**SNP Workshop Minutes**

Rydges South Bank Hotel

23 August 2008, 1 PM -- 5PM

Participants: Mark Sorrells (Cornell University); Rick Ward (DRRW
project); Gordon Cisar (DRRW project); Kathy Kahn (Bill & Melinda Gates
Foundation); Eduard Akhunov (Kansas State University); Sixin Liu
(University of Minnesota); Jorge Dubcovsky (UC Davis); Shiaoman Chao
(USDA-ARS); Jan Dvorak (UC Davis); Cal Qualset (UC Davis); Matthew
Hayden (Victorian AgriBiosciences Center); Dave Edwards (ACPFG); Jenny
Nelson (DRRW project)

Following introductions by the participants, Kathy Kahn addressed the
group. She talked about how the Bill and Melinda Gates Foundation (BMGF)
makes funding decisions, and she described the process of the
preparation of the DRRW proposal. The idea of a SNP platform for wheat
was discussed as part of the DRRW project, but in the end the BMGF did
not think it was their role to fund its development. First and foremost,
the BMGF is focused on agricultural development in Sub-Saharan Africa
and South Asia. Wheat is so important in the developed world that the
BMGF knows they are "never going it alone" on wheat research. However,
the BMGF does want to help catalyze an increase in investment in public
breeding in wheat, which is the rationale behind the advocacy component
of the DRRW project. The BMGF is not asking for a proposal on a SNP
platform for wheat at this time, but if there is something the
Foundation can to stimulate and/or coordinate with other donors, it will
consider that. She noted that she was participating in this workshop to
listen and learn.

Dave Edwards: There are other organizations that fund this work. How
does Gates see themselves interfacing with these?

Kathy: I have had initial conversations with GRDC, AusAid, and ACIAR
about where they might coordinate efforts. One issue of international
collaborations is how much data is shared outside of Australia. Is there
a way to leverage the work that GRDC and others do?

Mark Sorrells then introduced the workshop. The idea of the workshop is
to come up to speed on what everybody's doing, progress they've made,
roadblocks they've encountered, and methodologies we've found that work
or don't work. By working together, the wheat research community can
develop a SNP platform that will be more useful. He would like to see
the group reach some consensus on the frequency of SNPs and number
needed. He thanked the BMGF, who generously supported the workshop.

Wheat is far behind other crops in availability of high-throughput
markers. This is because it's a technically challenging crop,
polyploidy, low polymorphism. Out of this meeting, he hopes to come up
with a mechanism for sharing resources and progress in this group and
beyond. Another outcome of the meeting might be a proposal or
'prospectus' that could be shopped around to donors. Rick noted that the
DRRW project can help facilitate the development of a prospectus and
help find potential donors. If that comes up short, then Cornell could
potentially include it as component of phase II of DRRW project. The
target start date for Phase II of the DRRW project is Jan 2010. Rick is
personally interested in how strongly this group feels about whether a
SNP platform is useful -- the 'big picture' question.

Then the participants provided updates on their work and described their
methodologies and approaches.

Shiaoman: 2 platforms: genotype one SNP at a time. A lot of time we
focus on small \# SNPs but large number of samples. This system is
expensive -- each data point = sixty to eighty cents. Want to do more
multiplexing. The other system we use is large number of SNPs (1500 at a
time) but can only handle 96 samples at a time. Cost gets down to 3
cents per data point. This is very useful for genotyping germplasm,
mapping, and association genetics. Very robust. 3,000 SNPs on barley
breeding lines. To look for trait assoc. with SNPs. Each assays takes 3
days, 150,000 data points. Wants to use this on wheat. Wheat just does
not have enough markers -- only few thousand SSR markers. Not enough to
cover whole genome. We should move on to develop SNPs. Shiaoman's lab is
not engaged in SNP discovery.

Jorge: Several breeders decided to do preliminary tests on 2,000 to get
access to 40-50 lines and use part of their funding to create 240
springs, 240 winters. Germplasm was collected already.

Eduard: testing Illumina platform for markers. Can be used for HTP for
polyploidy genomes, using with homozygous lines.

Rick: How many SNPs are there right now in wheat?

Shiaoman: Florescence Polarization detects one at a time. Mostly using
for genotyping for mapping data and some breeding lines. Sixty cents is
expensive and the process is tedious. Breeders want data turned around
in 2 mos, I am not sure we can do that. Surveyed 400 on CAP project:
average polymorph was 20% (16% average, D genome is a lot less).

Jorge: This was on a Set of 20 adapted US germplasm. Percentages change
dramatically when you include a synthetic wheat genotype.

Shiaoman: German group -- IPK people, Marion Roder's group is developing
SNP markers.

Jorge: Most of polymorphism is deletion insertion polymorphism, not SNP.
Have to be better than DART. Markers have an identity; they are
sequence-based. Triticarte is to producing an intergrated DART map.
Thinks SNP will be better. At this moment DARTs are good because they
have targeted the most abundant polymorphisms. The rate of mutations is
100s of thousands times less than deletions.

Dave: DARTs are superb, for data point per dollar point of view, very
cost effective. Will be limitation. DART is valuable at the moment
because sequence info not needed up front. Will be a move from DARTs to
SNPs for crops that have sequence information.

Matt: Work is funded by GRDC. Been playing with SNP from USDA. Using
BeadExpress for detection (up to 384-plex). Low level of polymorphism in
domestic germplasm. 1) Ways of discovering new SNPs in their germplasm.
2) Genotyping SNPs . Did a complexity reduction similar to DARTS. Idea
is to discover SNPs on that, then microarray for mapping. Complexity
reduction = large scale. 2) Genotyping of SNPs -- grappling with
problem. How to prepare templates for large number of samples.
Scalability from small \# of markers to large number is problem. The 16
lines they use for discovery are core Australian varieties. All
hexaploids. Couple of synthetics (Dave Moody's work). Complexity
reduction method similar to DArTs. Selexa platform. 25,000-40,000
fragments according to AFLP. Not looking for haplotype, looking to do
genetic map. 1 divergent site every 100 base pairs. Only looking at
polymorphism between samples. Sequencing 1 individual at a time. Can
only be AB and D genomes. If there's a SNP in a contig, there must be a
paralog or a homolog. Looking for things that are polymorphic between
individuals. Tim Sorbridge doing the bioinformatics. Replaced Dave
Edwards.

Q: How many of your sequences are coming from repetitive sequences?
Matt: don't know yet, but can share the answers to that when we have it.

Jan: What is really essential for application is a highly (?) multiplex
Illumina platform.

Matt: on a Combimatrix. Microarray, extension based, not hybridization
based. Key Gene tried the complexity reduction approach.

Eduard Akhunov: Ridiculously high number of SNPs they say they
discovered. Matt: determining if same contig in this sample is same as
in that sample is the problem.

Dave Edwards: might be missing quite a few SNPs. There are ways of
getting around that but they have to be thought through.

Jan: NSF SNP project. Issue is what is the distribution of SNPs in wheat
in both ancestors and in breeding germplasm. Then the distribution
within genomes. Forces that determine that fate of variation. Is
important to realize the cost: 5-6 million dollars. Reasoning was SNPs
will be most useful in single copy DNA. Better to find single copy ...
and then find SNPs rather than find SNPs and be disappointed. 35 people
involved, following logic of EST project. First question is how much
diversity is there? How is it distributed among the genomes? Almost same
in A and B genomes. Intergenic DNA is evolving about 4 times as fast as
genic. Dicoccoides is highly diverse. Paradox is wheat has almost as
much polymorphism as barley. Hexaploid lost some diversity due to
bottleneck. Project Strategy was to go from genic regions in rice. (See
slides of JD.) Found interspecific polymorphism between genomes.
Developed genome specific primers. Reduced polyploidy system into
diploid system. Needed collaboration of 30 people to achieve this.
Mapped SNPs are far more useful than those that aren't. Accomplishments:
see slide. Very important points about distribution of SNPs in wheat.
Slide on Diversity map of wheat homoeologous group 2 (not published
anywhere). Used highly conserved markers, therefore the synteny with
rice is good. SNPs evolve very slowly. How much diversity has been
recovered with hybridization? Important question. Regions like this will
be impediment to use of SNPs in breeding work, but not in genetic work.
D genome is very low diversity compared to A and B. From breeding point
of view, very important to use synthetics.

Mark: There's a partial argument for multiple types of libraries. Could
fill these gaps.

Jorge: if you keep sequencing, you will find SNPs.

Mark: did you find any novel SNPs in hexaploids that didn't exist in the
progenitors?

Eduard: yes, about 30%.

Jorge: Do you use genes that are known to have specific effect? In
vernalization genes...

Rick/Eduard: Usually SNPs are diallelic (2 options).

Jan: frequency of SNPs, important to breeding. Tajima D for each
chromosome. If \>0, too few rare alleles; if \<o, too many rare alleles.

Dave: Any strategies for overcoming the inability to fine map?

Jan: You can do gene isolation using homologs, no problem.

Jorge: Can select synthetic that is most distant.

Dave: Related to synteny with rice -- is there more or less synteny with
brachypodium?

Jan: It's more or less the same. Also looked at sorghum, there is not
much of a difference. There are certain regions in wheat that rice does
not have. Because that evolved in wheat's lineage. However, brachypodium
has some of those, depends on the region. There is a reason for
optimism. There is a lot of polymorphism in dicoccoides, almost three
times as diverse as soybean. We made a strategic error in our project.
Using rice for selection of target. We focused on regions that were
conserved, which are the ones with least diversity. We stopped and then
took a different path. In the high recombination regions, we had few
SNPs. We should focus on finding SNPs in just some regions, high
recombination regions. Brachypodium is next best bet...because we can
use it to find loci that

Dave: My background is in bioinformatics, and I have not been working on
wheat long. I'm at the Australian Center for Plant Functional Genomics.
We work on abiotic stresses. We receive core funding from ARC, GRDC and
various state governments. Sequencing technology is key to SNPs. CMAP
databases: Grain Genes ad Gramene. New tools from ACPFG tool this allows
them to see maps in 3D. Two measures that SNPs are real: depth of
redundancy and co-segregation to define haplotype.

Jan: Big genomes have propensity to generate paralogs very recently with
around 90% of homology. How can you tell paralogs which are highly
similar from orthologs?

Dave: This is a main challenge. Have looked at lot of data in canola to
determine if we can look at different genomes. Probably not enough data
to differentiate between the genomes. Need to be able to capture the
variety of information.

Matt: We can necessarily work it out, but we sort it out in the wash.
Paralogs don't co-segregate.

How often do you find polymorphism on both genomes?

Matt: In brassica, about 80% (very high).

Eduard: Shared polymorphism between genomes is extremely low.

Dave: 454 sequencing will work for canola. But will be more of a
challenge for wheat. Make it user-friendly so that people can search
data. Can set up specific queries. Can search for keywords, etc. Took
whatever was in GenBank, so whatever was put in there over the years.
Could contribute this kind of work without additional resources, since
we're already doing it as part of ACPFG core work.

Eduard: See slides. 454 sequencing for SNP discovery in polyploidy
wheat. Sequenced cDNA libraries of 5 varieties (spelt, tauschii, urartu,
and 2 wheat varieties). Assembled contigs for diploid ancestors. Aligned
hexaploid 454 reads... (260-270 base pairs), and then 3^rd^ step (see
slide). Preliminary analysis: about 700 SNPs between Chinese Spring and
Jagger.

Mark: Once you detect paralog, it's still a useful SNP.

Jan: This is the best path: you know you are dealing with genic
sequences, got rid of illumina downstream problems. The only thing you
are missing is the introns. You can predict where the gene is.
Exploiting the synteny, you can predict that it is very likely the gene
is in this region.

Jorge: With a couple of runs like this, deposited in GenBank, everybody
in the world will be able to detect SNPs.

Matt: How do you go from SNPs to things you can genotype? How will
genotype except on OPA?

Dave: We've tried this in brassica and have found it's better to get
people together and do it at a larger scale.

What level of accuracy are you expecting?

Eduard: Most should be real, when you look at depth of data. Used
normalized cDNA libraries. Tissues: used 2 week seedlings. Hit almost
20,000 unique genes with Chinese Spring wheat.

Dave: have you seen many indels? Eduard: Didn't look at it closely, but
didn't see very many. There are some.

Shiaoman: what is quality of sequencing? Eduard: very high.

**COFFEE BREAK**

Kathy: How can we use this in breeding?

Mark: We are going to need the capability to do whole genome selection.
There is no other marker technology that will have the necessary
throughput. The companies are doing this already.

Dave: Whole genome selection is going to be the future. We just don't
have enough information available yet. The thing that's driving this is
economics. For many breeders, it's still cheaper to select in the field
or use SSRs. This is going to change. Need to think about what is going
to happen in 2-5 years time, because economics are going to shift, and
the amount of information that is there is going to shift.

With new sequencing technologies that's making it cheaper, we will get
more SNPs in all crops.

Once you've got a SNP platform developed, anybody can use it. Breeder
doesn't have to do anything. They can do whole genome selection without
any equipment. Selection is based on knowledge, association mapping,
etc. (See GSS?) Every breeder's germplasm is completely different. Soft
wheats are most variable. Train my markers on this, develop my
prediction equation on this. Genotype and phenotype, analyze data,
develop predication equation based on analysis method (+, - or zero).
International centers could facilitate this for marker classes. Kathy:
but they are not currently doing this, but why? What are the
roadbloacks?

Jan: Sharing is an issue.

Cal: We submitted a 2006 proposal to NSF on Illumina platform. Got US,
Canada and CIMMYT to agree to supply DNA to lab in Davis. This would
have been low cost except for running the Illumina. All the data would
be shared/made publicly available. The scheme was well accepted by the
collaborators and was well-reviewed by NSF, but not funded. Have to
settle on a system to develop the SNPs. IF you have 15, 20, 30 labs, you
can have multiple (need multiple). May need 5 different labs to do it 5
different ways.

Jan: SNPs are technology independent, so it doesn't matter which
platform. Different labs have different lengths. Only useful for
breeders in that lab. But anybody can do it in a lab, can enable MAS.
You do need a lab and people who can run gels, PCR, etc. SNPs only
require DNA -- the rest is bioinformatics.

DART is limiting because there is only one source of information. Lot of
work to convert DART marker to sequence tag: have to make primers for
DART sequence, then sequence different varieties. The polymorphism may
be outside the sequence...

Eduard: Infinium can run 6 million data points. 1 cent per SNP for human
genome.

Mark: McCouch said don't use Pearlgen for SNPs -- been problems from the
beginning in rice.

Need catalog for diversity for all genes.

Mark: In the last 40 minutes of this meeting, we want to:

1)  see if there is interest in a website for collecting info, sharing
    info

2)  see if there is interest in sharing libraries or sequence info.
    Dave: open software, but it's probably easier if we work with people
    to set up. But it's publicly available.

Kathy: What's different about Dave's software from the NSF proposal?
Cal: We were talking about assaying about 10,000 lines. Key is having
broad-based collaboration, all the way to breeders doing phenotypes. GCP
looking at drought. Multiple donors that are interested in this.

Dave: We need to link the data to the phenotypes. Have been analyzing a
lot of CIMMYT's info using ICIS. I think best approach is to link the
best available databases that are out there.

Cal: Lack of competency is at breeder level -- I'm one of them -- and a
lot of education is needed. Need to show to donors that not only will
work, but also how the capacity will be built up among the breeders so
that it will happen.

Mark: Wheat Phenome Atlas project is using the CIMMYT ESWYTs (Elite
Spring Wheat Yield Trials). Idea is to built phenomics database, DARrT
genotype everything and then do association mapping. Found regions of
genome that harbor stem rust that are known not to harbor major genes.

Jan: What is emerging from this discussion is not what is stucture of db
(algorthms, etc.) but what is the source of the data. Having
representaive germplasm with markers is a no-brainer. Basis of global
db. E.g., anybody who contributes to genotyping pool must make bag of
seed for everyone to use.

Cal: The reviewer said it wasn't good enough for breeder to maintain the
bag of seed from one plant, need to have centralized place.

Kathy: It makes sense for international centers to do that.

Jorge: But not all breeders would do this. In public programs majority
of people would do this.

Rick: They would have to agree to it (and their university lawyers would
too) before they join program.

Jan: One unique aspect of this SNP global db is that sooner or later the
pubs will be able to be in general background of markers.

Rick: Is one of the barriers the sharing of seed? All people
participating benefit from participating.

Kathy: Would this push wheat breeding too public and less sustainable?
What would be the incentive for private companies to participate?

Jorge: There are different business models that companies use.

Rick: I don't think it will dissuade private companies, will allow them
to move faster.

Jan: If I had my wish: genotype all the lines in the world. As a private
company, I would benefit from this resource by selecting the parents,
all I need to know is phenotype. I generate proprietary info. but in ten
years or so it will get genotyped again.

Cal: That's no different than what's been happening the last 75 years.
Anybody can access germplasm from genebanks, now it's just a database.

Rick: Is there a risk of companies patenting linkages/markers?

Cal: AFLPs were made in the private sector in Europe; that was the
impetus for the public effort.

Rick: Would anybody's organization or country take this on?

Jan: All the SNPs etc. are available from NSF project without any
restrictions.

Matt: GRDC requires leveraging and is interested in international
collaborations.

Cal: the NSF project was 2.2 million total (3 years). Genotyping with
available SNPs. Basic research on evolution of wheat. There are a number
of funders who should be interested: GCP should be interested,
CIMMYT/ICARDA, DRRW, GRDC. Not a huge amount of money (esp. considering
high overhead in NSF projects)

Jan: The US has put in a lot of money in purely altruistic outcomes, and
now feels the global community should come in and contribute.

Cal: Could get started with a relatively small contribution from the
BMGF.

Mark: CAP project, looking at phase 2, half as much money means less
people, looking to do association mapping.

Jan: Walnut people would not give money to further walnut breeding in
the world. These are purely altruistic that would be useful to everybody
on the planet. America has done enough, in their own opinion -- NSF
Plant Genome Project was not coming from lobbying by wheat
people/commissions.

Rick: So, will people in this room want to collaborate to do this?

Kathy: Analysis needs to be done about what's really needed to do the
SNP platform, which should be done by the people in this room. Need also
to explore among the donor community what they want to do.

Rick: Would be even more useful if someone were to distill a consensus
about where we are and where we need to be.

Kathy: It's very difficult in BMGF to advocate for inflated projects.
Might be better to put in terms of what can be done for a fixed amount
of money (reasonable amount, like 600k).

Rick: First need to explain the value, then the cost and way forward for
doing it.

Cal: Reminds me of ITMI, group came together. Prospectus was developed
that explained a potential project. Was instrumental in getting NSF
project on EST. We need to show that there is already investment, from
NSF and USDA and GRDC, in the prospectus. Second stage from here is to
get broader representation. Get 'general buy-in' from that wider
community and then get smaller group together to draft a prospectus.
Include genotyping centers.

Jorge: Developing mapping populations will go much faster (2 wks vs 6
mos), which is what we are doing in the rust work.

Jan: Once you discover genes and you want to use them, breeders need to
know polymorphism, and right now they don't know that.

Cal: You might craft a specific project, with a specific trait, and
describe how can you get it done better with SNP. Use DArT marker
results to help justify SNP marker development. Lack of markers for rust
is another justification. SNPs are immortal but the detection technology
will change over time.

Mark: Already have proof of concept: Crossa et al paper published about
regions associated with rust resistance but have no major known genes
for stem rust resistance.

Cal: That's the first step then. In GCP, drought is keystone there. Put
it in terms of project that is already funded, so easy for donor to
understand.

Sixin: We can have smaller goal to start, we can't do everything at
first.

Matt: True, but SNPs are still SNPs. Whether it's small or big effort is
the question.

Jan: The point is that we need globally to be able to respond to disease
etc. and the only way to do this is with a SNP platform.

Dave: Communication is so important. We can agree on general principles
and use those as the basis for proposals. Need a roadmap, agree on
principles, justification.

Mark: Here are some draft principles:

-   We need more SNPS

-   They need to be publicly available

-   We need to create a central way to access them

-   We need them applied to relevant genotypes

We can already start delivering some SNPs to stakeholders.

Rick: DRRW project has money for collaborative web tools to facilitate.

Mark: Let's put together a website for this effort.

It was suggested to have a follow up meeting at PAG in January 2009 with
broader representation. It would be good to know who around the world is
doing SNPs now. We need to work with the sequencing groups so they know
what this group is doing. We need to make sure we don't step on each
other's toes, and we should have a simple message about what we are
trying to do. For GRDC we could call it 'pre-breeding.' We should not
assume right away that it all has to be located in one place. We need to
germinate something, but whatever we do also needs to be managed. ITMI
could provide the infrastructure for a working group.

It was concluded that Mark and Eduard would work together to draft a
prospectus and it would be circulated to the group.

END

Jennifer Nelson

September, 9, 2008