Genome-wide association analysis of the anthocyanin and carotenoid contents of rose petals

Petal color is one of the key characteristics determining the attractiveness and therefore the commercial value of an ornamental crop. Here, we present the first genome-wide association study for the important ornamental crop rose, focusing on the anthocyanin and carotenoid contents in petals of 96 diverse tetraploid garden rose genotypes. Cultivated roses display a vast phenotypic and genetic diversity and are therefore ideal targets for association genetics. For marker analysis, we used a recently designed Axiom SNP chip comprising 68,000 SNPs with additionally 281 SSRs, 400 AFLPs and 246 markers from candidate genes. An analysis of the structure of the rose population revealed three subpopulations with most of the genetic variation between individual genotypes rather than between clusters and with a high average proportion of heterozygous loci. The mapping of markers significantly associated with anthocyanin and carotenoid content to the related Fragaria and Prunus genomes revealed clusters of associated markers indicating five genomic regions associated with the total anthocyanin content and two large clusters associated with the carotenoid content. Among the marker clusters associated with the phenotypes, we found several candidate genes with known functions in either the anthocyanin or the carotenoid biosynthesis pathways. Among others, we identified a glutathione-S-transferase, 4CL, an auxin response factor and F3’H as candidate genes affecting anthocyanin concentration, and CCD4 and Zeaxanthine epoxidase as candidates affecting the concentration of carotenoids. These markers are starting points for future validation experiments in independent populations as well as for functional genomic studies to identify the causal factors for the observed color phenotypes. Furthermore, validated markers may be interesting tools for marker-assisted selection in commercial breeding programmes in that they provide the tools to identify superior parental combinations that combine several associated markers in higher dosages.BMWi/ZI

Genomic regions in crop-wild hybrids of lettuce are affected differently in different environments: implications for crop breeding

Many crops contain domestication genes that are generally considered to lower fitness of crop–wild hybrids in the wild environment. Transgenes placed in close linkage with such genes would be less likely to spread into a wild population. Therefore, for environmental risk assessment of GM crops, it is important to know whether genomic regions with such genes exist, and how they affect fitness. We performed quantitative trait loci (QTL) analyses on fitness(-related) traits in two different field environments employing recombinant inbred lines from a cross between cultivated Lactuca sativa and its wild relative Lactuca serriola. We identified a region on linkage group 5 where the crop allele consistently conferred a selective advantage (increasing fitness to 212% and 214%), whereas on linkage group 7, a region conferred a selective disadvantage (reducing fitness to 26% and 5%), mainly through delaying flowering. The probability for a putative transgene spreading would therefore depend strongly on the insertion location. Comparison of these field results with greenhouse data from a previous study using the same lines showed considerable differences in QTL patterns. This indicates that care should be taken when extrapolating experiments from the greenhouse, and that the impact of domestication genes has to be assessed under field conditions

Abiotic stress QTL in lettuce crop–wild hybrids: comparing greenhouse and field experiments

The development of stress-tolerant crops is an increasingly important goal of current crop breeding. A higher abiotic stress tolerance could increase the probability of introgression of genes from crops to wild relatives. This is particularly relevant to the discussion on the risks of new GM crops that may be engineered to increase abiotic stress resistance. We investigated abiotic stress QTL in greenhouse and field experiments in which we subjected Recombinant Inbred Lines from a cross between cultivated Lactuca sativa cv. Salinas and its wild relative L. serriola to drought, low nutrients, salt stress, and above ground competition. Aboveground biomass at the end of the rosette stage was used as a proxy for the performance of plants under a particular stress. We detected a mosaic of abiotic stress QTL over the entire genome with little overlap between QTL from different stresses. The two QTL clusters that were identified reflected general growth rather than specific stress responses and co-located with clusters found in earlier studies for leaf shape and flowering time. Genetic correlations across treatments were often higher among different stress treatments within the same experiment (greenhouse or field), than among the same type of stress applied in different experiments. Moreover, the effects of the field stress treatments were more correlated to those of the greenhouse competition treatments than to those of the other greenhouse stress experiments, suggesting that competition rather than abiotic stress is a major factor in the field. In conclusion, the introgression risk of stress tolerance (trans-)genes under field conditions cannot easily be predicted based on genomic background selection patterns from controlled QTL experiments in greenhouses. Especially field data will be needed to assess potential (negative) ecological effects of introgression of these transgenes into wild relatives

New Developments in Molecular Techniques for Breeding in Ornamentals

In ornamental crops the development of genetic and molecular tools for breeding has been slow because of the large number of ornamental species, many of which are genetically complicated for breeding, being outbreeding crops, polyploid, and/or having a large genome.This is changing due to three recent developments: (i) next-generation sequencing can now generate large numbers of single nucleotide polymorphism (SNP) markers based on genomic or transcriptomic sequences, (ii) efficient and automated SNP detection systems render genotyping into an automated and relatively cheap process, and (iii) methods and software now exist to analyse these data, also in polyploid crops, to find associations with traits and to generate tools for marker-assisted breeding. The challenge for the coming years will be to implement these tools to speed up breeding.When more genome sequences of ornamental species or related species become available, it will also be possible to move from associated markers (for a trait or QTL region) to the underlying variation in the causal genes. Knowledge of the existing variation in functional alleles will make it possible to consider directing biosynthetic or regulatory pathways towards, e.g. different colour or scent combinations.New plant breeding techniques (also called ‘precision breeding techniques’) add new possibilities to direct the breeding process. Notably, gene editing (also called genome editing) using Crispr/Cas may be used to increase the pool of functional variation, but there are challenges to apply it in ornamentals, in terms of the availability of sequence information for the candidate genes and the existence of transformation and regeneration protocols

Genetic engineering at the heart of agroecology

We discuss whether genetic engineering and agroecology are compatible. For this, we investigated three cases of genetically engineered crops and considered agroecology as scientific discipline as well as a social movement. One case was the use of cisgenic modifications to make potato durably resistant to late blight, the second was the use of CRISPR/Cas to make rice resistant to bacterial blight and as a third case, we evaluated experiences with cultivating transgenic Bt crops. These cases demonstrated that genetic engineering offers opportunities to grow crops in novel integrated pest management (IPM) systems with, as direct benefit, a decrease in the use of chemical crop protection agents, and as indirect effect that the role of predators and biological control agents can become more important than in present conventional systems based on pesticides. We used a framework based on four concerns (both cons and pros) that were gathered from an extensive societal interaction organized around the Dutch research project DuRPh, which produced a proof-of-concept of a cisgenic late blight-resistant potato. We concluded that genetic engineering and agroecology certainly have synergy in the context of agroecology as science, when applied to making crops less vulnerable to pests and diseases and when combined with cultivation using IPM. By contrast, within the movement context, genetically engineered varieties may be welcomed if they include traits that contribute to successful IPM schemes and are socially benign. Whether they would actually be deemed desirable or acceptable will, however, vary depending on the norms and values of the social movements. We propose that some concerns may be reconcilable in a dialogue. Deontological arguments such as naturalness are more difficult to reconcile, as they relate to deeply felt ethical or cultural values. A step forward would be when also for these arguments everyone can make an informed choice and when these choices can coexist in a respectful manner.</p

Genome editing of polyploid crops : prospects, achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted

The use of intellectual property systems in plant breeding for ensuring deployment of good agricultural practices

Breeding innovations are relevant for sustainable agricultural development and food security, as new, resilient production systems require crop varieties optimally suited for these systems. In the societal debate around genetic engineering and other plant breeding innovations, ownership of patents on the technology used in the hands of large companies is often seen as a reason that small breeding companies are denied opportunities for further improving varieties or that farmers are restricted in using such varieties. However, intellectual property (IP) systems may also be used as tools to ensure the use of good agricultural practices when cultivating the resulting varieties. This paper explores documented cases in which IP systems (plant variety rights, patents and brand names) are used to promote that innovative varieties are grown according to good agricultural practices (GAP). These include effective disease resistance management regimes in innovative crop varieties of potato in order to prevent or delay pathogens from overcoming disease resistance genes, management regimes for transgenic insect-resistant Bt or herbicide-tolerant crops to prevent the development of resistant pests or weeds, respectively. The results are discussed with respect to the influence of breeders on GAP measures through various forms of IP and the contribution and role of other stakeholders, authorities and society at large in stimulating and ensuring the use of GAP.</p

Genetic engineering at the heart of agroecology

We discuss whether genetic engineering and agroecology are compatible. For this, we investigated three cases of genetically engineered crops and considered agroecology as scientific discipline as well as a social movement. One case was the use of cisgenic modifications to make potato durably resistant to late blight, the second was the use of CRISPR/Cas to make rice resistant to bacterial blight and as a third case, we evaluated experiences with cultivating transgenic Bt crops. These cases demonstrated that genetic engineering offers opportunities to grow crops in novel integrated pest management (IPM) systems with, as direct benefit, a decrease in the use of chemical crop protection agents, and as indirect effect that the role of predators and biological control agents can become more important than in present conventional systems based on pesticides. We used a framework based on four concerns (both cons and pros) that were gathered from an extensive societal interaction organized around the Dutch research project DuRPh, which produced a proof-of-concept of a cisgenic late blight-resistant potato. We concluded that genetic engineering and agroecology certainly have synergy in the context of agroecology as science, when applied to making crops less vulnerable to pests and diseases and when combined with cultivation using IPM. By contrast, within the movement context, genetically engineered varieties may be welcomed if they include traits that contribute to successful IPM schemes and are socially benign. Whether they would actually be deemed desirable or acceptable will, however, vary depending on the norms and values of the social movements. We propose that some concerns may be reconcilable in a dialogue. Deontological arguments such as naturalness are more difficult to reconcile, as they relate to deeply felt ethical or cultural values. A step forward would be when also for these arguments everyone can make an informed choice and when these choices can coexist in a respectful manner.</p

Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat

Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients.</p

De novo whole-genome assembly of Chrysanthemum makinoi, a key wild chrysanthemum

Chrysanthemum is among the top 10 cut, potted, and perennial garden flowers in the world. Despite this, to date, only the genomes of two wild diploid chrysanthemums have been sequenced and assembled. Here, we present the most complete and contiguous chrysanthemum de novo assembly published so far, as well as a corresponding ab initio annotation. The cultivated hexaploid varieties are thought to originate from a hybrid of wild chrysanthemums, among which the diploid Chrysanthemum makinoi has been mentioned. Using a combination of Oxford Nanopore long reads, Pacific Biosciences long reads, Illumina short reads, Dovetail sequences, and a genetic map, we assembled 3.1 Gb of its sequence into nine pseudochromosomes, with an N50 of 330 Mb and a BUSCO complete score of 92.1%. Our ab initio annotation pipeline predicted 95,074 genes and marked 80.0% of the genome as repetitive. This genome assembly of C. makinoi provides an important step forward in understanding the chrysanthemum genome, evolution, and history