ALTERNATIVE METHODS FOR STORING THE COLLECTION PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST

APPENDIX ALTERNATIVE METHODS FOR STORING THE COLLECTION PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST A Cryopreservation Pro Long term storage (Forsline, Towill et al 1998) Simple and space efficient(Forsline, Towill et al 1998) Low maintenance requirements (Forsline, Towill et al 1998) Desiccated dormant buds easily shipped (Forsline, Towill et al 1998) Grafted trees can be forced into early flowering (Forsline, Towill et al 1998) Con Less suited for low cold hardy varieties (Forsline, Towill et al 1998) US varieties more winter hardy (Forsline, Towill et al 1998) Different desiccation, freezing and thawing rates (cultivar specific) (Forsline, Towill et al 1998) Variable recovery rates (Forsline, Towill et al 1998) May lose some accessions Clonal integrity maintained (Forsline, Towill et al 1998) Desiccation damage (Volk and Walters 2003a) Material needs to be decontaminated (Volk and Walters 2003a) Some species need to go through tissueculture stage prior to cryopreservation Precultural conditions of both vegetative parent and explant material impact on success of cryopreservation (Helliot and deBoucaud 1997; Wu, Engelmann et al 1999; Wu, Zhao et al 2001; Volk and Walters 2003a) Cryopreservation diffusion rates in larger propagules may not be sufficient for survival after cryopreservation (Volk and Walters 2003a) Smaller explants susceptible to physical damage of extraction and exposure to toxic cryoprotectants (Volk and Walters 2003a) Physiological condition important for health and survival after cryopreservation (Wu, Engelmann et al 1999; Volk and Walters 2003a) Mature specimens not available for characterisation Back up systems needed in case of power/equipment failure Meristem cryopreservation necessary for less cold hardy species e.g Pyrus Expensive development work(Volk and Walters 2003a) Material not immediately available to growers and other users Long wait of years for production of flowers and pollen for breeding purposes (especially for Pyrus) Genetic changes/damage possible Not user friendly Development work on content of collection not possible Loss of staff to maintain field collection results in loss of technical backup and expert knowledge Cryopreservation of different types of material (Reed 2002) Dormant buds Readily available form field genebanks In vitro shoot tips Available at any time of the year Easy to manipulate physically and physiologically Embryogenic cultures Callus is generally easy to cryopreserve Degree of cold hardiness varies with reason and genotype Buds only available for a few moths during the winter Requires more storage space than other techniques Requires grafting and budding expertise for recovery Techniques are not developed for all plants Requires a laboratory and skilled workers Not all plants produce somatic embryos Genetic changes possible Techniques are not broadly applicable across species/accessions Embryonic axes Easy to remove and process In vitro systems needed to recover whole plants Cryopreservation; Future studies (Volk and Walters 2003a) • • • • Knowledge of biophysical properties of water within cells Physiological understanding of critical content for desiccation damage Hydraulic conductivity of water Thermal loads that effect cooling rates To determine the effect of the physiology of starting material on the cryopreservation of meristems Development work needed for each species to find optimum conditions for cryopreservation, thawing and recovery; (Slow freeze easier; large numbers can be processed; conditions less critical; toxicity lower and success rate usually higher Vitrification marginally better if carried out experienced person.) Understanding the stresses caused by drying, cooling, freezing and the effect of time on these processes A better understanding of the physiology and biochemistry of species to be cryopreserved and of meristem biology A better understanding of the effect of thawing on tissues Examination of genetic erosion: what happens during cryo storage and what happens during regeneration? Work on the nature of toxicity of cryoprotectants The investigation of beneficial elevated sugar levels in pre-culture The examination of the reason for vine buds being ‘very leaky’ The effect of electrolyte leakage on cryopreservability needs to be examined Evaluation of culture systems needed for recovery Acclimation - Do cells of accessions which can be cryopreserved, produce their own cryoprotectants? Do species which can be cryopreserved have the ‘right’ genes or susceptible species fail to turn on their genes Other questions: Ashworth, 1986 #74} How buds acclimate in the fall and increase in winter hardiness? What factors limit the hardiness of buds? What features distinguish a hardy from a non-hardy bud? What factors and conditions are required to maintain maximum bud hardiness? What controls the de-acclimation of flower buds and the progressive loss of cold hardiness? Notes US varieties may be more tolerant of cryopreservation because of genetic make up or exposure to harder winters producing hardening to a greater extent B Tissue Culture Pro Con Rapid multiplication possible if large numbers required Development work expensive and time consuming(Volk and Walters 2003a) No requirement for large land area High maintenance costs Not affected adverse weather Labour intensive Cultures easily transported International transport possible Accurate labelling at each subculture necessary (real danger of errors due to mislabelling) Pest and disease-free stocks maintained Propagation of recalcitrant species Danger of microbial contamination (Volk and Walters 2003b) Ease of rooting (especially for woody materials) Promotes endophytic bacteria (Reed, Buckley et al 1995) (Reed, Mentzer et al 1998) Rapid regeneration documented for many of NFC crops Stresses of media additives to control endophytic bacteria may decrease vigour and survival of cultures (Volk and Walters 2003a) Ease of germplasm exchange and shipment both within and between countries Mite/thrip infection possible Physiological changes with time/subcultures Somaclonal variation(Swartz, Galletta et al 1981) Ploidy problems Differences in growth and fruiting after regeneration Need light and temperature control Need sterile working area Delayed flowering Mature specimens not available for characterisation Future studies in tissue culture: Studies needed on the epigenetic effects (Phenotypic expression due to variation in expression) and on plant and hormone physiology Assessment of hormonal and nutritional needs required in vitro Further studies in developmental biology to examine the induction of roots, multiple shoots and flowers The control of endophytic bacteria The rejuvenation of woody species Episotic growth (eg in vitro Oak cultures) How to get recalcitrant species in culture Factors responsible for cold sensitivity of grape in vitro C Low temperature tissue culture Pro As for tissue culture above Maintenance and labour costs may be lower than for standard tissue culture above Less frequent subculture required Con As for tissue culture above, although maintenance and labour costs may be lower Need to go through multiplication and rooting media at 20oC (few months) before retuning to cold Still labour intensive Loss of some accessions Needs system for producing vigorous shots free of contaminants/endophytes prior to cold storage Development work: At present, Ribes found to be too ‘dirty’ to be stored in cold storage tissue culture Need to develop method of removing surface contaminants and endophytes Pers Comm Janine de Paz, (NCGR, Corvallis, Oregon, USA) Factors effecting cold hardiness Factors effecting bud break and survival at sub-optimum levels The effect of reducing nutrient and sugar levels D DEFRA National Field Collection at Brogdale Pro Con Morphological characteristics readily recorded Labour intensive Samples of propagating wood, leaves, fruit and pollen available High cost Large acreage Allows accessions to be characterised Prone to attack from pathogens & pests Allows users to select most appropriate material (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e would reduce major cost) Effected by extreme environmental conditions Risk of vandalism Climate changes may eventually effect fruiting due to lack of winter chill for flower initiation (User of material supplied responsible for import permits and quarantines etc) Position of accessions fixed in collection for many years with site maps giving location Need for re-propagation and parallel collections M9 need stakes to support scions Easier for public to see and appreciate collection which may result in more monetary support from foundations or the public-ie PR exercise Note: At NCGR, accessions in pear collection cut back annually; either 25% off the top, or left or right hand side of rows cut back with hedge trimmer on consecutive years to reduce cost of maintaining trees at a manageable size, and keep trees rejuvenated Development work: Need good genetic markers to identify accessions in field and those coming out of cryopreservation Research on genetic diversity can help define core collections for field collections, so reducing collection size and costs More reliable identification (fingerprinting) of accessions which are relatively easy and cheap to run, are needed Research is needed to develop health procedures during the collection and introduction of new accessions into field gene-banks Also faster and more efficient ways of screening and disease indexing new accessions Research is needed to improve management and maintenance of field collections Development of low-input management strategies can help reduce costs of maintaining field collections General procedures for field collections include propagation methods, selection of planting sites, planting procedures, cultivation practices, disease and pest management and harvest and storage of propagules Monitoring the genetic stability of the crop requires careful vigilance on the part of the curator and field staff, careful roguing, labelling and protection of the plants from biotic and abiotic dangers are important to the safety of the germplasm (Reed et al, 2004) E Ultra Dwarfing Rootstock (e.g M27 for apples) Pro Con Less land required Prone to pests and diseases Material readily available Prone to vandalism Morphological characteristics readily recorded Prone to climatic changes Samples of propagating wood, leaves, fruit and pollen available May not show all characteristics of full-sized plants Allows accessions to be characterised Fewer flowers (for breeders) Allows users to select most appropriate material Less material available than standard trees Position of accessions fixed in collection for many years with site maps giving location (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e would reduce major cost) (User of material supplied responsible for import permits and quarantines etc) May not produce enough fruit for crosses on trees Greater intensity of care necessary Smaller root balls M27 need wire frames to support scion Shorter life Doyenne du Comice interstock probably needed for pears More frequent re-propagation and parallel collections Effect of cherry and plum dwarfing rootstocks (Gisela and Pixy) on many scion cultivars not known Development work: Need good genetic markers to identify accessions in field and those coming out of cryopreservation Importance of secondary structure of proteins for dried or frozen material Effect of cherry and plum dwarfing rootstocks (Gisela and Pixy) on many scion cultivars is not known Before using these very dwarfing rootstocks research will be needed to look at the long term effects of growing a wide range of scions on such rootstocks F Cordons (for apples and pears) Pro Con Less land required Prone to pests and diseases Material readily available Prone to vandalism Morphological characteristics readily recorded Prone to climatic changes Samples of propagating wood, leaves, fruit and pollen available More susceptible to Fire Blight May not show all characteristics of full-sized plants Allows accessions to be characterised Allows users to select most appropriate material Fewer flowers (for breeders) Less material available than standard trees Position of accessions fixed in collection for many years with site maps giving location (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e would reduce major cost) May not produce enough fruit for crosses on trees (but could use pollen instead) Greater intensity of care necessary Posts and wires needed (User of material supplied responsible for import permits and quarantines etc) Cherry and plums not well suited to cordon cultivation Development work: Need good genetic markers to identify accessions in field and those coming out of cryopreservation G Potted Plants Pro Con Can be transported to new locations Prone to drying out Less land required High regular commitment to watering Morphological characteristics readily recorded Large mature plants not possible Samples of propagating wood, leaves, fruit and pollen available May not show all the characteristics of mature plant Allows accessions to be characterised Periodic re-potting (Annual?) Allows users to select most appropriate material Labour costs of re-potting etc (Use of genetic markers/fingerprinting would Loss of labels especially during re-potting (mislabelling causes masses of work) reduce time with parallel collections, i.e would reduce major cost) (User of material supplied responsible for import permits and quarantines etc) Can be kept in a screen house to protect from pests and extreme climatic conditions Restricted access to public because of danger of label theft Shorter life (more frequent re-propagation) Disease is more of a problem Effected by extreme environmental conditions Risk of vandalism Climate changes may eventually effect fruiting due to lack of winter chill for flower initiation Notes: At NCGR, the problem of label loss is counteracted by pushing a second label, upside-down, completely into the ground at the edge of the pot (so that it is not obvious to the general public, and can also be retrieved if the visible label falls out by accident) Genetic fingerprinting of the whole collection would also help to overcome label loss Any doubts could be checked with the stored information H Seed Storage Pro Con Long term storage May lose some accessions Simple and space efficient Mature specimens not available for characterisation Low maintenance requirements Easy to use for appropriate seed types (orthodox) i.e small seeded desiccation tolerant , cold tolerant, not clonally propagated accessions (Reed 2002) Not useful for large, cold sensitive or desiccation intolerant seeds (recalcitrant) or clonally propagated plants (Reed 2002) Back up systems needed in case of power/equipment failure Development work on content of collection not possible Loss of staff to maintain field collection results in loss of technical backup and expert knowledge Seed increase process necessary when number of seeds in accession falls below 100 or viability falls below acceptable level (eg tests indicate that only probably 30 viable seeds left)-Peters ,J., NCGR Seed increase process time consuming – Peters, J., NCGR Note: A seed collection would be complementary to, but not a replacement for a field collection Development work: Developing a way of storing recalcitrant seeds Importance of secondary structure of proteins for dried or frozen or frozen seeds Effect of lipid composition of storage of seeds How changes in volatiles can indicate deterioration of seeds 10 I Pollen Storage Pro Con Long term storage (Towill and Walters 2000) May lose some accessions Simple and space efficient (Towill and Walters 2000) Mature specimens not available for characterisation Low maintenance requirements Back up systems needed in case of power/equipment failure Desiccated dormant buds easily shipped (Forsline, Towill et al 1998) Development work on content of collection not possible Easy for many plant genera (Reed 2002) Loss of staff to maintain field collection results in loss of technical backup and expert knowledge Useful for breeding (Reed 2002) J Leaf Storage Pro Long term storage Con Mature specimens not available for characterisation Simple and space efficient Low maintenance requirements Material readily available for DNA extraction Back up systems needed in case of power/equipment failure Development work on content of collection not possible Loss of staff to maintain field collection results in loss of technical backup and expert knowledge 11 APPENDIX Table A2.1 – Number of Malus accessions cryopreserved at the USDAARS National Seed Storage Laboratory, Fort Collins, CO, showing acceptable viability levels in recovery grafting at the Plant Genetic Resource Unit, Geneva, NY Species M pumila M kansuensis M kirghisorum M x adstringens M x robusta M toringoides M sieboldii M asiatica M prunifolia M sargentii M x soulardii M hybrids (crabs) M ioensis M pumiia M sikkimensis M sylvestris M yunnanensis M sieversii M baccata M floribunda M hupehensis M spectabilis M coronaria M halliana M transitoria M micromalu M x magdeburgensis M fusca M angustifolia M honanensis M florentina M tschonoskii M trilobata Total Cryopreserved 487 1 5 42 10 5 20 13 3 642 Viability > 40% 452 1 5 40 4 15 1 1 0 575 From Forsline, P L., J R McFerson, et al (1999) Development of base and active collections of Malus germplasm with cryopreserved dormant buds Acta Horticulturae; Proceedings of EUCARPIA Symposium on Fruit Breeding and Genetics K R Tobutt, Alston, F.H Oxford, England 484: 75-77 12 Table A2.2 - Number of Malus accessions cryopreserved and recovered by grafting in the active collection at the USDA-ARS Plant Genetics Resources Unit, Geneva, NY Species M pumila M micromalus M hybrid M baccata M prunifolia M x magdeburgensis M spectabilis M honanensis M coronaria M x adstringens M florentina Total Cryopreserved 102 1 1 1 121 Viability > 40% 100 1 1 0 115 From Forsline, P L., J R McFerson, et al (1999) Development of base and active collections of Malus germplasm with cryopreserved dormant buds Acta Horticulturae; Proceedings of EUCARPIA Symposium on Fruit Breeding and Genetics K R Tobutt, Alston, F.H Oxford, England 484: 75-77 13 APPENDIX DECISION FLOW CHART FOR CRYOPRESERVED STORAGE OF CLONAL GERMPLASM IDENTIFY PRIORITY GERMPLASM DETERMINE BEST STORAGE FORM PRIORITIZE ACCESSIONS FOR STORAGE CHOOSE AN APPROPRIATE TECHNIQUE SET UP DATABASE OF NECESSARY INFORMATION SET UP TESTING AND STORAGE PROTOCOLS LOCATE A SAFE AND REMOTE STORAGE SITE PLAN LONG-TERM MONITORING INITIATE BASE STORAGE Fig Flow chart for cryopreserving clonal germplasm From Reed, B M (2002) Implementing Cryopreservation for long-term germplasm preservation in vegetatively propagated species Cryopreservation of Plant Germplasm II L E Towill and Y P S Bajaj Berlin, Springer-Verlag 50: 22-33 14 Table A3.1 Storage forms for cryopreserved germplasm Best Storage Form Plant groups Advantages Disadvantages Seed Small seeded, desiccation tolerant, cold tolerant, not clonally propagated Easy to use for appropriate seed types (orthodox) Not useful for large, cold sensitive or desiccation intolerant seeds (recalcitrant) or clonally propagated plants Pollen Many Easy for many plant genera, useful for plant breeding Preserves only half of the genome Dormant buds Temperate woody plants Dormant buds are readily available from field genebanks Degree of cold hardiness varies with season and genotype, requires more storage space than other techniques, requires grafting and budding expertise for recovery In vitro shoot tips Many Available at any time of year, easy to manipulate physically and physiologically Techniques are not developed for all plants, requires a laboratory and skilled workers Embryogenic cultures Various plant groups Callus is generally easy to cryopreserve Not all plants produce somatic embryos, techniques are not broadly applicable across species/accessions Embryonic axes Some marginally recalcitrant seed types Easy to remove and process In vitro systems are needed to recover whole plants Zygotic embryos Some marginally recalcitrant seed types Removal of the seed coat may improve recovery Time consuming and often technically difficult From Reed, B M (2002) Implementing Cryopreservation for long-term germplasm preservation in vegetatively propagated species Cryopreservation of Plant Germplasm II L E Towill and Y P S Bajaj Berlin, Springer-Verlag 50: 22-33 15 APPENDIX ALTERNATIVE METHODS FOR IDENTIFYING GENETIC RESOURCES PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST A RFLP techniques (Restricted Fragment Length Polymorphism) Pro Con Powerful for distinguishing apple accessions (Weeden and Lamb 1985; Nybom 1990; Mulcahy, Cresti et al 1993) Not easy (Korzun 2003) (Use of genetic markers/fingerprinting would reduce time with parallel collections, i.e would reduce major cost) Not easy to automate(Korzun 2003) Co-dominant: -i.e contain twice as much information, in a genetic cross, as dominant markers (eg RAPDs) Large amount of DNA required(Korzun 2003) Can use already-existing cDNA clones as probes Needs high level of skill Need high DNA quality(Korzun 2003) Time-consuming and expensive, compared to other marker methods ; high cost per analysis(Korzun 2003) Requires many previously-cloned genes Can re-probe Southern blots many times Often only polymorphism per probe Highly-reproducible between labs (Korzun 2003) Low development costs(Korzun 2003) Probably the lowest success rate of any method for detecting polymorphisms Need to send probes between labs Not really high resolution in terms of the number of RFLPs per base pair B RAPD techniques (Random Amplification of Polymorphic DNA) Pro Con Successful discrimination of sports in future (Pancaldi 1999) Not very efficient in locating genetic differences among mutants Easy to use (Korzun 2003) Failure to distinguish between sports and original cultivar (Harada , Matsukawa et al 1993; Mulcahy, Cresti et al 1993) Do not need to clone anything to mark a locus; primers applicable to any species Low development cost(Korzun 2003) Low cost per analysis(Korzun 2003) Unreliable; differs from lab to lab and from year to year i.e Useless (Ken Tobutt, pers com.) Not reproducible High quality DNA required 16 Probably cheapest and easiest method for labs just beginning to use molecular markers RAPD bands can often be cloned and sequenced to make SCAR (sequencecharacterized amplified region) markers that are highly reproducible Not easily reproducible between labs Dominant: only half the genetic information as co-dominant markers Null alleles not directly detected The lack of reproducibility of RAPDs can be overcome by converting them to SCARS Often several polymorphisms (1.5 to 50) per primer (Korzun 2003) Only primer sequences are needed to define a marker Cloned probes are not necessary Moderately amenable to automate (Korzun 2003) No previous knowledge of genome required (Goulao, Cabrita et al 2001) Very small amount of DNA required per analysis (Goulao, Cabrita et al 2001) C Micro-satellites (SSR) (Simple Sequence Repeat) Pro Con Highly-reproducible between labs Easy to use (Korzun 2003) Large number of polymorphisms per primer set Often multiple alleles in a population, which can be highly-informative Co-dominant Difficult to find good micro-satellite markers in each species This problem being solved and method becoming increasingly easy High development costs Moderate DNA quality required (Korzun 2003) Fail to discriminate between sports and original cultivar (Gianfranceschi ,Seglias et al.1998; Hokanson, Szewc-McFadden et al 1998) Cost effective deployment Highly amenable to automation (Korzun 2003) Highly reproducible (Korzun 2003) Low cost per analysis (Korzun 2003) Relatively simple 17 Most markers are monolucus and show Mendelian inheritance Highly informative High number of public SSR primer pairs available Amplifies specific marker Linked to specific allele Suitable for diversity studies EST (expressed Sequence Tag) Facilitates development of micro-satellite markers (Varshney, Graner et al 2005; Varshney, Sigmund et al 2005) D Incompatibility alleles (S alleles) Pro Con Easy to detect using PCR May not be appropriate for Ribes Allele specific primers available In Pyrus, nomenclature confusing or ambiguous (ie multiple names for same genotype) Highly polymorphic Easy to score and convert into genetic interpretation Relatively cheap (Similar to micro-satellite) Rapidly progressing field Suitable for diversity studies Development work needed: Need to establish uniform nomenclature for Pyrus, 18 E Retro Transposan Tags (RTT) Pro Con Highly variable polymers Development of appropriate primer sequences Good for forensic studies Need skill (for new taxa) Has much of genome represented High development costs Can be linked to genes of interest Not good for phylogenic/historical studies Good for fine scale genetic structure Useful for cultivar identification The application of LTR Retrotransposons as molecular markers in described in detail by Schulman, Flavell et al (2003) F SNP (Single Nucleotide Polymorphism) Pro Con Easy to use (Korzun 2003) Inappropriate for detecting duplicates Highly amenable to automation(Korzun 2003) Highly reproducible(Korzun 2003) Low cost per analysis(Korzun 2003) ; similar to SSR system- getting cheaper High development costs (eg $40,000 to develop system for11 sequences for grapes) Only one polymorph per probe Can be used to detect alleles if linked to specific genotype Can be used for haploid type analysis Sequence information available for grapes and apple PGRU is the only place in US where sequence based resolution has been worked out (May 2005) Suitable for diversity studies 19 G AFLP techniques (Amplified Fragment Length Polymorphism) Pro Con No sequence information required Very large number of polymorphisms per reaction (20-100) Probably the most polymorphisms for the cost Straightforward and reliable Easy to use Moderately amenable to automation(Korzun 2003) High reproducibility (Korzun 2003) Moderate development costs Moderate cost per analysis Not-reliable to convert AFLPs into SCARs Proprietary technology Null allele not detected Moderate DNA quality required(Korzun 2003) Technically rather difficult May need radioactivity or silver staining (environmentally suspect) for running gels on plates but not if capillary separation used Difficult to interpret Difficult to convert to simple genetic/arithmetic information Data may need to be stored as images Difficult to reproduce with 100% reliability Could be useful for confirmation Difficult to compare patterns May be useful for disciminating clones 20 APPENDIX DNA marker information (supplied by Dr.Cameron Peace) Rosaceae is also considered the model family (and Prunus the model genus) for fruit/nut trees, To DNA fingerprint the entire collection, DNA extraction needs to be done first This is a relatively large initial cost (If the cherries at least were previously fingerprinted, then perhaps DNA samples can be obtained from East Malling Research.) Each accession should have its DNA extracted in duplicate, then after a few initial DNA tests, any blanks re-extracted, until two replicate samples are available for each accession Replicating (and running each side-by-side in the DNA tests to allow easy scoring) is important for a long-term collection such as the NFC, where users will want to test over and over again for many things The next concern is the type of extraction method to use There are long extraction methods, which result in very good quality DNA samples but typically less than ten can be performed by a single person per day, and there are quick methods, which result in lower quality DNA that is fine for most PCR tests (but not so good where restriction is required like in RFLP or AFLP) and 50 or more samples could be extracted per person per day (even a hundred or more, if leaf material has been previously prepared - freeze-dried then ground to a fine powder in a ball mill or some such) Given such a massive collection, a quick method is recommended as long as equipment exists to easily powder the leaves The marker systems to be used for DNA fingerprinting the collection depend on the desired outcome First there is just general identity and genetic diversity analysis where identity analysis would identify duplicate accessions, and then when profiles are compared across all typed individuals, a picture of genetic relationships/diversity emerges This could use multiplex dominant marker systems such as RAF or AFLP, or single-locus marker systems such as microsatellites Microsatellites are hyper-variable and have already been developed for most of these species, and in most cases, work has been done to combine these so that several can be run at once For these reasons, I strongly recommend this marker system for identity and diversity analysis of the collection The second desired outcome for screening the collection is to identify diversity in specific useful genes This requires that useful genes and their specific sequences are already known Good examples are the S-allele test for cherries (and other selfincompatible Prunus) and, endoPG for Prunus fruit softening There are several interesting genes already known for apple (esp for disease resistance), several for Prunus, and probably Vitis too Many of these gene tests are probably transferable, so some testing could still be done in those cases Then in the future as researchers in each of these crops identify new useful genes and develop tests for them, the collection can be screened using the DNA library Costs DNA extraction costs are mostly labour, 21 The number of accessions divided by the number of samples that can be extracted per day, multiplied by 150%, equals the number of man-days required This can be done by a technician-level scientist Running the DNA tests depends on the technology available An automated system that can run many hundreds of samples per day given the size of the collection, is recommended More automated marker systems Capital costs (1$=£0.55 June, 2005) These costs are approximate and for the US, The automated systems compared here are an ABI3100 and LiCor, perhaps the two most commonly used brands/machines The ABI is more efficient and more accurate, and considered the best on the market The ABI can be set up as a 16 (better for a smaller lab) or 96 (more expensive, better if there are many many samples) capillary system Cost to buy: $130,000 (LiCor or ABI with 16 capillary system), $500,000 (ABI with 96 capillary system) Maintenance costs: $10,000 pa (LiCor or ABI/16, probably proportionally more for the ABI/96) - this is basically an ongoing warranty but considered to be worth it Running costs: LiCor (cheaper, as most can be prepared in the lab): approx $0.50 per sample ABI/16 (must buy gel and other necessities from the ABI company): approx $1.25 per sample Plus $1000 per "array", where one array will last for 400-500 runs (so I calculate from this that with about 570 samples per day [= per run], you'd need a new array every 250,000 samples, which is less than a cent a sample Basically negligible One array would last for something like 50 to 70 separate DNA tests per sample, i.e 25 to 35 DNA tests per accession when everything is done in duplicate) ABI/96: $1.25 per sample, $4000 per array Samples per day (with one trained technician): LiCor: 95 samples, or can multiplex tests at a time (i.e 190 samples per day) ABI (16 capillary): 190 samples, or can multiplex up to tests at a time (i.e 760 samples per day for x4 multiplex) ABI (96 capillary): 96 samples with 30-36 multiplexing (i.e 3000-3600 samples per day) IF there were 30 DNA tests (perhaps 20 SSRs and 10 candidate genes) that were desired to be run across all ~4000 accessions of the collection Assuming the machines were already available/bought, and the DNA already extracted in duplicate per accession, then the costs for running the 16 capillary ABI would be: * No samples = 8000 DNA extracts x 30 DNA tests = 240,000 * No samples per day = 500 (with multiplexing, and allowing for reruns where necessary) * No 1-person days required = 240,000 / 500 = 480 * Labour costs = 480 days x $100-$150 per day (£50-70.) = $48,000 to $72,000 22 * Running costs = 240,000 x $1.25 = $320,000 * Total cost = $368,000 to $392,000 plus the yearly maintenance cost 480 days of continuously running the ABI probably difficult especially if more than 30 DNA tests are desired, so the 96 capillary system would probably be the best way to handle so many samples Same calculations for the LiCor: * No samples = 240,000 * No samples per day = 150 (with multiplexing, and allowing for reruns where necessary) * No 1-person days required = 240,000 / 150 = 1600 * Labour costs = 1600 days x $100-$150 per day = $160,000 to $240,000 * Running costs = 240,000 x $0.50 = $120,000 * Total cost = $280,000 to $360,000 plus the yearly maintenance cost A little cheaper, but takes a lot longer Same calculations for ABI/96: * No samples = 240,000 * No samples per day = 2500 (with multiplexing, and allowing for reruns where necessary) * No 1-person days required = 240,000 / 2500 = 96 * Labour costs = 96 days x $100-$150 per day = $10,000 to $15,000 * Running costs = 240,000 x $1.25 = $320,000 * Total cost = $330,000 to $335,000 plus the yearly maintenance cost Similar price but so much quicker, and most desirable when many different DNA tests are to be conducted over the years Same calculations for simple polyacrylamide gels (for simplicity assuming silver staining rather than radioactivity): * No samples = 240,000 * No samples per day = 75 (with multiplexing, and allowing for reruns where necessary) * No 1-person days required = 240,000 / 75 = 3200 * Labour costs = 3200 days x $100-$150 per day = $320,000 to $480,000 * Running costs = 240,000 x $0.75 = $160,000 * Total cost = $480,000 to $640,000 It is possible with the LiCor and polyacrylamide to multi-load each gel, allowing 4x as many samples to be run in a day This would reduce the number of days for the LiCor to 400 and the polyacrylamide to 800, and so the total costs for Licor would be $200,000-$220,000, and $240,000-$280,000 for polyacrylamide For all of the above, labour needs probably aren't quite realistic, because to prepare more than 100 samples a day, you'd need more people on the job Maybe 500 samples could be prepared per day per person, assuming a couple of PCR machines (~$5000 each to purchase) are available per person So for 240,000 samples, it would take perhaps 480 person-days, costing $48,000-$72,000, just to prepare the samples for the machines 23 APPENDIX The following have expressed a wish to be involved in any future work which may arise as a result of recommendations made in this report on ‘Safeguarding the national fruit collection’ Dr Nahla V Bassil, NCGR, 33447 Peorria Road,Corvallis, Oregon 97331, ASA Dr Angela Baldo, PGRU, Cornell University, Geneva, NY 14456, USA Professor Phil Forsline, PGRU, Cornell University, Geneva, NY 14456, USA Dr Ken Tobutt, , East Malling Research, Maidstone, Kent Radovan Boskovic, East Malling Research, Maidstone, Kent Dr David Smith, Curator of Genetic Resources Collection, CABI Bioscience, Bakeham Lane, Egham, Surrey, TW20 9TY Dr Nigel Maxted, School of Biosciences, University of Birmingham, Birmingham B15 2TT Cameron Peace, Kearney agricultural Center, 9240 S Riverbend Av., Parlier, CA 93648, USA Professor David C.Lynch, Professor of Plant Biotechnology, Biological Sciences, School of Education, Health and Science, University of Derby, Kedleston Road, Derby, DE22 1GB Dr Keith Harding, Conservation & Environmental Chemistry Centre, School of Contemporary Sciences, Kydd Building, University of Abertay, Dundee, Bell Street, Dundee, Scotland,DD1 1HG, Dr Rex M Brennan, Fruit breeding Group, Genome Dynamic Programme, Scottish Crop Research Institute, Inergowie, Dundee Scotland DD1 5DD 24 ... better understanding of the physiology and biochemistry of species to be cryopreserved and of meristem biology A better understanding of the effect of thawing on tissues Examination of genetic erosion:... ALTERNATIVE METHODS FOR IDENTIFYING GENETIC RESOURCES PRELIMINARY LIST OF OPTIONS - POINTS FOR AND AGAINST A RFLP techniques (Restricted Fragment Length Polymorphism) Pro Con Powerful for distinguishing... careful vigilance on the part of the curator and field staff, careful roguing, labelling and protection of the plants from biotic and abiotic dangers are important to the safety of the germplasm (Reed