
SSCP Analysis of cDNA Markers Provides a Dense Linkage Map of the Aedes aegypti Genome
Authors: Fulton Ruth E , Salasek Michael L , DuTeau Nancy M , Black William C
Published: April 2021

An intensive linkage map of the yellow fever mosquito, Aedes aegypti, was constructed using single-strand conformation polymorphism (SSCP) analysis of cDNA markers to identify single nucleotide polymorphisms (SNPs). A total of 94 A. aegypti cDNAs were downloaded from GenBank and primers were designed to amplify fragments <500 bp in size. These primer pairs amplified 94 loci, 57 (61%) of which segregated in a single F1 intercross family among 83 F2 progeny. This allowed us to produce a dense linkage map of one marker every 2 cM distributed over a total length of 134 cM. Many A. aegypti cDNAs were highly similar to genes in the Drosophila melanogaster genome project. Comparative linkage analysis revealed areas of synteny between the two species. SNP polymorphisms are abundant in A. aegypti genes and should prove useful in both population genetics and mapping studies.
A Microsatellite-Based Genetic Linkage Map for Channel Catfish,Ictalurus punctatus
Authors: Waldbieser Geoffrey C , Bosworth Brian G , Nonneman Danny J , Wolters William R
Published: April 2021

Microsatellite loci were identified in channel catfish gene sequences or random clones from a small insert genomic DNA library. Outbred populations of channel catfish contained an average of eight alleles per locus and an average heterozygosity of 0.70. A genetic linkage map of the channel catfish genome (N = 29) was constructed from two reference families. A total of 293 microsatellite loci were polymorphic in one or both families, with an average of 171 informative meioses per locus. Nineteen type I loci, 243 type II loci, and one EST were placed in 32 multipoint linkage groups covering 1958 cM. Nine more type II loci were contained in three two-point linkage groups covering 24.5 cM. Twenty-two type II loci remained unlinked. Multipoint linkage groups ranged in size from 11.9 to 110.5 cM with an average intermarker distance of 8.7 cM. Seven microsatellite loci were closely linked with the sex-determining locus. The microsatellite loci and genetic linkage map will increase the efficiency of selective breeding programs for channel catfish.
High-Frequency Generation of Conditional Mutations Affecting Drosophila melanogaster Development and Life Span
Authors: Landis Gary , Bhole Deepak , Lu Lucy , Tower John
Published: April 2021

Genome sequencing reveals that a large percentage of Drosophila genes have homologs in humans, including many human disease genes. The goal of this research was to develop methods to efficiently test Drosophila genes for functions in vivo. An important challenge is the fact that many genes function at more than one point during development and during the life cycle. Conditional expression systems such as promoters regulated by tetracycline (or its derivative doxycycline) are often ideal for testing gene functions. However, generation of transgenic animals for each gene of interest is impractical. Placing the doxycycline-inducible (“tet-on”) promoter directed out of the end of the P transposable element produced a mobile, doxycycline-inducible promoter element, named PdL. PdL was mobilized to 228 locations in the genome and was found to generate conditional (doxycycline-dependent), dominant mutations at high frequency. The temporal control of gene overexpression allowed generation of mutant phenotypes specific to different stages of the life cycle, including metamorphosis and aging. Mutations characterized included inserts in the α-mannosidase II (dGMII), ash1, and pumilio genes. Novel phenotypes were identified for each gene, including specific developmental defects and increased or decreased life span. The PdL system should facilitate testing of a large fraction of Drosophila genes for overexpression and misexpression phenotypes at specific developmental and life cycle stages.
Genetic Analysis of the Drosophila Gsα Gene
Authors: Wolfgang William J , Hoskote Ashwini , Roberts Ian J H , Jackson Shannon , Forte Michael
Published: April 2021

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gsα, the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of “loss-of-function” mutations in the Drosophila Gsα gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gsα protein. Examination of mutant alleles representing loss-of-Gsα function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.
Population Dynamics of anAc-like Transposable Element in Self- and Cross-Pollinating Arabidopsis
Authors: Wright Stephen I , Le Quang Hien , Schoen Daniel J , Bureau Thomas E
Published: April 2021

Theoretical models predict that the mating system should be an important factor driving the dynamics of transposable elements in natural populations due to differences in selective pressure on both element and host. We used a PCR-based approach to examine the abundance and levels of insertion polymorphism of Ac-III, a recently identified Ac-like transposon family, in natural populations of the selfing plant Arabidopsis thaliana and its close outcrossing relative, Arabidopsis lyrata. Although several insertions appeared to be ancient and shared between species, there is strong evidence for recent activity of this element family in both species. Sequences of the regions flanking insertions indicate that all Ac-III transposons segregating in natural populations are in noncoding regions and provide no evidence for local transposition events. Transposon display analysis suggests the presence of slightly higher numbers of insertion sites per individual but fewer total polymorphic insertions in the self-pollinating A. thaliana than A. lyrata. Element insertions appear to be segregating at significantly lower frequencies in A. lyrata than A. thaliana, which is consistent with a reduction in transposition rate, reduction in effective population size, or reduced efficacy of natural selection against element insertions in selfing populations.
Size of Donor Chromosome Segments Around Introgressed Loci and Reduction of Linkage Drag in Marker-Assisted Backcross Programs
Authors: Hospital Frédéric
Published: April 2021

This article investigates the efficiency of marker-assisted selection in reducing the length of the donor chromosome segment retained around a locus held heterozygous by backcrossing. First, the efficiency of marker-assisted selection is evaluated from the length of the donor segment in backcrossed individuals that are (double) recombinants for two markers flanking the introgressed gene on each side. Analytical expressions for the probability density function, the mean, and the variance of this length are given for any number of backcross generations, as well as numerical applications. For a given marker distance, the number of backcross generations performed has little impact on the reduction of donor segment length, except for distant markers. In practical situations, the most important parameter is the distance between the introgressed gene and the flanking markers, which should be chosen to be as closely linked as possible to the introgressed gene. Second, the minimal population sizes required to obtain double recombinants for such closely linked markers are computed and optimized in the context of a multigeneration backcross program. The results indicate that it is generally more profitable to allow for three or more successive backcross generations rather than to favor recombinations in early generations.
Structure and Evolution of the hAT Transposon Superfamily
Authors: Rubin Eitan , Lithwick Gila , Levy Avraham A
Published: April 2021

The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new tools for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 hAT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.
SPO14 Separation-of-Function Mutations Define Unique Roles for Phospholipase D in Secretion and Cellular Differentiation in Saccharomyces cerevisiae
Authors: Rudge Simon A , Pettitt Trevor R , Zhou Chun , Wakelam Michael J O , Engebrecht JoAnne
Published: April 2021

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.
Bacterial Artificial Chromosome-Based Physical Map of the Rice Genome Constructed by Restriction Fingerprint Analysis
Authors: Tao Quanzhou , Chang Yueh-Long , Wang Jingzhao , Chen Huaming , Islam-Faridi M Nurul , Scheuring Chantel , Wang Bin , Stelly David M , Zhang Hong-Bin
Published: April 2021

Genome-wide physical mapping with bacteria-based large-insert clones (e.g., BACs, PACs, and PBCs) promises to revolutionize genomics of large, complex genomes. To accelerate rice and other grass species genome research, we developed a genome-wide BAC-based map of the rice genome. The map consists of 298 BAC contigs and covers 419 Mb of the 430-Mb rice genome. Subsequent analysis indicated that the contigs constituting the map are accurate and reliable. Particularly important to proficiency were (1) a high-resolution, high-throughput DNA sequencing gel-based electrophoretic method for BAC fingerprinting, (2) the use of several complementary large-insert BAC libraries, and (3) computer-aided contig assembly. It has been demonstrated that the fingerprinting method is not significantly influenced by repeated sequences, genome size, and genome complexity. Use of several complementary libraries developed with different restriction enzymes minimized the “gaps” in the physical map. In contrast to previous estimates, a clonal coverage of 6.0–8.0 genome equivalents seems to be sufficient for development of a genome-wide physical map of ~95% genome coverage. This study indicates that genome-wide BAC-based physical maps can be developed quickly and economically for a variety of plant and animal species by restriction fingerprint analysis via DNA sequencing gel-based electrophoresis.
Five Gametophytic Mutations Affecting Pollen Development and Pollen Tube Growth in Arabidopsis thaliana
Authors: Procissi Antonia , de Laissardière Solveig , Férault Madina , Vezon Daniel , Pelletier Georges , Bonhomme Sandrine
Published: April 2021

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as “early mutants”: the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as “late mutant” since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.
TCA1, a Single Nuclear-Encoded Translational Activator Specific for petA mRNA in Chlamydomonas reinhardtii Chloroplast
Authors: Wostrikoff K , Choquet Y , Wollman F-A , Girard-Bascou J
Published: April 2021

We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5′ untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b6f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation—known as the CES process—in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process.
The Divergent Caenorhabditis elegans β-Catenin Proteins BAR-1, WRM-1 and HMP-2 Make Distinct Protein Interactions but Retain Functional Redundancy in Vivo
Authors: Natarajan Lakshmi , Witwer Nina E , Eisenmann David M
Published: April 2021

β-Catenins function both in cell adhesion as part of the cadherin/catenin complex and in Wnt signal transduction as transcription factors. Vertebrates express two related proteins, β-catenin and plakoglobin, while Drosophila has a single family member, Armadillo. Caenorhabditis elegans expresses three β-catenin-related proteins, BAR-1, HMP-2, and WRM-1, which are quite diverged in sequence from each other and other β-catenins. While BAR-1 and WRM-1 are known to act in Wnt-mediated processes, and HMP-2 acts in a complex with cadherin/α-catenin homologs, it is unclear whether all three proteins retain the other functions of β-catenin. Here we show that BAR-1, like vertebrate β-catenin, has redundant transcription activation domains in its amino- and carboxyl-terminal regions but that HMP-2 and WRM-1 also possess the ability to activate transcription. We show via yeast two-hybrid analysis that these three proteins display distinct patterns of protein interactions. Surprisingly, we find that both WRM-1 and HMP-2 can substitute for BAR-1 in C. elegans when expressed from the bar-1 promoter. Therefore, although their mutant phenotypes and protein interaction patterns strongly suggest that the functions of β-catenin in other species have been segregated among three diverged proteins in C. elegans, these proteins still retain sufficient similarity to display functional redundancy in vivo.
technical knockout, a Drosophila Model of Mitochondrial Deafness
Authors: Toivonen Janne M , O'Dell Kevin M C , Petit Nathalie , Irvine Sharon C , Knight Gillian K , Lehtonen Marjo , Longmuir Mark , Luoto Kaisa , Touraille Sylvie , Wang Zongsheng , Alziari Serge , Shah Zahid H , Jacobs Howard T
Published: April 2021

Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko25t exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko25t mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.
Characterization of a Maize Chromosome 4 Centromeric Sequence: Evidence for an Evolutionary Relationship With the B Chromosome Centromere
Authors: Page Brent T , Wanous Michael K , Birchler James A
Published: April 2021

Previous work has identified sequences specific to the B chromosome that are a major component of the B centromere. To address the issue of the origin of the B and the evolution of centromere-localized sequences, DNA prepared from plants without B chromosomes was probed to seek evidence for related sequences. Clones were isolated from maize line B73 without B chromosomes by screening DNA at reduced stringency with a B centromeric probe. These clones were localized to maize centromere 4 using fluorescence in situ hybridization. They showed homology to a maize centromere-mapped sequence, to maize B chromosome centromere sequences, and to a portion of the unit repeat of knobs, which act as neocentromeres in maize. A representative copy was used to screen a BAC library to obtain these sequences in a larger context. Each of the six positive BACs obtained was analyzed to determine the nature of centromere 4-specific sequences present. Fifteen subclones of one BAC were sequenced and the organization of this chromosome 4-specific repeat was examined.
The Evolutionary Analysis of “Orphans” From the Drosophila Genome Identifies Rapidly Diverging and Incorrectly Annotated Genes
Authors: Schmid Karl J , Aquadro Charles F
Published: April 2021

In genome projects of eukaryotic model organisms, a large number of novel genes of unknown function and evolutionary history (“orphans”) are being identified. Since many orphans have no known homologs in distant species, it is unclear whether they are restricted to certain taxa or evolve rapidly, either because of a lack of constraints or positive Darwinian selection. Here we use three criteria for the selection of putatively rapidly evolving genes from a single sequence of Drosophila melanogaster. Thirteen candidate genes were chosen from the Adh region on the second chromosome and 1 from the tip of the X chromosome. We succeeded in obtaining sequence from 6 of these in the closely related species D. simulans and D. yakuba. Only 1 of the 6 genes showed a large number of amino acid replacements and in-frame insertions/deletions. A population survey of this gene suggests that its rapid evolution is due to the fixation of many neutral or nearly neutral mutations. Two other genes showed “normal” levels of divergence between species. Four genes had insertions/deletions that destroy the putative reading frame within exons, suggesting that these exons have been incorrectly annotated. The evolutionary analysis of orphan genes in closely related species is useful for the identification of both rapidly evolving and incorrectly annotated genes.
Empirical Evaluation of Genetic Clustering Methods Using Multilocus Genotypes From 20 Chicken Breeds
Authors: Rosenberg Noah A , Burke Terry , Elo Kari , Feldman Marcus W , Freidlin Paul J , Groenen Martien A M , Hillel Jossi , Mäki-Tanila Asko , Tixier-Boichard Michèle , Vignal Alain , Wimmers Klaus , Weigend Steffen
Published: April 2021

We tested the utility of genetic cluster analysis in ascertaining population structure of a large data set for which population structure was previously known. Each of 600 individuals representing 20 distinct chicken breeds was genotyped for 27 microsatellite loci, and individual multilocus genotypes were used to infer genetic clusters. Individuals from each breed were inferred to belong mostly to the same cluster. The clustering success rate, measuring the fraction of individuals that were properly inferred to belong to their correct breeds, was consistently ~98%. When markers of highest expected heterozygosity were used, genotypes that included at least 8–10 highly variable markers from among the 27 markers genotyped also achieved >95% clustering success. When 12–15 highly variable markers and only 15–20 of the 30 individuals per breed were used, clustering success was at least 90%. We suggest that in species for which population structure is of interest, databases of multilocus genotypes at highly variable markers should be compiled. These genotypes could then be used as training samples for genetic cluster analysis and to facilitate assignments of individuals of unknown origin to populations. The clustering algorithm has potential applications in defining the within-species genetic units that are useful in problems of conservation.
Anchored Reference Loci in Loblolly Pine (Pinus taeda L.) for Integrating Pine Genomics
Authors: Brown Garth R , Kadel Edward E , Bassoni Daniel L , Kiehne Kristine L , Temesgen Berhanu , van Buijtenen J P , Sewell Mitchell M , Marshall Kimberly A , Neale David B
Published: April 2021

Anchored reference loci provide a framework for comparative mapping. They are landmarks to denote conserved chromosomal segments, allowing the synthesis of genetic maps from multiple sources. We evaluated 90 expressed sequence tag polymorphisms (ESTPs) from loblolly pine (Pinus taeda L.) for this function. Primer sets were assayed for amplification and polymorphism in six pedigrees, representing two subgenera of Pinus and a distant member of the Pinaceae, Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). On average, 89% of primer sets amplified in four species of subgenus Pinus, 49% in one species of subgenus Strobus, and 22% in Douglas-fir. Polymorphisms were detected for 37–61% of the ESTPs within each pedigree. Comparative mapping in loblolly and slash pine (P. elliottii Englm.) revealed that ESTPs mapped to the same location. Disrupted synteny or significant disruptions in colinearity were not detected. Thirty-five ESTPs met criteria established for anchor loci. The majority of those that did not meet these criteria were excluded when map location was known in only a single species. Anchor loci provide a unifying tool for the community, facilitating the creation of a “generic” pine map and serving as a foundation for studies on genome organization and evolution.
Likelihoods and Simulation Methods for a Class of Nonneutral Population Genetics Models
Authors: Donnelly Peter , Nordborg Magnus , Joyce Paul
Published: April 2021

Methods for simulating samples and sample statistics, under mutation-selection-drift equilibrium for a class of nonneutral population genetics models, and for evaluating the likelihood surface, in selection and mutation parameters, are developed and applied for observed data. The methods apply to large populations in settings in which selection is weak, in the sense that selection intensities, like mutation rates, are of the order of the inverse of the population size. General diploid selection is allowed, but the approach is currently restricted to models, such as the infinite alleles model and certain K-models, in which the type of a mutant allele does not depend on the type of its progenitor allele. The simulation methods have considerable advantages over available alternatives. No other methods currently seem practicable for approximating likelihood surfaces.
A General Polyploid Model for Analyzing Gene Segregation in Outcrossing Tetraploid Species
Authors: Wu Rongling , Gallo-Meagher Maria , Littell Ramon C , Zeng Zhao-Bang
Published: April 2021

Polyploidy has played an important role in higher plant evolution and applied plant breeding. Polyploids are commonly categorized as allopolyploids resulting from the increase of chromosome number through hybridization and subsequent chromosome doubling or autopolyploids due to chromosome doubling of the same genome. Allopolyploids undergo bivalent pairing at meiosis because only homologous chromosomes pair. For autopolyploids, however, all homologous chromosomes can pair at the same time so that multivalents and, therefore, double reductions are formed. In this article, we use a maximum-likelihood method to develop a general polyploid model for estimating gene segregation patterns from molecular markers in a full-sib family derived from an arbitrary polyploid combining meiotic behaviors of both bivalent and multivalent pairings. Two meiotic parameters, one describing the preference of homologous chromosome pairing (expressed as the preferential pairing factor) typical of allopolyploids and the other specifying the degree of double reduction of autopolyploids, are estimated. The type of molecular markers used can be fully informative vs. partially informative or dominant vs. codominant. Simulation studies show that our polyploid model is well suited to estimate the preferential pairing factor and the frequency of double reduction at meiosis, which should help to characterize gene segregation in the progeny of autopolyploids. The implications of this model for linkage mapping, population genetic studies, and polyploid classification are discussed.
A Multivalent Pairing Model of Linkage Analysis in Autotetraploids
Authors: Wu Samuel S , Wu Rongling , Ma Chang-Xing , Zeng Zhao-Bang , Yang Mark C K , Casella George
Published: April 2021

Polyploidy has been recognized as an important step in the evolutionary diversification of flowering plants and may have a significant impact on plant breeding. Statistical analyses for linkage mapping in polyploid species can be difficult due to considerable complexities in polysomic inheritance. In this article, we develop a novel statistical method for linkage analysis of polymorphic markers in a full-sib family of autotetraploids. This method is established on multivalent pairings of homologous chromosomes at meiosis and can provide a simultaneous maximum-likelihood estimation of the double reduction frequencies of and recombination fraction between two markers. The EM algorithm is implemented to provide a tractable way for estimating relative proportions of different modes of gamete formation that generate identical gamete genotypes due to multivalent pairings. Extensive simulation studies were performed to demonstrate the statistical properties of this method. The implications of the new method for understanding the genome structure and organization of polyploid species are discussed.
The Kar3-Interacting Protein Cik1p Plays a Critical Role in Passage Through Meiosis I in Saccharomyces cerevisiae
Authors: Shanks Robert M Q , Kamieniecki Rebecca J , Dawson Dean S
Published: April 2021

Meiosis I in Saccharomyces cerevisiae is dependent upon the motor protein Kar3. Absence of Kar3p in meiosis results in an arrest in prophase I. Cik1p and Vik1p are kinesin-associated proteins known to modulate the function of Kar3p in the microtubule-dependent processes of karyogamy and mitosis. Experiments were performed to determine whether Cik1p and Vik1p are also important for the function of Kar3p during meiosis. The meiotic phenotypes of a cik1 mutant were found to be similar to those of kar3 mutants. Cells without Cik1p exhibit a meiotic defect in homologous recombination and synaptonemal complex formation. Most cik1 mutant cells, like kar3 mutants, arrest in meiotic prophase; however, in cik1 mutants this arrest is less severe. These data are consistent with the model that Cik1p is necessary for some, but not all, of the roles of Kar3p in meiosis I. vik1 mutants sporulate at wild-type levels, but have reduced spore viability. This loss in viability is partially attributable to vegetative chromosome loss in vik1 diploids. Cellular localization experiments reveal that Kar3p, Cik1p, and Vik1p are present throughout meiosis and are consistent with Cik1p and Vik1p having different meiotic roles.
Isolation of X and Y Chromosome-Specific DNA Markers From a Liverwort, Marchantia polymorpha, by Representational Difference Analysis
Authors: Fujisawa Masaki , Hayashi Kiwako , Nishio Tomohisa , Bando Tomoyuki , Okada Sachiko , Yamato Katsuyuki T , Fukuzawa Hideya , Ohyama Kanji
Published: April 2021

The liverwort Marchantia polymorpha has X and Y chromosomes in the respective female and male haploids. Here we report the successful exploitation of representational difference analyses to isolate DNA markers for the sex chromosomes. Two female-specific and six male-specific DNA fragments were genetically confirmed to originate from the X and Y chromosomes, respectively.
A Role for MMS4 in the Processing of Recombination Intermediates During Meiosis in Saccharomyces cerevisiae
Authors: de los Santos Teresa , Loidl Josef , Larkin Brittany , Hollingsworth Nancy M
Published: April 2021

The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.
Inherited Differences in Crossing Over and Gene Conversion Frequencies Between Wild Strains of Sordaria fimicola From “Evolution Canyon”
Authors: Saleem Muhammad , Lamb Bernard C , Nevo Eviatar
Published: April 2021

Recombination generates new combinations of existing genetic variation and therefore may be important in adaptation and evolution. We investigated whether there was natural genetic variation for recombination frequencies and whether any such variation was environment related and possibly adaptive. Crossing over and gene conversion frequencies often differed significantly in a consistent direction between wild strains of the fungus Sordaria fimicola isolated from a harsher or a milder microscale environment in “Evolution Canyon,” Israel. First- and second-generation descendants from selfing the original strains from the harsher, more variable, south-facing slope had higher frequencies of crossing over in locus-centromere intervals and of gene conversion than those from the lusher north-facing slopes. There were some significant differences between strains within slopes, but these were less marked than between slopes. Such inherited variation could provide a basis for natural selection for optimum recombination frequencies in each environment. There were no significant differences in meiotic hybrid DNA correction frequencies between strains from the different slopes. The conversion analysis was made using only conversions to wild type, because estimations of conversion to mutant were affected by a high frequency of spontaneous mutation. There was no polarized segregation of chromosomes at meiosis I or of chromatids at meiosis II.
A Genetic Map in theMimulus guttatusSpecies Complex Reveals Transmission Ratio Distortion due to Heterospecific Interactions
Authors: Fishman Lila , Kelly Alan J , Morgan Emily , Willis John H
Published: April 2021

As part of a study of the genetics of floral adaptation and speciation in the Mimulus guttatus species complex, we constructed a genetic linkage map of an interspecific cross between M. guttatus and M. nasutus. We genotyped an F2 mapping population (N = 526) at 255 AFLP, microsatellite, and gene-based markers and derived a framework map through repeated rounds of ordering and marker elimination. The final framework map consists of 174 marker loci on 14 linkage groups with a total map length of 1780 cM Kosambi. Genome length estimates (2011–2096 cM) indicate that this map provides thorough coverage of the hybrid genome, an important consideration for QTL mapping. Nearly half of the markers in the full data set (49%) and on the framework map (48%) exhibited significant transmission ratio distortion (α = 0.05). We localized a minimum of 11 transmission ratio distorting loci (TRDLs) throughout the genome, 9 of which generate an excess of M. guttatus alleles and a deficit of M. nasutus alleles. This pattern indicates that the transmission ratio distortion results from particular interactions between the heterospecific genomes and suggests that substantial genetic divergence has occurred between these Mimulus species. We discuss possible causes of the unequal representation of parental genomes in the F2 generation.
Mutations That Reduce Sinapoylmalate Accumulation in Arabidopsis thaliana Define Loci With Diverse Roles in Phenylpropanoid Metabolism
Authors: Ruegger Max , Chapple Clint
Published: April 2021

The products of phenylpropanoid metabolism in Arabidopsis include the three fluorescent sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine. The sinapoylmalate that accumulates in cotyledons and leaves causes these organs to appear blue-green under ultraviolet (UV) illumination. To find novel genes acting in phenylpropanoid metabolism, Arabidopsis seedlings were screened under UV for altered fluorescence phenotypes caused by changes in sinapoylmalate content. This screen identified recessive mutations at four Reduced Epidermal Fluorescence (REF) loci that reduced leaf sinapoylmalate content. Further analyses showed that the ref mutations affected other aspects of phenylpropanoid metabolism and some led to perturbations in normal plant development. A second class of mutations at the Bright Trichomes 1 (BRT1) locus leads to modest reductions in sinapate ester content; however, the most notable phenotype of brt1 mutants is the development of hyperfluorescent trichomes that appear to contain elevated levels of sinapate esters when compared to the wild type. These results indicate that at least five new loci affecting the developmentally regulated accumulation of phenylpropanoid secondary metabolites in Arabidopsis, and the cell specificity of their distribution, have been identified by screening for altered UV fluorescence phenotypes.
Diverse Variation of Reproductive Barriers in Three Intraspecific Rice Crosses
Authors: Harushima Yoshiaki , Nakagahra Masahiro , Yano Masahiro , Sasaki Takuji , Kurata Nori
Published: April 2021

Reproductive barriers are thought to play an important role in the processes of speciation and differentiation. Asian rice cultivars, Oryza sativa, can be classified into two main types, Japonica and Indica, on the basis of several characteristics. The fertility of Japonica-Indica hybrids differs from one cross to another. Many genes involved in reproductive barriers (hybrid sterility, hybrid weakness, and gametophytic competition genes) have been reported in different Japonica-Indica crosses. To clarify the state of Japonica-Indica differentiation, all reproductive barriers causing deviation from Mendelian segregation ratios in F2 populations were mapped and compared among three different Japonica-Indica crosses: Nipponbare/Kasalath (NK), Fl1084/Dao Ren Qiao (FD), and Fl1007/Kinandang puti (FK). Mapping of reproductive barriers was performed by regression analysis of allele frequencies of DNA markers covering the entire genome. Allele frequencies were explained by 33 reproductive barriers (15 gametophytic and 18 zygotic) in NK, 32 barriers (15 gametophytic and 17 zygotic) in FD, and 37 barriers (19 gametophytic and 18 zygotic) in FK. The number of reproductive barriers in the three crosses was similar; however, most of the barriers were mapped at different loci. Therefore, these reproductive barriers formed after Japonica-Indica differentiation. Considering the high genetic similarity within Japonica and Indica cultivars, the differences in the reproductive barriers of each cross were unexpectedly numerous. The reproductive barriers of Japonica-Indica hybrids likely evolved more rapidly than other genetic elements. One possible force responsible for such rapid evolution of the barriers may have been the domestication of rice.
ATP-Dependent Remodeling of the Spliceosome: Intragenic Suppressors of Release-Defective Mutants of Saccharomyces cerevisiae Prp22
Authors: Campodonico Eva , Schwer Beate
Published: April 2021

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.
Genetic Control of Extracellular Protease Synthesis in the Yeast Yarrowia lipolytica
Authors: Gonzalez-Lopez Claudia I , Szabo Roman , Blanchin-Roland Sylvie , Gaillardin Claude
Published: April 2021

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.