Phenotypic and Genetic Analysis of a Mutant of Heterorhabditis bacteriophora Strain HP88

Induction and characterization of a morphological mutant are described for Heterorhabditis bacteriophora strain HP88. A homozygous inbred line was used as the base population for mutagenesis and genetic analysis of mutations. Mutagenesis was induced by exposing young hermaphrodites to 0.05 M ethyl methanesulfonate. A dumpy mutant (designated Hdpy-l) was isolated from the F₂ generation of the mutagenized population. Morphological studies with light and scanning electron microscopy revealed that the head region of the adult stage was compressed. The head region of the infective juvenile was distorted and the mouth open. Backcross with the original population was successful only between mutant hermaphrodites and wild type males; 50-100 percent of the progeny of this cross maintained the dumpy phenotype, indicating that the ratio between self- and external fertilization of the eggs is > 1 and that the dumpy mutation is recessive.     

Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS

The frequency of males (5AA; XO) among the self progeny of wild-type Caenorhabditis elegans hermaphrodites (5AA; XX) is about one in 500. Fifteen him (for "high incidence of males") mutations have been identified that increase this frequency by a factor of ten to 150, as a result of increased X-chromosome nondisjunction. The mutations define ten complementation groups, which have been mapped: nine are autosomal, and one sex linked. Most of the mutants are superficially wild type in anatomy and behavior; however, him-4 mutants display gonadal abnormalities, and unc-86 mutants, which have a Him phenotype, exhibit a variety of anatomical and behavioral abnormalities. All the mutants segregate fertile 3X hermaphrodite progeny as well as XO male progeny. Some produce large numbers of inviable zygotes. Mutants in all ten genes produce diplo-X and nullo-X exceptional ova, and in the four strains tested, diplo-X and nullo-X exceptional sperm are produced by 2X "transformed" males. It appears likely that most of the mutants have defects in both gamete lines of the hermaphrodite. XO males of him strains other than him-4 and unc-86 are similar to wild-type males in anatomy and behavior, and all produce equal or almost equal numbers of haplo-X and nullo-X sperm, and no diplo-X sperm. Male fertility is reduced to varying extents in all him mutants. In four of the strains, nondisjunction during oogenesis has been shown to occur at a reductional division, and in three of these strains, abnormalities in recombination have been demonstrated. One mutant also exhibits autosomal nondisjunction, but many of the others probably do not. Therefore, the X chromosome of C. elegans may differ from the autosomes in the mechanisms controlling its meiotic behavior.——3X hermaphrodites are shorter and less fertile than 2X hermaphrodites, and they produce many inviable zygotes among their self progeny: these are probably 4X zygotes. Haplo-X and diplo-X ova are produced in 2:1 ratio by 3X hermaphrodites. him mutations are expressed in these animals, increasing the frequency of self-progeny males and 2X hermaphrodites.     

Systematic comparison of bacterial feeding strains for increased yield of Caenorhabditis elegans males by RNA interference-induced non-disjunction

Rare Caenorhabditis elegans males arise when sex chromosome non-disjunction occurs during meiosis in self-fertilizing hermaphrodites. Non-disjunction is a relatively rare event, and males are typically observed at a frequency of less than one in five hundred wild-type animals. Males are required for genetic crosses and phenotypic analysis, yet current methods to generate large numbers of males can be cumbersome. Here, we identify RNAi reagents (dsRNA-expressing bacteria) with improved effectiveness for eliciting males. Specifically, we used RNAi to systematically reduce the expression of over two hundred genes with meiotic chromosome segregation functions, and we identified a set of RNAi reagents that robustly and reproducibly elicited male progeny.     

An Autosomal Gene That Affects X Chromosome Expression and Sex Determination in CAENORHABDITIS ELEGANS

Recessive mutant alleles at the autosomal dpy-21 locus of C. elegans cause a dumpy phenotype in XX animals but not in XO animals. This dumpy phenotype is characteristic of X chromosome aneuploids with higher than normal X to autosome ratios and is proposed to result from overexpression of X-linked genes. We have isolated a new dpy-21 allele that also causes partial hermaphroditization of XO males, without causing the dumpy phenotype. All dpy-21 alleles show hermaphroditization effects in XO males that carry a duplication of part of the X chromosome and also partially suppress a transformer (tra-1) mutation that converts XX animals into males. Experiments with a set of X chromosome duplications show that the defects of dpy-21 mutants can result from interaction with several different regions of the X chromosome. We propose that dpy-21 regulates X chromosome expression and may be involved in interpreting X chromosome dose for the developmental decisions of both sex determination and dosage compensation.     

Mixed mating in androdioecious Mercurialis annua inferred using progeny arrays and diploid-acting microsatellite loci in a hexaploid background

Background and Aims

The frequency at which males can be maintained with hermaphrodites in androdioecious populations is predicted to depend on the selfing rate, because self-fertilization by hermaphrodites reduces prospective siring opportunities for males. In particular, high selfing rates by hermaphrodites are expected to exclude males from a population. Here, the first estimates are provided of the mating system from two wild hexaploid populations of the androdioecious European wind-pollinated plant M. annua with contrasting male frequencies.

Methods

Four diploid microsatellite loci were used to genotype 19–20 progeny arrays from two populations of M. annua, one with males and one without. Mating-system parameters were estimated using the program MLTR.

Key Results

Both populations had similar, intermediate outcrossing rates (t_m = 0·64 and 0·52 for the population with and without males, respectively). The population without males showed a lower level of correlated paternity and biparental inbreeding and higher allelic richness and gene diversity than the population with males.

Conclusions

The results demonstrate the utility of new diploid microsatellite loci for mating system analysis in a hexaploid plant. It would appear that androdioecious M. annua has a mixed-mating system in the wild, an uncommon finding for wind-pollinated species. This study sets a foundation for future research to assess the relative importance of the sexual system, plant-density variation and stochastic processes for the regulation of male frequencies in M. annua over space and time.     

Radiation-Sensitive Mutants of CAENORHABDITIS ELEGANS

Nine rad (for abnormal radiation sensitivity) mutants hypersensitive to ultraviolet light were isolated in the small nematode Caenorhabditis elegans. The mutations are recessive to their wild-type alleles, map to four of the six linkage groups in C. elegans and define nine new games named rad-1 through rad-9. Two of the mutants—rad-1 and rad-2—are very hypersensitive to X rays, and three—rad-2, rad-3 and rad-4—are hypersensitive to methyl methanesulfonate under particular conditions of exposure. The hypersensitivity of these mutants to more than one DNA-damaging agent suggests that they may be abnormal in DNA repair. One mutant—rad-5, a temperature-sensitive sterile mutant—shows an elevated frequency of spontaneous mutation at more than one locus; rad-4, which shows a cold-sensitive embryogenesis, reduces meiotic X-chromosome nondisjunction tenfold and partially suppresses some but not all mutations that increase meiotic X-chromosome nondisjunction; the viability of rad-6 hermaphrodites is half that of rad-6 males at 25°; and newly mature (but not older) rad-8 hermaphrodites produce many inviable embryo progeny. Meiotic recombination frequencies were measured for seven rad mutants and found to be close to normal.     

Mutations Causing Transformation of Sexual Phenotype in the Nematode CAENORHABDITIS ELEGANS

Ten mutations are described that transform genotypic hermaphrodites of the nematode Caenorhabditis elegans into phenotypic males. These fall into three autosomal complementation groups, termed tra-1, tra-2 , and tra-3. Two alleles of tra-1 produce almost complete transformation, to a fertile male phenotype; such transformed animals are useful for analyzing sex-linked genes. All alleles of tra-1 and tra-2 are recessive; the one known allele of tra-3 is both recessive and maternal in effect. Where tested, both XX and XXX hermaphrodites are transformed into males, but XO males (true males) are unaffected by these mutations. It is suggested that these genes are actually involved in hermaphrodite development and have no role in male development.     

Hermaphroditism in Meloidogyne hapla

Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females. Initially, hermaphrodite ovaries are filled with oocytes at various stages of development. Hermaphroditism is expressed later when young oocytes in the early pachytene region of the growth zone suddenly advance to diakinesis and proceed with maturation divisions, resulting in spermatid production. Spermatogenesis may be initiated shortly after the fourth molt, or later, after a female has produced some eggs. Spermatogenesis may occur in one or both gonads, and it may be initiated in one gonad before the other. Once initiated, spermatogenesis continues for the entire reproductive life of the hermaphrodite. Several thousand spermatozoa accumulate in the ovotestis. Because they do not pass through the oviduct into the spermatotheca, they do not take part in reproduction (nonfunctional hermaphroditism). Among the progeny of hermaphrodites, ca. 50% are hermaphroditic, and the remainder are apparently normal females which, however, produce about 50% hermaphroditic progeny. Two temperature regimes (20-23 C and 27-30 C) did not influence the percentage of hermaphrodites among the progeny. Hermaphroditism could not be transmitted to nonhermaphroditic isolates following attempted crosses between males of hermaphroditic and females of nonhermaphroditic isolates. Although this result suggests cytoplasmic rather than nuclear inheritance, this conclusion is not definitive.     

Maternal sex effects and inbreeding depression under varied environmental conditions in gynodioecious Fragaria vesca subsp. bracteata

Background and Aims

Gynodioecy (coexistence of females and hermaphrodites) is a sexual system that occurs in numerous flowering plant lineages. Thus, understanding the features that affect its maintenance has wide importance. Models predict that females must have a seed fitness advantage over hermaphrodites, and this may be achieved via seed quality or quantity. Females in a population of Fragaria vesca subsp. bracteata, a long-lived gynodioecious perennial, do not demonstrate a seed quantity advantage, so this study explored whether females produced better quality seed via maternal sex effects or avoidance of inbreeding depression (IBD).

Methods

Families of selfed and outcrossed seed were created using hermaphrodite mothers and families of outcrossed seed were created using female mothers. The effects of these pollination treatments were assessed under benign conditions early in life and under varied conditions later in life. To test for an effect of maternal sex, fitness components and traits associated with acclimation to variable environments of progeny of outbred hermaphrodites and females were compared. To test for expression of IBD, fitness parameters between inbred and outbred progeny of hermaphrodites were compared.

Key Results

Offspring of females were more likely to germinate in benign conditions and survive in harsh resource environments than outbred progeny of hermaphrodites. IBD was low across most life stages, and both the effect of maternal sex on progeny quality and the expression of IBD depended on both maternal family and resource condition of the progeny.

Conclusions

The effect of maternal sex and IBD on progeny quality depended on resource conditions, maternal lineage and progeny life stage. In conjunction with known lack of differences in seed quantity, the quality advantages and IBD observed here are still unlikely to be sufficient for maintenance of gynodioecy under nuclear inheritance of male sterility.     

A sex-determining gene,fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans

Sex in the nematode Caenorhabditis elegans is normally determined by the X chromosome to autosome (X:A) ratio, with XX hermaphrodites and XO males. Previous work has shown that a set of at least four autosomal genes (her-1, tra-2, tra-3, and tra-1) is signaled by the X:A ratio and appears to act in a regulatory pathway to determine sex. Twenty-one new recessive alleles of the gene fem-1(IV) (formerly isx-1) have been isolated. Seven of these may be null alleles; one of these is an amber mutation. The other 14 alleles are temperature sensitive. The putative null mutations cause both XO and XX animals to develop as females when the mother as well as the zygote is fem-1(?). Therefore, fem-1(+) is required (a) for the development of the male body and (b) for spermatogenesis in males and hermaphrodites. In addition, fem-1 shows a maternal effect: wild-type fem-1 product partially rescues the development of fem-1(?) progeny. By analyzing double mutants it has been shown that fem-1(+) is part of the sex-determination pathway and has two distinct functions: (1) in the soma it prevents the action of tra-1, thereby allowing male development to occur, and (2) in the germline it is necessary for spermatogenesis in both sexes.     

Gender plasticity and sexual system stability in Wurmbea

Background and aims

Sexually dimorphic populations are often located in drier habitats than cosexual populations. Gender plasticity (GP), whereby hermaphrodites alter female and male functions depending on resources, and sex-differential plasticity (SDP) between hermaphrodites and unisexuals are predicted to affect sexual system stability. Here, GP and SDP are evaluated in cosexual and gynodioecious Wurmbea biglandulosa and sub-dioecious and dioecious W. dioica.

Methods

GP was evaluated under two resource conditions, compared among sexual systems and assessed as to whether (1) males produced perfect flowers and (2) hermaphrodites altered investment in perfect (female function) and total (male function) flowers. SDP was assessed within sexual systems as differences between sex functions of hermaphrodites vs. unisexuals. Males and hermaphrodites were compared to assess whether size thresholds for female function differed among sexual systems. Plasticity costs were evaluated using correlations between female function and male traits in hermaphrodites, and in W. dioica by comparing hermaphrodite and male regressions between plant size and pollen production.

Key Results

In dioecious W. dioica no males exhibited GP, whereas 100 % did in gynodioecious and cosexual W. biglandulosa. In sub-dioecious W. dioica, resources affected GP (high, 66 %; low, 42 %). Hermaphrodites in all sexual systems reduced perfect but not total flowers under low resources. Unisexuals were unaffected, demonstrating SDP for female function only. Thresholds for female function were greater in sub-dioecious W. dioica than in W. biglandulosa. Plasticity costs were detected only in sub-dioecious W. dioica.

Conclusions

SDP for female function could assist female establishment in cosexual populations and maintain females in gynodioecious and sub-dioecious populations. Although the absence of male SDP should stabilize sub-dioecy, plasticity costs would render sub-dioecy unstable, favouring canalized males over hermaphrodites. This study highlights the importance of interactions between environmental conditions and hermaphrodite sex expression for the stability of dimorphic sexual systems.     

Effect of the Nematode Contortylenchus brevicomi on Gallery Construction and Fecundity of the Southern Pine Beetle

Field-collected Dendroctonus frontalis were reared in a controlled environment. Male-female beetle pairs retrieved from galleries 1, 2, or 5 wk after introduction into pine bolts were examined for nematode parasites. Data were obtained for each pair on gallery length, egg niche construction, egg viability, and progeny survival. In a separate study, beetle pairs were reared under laboratory conditions for 10 wk. The number of emerged adult progeny of each pair was recorded. Contortylenchus brevicomi, a nematode parasite, was found in 25% of all beetles that established galleries. After 2 and 3 wk, female beetles infected with the nematode had produced fewer eggs and shorter galleries than did uninfected females. Uninfected females mated with nematode-infected males showed similar trends, although the differences in the 2- and 3-wk tests were not significant. Progeny survival or egg viability was not affected by nematode parasitism of either parent beetle. Unikaryon minutum, a microsporidian parasite found in 65% of all colonizing beetles, had no effect on measured variables. The lower fecundity of beetles parasitized by C. brevicomi continued throughout the insect''s reproductive cycle. After 10 wk, nematode-infected beetle pairs produced fewer emerged adult progeny than did uninfected pairs.     

Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing

Background

High light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Outdoor cultivation requires the use of either wild-type or non-GMO derived mutant strains due to safety concerns. The identification and molecular characterization of such mutants derived from untagged forward genetics approaches was limited previously by the tedious and time-consuming methods involving techniques such as classical meiotic mapping. The combination of mapping with next generation sequencing technologies offers alternative strategies to identify genes involved in high light adaptation in untagged mutants.

Results

We used the model alga Chlamydomonas reinhardtii in a non-GMO mutation strategy without any preceding crossing step or pooled progeny to identify genes involved in the regulatory processes of high light adaptation. To generate high light tolerant mutants, wildtype cells were mutagenized only to a low extent, followed by a stringent selection. We performed whole-genome sequencing of two independent mutants hit1 and hit2 and the parental wildtype. The availability of a reference genome sequence and the removal of shared bakground variants between the wildtype strain and each mutant, enabled us to identify two single nucleotide polymorphisms within the same gene Cre02.g085050, hereafter called LRS1 (putative Light Response Signaling protein 1). These two independent single amino acid exchanges are both located in the putative WD40 propeller domain of the corresponding protein LRS1. Both mutants exhibited an increased rate of non-photochemical-quenching (NPQ) and an improved resistance against chemically induced reactive oxygen species. In silico analyses revealed homology of LRS1 to the photoregulatory protein COP1 in plants.

Conclusions

In this work we identified the nuclear encoded gene LRS1 as an essential factor for high light adaptation in C. reinhardtii. The causative random mutation within this gene was identified by a rapid and efficient method, avoiding any preceding crossing step, meiotic mapping, or pooled progeny. Our results open up new insights into mechanisms of high light adaptation in microalgae and at the same time provide a simplified strategy for non-GMO forward genetics, a crucial precondition that could result in the identification of key factors for economically relevant biological processes within algae.     

The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.     

Somatic damage to the X chromosome of the nematode Caenorhabditis elegans induced by gamma radiation

Summary Wild-type male embryos and young larvae of the nematode Caenorhabditis elegans were more sensitive than wild-type hermaphrodites to inactivation by gamma rays; wild-type males have one X chromosome per cell (XO), whereas wild-type hermaphrodites have two (XX). Furthermore, after transformation into fertile hermaphrodites by a her-1 mutation, XO animals were more radiosensitive than XX her-1 animals; and XX animals transformed into fertile males by a tra-1 mutation did not show increased radiosensitivity. It is concluded that wild-type males are more radiosensitive than wild-type hermaphrodites because they have one X chromosome rather than two, and the predominant mode of inactivation of XO animals involves damage to the single X chromosome. No sex-specific differences in survival were observed after UV irradiation.     

Facultative Vivipary is a Life-History Trait in Caenorhabditis elegans

Organisms partition their resources among growth, maintenance, and reproduction and, when resources become limiting, the allocation to one process necessitates reduced allocation to others. When starved, Caenorhabditis elegans adults retain progeny internally which then consume the parent body contents, and some of those larvae use the resources to reach the resistant, long-lived dauer stage. If starved under similarly extreme conditions, larvae from eggs laid outside of the body are unable to develop into dauers. We interpret this switch from ovipary, or laying eggs, to bearing live young as facultative vivipary. This switch is induced by starvation of late fourth-stage larvae, young adults, or gravid adults. In C. elegans, vivipary is the altruistic allocation of all available parental energy and nutrients to progeny, with the associated costs to adult hermaphrodites of truncated life span and fecundity. As a life-history trait, facultative vivipary is a survival-enhancing response to stress that may provide insights into the evolution of reproduction and longevity.     

Functional characterization of gynodioecy in Fragaria vesca ssp. bracteata (Rosaceae)

Background and Aims

Gynodioecy is a phylogenetically widespread and important sexual system where females coexist with hermaphrodites. Because dioecy can arise from gynodioecy, characterization of gynodioecy in close relatives of dioecious and sub-dioecious species can provide insight into this transition. Thus, we sought to determine whether Fragaria vesca ssp. bracteata, a close relative to F. chiloensis and F. virginiana, exhibits the functional and population genetic hallmarks of a gynodioecious species.

Methods

We compared reproductive allocation of females and hermaphrodites grown in the greenhouse and estimated genetic diversity (allelic diversity, heterozygosity) and inbreeding coefficients for field-collected adults of both sexes using simple sequence repeat (SSR) markers. We estimated mating system and early seed fitness from open-pollinated families of both sex morphs.

Key Results

Under greenhouse conditions, females and hermaphrodites allocated similarly to all reproductive traits except flower number, and, as a consequence, females produced 30 % fewer seeds per plant than hermaphrodites. Under natural conditions, hermaphrodites produce seeds by self-fertilization approx. 75 % of the time, and females produced outcrossed seeds with very little biparental inbreeding. Consistent with inbreeding depression, seeds from open-pollinated hermaphrodites were less likely to germinate than those from females, and family-level estimates of hermaphrodite selfing rates were negatively correlated with germination success and speed. Furthermore, estimates of inbreeding depression based on genetic markers and population genetic theory indicate that inbreeding depression in the field could be high.

Conclusions

The joint consideration of allocation and mating system suggests that compensation may be sufficient to maintain females given the current understanding of sex determination. Fragaria vesca ssp. bracteata exhibited similar sex morph-dependent patterns of mating system and genetic diversity, but less reproductive trait dimorphism, than its sub-dioecious and dioecious congeners.     

Suppression and Function of X-Linked Lethal and Sterile Mutations in CAENORHABDITIS ELEGANS

We have expanded our collection of recessive lethal and sterile mutants in the region of the X chromosome balanced by mnDp1(X;V), about 15% of the X linkage map, to a total of 54 mutants. The mutations have been mapped with respect to 20 overlapping deficiencies and five X duplications, and they have been assigned to 24 genes by complementation testing. Nine mutants are hermaphrodite-sterile: one of these is a sperm-defect mutant, two have abnormal gonadogeneses and six, in five genes, are maternally influenced mutants, producing inviable zygote progeny. One of the gonadogenesis mutants and two of the maternally influenced mutants are male fertile. All but one of the maternally influenced mutants give cross progeny when mated with wild-type males. Forty-three mutants were tested for suppression by homozygous sup-5(e1464), which is believed to be specific for null alleles. Ten mutants that were judged by independent criteria not to be null mutants are not suppressed. Nine of the other 33 mutants, in nine genes, are suppressed, five in both heterozygous and homozygous suppressor stocks and four only in homozygous suppressor stocks.