Chromosome Fragments in DICTYOSTELIUM DISCOIDEUM Obtained from Parasexual Crosses between Strains of Different Genetic Background

The first aneuploid strains of Dictyostelium discoideum have been unambiguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and V12 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have V12-derived centromeres, can be maintained in a NC4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group II and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group II.—We observed no deleterious effect on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group II. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (clyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group II encompassed by the fragment chromosome.     

Whole Genome Sequencing of Mutation Accumulation Lines Reveals a Low Mutation Rate in the Social Amoeba Dictyostelium discoideum

Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10⁻⁹, with a Poisson confidence interval of 4.1×10⁻⁹ − 9.5×10⁻⁹, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10⁻¹¹, with a Poisson confidence interval ranging from 7.4×10⁻¹³ to 1.6×10⁻¹⁰, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.     

Parasexual Genetic Analysis of Cell Proportioning Mutants of DICTYOSTELIUM DISCOIDEUM

Eight independently isolated mutants of Dictyostelium discoideum that differentiate exclusively into stalk cells make up one complementation group and carry single recessive mutations at the stalky locus, stkA, located on linkage group II. KY19, a previously described strain that differentiates into spores, but not stalk cells, was found to possess a recessive mutation defining the stalkless locus, stlA, located on linkage group VI. An analysis of the properties of these mutants, together with the phenotype of a haploid double mutant carrying stkA and stlA indicates that stlA results in poorly organized stalk tubes and incomplete stalk cell differentiation, while stkA causes all of the cells to differentiate into stalk cells, even when not enclosed in the stalk tube. The significance of these results is discussed in relation to current theories of pattern formation in D. discoideum.     

A Genetic Map of DICTYOSTELIUM DISCOIDEUM Based on Mitotic Recombination

A genetic map of the cellular slime mold Dictyostelium discoideum is presented in which 42 loci are ordered on five of the seven linkage groups. Although most of the loci were ordered using standing mitotic crossing-over techniques in which recessive selective markers were employed, use was also made of unselected recombined haploid strains. Consistent with cytological studies in which the chromosomes appear to be acrocentric, only a single arm has been found for each of the five linkage groups studied. The mating-type locus, matA, has been located in the tsgE-sprA interval on linkage group I on the basis of studies on diploids formed between strains of opposite mating type that have escaped from vegetative incompatibility.     

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

An Improved Brassica rapa Genetic Linkage Map and Locus-specific Variations in a Doubled Haploid Population

In this study, we describe the construction of an improved Chinese cabbage genetic linkage map by integrating simple sequence repeats (SSRs) and insertion/deletion polymorphisms (InDels) into a previously published map of a doubled haploid (DH) population. The population was derived from a cross between the Chinese cabbage line BY (Brassica rapa ssp. pekinensis) and a European turnip line MM (Brassica rapa L. ssp. rapifera). A total of 629 markers were aligned to ten linkage groups, with a total map length of 1,173.8 cM, and an average distance between markers of 1.87 cm. Of the 126 SSRs and 133 InDels mapped, 46 and 34 were novel, respectively. A comparison of the linkage map with the B. rapa genome showed that more than 93 % of the markers, including 112 SSRs and 129 InDels, could be anchored unambiguously to a specific location on one of the ten chromosomes. In most cases, the order of markers on the linkage map and physical map was similar; however, the majority of linkage groups contained a number of markers whose positions were either transposed or had moved slightly forwards or backwards. During microspore culture, it was observed that 11 SSRs and one InDel showed either variation in size, or the appearance of new marker bands in the DH lines. As a first step to addressing this SSR/InDel marker instability, six SSR and one InDel loci were sequenced, which revealed that the size variation was due mainly to changes in repeat-motif number or to the insertion/deletion of new fragments of DNA.     

QTL analysis for resistance to bacterial wilt (Burkholderia caryophylli) in carnation (Dianthus caryophyllus) using an SSR-based genetic linkage map

Bacterial wilt (Burkholderia caryophylli (Burkholder) Yabuuchi et al.) is one of the most damaging diseases during carnation (Dianthus caryophyllus L.) cultivation in Japan. To find molecular markers for use in marker-assisted selection, we constructed a simple sequence repeat (SSR)-based genetic linkage map of carnation using an F₂ population of 90 plants derived from a cross between a highly resistant line (85-11) and a susceptible cultivar (Pretty Favvare). To develop a large number of SSR markers, we constructed four new SSR-enriched genomic libraries and conducted expressed sequence tag analysis. We mapped 178 SSR loci into 16 linkage groups. The map covered 843.6?cM, with an average distance of 6.5?cM between two loci. This is the first report of a genetic linkage map based mainly on SSR markers in the genus Dianthus. Quantitative trait locus (QTL) analysis identified one locus for resistance to bacterial wilt in linkage group (LG) B4. The locus explained 63.0% of the phenotypic variance for resistance to bacterial wilt. The SSR markers CES1161 and CES2643 that were closest to the QTL were efficient markers for selecting lines with resistance derived from line 85-11. A positional comparison using SSR markers as anchor loci revealed that LG B4 corresponded to LG A6 in a previously constructed map. We found that the position of the resistance locus derived from line 85-11 was similar to that of the major resistance locus observed for a highly resistant wild species, Dianthus capitatus ssp. andrzejowskianus.     

Linkage Analysis of Nystatin Resistance Mutations in DICTYOSTELIUM DISCOIDEUM

Earlier linkage analyses of nystatin resistance loci in Dictyostelium discoideum tentatively mapped the nysB and nysC loci to the previously unmarked linkage group V. The data presented here establishes that nysB maps to linkage group VI and that nysC maps to linkage group IV. The third nystatin resistance locus, nysA, maps to linkage group II.     

Linkage analysis of developmental mutations in aggregation-deficient mutants of Dictyostelium discoideum

Summary Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 °C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e., PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.     

Amino Acid Repeats Cause Extraordinary Coding Sequence Variation in the Social Amoeba Dictyostelium discoideum

Protein sequences are normally the most conserved elements of genomes owing to purifying selection to maintain their functions. We document an extraordinary amount of within-species protein sequence variation in the model eukaryote Dictyostelium discoideum stemming from triplet DNA repeats coding for long strings of single amino acids. D. discoideum has a very large number of such strings, many of which are polyglutamine repeats, the same sequence that causes various human neurological disorders in humans, like Huntington’s disease. We show here that D. discoideum coding repeat loci are highly variable among individuals, making D. discoideum a candidate for the most variable proteome. The coding repeat loci are not significantly less variable than similar non-coding triplet repeats. This pattern is consistent with these amino-acid repeats being largely non-functional sequences evolving primarily by mutation and drift.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Seed colour loci, homoeology and linkage groups of the C genome chromosomes revealed in Brassica rapa-B. oleracea monosomic alien addition lines

Background and Aims

Brassica rapa and B. oleracea are the progenitors of oilseed rape B. napus. The addition of each chromosome of B. oleracea to the chromosome complement of B. rapa results in a series of monosomic alien addition lines (MAALs). Analysis of MAALs determines which B. oleracea chromosomes carry genes controlling specific phenotypic traits, such as seed colour. Yellow-seeded oilseed rape is a desirable breeding goal both for food and livestock feed end-uses that relate to oil, protein and fibre contents. The aims of this study included developing a missing MAAL to complement an available series, for studies on seed colour control, chromosome homoeology and assignment of linkage groups to B. oleracea chromosomes.

Methods

A new batch of B. rapa–B. oleracea aneuploids was produced to generate the missing MAAL. Seed colour and other plant morphological features relevant to differentiation of MAALs were recorded. For chromosome characterization, Snow''s carmine, fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used.

Key Results

The final MAAL was developed. Morphological traits that differentiated the MAALs comprised cotyledon number, leaf morphology, flower colour and seed colour. Seed colour was controlled by major genes on two B. oleracea chromosomes and minor genes on five other chromosomes of this species. Homoeologous pairing was largely between chromosomes with similar centromeric positions. FISH, GISH and a parallel microsatellite marker analysis defined the chromosomes in terms of their linkage groups.

Conclusions

A complete set of MAALs is now available for genetic, genomic, evolutionary and breeding perspectives. Defining chromosomes that carry specific genes, physical localization of DNA markers and access to established genetic linkage maps contribute to the integration of these approaches, manifested in the confirmed correspondence of linkage groups with specific chromosomes. Applications include marker-assisted selection and breeding for yellow seeds.     

Variation,Sex, and Social Cooperation: Molecular Population Genetics of the Social Amoeba Dictyostelium discoideum

Dictyostelium discoideum is a eukaryotic microbial model system for multicellular development, cell–cell signaling, and social behavior. Key models of social evolution require an understanding of genetic relationships between individuals across the genome or possibly at specific genes, but the nature of variation within D. discoideum is largely unknown. We re-sequenced 137 gene fragments in wild North American strains of D. discoideum and examined the levels and patterns of nucleotide variation in this social microbial species. We observe surprisingly low levels of nucleotide variation in D. discoideum across these strains, with a mean nucleotide diversity (π) of 0.08%, and no strong population stratification among North American strains. We also do not find any clear relationship between nucleotide divergence between strains and levels of social dominance and kin discrimination. Kin discrimination experiments, however, show that strains collected from the same location show greater ability to distinguish self from non-self than do strains from different geographic areas. This suggests that a greater ability to recognize self versus non-self may arise among strains that are more likely to encounter each other in nature, which would lead to preferential formation of fruiting bodies with clonemates and may prevent the evolution of cheating behaviors within D. discoideum populations. Finally, despite the fact that sex has rarely been observed in this species, we document a rapid decay of linkage disequilibrium between SNPs, the presence of recombinant genotypes among natural strains, and high estimates of the population recombination parameter ρ. The SNP data indicate that recombination is widespread within D. discoideum and that sex as a form of social interaction is likely to be an important aspect of the life cycle.     

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.     

Interspecies linkage analysis of <Emphasis Type="Italic">mo,</Emphasis> a <Emphasis Type="Italic">Bombyx mori</Emphasis> locus associated with mosaicism and gynandromorphism

The mo (hereditary mosaic) mutation is one of the most famous and interesting mutations of the silkworm, Bombyx mori. Females homozygous for mo generate mosaic and gynandromorphic offspring due to non-elimination of polar bodies and subsequent double fertilization events, irrespective of the genotype of the mated males. Although mo was first reported in 1927, the locus has not been mapped to linkage groups, as the mutation is unstable and appears to be sensitive to genetic background. In this study, linkage analysis of mo was performed using PCR-based markers on single nucleotide polymorphism linkage maps. Bombyx mandarina was used as the mating partner for the B. mori mo strain, as it is easier to identify polymorphic markers between B. mori and B. mandarina than within B. mori strains. Surprisingly, we identified two homozygous linkage groups (LGs) in all of the 12 B₁ (first backcross generation) moths that had deposited mosaic eggs. It was revealed that + ^mo is located on the M chromosome of B. mandarina, which corresponds to two linkage groups of B. mori, LG 14 and 27. Based on further linkage analysis using B. mori as a mating partner, mo was mapped to LG 14. Additionally, we found that mo activity could be modified by a gene(s) on LG 17.     

Rapid identification of non-allelic nystatin resistance mutations inDictyostelium discoideum

Spontaneous nystatin resistance mutations inDictyostelium discoideum fall into three complementation groups;nys A. nys B andnys C. We demonstrate three methods for rapidly distinguishing mutations in the three complementation groups. In the first methodnys B andnys C mutations are identified by their sensitivity to the sterol biosynthesis inhibitors azasterol A25822B and fenarimol respectively. The second method exploits the differential sensitivities of thenys mutations to the polyene antibiotic pimaricin. In the last method we show thatnys C and non-nys C mutants can be distinguished on the basis of the Lieberman-Burchard color reaction for sterols.     

Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum

Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms.     

From Grazing Resistance to Pathogenesis: The Coincidental Evolution of Virulence Factors

To many pathogenic bacteria, human hosts are an evolutionary dead end. This begs the question what evolutionary forces have shaped their virulence traits. Why are these bacteria so virulent? The coincidental evolution hypothesis suggests that such virulence factors result from adaptation to other ecological niches. In particular, virulence traits in bacteria might result from selective pressure exerted by protozoan predator. Thus, grazing resistance may be an evolutionarily exaptation for bacterial pathogenicity. This hypothesis was tested by subjecting a well characterized collection of 31 Escherichia coli strains (human commensal or extra-intestinal pathogenic) to grazing by the social haploid amoeba Dictyostelium discoideum. We then assessed how resistance to grazing correlates with some bacterial traits, such as the presence of virulence genes. Whatever the relative population size (bacteria/amoeba) for a non-pathogenic bacteria strain, D. discoideum was able to phagocytise, digest and grow. In contrast, a pathogenic bacterium strain killed D. discoideum above a certain bacteria/amoeba population size. A plating assay was then carried out using the E. coli collection faced to the grazing of D. discoideum. E. coli strains carrying virulence genes such as iroN, irp2, fyuA involved in iron uptake, belonging to the B2 phylogenetic group and being virulent in a mouse model of septicaemia were resistant to the grazing from D. discoideum. Experimental proof of the key role of the irp gene in the grazing resistance was evidenced with a mutant strain lacking this gene. Such determinant of virulence may well be originally selected and (or) further maintained for their role in natural habitat: resistance to digestion by free-living protozoa, rather than for virulence per se.     

Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein

The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions. We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein. This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba. It is expressed constitutively during vegetative growth and throughout the differentiation cycle. The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa. It shows the characteristic three-domain structure of NSF proteins. A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences. The D. discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs. The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weight of approx. 80 000 in a D. discoideum crude extract and the recombinant D. discoideum His₆-NSF expressed in Escherichia coli.