WS1 Applying
Modern Genomic Tools to the Management and Characterization
of Plant Genetic Resources
A. Preliminary program (abstracts below)
Morning
session – ‘setting the scene’ - 8:00 – 8:05 Welcome address
- 8:05 – 8:40
Keynote address: Dr. Christopher M. Richards: The influence
of large-scale genomics
and the changing role
of ex situ collections
- 8:40 – 9:00 Dr.
David Spooner: DNA barcoding: An oversimplified
solution to a complex problem
- 9:00 – 9:20
Dr. Jan Engels: Towards a rational, secure
and effective long-term conservation strategy
- 9:20 – 9:40 Dr. Ken Richards: Challenges of applying
molecular techniques to PGR management – a
Canadian perspective
- 9:40 – 10:00 Q & A
session
- 10:00 – 10:30 TEA and COFFEE
- 10:30 – 11:10
Keynote address: Dr. Loren Rieseberg: Population genetic
challenges and
the potential of
modern genomics technologies for the
management and characterization
of plant genetic resources
- 11:10 – 11:30
Dr. Rich Cronn/ Dr. Aaron Liston: Multiplex
Sequencing of Plant Chloroplast
Genomes Using
Illumina/Solexa
Sequencing-By-Synthesis Technology
- 11:30 – 11:50
Dr. Nolan Kane: Chloroplast genome sequencing
using Solexa and SOLiD
- 11:50 – 12:10
Dr. Katrina Dlugosch: Prospects and challenges
of 454 sequencing for PGR characterization
- 12:10 – 12:30
Q & A session
- 12:30 – 13:30 LUNCH Sponsored by Applied Biosystems
Afternoon session – ‘opportunities
through genomics’
- 13:30 – 14:30 Speakers from
the private sector
- 14:30 – 15:00
Q&A session
on genomics technologies
- 15:00 – 15:30
TEA and COFFEE
- 15:30 – 16:30
Discussion session
- 16:30 – 17:00
Conclusions and wrap-up (Chair:
Quentin Cronk,
Professor
in Plant
Science, UBC)
B. Talk abstracts (in order of program)
Keynote: The influence of large-scale genomic and the changing
role of ex situ collections
Christopher M. Richards
The development of large scale genomics resources in non-model
organisms promises to have a fundamental impact on the utilization
of genetic resources. Technical innovation in high through-put
sequencing has reduced the cost to a point where genome-wide
SNP development is feasible across a range of taxa including
wild relatives of domesticated cultivars. While the focus
of these efforts are clearly on gene discovery and assessment
of candidate loci for crop improvement, methods and markers
developed here will be important in genebank management.
Large scale surveys of wider genetic variation with significantly
more biological complexity that model organisms will present
a number of technical and analytical challenges. Increasingly,
the technical work of crop improvement programs making use
of genetic resources draws on the disciplines of phylogeography,
molecular evolution and ecological genetics. I will describe
how development of novel analytical approaches for population
genomics will in turn influence the way we collect, and monitor
and database the diversity in large ex situ collections and
I will present ideas on the future roles genebanks may play
as both providers of viable germplasm and associated data.
DNA barcoding: An oversimplified solution to a complex problem
David M. Spooner
DNA barcoding (“barcoding”) has been proposed
as a rapid and practical molecular tool to identify species
via diagnostic variation in short orthologous DNA sequences
from one or a few universal genomic regions. It seeks to
overcome the “taxonomic impediment” caused by
a greater need for species identifications than the supply
of taxonomic specialists. A number of barcoding regions have
been proposed for plants, including the internal non-transcribed
spacer of nuclear ribosomal DNA (ITS), and the plastid markers
trnH-psbA intergenic spacer, matK, and other plastid regions,
with the first three being the most variable. This study
tests the utility of barcoding with these three regions in
a complicated plant group, Solanum sect. Petota; wild potatoes.
These DNA regions fail to provide species-specific markers
in sect. Petota because ITS has too much intraspecific variation
and the plastid markers lack sufficient polymorphism. Wild
potatoes are not alone in failing to work with barcoding
regions. Addressing the taxonomic impediment will require
a comprehensive and integrative program of research and training
using a variety of data sets appropriate to different species
groups. Barcoding, in contrast, is impeded by common complicating
biological phenomena, is a retroactive procedure that relies
on well defined species to function, is based solely on DNA
sequences that are often inappropriate at the species level,
has been poorly tested with replicate samples, and ignores
substantial practical and theoretical problems in defining
species.
Towards a rational, secure and effective long-term conservation
strategy
Johannes M.M. Engels (presenter) and Robbert van Treuren
A rough analysis of the history of how most of the existing
germplasm collections have been established, and comparing
the outcome of this analysis with what one would expect that
such collections should contain in terms of genetic diversity
for a given genepool, allows the conclusion that the content
of existing ex situ collections leaves room for improvement,
especially from a long-term conservation perspective. Many
of these collections have grown out of breeders’ working
collections that consisted of a selected set of accessions
and/or have been established by countries and/or national
or institutional genebanks with the aim of providing genetic
diversity, in particular specific traits, to users (i.e.
predominantly plant breeders) of those collections in a given
country. This approach has resulted in considerable redundancy
and in genetic diversity gaps, both from a genetic diversity
as well as from a geographic perspective.
A long-term global or regional ex situ germplasm collection
for a given crop genepool should contain an adequate representation
of the total existing genetic diversity in that genepool
(both, in situ as well as ex situ) in as few as possible
samples (i.e. accessions) in order to be rational. This principle
begs the question if a long-term conservation collection
should aim at storing genotypes or genes/alleles.
Modern genomic and information management tools allow now
more efficient and effective conservation approaches and
methodologies to be applied and this results among others
in:
o better monitoring of routine conservation activities (e.g.
collecting and regeneration);
o attempts to work towards more adequately composed collections
for long-term conservation, including the identification
of collection gaps, unwanted duplicates and genetic redundancy
(e.g. proposed approach to establish a global strategic base
collection for cacao);
o better coordinated and more complementary conservation
efforts between in situ (natural habitats and on-farm) and
ex situ conservation programmes;
o more efficient collaboration efforts between genebanks,
countries as well as between regions (e.g. rationalizing
Allium, predominantly garlic collections; establishment and
operation of a virtual European genebank system, i.e. AEGIS;
rationalisation efforts of the Global Crop Diversity Trust);
o More rational and cost efficient global or regional conservation
efforts;
o Better services to users (core collection and core selection
formation).
Challenges of applying molecular
techniques to PGR management – a
Canadian perspective
Ken Richards
Genetic resources are playing an increasingly important role
in Canadian agriculture for the betterment of Canadian and
world societies. Recently Agriculture and Agri-Food consulted
with national stakeholders about research priorities and
determined one to be: “Understanding and conserving
Canadian bioresources”. In response to this national
priority the Canadian Genetic Resources Program developed
long-term objectives: to protect and conserve the genetic
diversity of Canadian bioresources, contribute to the security,
protection and safety of the food system, enhance the environmental
performance of the Canadian agricultural system, contribute
to the development of new opportunities for agriculture,
thereby enhancing food and feed quality, Canadian health
and wellness, and economic benefits for the industry, and
support bioresource-related regulatory requirements. The
Program also developed specific short-term objectives:
a) develop new techniques to conserve and regenerate plant,
animal and microbial germplasm to maintain genetic integrity
and minimize genetic erosion;
b) create new phenotypic and genotypic information including
identifying new sources of disease resistance, abiotic stress
resistance, nutritional quality and bioactive compounds,
through characterization and evaluation of bioresource attributes;
c) assess genetic diversity changes in domesticated plant
and animal germplasm;
d) improve the structure of the GRIN-CA database for delivery
of bioinformation; and
e) contribute to access and benefit sharing regimens (acquire,
donate, maintain, regenerate germplasm) consistent with Canada's
commitments to international treaties, e.g. Convention on
Biological Diversity (CBD) and the FAO International Treaty
on Genetic Resources for Food and Agriculture (ITGRFA).
Plant Gene Resources of Canada has applied various molecular
techniques to help meet some of the above objectives, namely
those associated with characterization and diversity changes
of plant germplasm. Examples from diverse crop and wild species
will illustrate the advances made in use of techniques and
also some of the limitations experienced.
Keynote: Population genetic challenges
and the potential of modern genomics technologies for the
management and characterization
of plant genetic resources
Loren Rieseberg
The development of molecular diagnostic tools for the management
and characterization of crop germplasm such as landraces,
breeder’s varieties, as well as populations of wild
relatives is useful for several reasons. An appropriate method
could provide a standardized means for identifying and categorizing
germplasm across species and across institutions. It could
also be used to reduce unwanted duplication in germplasm
repositories, assess genetic relationships, develop a more
stable classification of domesticated and wild populations,
and detect contaminated or admixed samples. Furthermore,
if biologically relevant molecular variation were assayed,
it might be feasible to predict the likely value of germplasm
for breeding and crop improvement. A variety of different
approaches are currently being employed to characterize germplasm
in different crops, ranging from allozymes to microsatellites
to single nucleotide polymorphisms (SNPs) in nuclear loci.
Also, DNA-barcoding approaches, including whole plastome
sequencing, are now being considered for analyses of clonal
and selfing crops. I will explore the strengths and weaknesses
of the primary methods currently being employed (or that
have recently become technically feasible) for germplasm
characterization. I will also discuss the population genetic
challenges associated with the development of a widely applicable,
stable, and cost-effective strategy for analyzing crops that
vary in mating system, ploidy level, and means of propagation.
When assessing different approaches, I will do so in the
light of rapid advances in sequencing and SNP genotyping
technologies that are providing new technological solutions
to old problems.
Multiplex Sequencing of Plant Chloroplast Genomes Using
Illumina/Solexa Sequencing-By-Synthesis Technology
Richard Cronn, Aaron Liston
Chloroplast and mitochondrial organellar genomes are widely
used in plant germplasm characterization because they offer
a simple means to evaluate cytoplasmic diversity, germplasm
differentiation, and taxonomic affinities. Due to their haploid
nature and (typically) uniparental transmission, these genomes
are highly responsive to drift. These positive attributes
are counterbalanced by two undesirable features; a large
size and highly conservative mutation rate. Haplotype variation – when
present – is rarely found in a single mutational ‘hotspot’,
but is usually dispersed across the genome in simple repeats,
small rearrangements, and single nucleotide polymorphisms.
Because of the limited variation detected in most taxa, a
host of genes, spacers, introns, and microsatellite repeats
are frequently pre-screened to identify “tortoises” and “hares” so
that genotyping efforts can be tailored to specific taxonomic
questions.
An alternative to this endless pursuit is to sequence entire
genomes and evaluate all mutational classes genome-wide.
In this presentation, we show how multiplex “sequencing-by-synthesis” (MSBS)
on the Illumina Genome Analyzer is one way to achieve this
goal. We have successfully used MSBS to sequence PCR-amplified
plastomes from 4 to 6 species of Pinus simultaneously. By ‘tagging’ each
genome with a unique adapter, microreads (36 - 40 bp) can
be sorted and independently assembled using a combination
of de novo and reference-guided steps. Results to date show
that draft genomes can be rapidly assembled that are 85%
to 98% complete, with an average sequencing depth over 50X.
The power of this approach is highlighted with Pinus torreyana,
a species that has yet to reveal intraspecific cpDNA divergence
in previous RFLP and cpSSR studies. In a comparison of two
individuals, we identified 5 SNPs in 101 kb of chloroplast
DNA. We conclude this talk by considering how MSBS might
be applied to population-level screening of additional genes
and genomes.
Chloroplast genome sequencing using Solexa and SOLiD
Nolan Kane
Next-generation sequencing technology enables rapid, inexpensive
characterization of small genomes, but cannot easily deal
with the highly complex genomes of most eukaryotes. However,
the smaller organellar genomes can be isolated and sequenced,
enabling these technologies to be applied to eukaryotic systems.
Here we report the use of Solexa and SOLiD to sequence whole
chloroplast genomes from several species from the Compositae
(Asteraceae), the largest plant family. The advantages of
each of these technologies is discussed, and several potential
uses are examined.
Prospects and challenges of 454 sequencing for PGR characterization
Katrina Dlugosch
Roche 454 Life Science GS FLX sequencing is a next-generation
technology that currently yields ~100 megabases of sequence
per run, in ~250 bp lengths. These are relatively long reads
among the next-gen approaches available, and they offer the
potential to reconstruct complex (repetitive) nuclear genomic
sequence without the use of an existing template for assembly.
Where a template is available to aid assembly, long reads
also offer the possibility of obtaining large amounts of
sequence at low coverage. I will relate some of our own experiences
with comparative genomics from low-coverage GS FLX data,
and I will detail the laboratory and bioinformatic requirements
of using this approach to manage plant genetic resources.
C. Workshop outline
As new sequencing technologies become rapidly available,
the price for sequencing is predicted to drop continuously.
The human genomics community in particular is pressing
hard for cheaper and faster sequencing methods as they
promise new and improved treatments in the area of medicine.
The great potential of these technologies for the field
of plant genetic resource management have so far remained
largely untapped. However, sequencing large areas of the
genome in order to obtain information about inter-and intra-specific
variability is about to become a reality due to the ever
decreasing cost of sequencing technologies. Soon, germplasm
will become distinguishable on the level of varieties and
land races with standardized methods that are fast, reliable
and affordable. Such methods could include the use of massively
parallel sequencing to decipher the genetic code of whole
plastids and/or chip based approaches that could survey
SNP variation at many nuclear loci for many individuals.
This will allow researchers to tackle problems such as
landrace genotyping, species level identification of wild
relatives in a genebank setting, the detection of duplicate
accessions, greater efficiency of germplasm management
and a standardized molecular characterization protocol
between different genebanks across the globe.
The great opportunities for plant genetic resource management
that arrive with these new technologies need to be explored.
Challenges of already established methods, such as plant
DNA-barcoding, should be addressed and limitations of such
techniques should be discussed in the context of plant genetic
resource management. The diversity of crops regarding their
reproductive biology, agricultural management and genetic
make-up, poses a particular challenge that needs special
consideration for the development of global standards. Furthermore,
the generation of tools, such as a centralized database where
standardized methods can be documented and characterization
results can be submitted, ought to be a topic of discussion.
At this workshop, we aim to discuss how best to make use
of these emerging possibilities and how to actively influence
the development of accompanying bioinformatics methods as
to adapt them to suit the plant genetic resource community’s
needs. The debate about the usefulness of many of these methods
needs to be moved from the informal setting of ‘institutional
hallways’ to an inter-institutional level in order
to work on a common strategy to capitalize on these rapidly
emerging opportunities for the management of plant genetic
resources.
The workshop will consist of a series of lectures, ranging
from technical and theoretical viewpoints to more applied
aspects. We are also planning several ‘breakout sessions’,
in which the participants will be able to get first-hand
experience with some of the new methods and analysis techniques
under the guidance of experts in the field and representatives
of the private sector. Furthermore a mediated discussion
forum is envisioned, where scientists can freely exchange
their ideas on this topic and debate controversial issues.
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