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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