Endosperm and seed transcriptomes reveal possible roles for small RNA pathways in wild tomato hybrid seed failure

Crosses between the wild tomato species Solanum peruvianum and S. chilense result in hybrid seed failure (HSF), characterized by endosperm misdevelopment and embryo arrest. We previously showed that genomic imprinting, the parent-of-origin–dependent expression of alleles, is perturbed in hybrid endosperm, with many of the normally paternally expressed genes losing their imprinted status. Here, we report transcriptome-based analyses of gene and small RNA expression levels. We identified 2,295 genes and 468 small RNAs (sRNAs) as differentially expressed (DE) when comparing reciprocal hybrid seed to seeds and endosperms from the two within-species crosses. Our analyses uncovered a pattern of overdominance in endosperm gene expression in both cross directions, in marked contrast to the patterns of sRNA expression in whole seeds. Intriguingly, patterns of increased gene expression resembled the previously reported increased maternal expression proportions in hybrid endosperms. We identified physical clusters of sRNAs; DE sRNAs exhibited reduced levels of expression in hybrid seeds from both cross directions. Moreover, sRNAs mapped to genes coding for key proteins involved in epigenetic regulation of gene expression, suggesting a regulatory feedback mechanism. We describe examples of genes that are targets of sRNA-mediated gene silencing; in these cases, reduced sRNA expression was concomitant with increased gene expression in hybrid seeds. Our analyses also show that S. peruvianum dominance impacts gene and sRNA expression in hybrid seeds. Overall, our study indicates roles for sRNA-mediated epigenetic regulation in HSF between closely related wild tomato species

Incidence and developmental timing of endosperm failure in post-zygotic isolation between wild tomato lineages

Background and AimsDefective hybrid seed development in angiosperms might mediate the rapid establishment of intrinsic post-zygotic isolation between closely related species. Extensive crosses within and among three lineages of wild tomatoes (Solanum section Lycopersicon) were performed to address the incidence, developmental timing and histological manifestations of hybrid seed failure. These lineages encompass different, yet fairly recent, divergence times and both allopatric and partially sympatric pairs.MethodsMature seeds were scored visually 2 months after hand pollinations, and viable-looking seeds were assessed for germination success. Using histological sections from early-developing seeds from a sub-set of crosses, the growth of three major seed compartments (endosperm, embryo and seed coat) was measured at critical developmental stages up to 21 d after pollination, with a focus on the timing and histological manifestations of endosperm misdevelopment in abortive hybrid seeds.Key ResultsFor two of three interspecific combinations including the most closely related pair that was also studied histologically, almost all mature seeds appeared ‘flat’ and proved inviable; histological analyses revealed impaired endosperm proliferation at early globular embryo stages, concomitant with embryo arrest and seed abortion in both cross directions. The third interspecific combination yielded a mixture of flat, inviable and plump, viable seeds; many of the latter germinated and exhibited near-normal juvenile phenotypes or, in some instances, hybrid necrosis and impaired growth.ConclusionsThe overall results suggest that near-complete hybrid seed failure can evolve fairly rapidly and without apparent divergence in reproductive phenology/biology. While the evidence accrued here is largely circumstantial, early-acting disruptions of normal endosperm development are most probably the common cause of seed failure regardless of the type of endosperm (nuclear or cellular)

Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components

Estimating Parameters of Speciation Models Based on Refined Summaries of the Joint Site-Frequency Spectrum

Understanding the processes and conditions under which populations diverge to give rise to distinct species is a central question in evolutionary biology. Since recently diverged populations have high levels of shared polymorphisms, it is challenging to distinguish between recent divergence with no (or very low) inter-population gene flow and older splitting events with subsequent gene flow. Recently published methods to infer speciation parameters under the isolation-migration framework are based on summarizing polymorphism data at multiple loci in two species using the joint site-frequency spectrum (JSFS). We have developed two improvements of these methods based on a more extensive use of the JSFS classes of polymorphisms for species with high intra-locus recombination rates. First, using a likelihood based method, we demonstrate that taking into account low-frequency polymorphisms shared between species significantly improves the joint estimation of the divergence time and gene flow between species. Second, we introduce a local linear regression algorithm that considerably reduces the computational time and allows for the estimation of unequal rates of gene flow between species. We also investigate which summary statistics from the JSFS allow the greatest estimation accuracy for divergence time and migration rates for low (around 10) and high (around 100) numbers of loci. Focusing on cases with low numbers of loci and high intra-locus recombination rates we show that our methods for the estimation of divergence time and migration rates are more precise than existing approaches

Enhanced robustness digital holographic microscopy for demanding environment of space biology

We describe an optimized digital holographic microscopy system (DHM) suitable for high-resolution visualization of living cells under conditions of altered macroscopic mechanical forces such as those that arise from changes in gravitational force. Experiments were performed on both a ground-based microgravity simulation platform known as the random positioning machine (RPM) as well as during a parabolic flight campaign (PFC). Under these conditions the DHM system proved to be robust and reliable. In addition, the stability of the system during disturbances in gravitational force was further enhanced by implementing post-processing algorithms that best exploit the intrinsic advantages of DHM for hologram autofocusing and subsequent image registration. Preliminary results obtained in the form of series of phase images point towards sensible changes of cytoarchitecture under states of altered gravity

Mapping of QTL for Resistance against the Crucifer Specialist Herbivore Pieris brassicae in a New Arabidopsis Inbred Line Population, Da(1)-12×Ei-2

In Arabidopsis thaliana and other crucifers, the glucosinolate-myrosinase system contributes to resistance against herbivory by generalist insects. As yet, it is unclear how crucifers defend themselves against crucifer-specialist insect herbivores.We analyzed natural variation for resistance against two crucifer specialist lepidopteran herbivores, Pieris brassicae and Plutella xylostella, among Arabidopsis thaliana accessions and in a new Arabidopsis recombinant inbred line (RIL) population generated from the parental accessions Da(1)-12 and Ei-2. This RIL population consists of 201 individual F(8) lines genotyped with 84 PCR-based markers. We identified six QTL for resistance against Pieris herbivory, but found only one weak QTL for Plutella resistance. To elucidate potential factors causing these resistance QTL, we investigated leaf hair (trichome) density, glucosinolates and myrosinase activity, traits known to influence herbivory by generalist insects. We identified several previously unknown QTL for these traits, some of which display a complex pattern of epistatic interactions.Although some trichome, glucosinolate or myrosinase QTL co-localize with Pieris QTL, none of these traits explained the resistance QTL convincingly, indicating that resistance against specialist insect herbivores is influenced by other traits than resistance against generalists

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals

The Relationship of Nucleotide Polymorphism, Recombination Rate and Selection in Wild Tomato Species

We analyzed the effects of mating system and recombination rate on single nucleotide polymorphisms using 14 single-copy nuclear loci from single populations of five species of wild tomatoes (Solanum section Lycopersicon). The taxa investigated comprise two self-compatible (SC) and three self-incompatible (SI) species. The observed reduction in nucleotide diversity in the SC populations compared to the SI populations is much stronger than expected under the neutral effects of the mating system on effective population size. Importantly, outgroup sequences available for 11 of the 14 loci yield strong positive correlations between silent nucleotide diversity and silent divergence, indicative of marked among-locus differences in mutation rates and/or selective constraints. Furthermore, using a physical estimate of local recombination rates, we find that silent nucleotide diversity (but not divergence) is positively correlated with recombination rate in two of the SI species. However, this correlation is not nearly as strong as in other well-characterized species (in particular, Drosophila). We propose that nucleotide diversity in Lycopersicon is dominated mainly by differences in neutral mutation rates and/or selective constraints among loci, demographic processes (such as population subdivision), and background selection. In addition, we hypothesize that the soil seed bank plays an important role in the maintenance of the large genetic diversity in the SI species (in particular L. peruvianum)