Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Outcrossing rates in an experimentally admixed population of self-compatible and self-incompatible Arabidopsis lyrata

The transition to self-compatibility from self-incompatibility is often associated with high rates of self-fertilization, which can restrict gene flow among populations and cause reproductive isolation of self-compatible (SC) lineages. Secondary contact between SC and self-incompatible (SI) lineages might re-establish gene flow if SC lineages remain capable of outcrossing. By contrast, intrinsic features of SC plants that reinforce high rates of self-fertilization could maintain evolutionary divergence between lineages. Arabidopsis lyrata subsp. lyrata is characterized by multiple origins of self-compatibility and high rates of self-fertilization in SC-dominated populations. It is unclear whether these high rates of selfing by SC plants have intrinsic or extrinsic causes. We estimated outcrossing rates and examined patterns of pollinator movement for 38 SC and 40 SI maternal parents sampled from an admixed array of 1509 plants sourced from six SC and six SI populations grown under uniform density. Although plants from SI populations had higher outcrossing rates (mean tm = 0.78 ± 0.05 SE) than plants from SC populations (mean tm = 0.56 ± 0.06 SE), outcrossing rates among SC plants were substantially higher than previous estimates from natural populations. Patterns of pollinator movement appeared to contribute to lower outcrossing rates for SC plants; we estimated that 40% of floral visits were geitonogamous (between flowers of the same plant). The relatively high rates of outcrossing for SC plants under standardized conditions indicate that selfing rates in natural SC populations of A. lyrata are facultative and driven by extrinsic features of A. lyrata, including patterns of pollinator movement.

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Morpho-phenological Variability of Flowers Traits and Hybridization of Five Inbred Lines of Cowpea [Vigna unguiculata (L.) Walp.] in Côte d'Ivoire

Knowledge of the morphology and floral phenology and the mastery of a manual pollination technique in a plant species are essential for its genetic improvement. The objective of this work will be to determine the morpho-phenological characteristics of the flowers of five self-fertilization lines of cowpea and to identify an indicated manual pollination technique. The study was carried out in the Botanical Garden of University Peleforo GON COULIBALY, Côte d’Ivoire where 10 morpho-phenological characters of the flower were evaluated on five lines of cowpea self-fertilization. Thus, three manual pollination techniques (A, B and C) were tested by evaluating traits such as knotting rates, filling rates and maternal and paternal effects on fruit yields. The results revealed four stages of flower development in cowpea. These are successively stage of “floral button initiation”, “dark green floral button”, “pale green or pale-yellow floral button” and “blooming flower”. Of the three manual pollination techniques tested, technique C resulted in higher rates of knotting (45.38%) and pod filling (58.03%). Results also showed significant maternal and xenia effects on fruit yields in the cowpea. On the basis of the results generated on the floral biology of the cowpea, it appears that a hybridization program can now be conducted at the UPGC Botanical Garden for the creation of high-performance varieties adapted to climate change for the benefit of producers in Côte d’Ivoire.

Evaluation of the Morphological Quality of Hybrid Seeds and the Vegetative Development of Plants from the Two Reproductive Systems of Elite Dura Genitors from Oil Palm (Elaeisguineensis Jacq) Seed Fields in Côte d'Ivoire

The production of quality oil palm plant material (Elaeisguineensis Jacq.) necessarily involves the creation of seed fields made up of elite Dura parents. Two methods of reproduction of these elite Duragenitors were adopted for the establishment of current seed fields by the National Center for Agronomic Research (CNRA). The first group is made up of elite first-cycle parents who have undergone two successive generations of [(G1) AF] AF type self-fertilization, noted AFAF. The second group is composed of genitors having undergone a prior self-fertilization followed by AFSIB-type recombination cycle of recombination between genitors of the type (G1 × G2) AF, noted AFSIB. This study involves evaluation of morphological quality of seeds from the two breeding systems of the parents and their quality of vegetative development. For this, the seeds from 553 Dura parents distributed among eight (8) descendants and grouped within the category C1001 F were used.The resultsderived from this study have shown that the seeds obtained from the parents through double self-fertilization of AFAF-type parents are characterized by a thin shell and a high percentage of germination. After the elimination of the abnormal plants at the end of the nursery, various traits viz.circumference at the crown, height of the plants and the number of leaves emitted from the plants on the healthy plants showed that the plants resulting from the seeds obtained from self-fertilization followed by AFSIB-type recombination showed good vegetative development and vigor compared to those of AFAF type respectively.

Establishment of local adaptation in partly self-fertilizing populations

Populations often inhabit multiple ecological patches and thus experience divergent selection, which can lead to local adaptation if migration is not strong enough to swamp locally adapted alleles. Conditions for the establishment of a locally advantageous allele have been studied in randomly mating populations. However, many species reproduce, at least partially, through self-fertilization, and how selfing affects local adaptation remains unclear and debated. Using a two-patch branching process formalism, we obtained a closed-form approximation under weak selection for the probability of establishment of a locally advantageous allele (P) for arbitrary selfing rate and dominance level, where selection is allowed to act on viability or fecundity, and migration can occur via seed or pollen dispersal. This solution is compared to diffusion approximation and used to investigate the consequences of a shift in a mating system on P, and the establishment of protected polymorphism. We find that selfing can either increase or decrease P, depending on the patterns of dominance in the two patches, and has conflicting effects on local adaptation. Globally, selfing favors local adaptation when locally advantageous alleles are (partially) recessive, when selection between patches is asymmetrical and when migration occurs through pollen rather than seed dispersal. These results establish a rigorous theoretical background to study heterogeneous selection and local adaptation in partially selfing species.

Seed Paternity Analysis Using SSR Markers to Assess Successful Pollen Donors in Mixed Olive Orchards

The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.

Deforestation is the turning point for the spreading of a weedy epiphyte: an IBM approach

The rapid spread of many weeds into intensely disturbed landscapes is boosted by clonal growth and self-fertilization strategies, which conversely increases the genetic structure of populations. Here, we use empirical and modeling approaches to evaluate the spreading dynamics of Tillandsia recurvata (L.) L. populations, a common epiphytic weed with self-reproduction and clonal growth widespread in dry forests and deforested landscapes in the American continent. We introduce the TRec model, an individual-based approach to simulate the spreading of T. recurvata over time and across landscapes subjected to abrupt changes in tree density with the parameters adjusted according to the empirical genetic data based on microsatellites genotypes. Simulations with this model showed that the strong spatial genetic structure observed from empirical data in T. recurvata can be explained by a rapid increase in abundance and gene flow followed by stabilization after ca. 25 years. TRec model’s results also indicate that deforestation is a turning point for the rapid increase in both individual abundance and gene flow among T. recurvata subpopulations occurring in formerly dense forests. Active reforestation can, in turn, reverse such a scenario, although with a milder intensity. The genetic-based study suggests that anthropogenic changes in landscapes may strongly affect the population dynamics of species with ‘weedy’ traits.

The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis

Entomopathogenic nematodes, including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as producing hermaphrodites in the first generation. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

The distribution of mutational effects on fitness in Caenorhabditis elegans inferred from standing genetic variation

The distribution of fitness effects for new mutations is one of the most theoretically important but difficult to estimate properties in population genetics. A crucial challenge to inferring the distribution of fitness effects (DFE) from natural genetic variation is the sensitivity of the site frequency spectrum to factors like population size change, population substructure, genome structure, and non-random mating. Although inference methods aim to control for population size changes, the influence of non-random mating remains incompletely understood, despite being a common feature of many species. We report the distribution of fitness effects estimated from 326 genomes of Caenorhabditis elegans, a nematode roundworm with a high rate of self-fertilization. We evaluate the robustness of DFE inferences using simulated data that mimics the genomic structure and reproductive life history of C. elegans. Our observations demonstrate how the combined influence of self-fertilization, genome structure, and natural selection on linked sites can conspire to compromise estimates of the DFE from extant polymorphisms with existing methods. These factors together tend to bias inferences towards weakly deleterious mutations, making it challenging to have full confidence in the inferred DFE of new mutations as deduced from standing genetic variation in species like C. elegans. Improved methods for inferring the distribution of fitness effects are needed to appropriately handle strong linked selection and selfing. These results highlight the importance of understanding the combined effects of processes that can bias our interpretations of evolution in natural populations.