LS Plant Breeding (United Kingdom)

Abstract Multiple studies have identified genes affecting grain morphology, yet their capacity to deliver yield gains under field conditions remains unclear. We performed a multiyear, multilocation factorial evaluation of GRAIN WIDTH2 ( TaGW2 ) mutants in hexaploid wheat using BC 4 near-isogenic lines, sowing-density treatments and semi-dwarfing RHT1 backgrounds. Loss-of-function mutations in TaGW2 increased grain size and thousand grain weight (TGW) additively; with the aaBBDD single mutant showing the most stable singlelocus effect, while the aabbdd triple mutant achieved ~20% higher TGW across twelve field trials. However, overall grain yield remained unchanged or slightly reduced, reflecting a compensatory trade-off with grain number. Spike phenotyping of both main and secondary tillers showed comparable increases in TGW and spike yield despite fewer grains per spike, indicating that limited yield gain primarily reflects reduced spike number per unit area rather than decreased spike-level productivity. Effects were stable across sowing densities, whereas interactions with semi-dwarfing alleles were allele-specific: RHT-B1b partially suppressed TGW gains and accentuated yield penalties, whereas RHT-D1b maintained the large-grain phenotype and productivity. Across experiments, the TaGW2-A1D1 double mutant increased TGW (~14%) while maintaining yield stability, identifying it as a promising genotype for breeding. We conclude that TaGW2 is a reliable modifier of grain size but not yield in isolation.

Leaves of shade-avoiding plants such as Arabidopsis (Arabidopsis thaliana) change their growth pattern and position in response to low red to far-red ratios (LRFRs) encountered in dense plant communities. Under LRFR, transcription factors of the phytochrome-interacting factor (PIF) family are derepressed. PIFs induce auxin production, which is required for promoting leaf hyponasty, thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty, with gene expression patterns suggesting that LRFR regulates the ABA response. Here, we show that LRFR leads to a rapid increase in ABA levels in leaves. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves than wild-type Arabidopsis under white light but respond less to LRFR. Consistent with this, ABA application decreases leaf angle under white light; however, this response is inhibited under LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate that LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty under white light than under LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position.

Fruit quality is represented by a series of genetically controlled features that change throughout the entire ontogenic life. Among the several quality traits, texture plays a crucial role, impacting both consumers’ appreciation and postharvest performance. In order to decipher its regulation a multidisciplinary approach was employed. Initially, the texture performance was measured with a high resolution phenotyping device, represented by a texture analyzer equipped with an acoustic device. In the first attempt to dissect the fruit texture genetic control, two QTL mapping strategies were used. The first approach employed six bi-parental families linked by a common pedigree scheme, known as pedigree based analysis. The joint analysis of the phenotypic and genotypic data set through a Bayesian statistics identified a series of genomic regions related to both mechanical and acoustic signatures. These regions were further validated with a genome wide association study approach, which considered a much larger phenotypic and genotypic variation. To complement the genetic information, a whole transcriptome analysis was also carried out. To this end, two microarray platforms were designed and used to unravel the functional machinery ongoing during the fruit development and ripening phases, especially with regards to the plant hormone ethylene. In this study, the role of this hormone was dissected applying 1-MCP, a molecule competing with ethylene at receptor level. The combination of these resources provides a valuable source of information, essential to step forward in the comprehension of the genetic and physiological regulation of the fruit texture in apple. This knowledge would enable, in a close future, a more accurate and precise selection of the most favourable and valuable new apple accessions distinguished by a superior fruit quality.

Proof of concept of Bayesian integrated QTL analyses across pedigree-related families from breeding programs of an outbreeding species. Results include QTL confidence intervals, individuals' genotype probabilities and genomic breeding values. Bayesian QTL linkage mapping approaches offer the flexibility to study multiple full sib families with known pedigrees simultaneously. Such a joint analysis increases the probability of detecting these quantitative trait loci (QTL) and provide insight of the magnitude of QTL across different genetic backgrounds. Here, we present an improved Bayesian multi-QTL pedigree-based approach on an outcrossing species using progenies with different (complex) genetic relationships. Different modeling assumptions were studied in the QTL analyses, i.e., the a priori expected number of QTL varied and polygenic effects were considered. The inferences include number of QTL, additive QTL effect sizes and supporting credible intervals, posterior probabilities of QTL genotypes for all individuals in the dataset, and QTL-based as well as genome-wide breeding values. All these features have been implemented in the FlexQTL(™) software. We analyzed fruit firmness in a large apple dataset that comprised 1,347 individuals forming 27 full sib families and their known ancestral pedigrees, with genotypes for 87 SSR markers on 17 chromosomes. We report strong or positive evidence for 14 QTL for fruit firmness on eight chromosomes, validating our approach as several of these QTL were reported previously, though dispersed over a series of studies based on single mapping populations. Interpretation of linked QTL was possible via individuals' QTL genotypes. The correlation between the genomic breeding values and phenotypes was on average 90 %, but varied with the number of detected QTL in a family. The detailed posterior knowledge on QTL of potential parents is critical for the efficiency of marker-assisted breeding.

Phloem feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, but also by vectoring plant viruses. During their evolution plants have developed a variety of defense traits to combat insects. These plant resistance traits can be antixenotic and/or antibiotic. Antixenosis is the first line of defense that prevents insects from landing and settling, while antibiosis reduces the population development of the colonizing insects.In this project we aimed at identifying genes that can increase resistance towards phloem feeding insects and also prevent, as far as possible, transmission of viruses. Acknowledging that changing the expression level or expression localization of genes might increase resistance, we screened an Arabidopsis thaliana activation tag gain-of-function mutant collection for increased resistance towards the green peach aphid (Myzus persicae). In these mutants, tagged genes are overexpressed by the strong 35S enhancer adjacent to the natural promoter that results in a dominant gain-of-function phenotype. The overexpression of a particular gene in such mutants may result in enhanced resistance to aphids and other phloem feeding insects. To identify mutants with increased insect resistance efficient and reproducible screening methods needed to be developed first. Based on the hypothesis that there is a trade-off between plant fitness and plant resistance, we first screened a subset of 170 mutants that were previously selected based on their reduced growth to increase the chance of identifying mutants with increasedresistance. In this screening we usedchoice assays and selected one mutant that displays enhanced antixenosis based resistance towards aphids. Further characterization of this mutant revealed that that the antixenosis is phloem based and requires intact plants. To evaluate aphid resistance of a larger number (>5000) of activation tag mutants, we established a high throughput screening system in which plant resistance against aphids is inferred from a reduced transmission of the circulative Turnip yellows virus(TuYV). This virus can only be transmitted into a plant after virus-infected aphids feed for a prolonged (> 10min) time from the phloem sap. In the initial screening 13 virus-free mutant lines were identified. The putative candidate mutant lines were re-evaluated and characterized, resulting in nine mutants on which aphids showed a reduced population development. Molecular analysis of two of these mutants revealed that the genes underlying the resistance were IRM1(Increased ResistancetoMyzus persicae1,At5g65040)and SKS13 (SKU5Similar13, At3g13400). In wild type plants,IRM1is strongly expressed in xylem and extremely low expressed in other plant tissue whereas SKS13 is exclusively expressed in pollen. We show that constitutive overexpression of these genes in all plant tissues confers enhanced resistance towards aphids. Analysis of aphid feeding behavior showed that the resistance conferred by IRM1and SKS13affect the aphids differently. On the IRM1 overexpressing mutant aphids encounter difficulties in reaching the phloem, indicating that resistance factors are located between the cell surface and the phloem. On the SKS13overexpressingmutant the phloem feeding of aphids is severely affected, indicating that resistance factors are phloem based. Further analysis strongly suggests the involvement of Reactive Oxygen Species (ROS) in the reduced aphid performance on the SKS13overexpressingmutant. We also show that the resistances are not aphid specific, as the performance of the cabbage aphid (Brevicoryne brassicae)is also affectedon both overexpressing mutants. The results obtained in this thesis show that plant resistance to insects can be increased by expressing genes that are assigned for other biological functions. Characterization of the identified mutants revealed twogenes conferring enhanced aphid resistance via different mechanisms. These findings lead to a better understanding of plant-aphid interactions on the molecular level. Furthermore, such knowledge obtained from the model plant A.thalianashould be applied in crop plants, which can be achieved by transgenic and genetic studies in combination with newly developed techniques, such as RNAi and TILLING.

Salinity is the most severe abiotic stress perceived by plants and affects about 800 million hectares of land worldwide, including 20% of the world’s highly productive irrigated land. Significant crop yield losses are observed due to salinity. Salinization is increasing because of poor irrigation management and climate change. Improving salt tolerance in crops is for these reasons an important target for plant breeding in the near future. However, salinity tolerance in plants is not easy to breed for due to its interaction with many physiological processes controlled by many genes and their interaction with the environment. Barley is a good model crop to study different mechanisms conferring salt tolerance in cereals. A traditional QTL mapping approach in combination with a new association mapping method allowed us to efficiently explore the genetics and genetic diversity of salt tolerance in barley. Improvements of the association mapping technology highly increased detection power and mapping accuracy. The traits and QTLs identified in this thesis point out both osmotic and ionic stress tolerant genes as important targets for salt tolerance breeding. This thesis provides tools to plant breeders for the application of marker-assisted introgression breeding of salt tolerance genes in their breeding programs. Some QTLs were found to be syntenic with the important QTLs/genes for salt tolerance found in wheat and rice such as Na+ and K+ transporter gene families. Other QTLs were new and suggest the presence of novel genes that play an important role in plant ion homeostasis, transportation of Cl- and Ca2+ and osmotic tolerance. We demonstrated that association mapping can be a powerful approach to dissect the complexity of salt tolerance in barley. The newly available high-density SNP map of barley and the barley genome sequence in the near future further increases the accuracy of mapping studieswith the association panel and will greatly facilitate the cloning of the genes underlying salt tolerance in barley. This thesis thus contributes to better a understanding of the physiological and genetic basis of salt tolerance and improved breeding strategies for the development of salt tolerant varieties.

The Tailoring Food Sciences to Endogenous Patterns of Local Food Supply for Future Nutrition (TELFUN) project aimed to support people to choose their own way of producing, processing, and consuming local foods that fit best in their local conditions. The central research question was: how do technological practices, developed from within food networks, enhance food-sovereignty and the nutritional status of people? TELFUN was an interdisciplinary and comparative research program based on twelve research projects, divided in four disciplines and carried out in three geographical locations. Its scientific development was divided in four disciplines - plant breeding, food technology, human nutrition, and sociology of science and technology - for a more complete understanding of the production, processing, utilization and consumption of lupin in Ecuador. Similar studies took place in West Africa and cowpea and India with mungbean (www.telfun.info). The lupin (Lupinus mutabilis) is a native legume of the Andean zone. Like other species of lupin it has easily adapted to marginal regions with limited water supply and poor nourished soils. As compared to other legumes used in human nutrition, lupin seeds have high protein, and variable iron and zinc content. These are just a few attributes that make the Andean lupin be considered a food of the future (www.telfun.info). Disadvantages of lupine are its high susceptibility to anthracnose and its high content of alkaloids (which have to be removed by processing). A specific research question to tackle lupin production in Ecuador was studied in the breeding discipline: How do exploratory studies, both on the pathogen and on the lupin genetic diversity, contribute to the development of better lupin varieties in the Cotopaxi province - Ecuador? Chapter 1 presents the importance, nutritional value, agronomical characteristics and breeding for anthracnose resistance of lupin in Ecuador. In Chapter 2, the actual lupin production, a number of agronomic characteristics, the nutritional value and anthracnose susceptibility of a set of lupin cultivars were determined. The importance of characterizing genetic resources of L. mutabilis for crop improvement is outlined. In Chapter3, morphological, molecular and pathological methods were developed to identify the causal agent of anthracnose in lupin and tamarillo (another Andean native crop). Anthracnose diversity and the phylogenetic relationships between isolates of both hosts were studied. The techniques developed can be used to identify and monitor the dominant Colletotrichum species in the multi cropping Andean system to establish an appropriate disease management and breeding strategies. In Chapter 4, anthracnose susceptibility and the disease development of lupin cultivars was assessed under natural infection of C. acutatum. Seed samples of naturally infected plants were used to determine the level of anthracnose infection in seeds. Recommendations are made to reduce the impact of the disease in the field and for the appropriate local management of lupin seed until new anthracnose resistant lupin varieties are developed. In Chapter 5, the development of methods for screening anthracnose resistance in individual lupin plants are described. Inoculation methods, phenological stages and the relation of resistance and alkaloid content are discussed. In Chapter 6, we put in perspective our findings and the appropriate methodologies we developed for starting an Andean lupin breeding program with emphases on anthracnose resistance. In conclusion, lupin can play an important role in increasing living conditions of the poor farmers in Ecuador. It is needed that the agro-ecological production systems are maintained by using sustainable farming methods. The use of disease free lupin seed is of utmost importance and a good understanding of the plant– pathogen interactions can increase yield. Lupin breeding should aim at anthracnose resistance, high yielding varieties, and cultivars with high levels of protein and micronutrients. Having high-quality lupin cultivars will, together with the results in the other three disciplines - food technology, human nutrition, and sociology of science and technology –contribute to the food sovereignty concept, that is to connect local networks chains for a better production, processing and consumption of lupin (www.telfun.info).

Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases on potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late blight resistant cultivars, but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized. R-gene based resistance to any plant pathogen has been conceptualized according to a model known as gene-for-gene interaction. When matching avirulence (Avr) and resistance (R) proteins are produced by the pathogen and the plant respectively, a resistance response is triggered resulting in a hyper-sensitive response (HR) causing necrosis and cell death at the infection site. If one of these components is missing the plant-pathogen interaction is compatible and will result in completion of the life cycle of the pathogen. This thesis describes the cloning and the characterization of the resistant alleles Rpi-vnt1.1, Rpivnt1.2 and Rpi-vnt1.3 from Solanum venturii and their counterpart Avr-vnt1 from Phytophthora infestans. The Rpi-/Avr- genes pair Rpi-vnt1/Avr-vnt1 along with R3a/Avr3a and Rpi-blb3/Avr2 have been used to study the genetic and molecular mechanisms behind tuber blight resistance. The cloning of Rpi-vnt1 alleles (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) was achieved by the combination of long range PCR (Chapter 2) and a classical map based cloning strategy (Chapter 3). The long range PCR made use of Tm2 homologous PCR primers, upon identification of Tm2 sequence homology in associated markers generated with an NBStargeted fingerprinting technique. Rpi-vnt1 alleles belong to the CC-NBS-LRR class of plant R genes and encode predicted peptides of 891 and 905 amino acids, respectively, which share 75% amino acid (a.a.) identity with the ToMV resistance protein Tm-22 from tomato. Compared to Rpi-vnt1.1, the allele Rpi-vnt1.3 harbors a 14 amino acid insertion in the Nterminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum. An allele mining study of Rpi-vnt1 alleles across Solanum section Petota showed that the three functional alleles were confined within S. venturii as only two accessions from the closely related species S. weberbaueri and S. mochiquense carried Rpi-vnt1.1 (Chapter 4). Subsequent alignment of Rpi-vnt1-like homologs with Rpi-vnt1 alleles revealed the presence of illegitimate recombination (IR) signatures suggesting that two successive deletion events might have occurred in the CC domain. Meanwhile, the construction of a Neighbor Joining tree, based on AFLP data from all the accessions carrying Rpi-vnt1 alleles or Rpi-vnt1-like homologs showed that Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 alleles belong to a monophyletic clade. Signatures of illegitimate recombination and the monophyletic grouping of Rpi-vnt1 alleles suggested how Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 could have evolved. Extensive phenotyping with various Phytophthora isolates identified another Rpi gene in S. venturii named Rpi-vnt2, complementing the Rpi-vnt1 allelic resistance spectrum. The genetic position of this second independent locus is not yet identified. The identification of the matching avirulence factor from the pathogen, Avr-vnt1, was achieved by using an efficient and high throughput effector screen of resistant wild potato species (Chapter 5). Avr-vnt1 encodes a typical RXLR-EER effector which expression is induced 2 days post inoculation. Avr-vnt1 is located on a single locus in the reference strain T30-4. Among nine isolates, four alleles were identified. The virulent strain EC1 carries a functional coding sequence of Avr-vnt1 but fails to express the gene. In Chapter 6, the genetic and molecular mechanisms of tuber late blight have been investigated. Using transgenic cv. Desiree plants transformed with Rpi-vnt1.1, R3a or Rpiblb3 tuber blight resistance could be studied in an identical genetic background. First, we demonstrated that transient co-expression of the matching Avr- genes in these transgenic tuber slices trigger a hypersensitive responses (HR), showing that the presence and interaction of both proteins is sufficient to establish tuber blight resistance. Second, phenotypic and molecular analysis of a panel of transformants for Rpi-vnt1.1, R3a and Rpi-blb3, and transcriptional analysis of the corresponding effectors (Avr-vnt1, Avr3a and Avr2 respectively) during leaf and tuber infection showed that the expression level of a given Rgene should equal or exceed the expression level of the matching effector in order to trigger an efficient resistance response in the tuber. Therefore, foliar and tuber late blight resistance are controlled by similar genetic mechanisms. The perceived lack of phenotypic correlation between foliage and tuber blight resistance is thus solely due to the tissue specific expression level of the Rpi-gene. In the general discussion (Chapter 7), results from the experimental chapters are discussed in a broader perspective.

Producing starches with altered composition, structure and novel physico-chemical properties in planta by manipulating the enzymes which are involved in starch metabolism or (over)expressing heterologous enzymes has huge advantages such as broadening the range of starch applications and reducing the costs of the post-harvest starch modification. The starch binding domain (SBD) technology has been extensively explored in our lab for modifying starch in planta and producing so-called “tailored starches”. In this thesis, two different microbial enzymes, E. coli glycogen branching enzyme (GlgB) and Neisseria polysaccharea amylosucrase, were chosen and expressed in potato to modify starch composition or structure by fusing with an SBD. Expression of these different fusion proteins in potato resulted in starch granule morphology and structure alteration severely in different ways. Moreover, expression of SBD-amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a 2-fold increase in median granule size, and altered physico-chemical properties including higher end viscosity and higher enzymatic digestibility. Furthermore, earlier produced MAT/SBD transformants were analyzed at the transcriptional level using the POCI array. It showed that although expression of MAT in potato resulted in amalgamated starch granules, it did not affect the transcript level of other genes in the starch biosynthetic pathway. Next to these experiments, a tandem CBM25 domain of amylase from Microbacterium aurum was introduced in potato and the results suggested that it can be used as a novel tool for targeting proteins to granules during starch biosynthesis without causing side-effects. All these studies in this thesis demonstrated that the feasibility of modification of starch in planta by expression of heterologous enzymes fused with SBD.

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