Genome editing of woody perennial trees

This chapter discusses the challenges facing woody perennial trees, namely the heterozygosity of outcrossing species and limited genomic resources. Gene knockouts (KO) represent the predominant applications of the CRISPR technology in woody perennial crops to date and will be the focus of this chapter. The issues considered for gene KO are generally applicable to other CRISPR applications targeting regulatory sequences or non-coding genes. Case studies from the Populus 4-coumarate:CoA ligase (4CL) family are presented to demonstrate the power of CRISPR in elucidating functional redundancy as well as specificity of duplicated genes in a perennial woody species.

fullsibQTL: an R package for QTL mapping in biparental populations of outcrossing species

ABSTRACTAccurate QTL mapping in outcrossing species requires software programs which consider genetic features of these populations, such as markers with different segregation patterns and different level of information. Although the available mapping procedures to date allow inferring QTL position and effects, they are mostly not based on multilocus genetic maps. Having a QTL analysis based in such maps is crucial since they allow informative markers to propagate their information to less informative intervals of the map. We developed fullsibQTL, a novel and freely available R package to perform composite interval QTL mapping considering outcrossing populations and markers with different segregation patterns. It allows to estimate QTL position, effects, segregation patterns, and linkage phase with flanking markers. Additionally, several statistical and graphical tools are implemented, for straightforward analysis and interpretations. fullsibQTL is an R open source package with C and R source code (GPLv3). It is multiplatform and can be installed from https://github.com/augusto-garcia/fullsibQTL.

Optimal contribution selection in highly fecund species with overlapping generations

Optimal contributions approaches to parental selection in closed breeding populations aim to maximise genetic gains, while restraining long-term inbreeding. The adoption of optimal contribution selection (OCS) in highly-fecund outcrossing species presents a number of challenges not applicable to species of low fecundity (e.g. livestock) for which they were developed. This is particularly true if overlapping-generations or rolling-front breeding strategies are applied, in which case the number of individuals per family in juvenile (i.e. sexually immature) age groups is not necessarily known but is likely to be large. In these circumstances, conventional OCS procedures must be modified or a large number of dummy individuals defined, making computations onerous. Here, an approach to OCS is presented that involves the use of ‘between-family relationship matrices’ instead of ‘between-individual relationship matrices’. The method is applicable to breeding programs involving highly fecund outcrossing species with overlapping generations, including circumstances where the number of juveniles per family is unknown but large.

The selfing syndrome overshadows other differences when comparing fitness across Capsella species

SUMMARYSelf-fertilization has recurrently evolved from outcrossing. Self-fertilization provides an advantage in the short-term as individuals do not require a mate to reproduce, but self-fertilization is also associated with both decreased genetic diversity and accumulation of weakly deleterious mutations, which could, however, be alleviated in polyploid selfers. If pollinators are not limited, individual fitness is thus expected to be higher in outcrossers than in selfers. We measured several life history traits in four Capsella species under two different treatments (disturbed and undisturbed) to assess the effects of mating system and ploidy level on reproductive, vegetative and phenological traits. The experiment was carried out outdoor in Northwest Greece, within the range of the obligate outcrossing species, C. grandiflora, so it could be naturally pollinated and its fitness directly compared to that of its self-fertilizing relatives. Disturbance of the environment did not affect the phenotype in any of the four species. However, for most traits the obligate outcrossing species performed better than all selfing ones. In contrast, polyploidy did not seem to confer an advantage in terms of survival or reproduction compared to diploidy. Finally, plants from Asia and northern Europe had lower performances than accessions from southern Europe and the Middle-East.

Genome-Wide Diversity Analysis of Valeriana officinalis L. Using DArT-seq Derived SNP Markers

Common valerian (Valeriana officinalis L.) is one of the most important medicinal plants, with a mild sedative, nervine, antispasmodic and relaxant effect. Despite a substantial number of studies on this species, the genetic diversity and population structure have not yet been analyzed. Here, we use a next-generation sequencing-based Diversity Array Technology sequencing (DArT-seq) technique to analyze Polish gene bank accessions that originated from wild populations and cultivars. The major and, also, the most astounding result of our work is the low level of observed heterozygosity of individual plants from natural populations, despite the fact that the species is widespread in the studied area. Inbreeding in naturally outcrossing species such as valerian decreases reproductive success. The analysis of the population structure showed the potential presence of a metapopulation in the central part of Poland and the formation of a distinct gene pool in the Bieszczady Mountains. The results also indicate the presence of the cultivated gene pool within wild populations in the region where the species is cultivated for the needs of the pharmaceutical industry, and this could lead to structural and genetic imbalances in wild populations.

Balancing selection maintains ancient genetic diversity in C. elegans

Summary paragraphThe mating system of a species profoundly influences its evolutionary trajectory1–3. Across diverse taxa, selfing species have evolved independently from outcrossing species thousands of times4. The transition from outcrossing to selfing significantly decreases the effective population size, effective recombination rate, and heterozygosity within a species5. These changes lead to a reduction in the genetic diversity, and therefore adaptive potential, by intensifying the effects of random genetic drift and linked selection6,7. Selfing has evolved at least three times independently in the nematode genus Caenorhabditis8, including in the model organism Caenorhabditis elegans, and all three selfing species show substantially reduced genetic diversity relative to outcrossing species8,9. Selfing and outcrossing Caenorhabditis species are often found in the same niches, but we still do not know how selfing species with limited genetic diversity can adapt to and inhabit these same diverse environments. Here, we discovered previously uncharacterized levels and patterns of genetic diversity by examining the whole-genome sequences from 609 wild C. elegans strains isolated worldwide. We found that genetic variation is concentrated in punctuated hyper-divergent regions that cover 20% of the C. elegans reference genome. These regions are enriched in environmental response genes that mediate sensory perception, pathogen response, and xenobiotic stress. Population genomic evidence suggests that these regions have been maintained by balancing selection. Using long-read genome assemblies for 15 wild isolates, we found that hyper-divergent haplotypes contain unique sets of genes and show levels of divergence comparable to that found between Caenorhabditis species that diverged millions of years ago. Taken together, these results suggest that ancient genetic diversity present in the outcrossing ancestor of C. elegans has been maintained by long-term balancing selection since the evolution of selfing. These results provide an example for how species can avoid the evolutionary “dead end” associated with selfing by maintaining ancestral genetic diversity.

Genome-wide characterization of genetic diversity and population structure of Valeriana officinalis L.

Background: Valeriana officinalis L. is one of the most important medicinal plant with a mild sedative, nervine, antispasmodic and relaxant effect. Despite a substantial number of studies on this species, population genomics has not yet been analyzed. The main aim of this study was: characterization of genetic variation of natural populations of V. officinalis in Poland and comparison of variation of wild populations and the cultivated form using Next Generation Sequencing based DArTseq technique. We also would like to establish foundations for genetic monitoring of the species in the future and to develop genetic fingerprint profile for samples deposited in gene bank and in natural sites in order to assess the degree of their genetic integrity and population structure preservation in the future.Results: The major and also the most astounding result of our work is the low level of observed heterozygosity of individual plants from natural populations despite the fact that the species is widespread in the studied area. Inbreeding, in naturally outcrossing species such as valerian, decreases the reproductive success. The analysis of the population structure indicated the potential presence of metapopulation in a broad area of Poland and the formation of a distinct gene pool in Bieszczady Mountains. The results also indicate the presence of individuals of the cultivated form in natural populations in the region where the species is cultivated for the needs of the pharmaceutical industry and this could lead to structural and genetic imbalance in wild populations.Conclusions: The DArTseq technology can be applied effectively in genetic studies of V. officinalis. The genetic variability of wild populations is in fact significantly lower than assumed. Individuals from the cultivated population are found in the natural environment and their impact on wild populations should be monitored.

Genetic Diversity and Population Structure of Rhododendron canescens, a Native Azalea for Urban Landscaping

Rhododendron canescens is a deciduous azalea native to the southeastern United States that is used in landscaping due to its ornamental qualities. A genotyping-by-sequencing (GBS) approach was taken to characterize the genetic structure and diversity of a R. canescens germplasm collection. Single nucleotide polymorphisms (SNPs) were identified by two software platforms, STACKS and GBS-SNP-CROP. Three distinct R. canescens populations were detected by STRUCTURE analysis with GBS-SNP-CROP data, whereas two populations were distinguished using STACKS data. Principal component analysis (PCA) with data from both SNP pipelines supported the presence of three populations. Statistical results indicated that there was low genetic differentiation between the populations, but relatively high genetic diversity within populations. The inbreeding coefficient of the R. canescens accessions was low, which would be expected with an outcrossing species. These results suggest that there may be a significant level of gene flow between populations of R. canescens.

Can Sexual Selection Cause Divergence in Mating System-Related Floral Traits?

Premise of the ResearchThe wide diversity of floral traits seen among plants is shaped by neutral and selective evolutionary processes. In outcrossing species, sexual selection from competing pollen donors is expected to be important for shaping mating system-related traits but empirical evidence is scarce. In a previous evaluation of experimental evolution lines crossed with either one or two pollen donors (monogamous, M, or polyandrous, P, lines) at early floral stages in mixed-mating Collinsia heterophylla (Plantaginaceae), P showed enhanced pollen competitive ability and reduced maternal seed set compared to M, in accordance with sexually antagonistic evolution of pollen. Here, we asked whether the presence of sexual selection during pollen competition affect mating system-related floral traits in the same lines.MethodologyWe compared flowering start, timing of anther-stigma contact (as an indication of timing of self-pollination), timing of stigma receptivity and first seed set between M and P, and with a source line, S (starting material). The former three traits are later in outcrossers than in selfers of Collinsia. The latter trait was expected to be earlier in P than in M because of sexual selection for early seed siring of pollen.Pivotal ResultsArtificial polyandry for four generations resulted in later flowering start and later anther-stigma contact in P compared to M, and the latter trait was intermediate in S. Thus, P appeared more ‘outcrossing’ than M. Stigma receptivity did not differ between lines. First seed set was earlier in P than in M, as expected from sexual selection.ConclusionsOur results from C. heterophylla experimental evolution lines suggest that a component of sexual selection during outcross pollination could enhance the patterns of floral divergence commonly found between outcrossers and selfers.