Molecular and other biotech

Dag Lindgren

Uppsala 14 maj

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.

• 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 >20 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.

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)

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.

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.

• 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.

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.

”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.

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.

• 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.

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).

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.

Markers for associations

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

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…

Not extreme close linkage

• Cis- and trans- different in different families

• Different associations in different materials

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.

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.

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.)

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.

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.

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.

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.

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.

UPSC has now a ”release” withGMO trees at Umeå (Stefan Jansson)

Perhaps the only outside GMO trees in whole EU 2006?

The genotypes are not hardy, in greenhouse in winter, so it is only a semi-release!

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.

• 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.

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.

GMO trees under attackThe 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.

• 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.

• 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.

• 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.

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.

A million SE plants. Plum Creek Nursery in Georgia

Photo Dag Lingren Sept 06

Plantation with SE trees Sept 06 in Georgia

Photo Dag Lingren Sept 06

Transplantation maching, SE miniplugs are transplanted to open

land. Nursery in Georgia

Photo Dag Lingren Sept 06

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.

• 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.