Realistic population and molecular genetic tools for genetic assessment (a simple minded, but pragmatic view!) Brian Ford-Lloyd

What will I talk about? 1. Definitions 2. Molecular and Popgen Background 3. The CWR list 4. Easy guides to erosion 5. Genetic pollution 6. Application of methodologies 7. CBD targets 8. When to do Molpopgen?

1. Definitions Genetic erosion –a permanent reduction in richness or evenness of common localized alleles –or the loss of combinations of alleles over time in a defined area ( after Guarino) Genetic pollution –gene flow from transgenic (or non- transgenic ?) crops to natural populations

2. Some background sampling variation or drift causes loss of genetic variation in small populations effective size, N e, not actual size N, determines the rate of this loss in natural populations N e is less than N difficult to obtain an estimate of the ratio N e /N

An example of N e /N Papaver dubium: –50% of all seed set from 2% of plants –This gives a ratio of N e /N of 0.07 –Papaver plants set 75% of seed by self- pollination –Ratio reduced to 0.024

Minimum viable population (MVP) ‘The minimum size of a population which will allow us to reduce the loss of genetical variation and heterozygosity by the inbreeding caused by drift to an acceptable level’ (there are several other definitions)

In situ conservation for species like P. dubium a population size of N = 5000 is reasonably safe populations of herbaceous CWR such as wild wheat, in which the density of plants is often around 10 per m 2, occupy only about 500 m 2 of ground for a tropical forest dipterocarp (density can be as low as only 2 per km 2), a population of this size would require a reserve of 2500 km 2 !!! wild Beta N e ? wild Beta N e ?

Important information at the gene level Effective population size –Level of heterozygosity –Inbreeding Change in allele frequency Genetic diversity Allele richness Gene flow Genetic drift –genetic erosion Natural selection –erosion and pollution Migration –pollution (erosion) or replenishment

Molecular markers/DNA profiling Arbitrarily primed markers (RAPD/ISSR) -? AFLPs -? SNPs/DNA sequence -? EST based markers -? isozymes/allozymes SSRs (microsatellites) –Yes because they are co-dominant, but.....

Are there primers available for species on the CWR list? Out of 160 random CWR taxa (genera) surveyed: 29% had SSR primers available in the published literature

Acer Aegilops Albizia Allium Arachis Armeniaca Asparagus Avena Begonia Cannabis Castanea Citrullus Corylus Cynodon Dianthus Diplotaxis Elymus Eruca Eryngium Festuca Ficus Fragaria Geum Gossypium Iris Lactuca Lolium Lupinus Malus Nigritella Olea Pelargonium Pinus Plantago Prangos Prunus Pterocarya Ribes Rosa Salix Sinapis Sophora Sorbus Taraxacum Tripolium Vicia Vitis Zostera CWR genera with SSR primers:

Abutilon Achillea Aconitum Agave Agrostis Alternanthera Amygdalus Anthericum Apium Aquilegia Artemisia Atriplex Atropa Axonopus Berberis Bituminaria Broussonetia Calliandra Capparis Carum Ceratonia Chamaecytisus Chamaemelum Chrysanthemoides Cichorium Cleome Colocasia Consolida Convallaria Cordia Corynephorus Crocus Cryptotaenia Cynara Cyperus Dalbergia Daucus Digitalis Drosophyllum Elaeagnus Epimedium Fagopyrum Flaveria Furcraea Galega Halimodendron Hedera Helleborus Hippocrepis Hydrocotyle Iberis Imperata Isatis Juglans Juncus Laurus Lavandula Lens Leucaena Limodorum Linum Lotus Ludwigia Maclura Matteuccia Melissa Mentha Mercurialis Monochoria Myrrhis Narcissus Nasturtium Nigella Onobrychis Ornithopus Osmunda Papaver Parietaria Phacelia Phalaris Phleum Phoenix Phyla Poa Polemonium Portulaca Ranunculus Rhododendron Rubus Rumex Sagittaria Salvia Sambucus Santolina Scilla Securigera Sesleria Silphium Stachys Syringa Tetragonia Thymus Trapa Trigonella Tulipa Vaccinium Valerianella Vallisneria Verbena Vincetoxicum Viola Vulpia Xanthium CWR genera without primers:

3. The CWR list The CWR list has around 20,000 species In theory we could undertake detailed genetic assessment of genetic erosion/pollution on over 6000 taxa using SSRs If we wanted to, and had the resources and

The key issues might be: How can we assess the majority of our CWR species simply and easily? –and minimise genetic erosion/pollution –and maximise genetic diversity in in situ conservation How do we prioritise the taxa for molecular population genetic intensive study? –(workshop 2?) A huge task!

4. Simple and easy guides? Information on breeding system –around 80% of diversity is within populations of outbreeders –most diversity is among populations of inbreeders

Further simple guides: Effective and actual population sizes are not the same But, actual population size can be a rough guide Will give us an idea about erosion if actual population is getting smaller

Resampling? If populations are staying the same size, then molecular population genetic analysis may be needed only once If population size is decreasing, then may need resampling - when? how often?

Other simple guides: Taxonomic diversity –assuming diversity is spread across taxa, ensuring that subspecific taxa are conserved should ensure that diversity is conserved Ecogeographic diversity –populations that have different adaptive norms will be genetically diverse Red data listing –what genetic information is revealed?

5. Genetic pollution “It is clear that spontaneous hybridisation and introgression of genes from domesticated plants into wild relatives is a common characteristic of domesticated plants” –Ellstrand, 1999 conventional or transgenic

Genetic pollution: The Gene Pool Concept will provide an indicator of the CWR species that are vulnerable, but 22 out of 25 of the World’s most important crops have evidence of natural hybridisation with one or more wild relative This could extrapolate to over 18,000 (90%) of our CWR species wheat rice maize soybean barley sorghum millet cotton rape beans sunflower potato sugarcane cassava oats coconut coffee cowpea rye oil palm sweet potato olive grape wheat rice maize soybean barley sorghum millet cotton rape beans sunflower potato sugarcane cassava oats coconut coffee cowpea rye oil palm sweet potato olive grape

Can genetic pollution affect genetic diversity? gene flow can cause change in genetic diversity –in 12 different studies, diversity in introgressed populations was greater can gene flow cause extinction? –more data are needed –it is ‘speculated’ that hybridisation may have caused extinction of CWR of Capsicum, date palm, hemp, maize, sweet pea Data not involving transgenes Data not involving transgenes

6. How to apply assessment methodologies? Other prioritisations first, then - are any subspecific taxa seriously threatened? are any major habitats/regions threatened? are most populations’ sizes declining (outbreeding species) ? are some populations’ sizes declining (inbreeding species) ? do sampled populations contain significant genetic diversity? –if yes, then only re-sample if change in population size

Monitoring genetic pollution? Bottom line - measure gene flow –need F ST and molecular markers Could assess: –occurrence of hybrids and hybrid derivatives (morphological) –fitness of hybrids/hybrid derivatives –spread of hybrids/hybrid derivatives Must be over large timescale, large geographical area, large sample size

7. CBD 2010 targets and WS5 A: Focal area Status and trends of the components of biological diversity Threats to biodiversity A: Focal area Status and trends of the components of biological diversity Threats to biodiversity B: Indicator for immediate testing Trends in abundance and distribution of selected species B: Indicator for immediate testing Trends in abundance and distribution of selected species C: Possible indicators (require further development) Trends in genetic diversity of.... cultivated plants... Number and cost of alien invasions C: Possible indicators (require further development) Trends in genetic diversity of.... cultivated plants... Number and cost of alien invasions CWR list & Euro+Med CWR list & Euro+Med

8. When to do molecular population genetics? if most populations’ sizes are declining (outbreeding species) ? if some populations’ sizes are declining (inbreeding species) ? –and/or because any one major habitat/region is threatened –and/or because any subspecific taxon is seriously threatened then sample and do molpopgen to establish whether populations in protected areas are adequate, or which populations to protect Red Data Listing? Red Data Listing?

Don’t - plan to do molpopgen first Do - molpopgen last or even not at all –when other guides have been examined –when other assessments have been done Do - use molpopgen as last resort to: –select best populations for in situ conservation –monitor populations or critical situations Don’t - use molpopgen to prioritise CWR list! A realistic message?