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Partnership STW - Rijk Zwaan September 2011 STW – Rijk Zwaan Zaadteelt en Zaadhandel BV Program Plan Title: Managing meiotic recombination for plant breeding 1. Introduction and industrial relevance The Dutch technology foundation (STW) initiated the STW Partnership Programs to stimulate effective cooperations between academia and industries. This particular program is an initiative in which Rijk Zwaan Zaadteelt en Zaadhandel BV proposes the program theme, and universities are invited to submit dedicated research proposals in a tender. The research is carried out under the guidance of both university professors and industry staff, at university and industrial laboratories, and in a practical/hospital environment. Rijk Zwaan is a privately owned company with about 2000 employees worldwide. The headquarters is in De Lier and the biotechnology centre is located in Fijnaart (West Brabant). The management of the company is highly research oriented. Rijk Zwaan group is focused on the development of science driven new concepts aimed at rationalization of plant breeding. It executes research and development of fundamental mechanisms in meiosis. The major focus areas of Rijk Zwaan group are linked to quantitative genetics, bioinformatics, cytogenetics, mutagenesis (targeted), recombination suppression, and recombination stimulation. A large part of the Rijk Zwaan R&D departments are located in Fijnaart and De Lier in the Netherlands. This research plan has been developed in consult with -and through contributions fromnational and international top research groups in the field. 2. Focus, objectives and applications Focus Development and understanding of new concepts in plant genetics and their repercussions for plant breeding. Meiotic divisions drive chromosome recombination and segregation, and are therefore the core mechanism of genetics. In their turn, the „rules‟ in plant genetics are the steering force for plant breeding: the making of new plant varieties. During meiosis, two divisions take place. The first, is a reductional division whereby at the same time, genetic information is exchanged between non-sister chromatids, the second division is a mitosis like division that separates sister chromatids. Control of recombination, either occurring during meiosis or as a consequence of site directed mutagenesis (gene targeting), will enable modern plant breeders to reduce time and financial investments for improving their vegetables/crops of interest. The presently available genotypes have already been selected for a variety of desired traits, which should be kept. The margins for improvement are however considerable. The global activity of breeding companies further necessitates rapid breeding for optimized growth under local conditions. Also greenhouse technologies are constantly under development, reducing costs and energy loss, but thereby requiring crop adaptation as well. In order to be able to engineer or reengineer (as in Reverse Breeding) the optimized genotypes in a time and cost efficient manner, enhancing or reducing recombination is pivotal. Objectives Plant breeding can make a quantum leap by understanding, interfering and steering meiotic recombination and its cell divisions. Plant breeders aim to manage genetic variation, in order to create and reproduce favourable hybrids at will and in the shortest and most labour efficient way. A greater part of this variation is generated at meiosis, where two different divisions occur. The first division involves the formation of crossovers between homologous chromosomes that lead to recombinant chromatids in the gametes. The second process includes random orientation of chromosome pairs at metaphase I leading to equal and balanced assortment of parental chromosomes to the gametes. Each set of parental chromosomes has at least one crossover that is essential for a proper orientation and separation of the chromosome pairs. Number and position of the crossovers are strictly controlled by a complex regime of genetic and chromosomal factors, and can be modified by down-regulation or over-expression of genes involved in the crossover pathway. By completely knocking down the crossover machinery an a-synaptic meiosis takes place in which non-recombinant chromosomes are distributed randomly to the poles. This principle is the basis of Reverse Breeding: generating complementary genotypes from any heterozygous F1, by knocking down crossovers and generating viable balanced gametes to doubled haploids. The proof of principle was demonstrated in an Arabidopsis Columbia x Landsberg erecta F1, transformed by an RNAi :: DMC1silencing construct and a CenH3 haploid inducer system to obtain doubled haploid progeny from the viable spores. With a powerful co-dominant genetic screening method with markers covering all chromosomes we now can select complementary parents with any given chromosome set from the starting hybrid. The transgene needed to suppress crossovers is required for only a single meiosis and can, using hybrids from different transformants easily be removed. The possibility of simplifying modes of inheritance has revolutionized the way on how we think of plant breeding using the plethora of novel molecular and genetic toolkits. Applications The results of the research will be applied in both the making of new varieties as well as the elucidation of complex traits and genetic interactions that were not possible or tedious with existing technologies. 3. Major Research Areas The Partnership research program will focus on the combination of three major development/research areas and their interactions. 1) Quantitative aspects of Reverse Breeding and Reverse Progeny Mapping. In most quantitative genetic analyses, including statistical methods for the detection of Quantitative Trait Loci (QTLs), it is assumed that the probability of a crossover event is constant along the chromosome, and that the crossovers are independent. In general, this is a reasonable assumption for standard crosses. However, in a situation where meiotic recombination can be managed, a far more detailed mathematical model of the meiotic process is needed, which takes into account the influence of both the genotype and the environment. Partnership STW – Rijk Zwaan / Program Plan; Page 2 of 7 Another important quantitative aspect is the development of new statistical tools for QTL detection, which takes into account the variation in recombination frequency. So far, no statistical software has been developed that allows QTL mapping on lines and combinations of lines of Reverse Breeding experiments, or for the QTL fine mapping in RPM (Reverse Progeny Mapping) families. Another important statistical aspect is the detection of QTLs controlling the recombination frequency, and the response of those QTLs to environmental conditions. Using Reverse Breeding, as described above, it now becomes possible to create lines that are heterozygous for only one chromosome pair, which by backcrossing, respectively inbreeding will give offspring, that are heterozygous for only a small chromosome region; these are „introgression libraries‟, respectively „recombinant inbred libraries‟ for only one chromosome. These monosomic chromosome substitutions and their corresponding mapping populations are outstanding material for studying gene-gene-environment interactions and can disentangle the complex nature of QTLs. In Reverse Breeding, it is possible to make genetic analysis chromosome per chromosome. So far, no mathematical algorithms respectively software has been developed that allows QTL mapping on lines and combination of lines of Reverse Breeding experiments. Such (RB) lines are already available from an Arabidopsis Columbia x Landsberg erecta F1. The F1 hybrid can be reconstructed by several combinations of chromosome substitution panels, but in addition, sub-hybrid families can be made that differ from the original F1 hybrid in that for individual or multiple chromosome numbers, the heterozygous chromosomes of the original F1 hybrids were substituted by homozygous chromosomes of respectively either parent. An extremely important new quantification/estimation parameter is the contribution of epigenetics towards a phenotype: Reverse Breeding allows reconstruction of the genetically identical hybrid with different parental combinations. If phenotypic difference can be found between the genetically identical reconstructed hybrids, then only epigenetic influence can be accounted for this phenomenon. For the first time in plant genetics, the quantification of the contribution of epi-genetics and genetics to a certain phenotype can be quantified. In yet another discovery (Reverse Progeny Mapping) a complementary (to RB) mapping population of lines is generated. This mapping panel is obtained by modifying the second meiotic division (omission of the division): spores that were formed without undergoing the second meiotic division are diploid and plants from such spores form SDR (Second Division Restitution) panels. In this case, the skipping of the second division of meiosis leads to lines that contain residual amounts of heterozygosity on all chromosomes. In fact such lines are almost the equivalent of Doubled Haploids but they retain a small amount of heterozygous loci that were derived from dissimilar loci from original parental lines and retained by crossover. The mapping power of such lines lies in the accuracy of phenotyping. Segregation for a certain trait in the progeny of a (primary RPM-0) line =RPM-1 line, can only take place if the original parents were heterozygous for the given trait and if recombination took place at the locus ruling the trait. RPM mapping populations have so far never been used in plant genetics; except for only one type of populations has similar features (Heterogeneous Inbred Families). To make HIF‟s, is however, a very costly and tedious exercise. To take advantage of technologies in which we can manage the recombination process, there is also a need to develop new efficient selection strategies in plant breeding. For this, new statistical and mathematical methods have to be developed, using a combination of statistical optimal design and mathematical optimization. The amount of DH‟s to be produced that did not undergo recombination for F1 reconstruction purposes, respectively the making of chromosome substitution lines, is dependent on the haploid chromosome number of the species. The use of RPM is both dependent on the haploid chromosome number of the species and in addition of the frequency of recombination. Partnership STW – Rijk Zwaan / Program Plan; Page 3 of 7 The result of the research in this domain will be ready to use algorithms and software that allows the user to determine when to use RB, RPM and give recommendations in term of number of lines to use in combination with probabilities. 2) Discovery of variation for meiotic recombination frequencies. A new area of meiosis research can be entered by finding plant variants (“lines”, “races”, “ecotypes”, or “evoked mutants”) that show either an increased or a decreased frequency in meiotic recombination. Increased recombination is very important for genetic mapping strategies, because this leads to larger genetic map distances, finer maps and easier map-based cloning. For practical breeding, control of recombination can help to accurately remove undesired linkage drag in introgression libraries. Because of the availability of genomic information and genome wide association populations, this theme should be worked out first in the model species Arabidopsis thaliana. Decreased recombination allows improvement of Reverse Breeding technologies. In spite of its enormous impact in plant genetics and plant breeding, Reverse Breeding currently suffers from the drawback that it can only be applied for crops with a relative low chromosome number (max. 12 chromosomes per haploid genome). If „lines‟ can be found that suppress recombination without serious impact on fertility and chromosome segregation, it will render „Reverse Breeding‟ applicable for crops with high chromosome numbers such as wheat, cotton, potato, alfalfa, and so on. A valid alternative for finding naturally occurring variants with increased or decreased recombination frequencies is mutagenesis f.i. using zinc finger based artificial transcription factors (ZF-ATFs). This type of mutagenesis, also called “genome interrogation”, allows finding an evoked mutant phenotype already in the M1 populations, due to the dominant nature of the trans-acting ZF-ATF. By combining genome interrogation and natural variation, efficient screening schemes should be worked out that allow identification of plants with altered recombination frequency. The outcome of this work package is knowledge of genes that control recombination frequencies, which, in addition provides molecular markers that allow „breeding‟ with alleles that have either an increase of recombination frequency or suppression. 3) Targeted mutagenesis during meiosis. Methodology for targeted mutation and DNA integration („gene targeting‟), by which alleles in the plant genome can be altered as wished, are becoming more and more realistic. Methods by which this is feasible have been developed, but the frequencies by which the events can be obtained are still too low for routine application. The methods that have been developed rely largely on the natural process of homologous recombination, which cells use for DNA repair and for meiosis. In yeast, but also in lower plants such as moss, homologous recombination (HR) is used not only for meiosis but also for DNA repair somatic cells. This homology-directed repair can facilitate precise integration of DNA templates at a genetic locus of interest in case that a break in the DNA would occur. Unfortunately, in higher plants (and mammals) DNA breaks are predominantly repaired by nonhomologous end-joining (NHEJ), a process that does not require any homology between DNA templates, but just serves to seal the break. Hence, gene targeting (GT) in plants is still a very inefficient and rarely occurring process and means to achieve a breakthrough are required. One aspect that could help to improve GT efficiency might reside in developing a method to deliver DNA templates that contain the desired allele (mutation) into plant meiocytes. Delivery of DNA into the meiocyte has so far not been achieved. The development of such a method has to be part of this framework. In addition, the study of meiotic „targeted mutagenesis‟ or „allele replacement‟ will result in an alternative method to somatic (mitotic) methods. It will be determined, if, and how more efficient, homologous recombination in the meiocyte will be, and what further perspective this will offer. Partnership STW – Rijk Zwaan / Program Plan; Page 4 of 7 4. Scientific challenges The major scientific challenge is the use of the potential of and to interfere with the divisions taking place during meiosis. Technically interfering in the divisions has to be done experimentally, such as, (but not limited to) screening for genetic variants or simulated mutants (Zn finger mutagenesis). The focus should be oriented on at least two overlapping circles as depicted in figure 1. Project proposals should only be submitted when the research is conducted in the overlap area between at least two but preferably all three overlapping areas. During early phases every discipline has to work out its own work plan, however should inform the members of the other core projects. The tight interplay between meiotic divisions, the omission of the first, respectively the second and its consequences for genetic mapping and the structure of the mapping population is of outmost importance. The cytogenetics of chiasmata and recombination and the screening for variants that favor chiasmata with diminished recombination has to be fitted in the mathematical modeling. 5. Fit of research proposals into the program Project proposals that meet the following fit-to-the-program criteria are welcome: The project proposals should address the interplay between cytogenetics, molecular biology and quantitative genetics related to plant meiosis (see Figure 1). Projects should be submitted from at least two research groups from different universities or scientific disciplines. Projects should be multidisciplinary and should address at least two areas (circles) as depicted in Figure 1. However, proposals within the overlapping three areas will have the highest priority. Projects should focus on application/valorisation into plant genetics, respectively plant breeding. Not part of this program are project proposals covering: Research on yeasts, insects, nematodes, animals, or lower plants such as mosses or ferns. Just one of the areas depicted in Figure 1. Traditional QTL analysis Classical mutagenesis (such as EMS and fast neutrons) Only effects on mitosis: mitosis can be part of the project, but never alone, always in comparison or relation with meiosis. Development of (doubled) haploid systems or tissue culture Research and development on the basis of existing patents that are not owned by the university applicants themselves. Research topics on which there is a conflict of interest of one of the participants as a result of affiliations with Rijk Zwaan competitor industries. 6. Unique character of the program The uniqueness of the program is that Rijk Zwaan research works closely together in one team consisting of a number of top university groups as well. The challenge as well as the Partnership STW – Rijk Zwaan / Program Plan; Page 5 of 7 opportunity is to build and manage one of the largest (internationally linked) Dutch scientific community within the Netherlands on specialized meiosis. Since the majority of breeding traits have a multifactorial origin, within this research network a unique combination is organized linking phenotyping, mutagenesis, quantitative genetics, and (epi-) genetics and chromosome biology together. The Dutch universities collectively will –through this partnership program- form a new and unique scientific community on the role of specialized meiosis management. Currently there are no other programs running in the Netherlands on this specific program topic. For the purpose of maintaining competitive advantage, Rijk Zwaan Zaadteelt en Zaadhandel BV sees this program as a spearhead to further sustain its commercial future. The unique multidisciplinary approach generates a competitive advantage both for an industry and an academic perspective for the Dutch participants in this program. 7. Duration and budget The proposed duration of the program is 5 years, the budget is M€ 3 funded as 50% vs. 50% by STW and Rijk Zwaan Zaadteelt en Zaadhandel BV. The universities contributions are additional and implicitly on a pm base by making available infrastructure, use of equipment and professor + research staff guidance. 8. Program Committee The program will be managed by a program committee (PC) consisting of six persons: Dr. E. Gutteling, Dr. Kees van Dun, and Ir. J. de Wit by Rijk Zwaan Breeding BV, and three members nominated by STW. The members of the PC have skills and expert knowledge appropriate to assess the proposals and have a high level and recognition of experience in the particular field. PC members may invite other advisory members to the PC meetings, although these will not have any voting rights. The PC is responsible for the overall directions and management of the program. PC meetings will be organized/planned twice a year unless PC decides differently based on needs. All members of the PC are subject to confidentiality restrictions to protect any ideas laid down in the university project proposals. STW shall appoint a program manager to the PC who shall not have voting rights but will assist regarding administration/organization. This program manager will not be funded from the project. Partnership STW – Rijk Zwaan / Program Plan; Page 6 of 7 Fig. 1 Meiosis Works: interactions between disciplines. Mathematical modeling Recombination variaton Targeted mutagenesis Partnership STW – Rijk Zwaan / Program Plan; Page 7 of 7