Molecular and other biotech

1
Molecular and other biotech

• Dag Lindgren
• Uppsala 14 maj

2
Reservation I am not very suitable to make this
presentation or task, because I am too negatively
biased. It costs non-biotech genetics at SLU a
high price is less relevance for coming
generations and Swedish forestry.
3

• Normally it takes long time before an idea to
  practice.
• Practice must normally start slow in small scale
  before it becomes a main method.
• The small development scale is not
  implementation, but research funded by research
  grants or development money, not routine
  breeding.
• It is first when something is seen a routine
  breeding financed by routine breeding it is
  implemented.
• To be implemented in routine breeding in ten
  years and thus qualify for preparations according
  to this document, most methods should already now
  been used for development in compartments of the
  breeding population (like pine cuttings for
  breeding). To really trust in a new method it
  should be used in a compartment for a cycle, that
  is gt20 years! Research with external funding,
  where normally the driving force is University
  scientists is not implementation.Promising
  progress does not mean breeding routine within
  10 years.
• New methods will usually complement old ones. Not
  replace, but just add something. The competence
  to handle trees in experiment which are managed
  and measured about as it is done now will still
  be needed.

4
Biotech progress is often overexegarated

• We placed markers on gene maps of Scots pine
  almost four decades ago.
• We could identify individuals of the same clone
  by markers three decades ago.
• We measured pollen contamination with markers two
  decades ago.
• We talked about GMOs two decades ago.
• We have worked towards clonal forestry for spruce
  in four decades. (Clonal forestry seems as the
  horizon, it does not come closer by walking
  towards it)

5
Molecular markers - identification

• The molecular markers become more powerful during
  the decades. But everything is not a revolution
  by several magnitudes, but say a factor 3 in 20
  years as price tag for the same pollen
  contamination investigation and the same
  competence needed. Larger factor if big scaled
  (five man-years) and for a few specific purposes.
  It is seldom low-cost.

6
Parentage

• It is possible to decide which (among suspects)
  are the parents to a tree. That could find
  immediate applications in operative breeding.
• Reliability is high enough and increase.
• Costs is probably affordable for some
  applications and decrease.

7

• Markers can experimentally estimate effective
  size (status number, coancestry, group
  coancestry, gene diversity), which actually is a
  requirement in BC. (Similar to Wrights
  F-statistics, FST) Can monitor changes of these
  parameters over time. Routine monitoring by
  Skogforsk could be discussed, I think it may be
  too early to make it routine the next decade. But
  some scientists are keen to measure real
  materials by research grants.
• Inbreeding and relatedness will be better
  monitored.

8
Seed orchard contamination, dysfunction and male
fertility can be better studied

• This has been possible some decades, but a study
  now probably get 3 times more results more
  certain at the same cost as some decades ago.
• We have got funding from föreningen for a seed
  orchard study (Västerhus) and the first results
  (300 seeds) has come. The pollen contamination is
  about 50, confirming results decades ago with
  less reliable markers. I had hoped that we
  overestimated the problem because of inadequate
  technique, but it seems we did not.
• David Hall is planning a contamination study, but
  he has done so some years without execution.
• It feels too early to comment on the feasibility,
  it was more problematic than I thought when
  planning the study.

9
Tag error elimination

• The breeding population could be checked for
  errors in pedigree.
• Erronously tagged clones occur. Yoshinary
  suggests 10 on average and 50 in special cases.
  Johan checked an Umeå seed orchard material and
  found no error in 36 ramets. I personally checked
  a Turkish seed orchard and found at least one
  error among 75 ramets.
• Of the magnitude almost 1 of trees in the Seed
  orchard Västerhus are rootstocks which have
  replaced a dead graft. We found with markers 1
  mislabelled graft among 84.
• I believe Swedish breeders are extremely clever
  and careful, so the error-rate in breeding
  material is certainly below 5. But this figure
  could be good to verify!
• Probably it is not justified to make it a
  routine, but at least it is something which
  requires more discussion.
• I suggest Skogforsk initiates a project to
  estimate different type of identification errors
  connected to breeding and seed orchards. With the
  results, it can be discussed if some type of
  routine.

10
Routine DNA sampling?

• It may be a good idea to routinely store a DNA
  sample of every founder of the breeding
  population forever? (Gene conservation). To be
  able to do investigations in the future and check
  impact of the founders on the Swedish forest in
  some distant future. The option will be gone
  forever soon to sample the founders. I guess it
  is cheap, it just mean collection and storage of
  a small piece of tissue.
• I suggest a feasibility study is done by someone
  at Skogforsk.
• Do not forget that it is also politically correct.

11

• Markers help to study gene-flow, alien genes,
  domestication of genes, pollen flight in natural
  forests, genetic structure of natural forests,
  immigration history, autochtony. Skogforsk could
  stimulate that such studies by Universities give
  relevant information for forestry.

12
Breeding without breedingPolycross breeding etc.

• I talked about that in Ekebo May 2007 and
  presented ideas in detail, I will not repeat that
  now, but magnification of some details will be
  given in Ekebo May 2008.
• I work on it with Xiao-Ru Wang, we got funds for
  a minor Scots pine study, which is going on, some
  material is planned to be collected outside Umeå
  10 June, and we apply for a Norway spruce study
  and got funding for some pilot work (e.g.
  material collection), we have bought tickets for
  Ekebo 28-29 May to collect material (Bo
  Karlsson).
• It is possible to use polycrosses for selection
  forwards. I do not recommand to think about this
  as a major strategy, but I recommand to compose
  pollen mixes so this is feasible, if PC anyway is
  done (I told how a year ago).

13
Rekommendation för BWB

• Det är ganska troligt specialfall av BWB kommer
  till praktisk användning i operativ förädling
  inom tio år thus within the period of study.
• Genomslag kan ske snabbt (no real risk involved,
  need not wait a full breeding cycle for full
  scale implementation, actually some ideas can be
  immediatly implemented if they work)
• I do not predict a large scale break-through
  within the next four years, but it is not
  excluded.
• Skogforsk understöd forskning och stimulera
  forskning på skarpa material. Have a
  preparedness to implement it for real breeding.

14
Markers for associations
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Marker aided selection

• Important genes may not be identifyable and
  reproducible, it may just appear so.
• Negative genes (inbreeding) may seem important,
  thus MAS effort may result in weeding of
  unimportant genes rather than selection for
  important
• Important gene in one genetic and physical
  environment may not be important in another (thus
  a lot of possible interactions not considered in
  simple experiments).
• Unstable genes
• Field performance is the added action of all
  important genes, MAS gives just a small part of
  them.
• Higher selection effort for one thing means lower
  for all others. The breeding stock is usually a
  limited resource.
• A large added administrative trouble, costs for
  sampling and preparation, costs for analyses,
  costs for compiling data from different sources.
• If one start select for good DNA-pieces, it is an
  evident risk of faster accumulation of coancestry
  and subsequent inbreeding.
• Growth in mature age may be the most important,
  and probably too composite and complex to locate
  on the gene map.
• Not sufficiently dense maps yet for Swedish
  conifers Insufficient polymorphy.
• Riskier to change influence of one piece on the
  genome beyond the natural variation, than to get
  the same effect on production value by many small
  changes in different parts of the genome
• Genes may have many effects so a MAS indication
  does not eliminate the need for field test,
• Yet forbiddingly expensive.
• Etc

16
MAS

• 20 years of QTL research gave us good insight
  about genome organization, but no meaningful QTL
  to hang the hat on.
• FAO conference 2003 on MAS. From summary 2.7
  Practical applications of MAS Although
  documentation was limited, the current impact
  seemed small   
• Efforts have been going on for at least a decade
  for forest trees in Sweden, without coming near
  to an application.
• At least one scientist reasearching it in Sweden
  (Reza Yasdani) got the sack and few of the
  pioneers in MAS applying it to forest tree
  breeding is with us any more

17
Not extreme close linkage

• Cis- and trans- different in different families
• Different associations in different materials

18
Associations (populationwide linkage
disequilibrium)

• Usually not large effects
• It will probably take some time (10 years) till
  the pine and spruce genome will be sufficiently
  densily covered.
• Not that mature field, so remains to see what
  will come. But linkage disequilibrium may mark
  more than a family, it may be enough to get the
  fine scaled gene mapping on the population level.
• It may give us multi-locus markers, which would
  be much more powerful for e.g. BWB than e.g.
  micro-satellites.

19
I searched my 50 first hits on the webI also
looked in Wicopedia

• Association mapping I found no successful
  breeding operation
• QTL I found no successful breeding operation
• (the limit to successful may be debated)
• Neither any student in the research school could
  point at a successful commercial application.

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Place in system

• MAS research should not be seen as applied and
  motivated by the possible practical applications.
• Instead it should be viewed as basic research.
• Guess it is on way down internationally as both
  basic and applied research. (My feeling only.)

21
Domestic animals

• Still large commercial investments are done
  relying on MAS variants in animals, but the real
  result is uncertain and badly documented.
• The documented positive experience with animals
  is rather meager, mainly single genes connected
  to resistance to diseases.
• Patrik gave a rather pessimistic view at his
  docentföreläsning.

22
Agricultural Plant Breeding

• Usually they use nearly homozygous lines which
  are crossed rather seldom. Markers helps to keep
  track of known genes after such recombinations.
  But foresters do not use lines or known genes.
• A rather large share of the breeding efforts is
  to make varieties marketable, thus homogenous,
  stable and distinct, and markers help in that
  respect. But such demands are not pressing for
  forest varieties.

23
MAS conclusion

• Will not be implemented in operative breeding
  within ten years
• The prognosis considering the large investments
  under long time seems not good. Suggest it is
  seen as basic research, not applied.
• Associations (disequilibriums) is a possible
  development, but still farer away to operative
  breeding.
• Associated genes may be useful as markers but
  maps are still not dense enough
• I suggest no support, not even mentally, from
  Skogforsk. Off course Skogforsk is always helpful
  with being a discussion partner, in particular
  help with identifying and access to material, and
  is willing to assist practically for costs paid,
  but not beyond that.

24
Molecular diagnostic tools

• The state of plants not always directly coupled
  to the genes that control the physiological
  process will be easier to record with molecular
  symptoms. New measurement devices for relevant
  characters can be foreseen. Then they appear and
  has left the development stage Skogforsk can test.

25
GMO

• That GMO will not be implemented in operative
  breeding within the next decade I consider so
  uncontroversial within Skogforsk, so I will not
  talk much about the opportunities with GMO.
• There are full GMO presentations on my web if
  someone is interested, and I have given them, so
  many of you have listened. My time for
  presentation is limited.
• I will instead talk about what I think Skogforsk
  should do the next ten years.

26
UPSC has now a release withGMO trees at Umeå
(Stefan Jansson)
The genotypes are not hardy, in greenhouse in
winter, so it is only a semi-release!
Perhaps the only outside GMO trees in whole EU
2006?
27
GMO is bad PR, therefore Skogforsk should not be
associated with the first batches of GMO field
trials

• As there are no field trials and no plans for
  real field trials it is no sacrifice. Just that
  there are no field trials yet is a sufficient
  argument that GMO will not be in operative
  breeding in ten years.
• Swedish GMO field trials should first be managed
  by governmental institutions (SLU and its
  Research Parks).
• To manage GMO trials is a skill and competence,
  which need to be developed at SLU before
  Skogforsk takes over and learn from SLU
  experiences.

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• The first batches of GMO field trials has a too
  basic character to be of interest for the applied
  Skogforsk.
• If Skogforsk becomes associated with GMO field
  tests, it will give the public and the granting
  agencies the false impression that practical
  implementation is near and supported by Skogforsk
  and its breeders. The propaganda from the
  Scientists will magnify that impression and to
  overselling of the possibilities.
• GMO field trials are likely to get unwelcome
  attention and visits.

29
GMO trees under attack
The only field trial with GMO trees of relevance
for Scandinavian forestry (birch at Punkaharju)
was visited by activists in July 2002. Warning
signs were posted, but no other harm.
30
GMO trees under attack
The trial was destroyed June 20, 2004. The
seedlings were broken off or ripped from the
ground.
When the vandalism was reported to the police,
local greenies reciprocated by reporting the
Institute for unsafe management and for
unreported material in this field experiment.
31

• In BC seed orchards and Yousry El-Kassabys car
  were vandalized as greenies thought these in some
  way were associated with GMO. Therefore the
  street address of the BC manager visiting our
  seed orchard conference is secret.
• Green certification generally do not approve GMO.

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• State and governmental agencies can deal with
  these PR problems. For university scientists
  funding or reputation it seem not to be a
  problem, they can trustworthy claim that it basic
  research and they got the advantages of appearing
  futuristic and in the frontline.
• For associations like Skogforsk close to forestry
  with real forests, GMO can cause large problems,
  and not just for those directly involved, but
  innocent bystanders may also be hit.

33

• Actually GMO IS the future, but the VERY far
  future. To evaluate the GMOs and bring them to
  use we need field trials. So verbally Skogforsk
  may encourage GMO field trials and development of
  competence to manage them at SLU.
• When the first batches of real GMO field trials
  has been out a number of years without trouble,
  Skogforsk may revise the policy.

34
Vegetative propagation somatic embryogenesis of
Norway spruce

• Hundreds of SE clones are under testing
• One percent of the southern US pine market is SE.
  The plants are sold to the public for 2-3 SEK.
• Field trials with SE southern pines develop
  normal
• STT invests in somatic embryogenesis.
• I cannot see a reason why STT could not set up a
  unit in Sweden if forestry pays 2 SEK more for a
  plant which increases production 20. The unit
  can be build immediately, as the biggest problem
  is if forestry wants to pay for the gain, there
  is not reason waiting. The largest and safest
  gain comes from mass-multiplication of families
  with selected parents, do not wait for tested
  clones! The mass-multiplied families will plow
  the way for the tested clones.

35
A million SE plants. Plum Creek Nursery in Georgia
Photo Dag Lingren Sept 06
36
Plantation with SE trees Sept 06 in Georgia
Photo Dag Lingren Sept 06
37
Transplantation maching, SE miniplugs are
transplanted to open land. Nursery in Georgia
Photo Dag Lingren Sept 06
38
Skogsbruk med SE-kloner

• Det torde vara tekniskt och biologiskt möjligt
  att använda SE-kloner av gran i operativt
  skogsbruk inom några år så att produktionen
  förbättras. Merkostnaden torde kunna pressas till
  vad den är på marknaden i USA, 2 kronor/planta
• En eller några procent av plantmarknaden är
  ekonomiskt tillgänglig med dagens teknik. Resten
  tycker det är för dyrt och komplicerat. Den
  tillgängliga marknaden är för liten för att vara
  värd att investera för.
• För att marknaden skall bli tillräckligt stor
  fordras
• antingen att merkostnaden blir obetydlig eller
  noll. Det är ingen orimlig tanke att artificiella
  klonade industriellt framställda frön skall kunna
  blir billigare än fröplantagefrö. Så detta är
  värt att satsa på långsiktigt!
• eller att skogsbruket radikalt uppvärderar vad
  den genetiska vinsten är värd. Det är ingen
  orimlig tanke att det går att framställa
  SE-system så detta går.
• Det är realistiskt att tro att man om man
  omedelbart investerade på en SE anläggning med
  CellFor modellen skulle kunna få fram 10 miljoner
  granplantor per år 2011 som kunde säljas för 3
  kronor styck och därigenom göra investeringen
  lönsam. Jag föreslår att storskogsbruket nu
  tecknar kontrakt med STT att köpa 2 millioner
  småplantor per år för omskolning till containers
  under 2010-2016 för 3 kronor per omskolningsbar
  småplanta för att driva fram en tekniskt
  utveckling som kan användas när de testade SE
  klonerna kommer. Jag föreslår att detta system
  planeras för nordeuropa och inte enbart Sverige,
  så man får en större marknad för dyra plantor.
  Pluggarna transporteras i kyltransporter över den
  amerikanska kontinenten, man kan göra en liknande
  distribution i Europa. Iden på kort sikt är
  uppblåsning av de bästa fröna som ändå kommit
  fram i granförädlingen 2006. Övergången till
  testade kloner kan ske gradvis allteftersom
  säkrare testresultat kommer fram.

39

• If SE reaches the praxis in some scale I also
  suggest that Skogforsk consider changing
  technique for clone test to SE.
• I suggest that Skogforsk also encourages forestry
  to classical vegetative propagation by cuttings.