Responsible Innovation in Plant Breeding: The Case of Hybrid Potato Breeding.

As an emerging innovation, hybrid potato breeding raises high expectations about faster variety development and clean true potato seed as a new source of planting material. Hybrid breeding could, therefore, substantially contribute to global food security and other major sustainable development goals. However, its success will not only depend on the performance of hybrid potato in the field, but also on a range of complex and dynamic system conditions. This article is based on a multidisciplinary project in which we have studied the innovation dynamics of hybrid potato breeding and explored how these dynamics may shape the future of hybrid potato. Inspired by the approach of responsible innovation, we closely involved key players in the Dutch and international potato sector and other relevant actors in thinking about these potato futures. An important and recurrent theme in our work is the tension between the predominant commercial innovation dynamics in plant breeding and promises to respond to the global challenges of food security, agrobiodiversity and climate change. In this article, we, therefore, discuss responsible innovation strategies in (hybrid) potato breeding, which may help to bridge this tension and finally reflect on the implications for the field of plant breeding in general.

Converting Hybrid Potato Breeding Science into Practice.

Research on diploid hybrid potato has made fast advances in recent years. In this review we give an overview of the most recent and relevant research outcomes. We define different components needed for a complete hybrid program: inbred line development, hybrid evaluation, cropping systems and variety registration. For each of these components the important research results are discussed and the outcomes and issues that merit further study are identified. We connect fundamental and applied research to application in a breeding program, based on the experiences at the breeding company Solynta. In the concluding remarks, we set hybrid breeding in a societal perspective, and we identify bottlenecks that need to be overcome to allow successful adoption of hybrid potato.

Crucial factors for the feasibility of commercial hybrid breeding in food crops.

There is an ongoing societal debate about plant breeding systems and their impact on stakeholders in food systems. Hybrid breeding and hybrid seed have become controversial topics as they are believed to mostly serve high-tech agricultural systems. This article focuses on the perspective of commercial plant breeders when developing new cultivars of food crops. Arguably, hybrid breeding is the most effective breeding system for genetic improvement of crops, enhancing yields, improving product quality and increasing resistance against (a)biotic stresses. Nonetheless, hybrid breeding is not commercially applied in all crops. We analyse how biological and economic factors determine whether a commercial plant breeder opts for the hybrid system or not. We show that the commercial feasibility of hybrid breeding depends on the crop and business case. In conclusion, the commercial application of hybrid breeding in crops seems to be hampered mostly by high costs of seed production. Case studies regarding the hybrid transitions in maize, wheat and potato are included to illustrate these findings.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato.

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

Solyntus, the New Highly Contiguous Reference Genome for Potato (Solanum tuberosum).

With the rapid expansion of the application of genomics and sequencing in plant breeding, there is a constant drive for better reference genomes. In potato (Solanum tuberosum), the third largest food crop in the world, the related species S. phureja, designated "DM", has been used as the most popular reference genome for the last 10 years. Here, we introduce the de novo sequenced genome of Solyntus as the next standard reference in potato genome studies. A true Solanum tuberosum made up of 116 contigs that is also highly homozygous, diploid, vigorous and self-compatible, Solyntus provides a more direct and contiguous reference then ever before available. It was constructed by sequencing with state-of-the-art long and short read technology and assembled with Canu. The 116 contigs were assembled into scaffolds to form each pseudochromosome, with three contigs to 17 contigs per chromosome. This assembly contains 93.7% of the single-copy gene orthologs from the Solanaceae set and has an N50 of 63.7 Mbp. The genome and related files can be found at https://www.plantbreeding.wur.nl/Solyntus/ With the release of this research line and its draft genome we anticipate many exciting developments in (diploid) potato research.

Chlorophyll fluorescence imaging reveals genetic variation and loci for a photosynthetic trait in diploid potato.

Physiology and genetics are tightly interrelated. Understanding the genetic basis of a physiological trait such as the quantum yield of the photosystem II, or photosynthetic responses to environmental changes will benefit the understanding of these processes. By means of chlorophyll fluorescence (CF) imaging, the quantum yield of photosystem II can be determined rapidly, precisely and non-invasively. In this article, the genetic control and variation in the steady-state quantum yield of PSII (ΦPSII ) is analyzed for diploid potato plants. Current progress in potato research and breeding is slow due to high levels of heterozygosity and complexity of tetraploid genetics. Diploid potatoes offer the possibility of overcoming this problem and advance research for one of the globally most important staple foods. With the help of a diploid genetic mapping population two genetic loci that were strongly associated with differences in ΦPSII were identified. This is a proof of principle that genetic analysis for ΦPSII can be done on potato. The effects of three different stress conditions that are important in potato cultivation were also tested: salt stress, low temperature and deficiency in the macronutrient phosphate. For the last two stresses, significant decreases in photosynthetic activity could be shown, revealing potential for stress detection with CF based tools. In general, our findings show the potential of high-throughput phenotyping for physiological research and breeding in potato.

Characterization of polygenic resistance to powdery mildew in tomato at cytological, biochemical and gene expression level.

Extensive research in the area of plant innate immunity has increased considerably our understanding of the molecular mechanisms associated with resistance controlled by a dominant resistance gene. In contrast, little is known about the molecular basis underlying the resistance conferred by quantitative trait loci (QTLs). In this study, using the interaction of tomato (Solanum lycopersicum) with Oidium neolycopersici, we compared the cytological, biochemical and molecular mechanisms involved in both monogenic and polygenic resistances conferred by a dominant gene (Ol-1) and three QTLs (Ol-qtls), respectively. Our results showed that the three Ol-qtls jointly confer a very high level of broad-spectrum resistance and that the resistance is associated with both the hypersensitive response and papillae formation, with the hypersensitive response being prevalent. Both H(2)O(2) and callose accumulation, which are coupled with Ol-1-mediated resistance, are also associated with the resistance conferred by Ol-qtls. Further, we analysed the pathogen-induced transcript profiles of near-isogenic lines carrying the three Ol-qtls and the Ol-1 gene. Transcript profiles obtained by cDNA-amplified fragment length polymorphism analysis showed that, on fungal challenge, about 70% of the transcript-derived fragments are up-regulated in both susceptible and resistant genotypes. Most of the sequenced transcript-derived fragments showed homology to genes with functions in defence responses, suggesting that defence-responsive genes responsible for basal defence are involved in both monogenic and polygenic resistances conferred by Ol-1 and Ol-qtls, respectively. Although about 18% of the identified transcript-derived fragments are specific for either monogenic or polygenic resistance, their expression patterns need to be further verified by quantitative reverse transcriptase-polymerase chain reaction.

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes.

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid.

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis.

Naturally occurring broad-spectrum powdery mildew resistance in a Central American tomato accession is caused by loss of mlo function.

The resistant cherry tomato (Solanum lycopersicum var. cerasiforme) line LC-95, derived from an accession collected in Ecuador, harbors a natural allele (ol-2) that confers broad-spectrum and recessively inherited resistance to powdery mildew (Oidium neolycopersici). As both the genetic and phytopathological characteristics of ol-2-mediated resistance are reminiscent of powdery mildew immunity conferred by loss-of-function mlo alleles in barley and Arabidopsis, we initiated a candidate-gene approach to clone Ol-2. A tomato Mlo gene (SlMlo1) with high sequence-relatedness to barley Mlo and Arabidopsis AtMLO2 mapped to the chromosomal region harboring the Ol-2 locus. Complementation experiments using transgenic tomato lines as well as virus-induced gene silencing assays suggested that loss of SlMlo1 function is responsible for powdery mildew resistance conferred by ol-2. In progeny of a cross between a resistant line bearing ol-2 and the susceptible tomato cultivar Moneymaker, a 19-bp deletion disrupting the SlMlo1 coding region cosegregated with resistance. This polymorphism results in a frameshift and, thus, a truncated nonfunctional SlMlo1 protein. Our findings reveal the second example of a natural mlo mutant that possibly arose post-domestication, suggesting that natural mlo alleles might be evolutionarily short-lived due to fitness costs related to loss of mlo function.

Allelic variation in the effector genes of the tomato pathogen Cladosporium fulvum reveals different modes of adaptive evolution.

The allelic variation in four avirulence (Avr) and four extracellular protein (Ecp)-encoding genes of the tomato pathogen Cladosporium fulvum was analyzed for a worldwide collection of strains. The majority of polymorphisms observed in the Avr genes are deletions, point mutations, or insertions of transposon-like elements that are associated with transitions from avirulence to virulence, indicating adaptive evolution of the Avr genes to the cognate C. fulvum resistance genes that are deployed in commercial tomato lines. Large differences in types of polymorphisms between the Avr genes were observed, especially between Avr2 (indels) and Avr4 (amino-acid substitutions), indicating that selection pressure favors different types of adaptation. In contrast, only a limited number of polymorphisms were observed in the Ecp genes, which mostly involved synonymous modifications. A haplotype network based on the polymorphisms observed in the effector genes revealed a complex pattern of evolution marked by reticulations that suggests the occurrence of genetic recombination in this presumed asexual fungus. This, as well as the identification of strains with identical polymorphisms in Avr and Ecp genes but with opposite mating-type genes, suggests that development of complex races can be the combined result of positive selection and genetic recombination.

Biochemical and molecular mechanisms involved in monogenic resistance responses to tomato powdery mildew.

The monogenic genes Ol-1, ol-2, and Ol-4 confer resistance to tomato powdery mildew Oidium neolycopersici via different mechanisms. The biochemical mechanisms involved in these monogenic resistances were studied by monitoring through time the association of H2O2 and callose accumulation with hypersensitive response (HR) and papilla formation. Our results showed that H2O2 and callose accumulation are coupled with both Ol-1- and Ol-4-mediated HR-associated resistance as well as with the ol-2-mediated papillae-associated resistance. Further, the transcriptomal changes related to these monogenic resistances were studied by using cDNA-amplification fragment length polymorphism. The expression profiling clarified that 81% of DE-TDF (differentially expressed transcript-derived fragments) were up-regulated upon inoculation with O. neolycopersici in both the compatible and Ol-1-mediated incompatible interactions, though with a difference in expression timing. Of these DE-TDF, more than 70% were not detected in the Ol-4-mediated resistance, while 58% were expressed in the ol-2-mediated resistance, generally at later timepoints. Sequence information suggested that most of these DE-TDF are related to genes involved in either basal defense or establishment of compatibility. In addition, DE-TDF (19%) specifically expressed in different incompatible interactions were identified. Expression patterns of some DE-TDF and marker gene GluB suggested that papillae-associated resistance exploits a different defense pathway from that of HR-associated resistance.

Domestication and breeding of tomatoes: what have we gained and what can we gain in the future?

BACKGROUND: It has been shown that a large variation is present and exploitable from wild Solanum species but most of it is still untapped. Considering the thousands of Solanum accessions in different gene banks and probably even more that are still untouched in the Andes, it is a challenge to exploit the diversity of tomato. What have we gained from tomato domestication and breeding and what can we gain in the future? SCOPE: This review summarizes progress on tomato domestication and breeding and current efforts in tomato genome research. Also, it points out potential challenges in exploiting tomato biodiversity and depicts future perspectives in tomato breeding with the emerging knowledge from tomato-omics. CONCLUSIONS: From first domestication to modern breeding, the tomato has been continually subjected to human selection for a wide array of applications in both science and commerce. Current efforts in tomato breeding are focused on discovering and exploiting genes for the most important traits in tomato germplasm. In the future, breeders will design cultivars by a process named 'breeding by design' based on the combination of science and technologies from the genomic era as well as their practical skills.

The construction of a Solanum habrochaites LYC4 introgression line population and the identification of QTLs for resistance to Botrytis cinerea.

Tomato (Solanum lycopersicum) is susceptible to grey mold (Botrytis cinerea). Partial resistance to this fungus has been identified in accessions of wild relatives of tomato such as Solanum habrochaites LYC4. In a previous F(2) mapping study, three QTLs conferring resistance to B. cinerea (Rbcq1, Rbcq2 and Rbcq4a) were identified. As it was probable that this study had not identified all QTLs involved in resistance we developed an introgression line (IL) population (n = 30), each containing a S. habrochaites introgression in the S. lycopersicum cv. Moneymaker genetic background. On average each IL contained 5.2% of the S. habrochaites genome and together the lines provide an estimated coverage of 95%. The level of susceptibility to B. cinerea for each of the ILs was assessed in a greenhouse trial and compared to the susceptible parent S. lycopersicum cv. Moneymaker. The effect of the three previously identified loci could be confirmed and seven additional loci were detected. Some ILs contains multiple QTLs and the increased resistance to B. cinerea in these ILs is in line with a completely additive model. We conclude that this set of QTLs offers good perspectives for breeding of B. cinerea resistant cultivars and that screening an IL population is more sensitive for detection of QTLs conferring resistance to B. cinerea than the analysis in an F(2) population.

Three QTLs for Botrytis cinerea resistance in tomato.

Tomato (Solanum lycopersicum) is susceptible to grey mold (Botrytis cinerea). Partial resistance to this fungus was identified in accessions of wild relatives of tomato such as S. habrochaites LYC4. In order to identify loci involved in quantitative resistance (QTLs) to B. cinerea, a population of 174 F(2) plants was made originating from a cross between S. lycopersicum cv. Moneymaker and S. habrochaites LYC4. The population was genotyped and tested for susceptibility to grey mold using a stem bioassay. Rbcq1, a QTL reducing lesion growth (LG) and Rbcq2, a QTL reducing disease incidence (DI) were identified. Rbcq1 is located on Chromosome 1 and explained 12% of the total phenotypic variation while Rbcq2 is located on Chromosome 2 and explained 15% of the total phenotypic variation. Both QTL effects were confirmed by assessing disease resistance in two BC(2)S(1) progenies segregating for either of the two QTLs. One additional QTL, Rbcq4 on Chromosome 4 reducing DI, was identified in one of the BC(2)S(1) progenies. F(2) individuals, homozygous for the Rbcq2 and Rbcq4 alleles of S. habrochaites showed a reduction of DI by 48%. QTLs from S. habrochaites LYC4 offer good perspectives for breeding B. cinerea resistant tomato cultivars.

Mating-type genes and the genetic structure of a world-wide collection of the tomato pathogen Cladosporium fulvum.

Two mating-type genes, designated MAT1-1-1 and MAT1-2-1, were cloned and sequenced from the presumed asexual ascomycete Cladosporium fulvum (syn. Passalora fulva). The encoded products are highly homologous to mating-type proteins from members of the Mycosphaerellaceae, such as Mycosphaerella graminicola and Cercospora beticola. In addition, the two MAT idiomorphs of C. fulvum showed regions of homology and each contained one additional putative ORF without significant similarity to known sequences. The distribution of the two mating-type genes in a world-wide collection of 86 C. fulvum strains showed a departure from a 1:1 ratio (chi(2)=4.81, df=1). AFLP analysis revealed a high level of genotypic diversity, while strains of the fungus were identified with similar virulence spectra but distinct AFLP patterns and opposite mating-types. These features could suggest the occurrence of recombination in C. fulvum.

High-resolution fine mapping of ps-2, a mutated gene conferring functional male sterility in tomato due to non-dehiscent anthers.

Functional male sterility is an important trait for the production of hybrid seeds. Among the genes coding for functional male sterility in tomato is the positional sterility gene ps-2. ps-2 is monogenic recessive, confers non-dehiscent anthers and is the most suitable for practical uses. In order to have tools for molecular-assisted selection (MAS) we fine mapped the ps-2 locus. This was done in an F(2) segregating population derived from the interspecific cross between a functionally male sterile line (ps-2/ps-2; Solanum lycopersicum) and a functionally male fertile line (S. pimpinellifolium). Here we report the procedure that has led to the high-resolution fine mapping of the ps-2 locus in a 1.65 cM interval delimited by markers T0958 and T0635 on the short arm of Chromosome 4. The presence of many COS markers in the local high-resolution map allowed us to study the synteny between tomato and Arabidopsis at the ps-2 locus region. No obvious candidate gene for ps-2 was identified among the known functional male sterility genes in Arabidopsis.

Tomato defense to the powdery mildew fungus: differences in expression of genes in susceptible, monogenic- and polygenic resistance responses are mainly in timing.

Oidium neolycopersici is a causal agent of tomato powdery mildew. In this paper, gene expression profiles were investigated of susceptible, monogenic- and polygenic resistant tomato genotypes in response to O. neolycopersici infection by using cDNA-AFLP. Around 30,000 TDFs (Transcript Derived Fragments), representing approximately 22% of the transcriptome based on in silico estimation, were identified and 887 TDFs were differentially expressed (DE-TDFs) upon inoculation with O. neolycopersici spores. Forty-two percent of the identified DE-TDFs were detected in both the compatible and incompatible interactions, a subset of these were studied for their temporal patterns. All of these common induced DE-TDFs displayed an expression peak at 7 days post incoluation in monogenic resistant response but sustained up-regulation in the susceptible and the polygenic resistant response. While more than half of these common DE-TDFs showed earlier timing in incompatible interactions compared to compatible interaction. Only 2% of the identified DE-TDFs were specific to either the monogenic or the polygenic resistant response. By annotation of the 230 sequenced DE-TDFs we found that 34% of the corresponding transcripts were known to be involved in plant defense, whereas the other transcripts played general roles in signal transduction (11%), regulation (24%), protein synthesis and degradation (11%), energy metabolism (12%) including photosynthesis, photorespiration and respiration.

Construction of a genetic linkage map of Thlaspi caerulescens and quantitative trait loci analysis of zinc accumulation.

Zinc (Zn) hyperaccumulation seems to be a constitutive species-level trait in Thlaspi caerulescens. When compared under conditions of equal Zn availability, considerable variation in the degree of hyperaccumulation is observed among accessions originating from different soil types. This variation offers an excellent opportunity for further dissection of the genetics of this trait. A T. caerulescens intraspecific cross was made between a plant from a nonmetallicolous accession [Lellingen (LE)], characterized by relatively high Zn accumulation, and a plant from a calamine accession [La Calamine (LC)], characterized by relatively low Zn accumulation. Zinc accumulation in roots and shoots segregated in the F3 population. This population was used to construct an LE/LC amplified fragment length polymorphism (AFLP)-based genetic linkage map and to map quantitative trait loci (QTL) for Zn accumulation. Two QTL were identified for root Zn accumulation, with the trait-enhancing alleles being derived from each of the parents, and explaining 21.7 and 16.6% of the phenotypic variation observed in the mapping population. Future development of more markers, based on Arabidopsis orthologous genes localized in the QTL regions, will allow fine-mapping and map-based cloning of the genes underlying the QTL.

Tomato defense to Oidium neolycopersici: dominant Ol genes confer isolate-dependent resistance via a different mechanism than recessive ol-2.

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.