Seed Sampling Techniques and Population Size V.L. Bradley and R.C. Johnson Western Regional Plant Introduction Station

Bulk sampling was used in grass regeneration nurseries until Central Ferry, Washington Grass Nursery, 1997

Bulk Sampling All seeds from each plant are combined

Lolium multiflorum study Annual grass Three accessions Original seed (R0) regenerated for 3 yrs (R1, R2, R3) Seed sampling methods: Bulk Balanced

Types of Sampling Bulk sampling: plants are harvested without regard to variation in seeds per plant. This is a major factor reducing Ne well below Nc. Balanced sampling: an equal number of seeds are collected from each plant. Controls maternal effects but not paternal effects (pollen). Helps to maintain Ne.

Balanced Sampling Same number of seeds from each plant

Lolium multiflorum study Analysis of 8 isozymes completed on each population Calculated: Allele frequency Heterozygosity Average alleles per locus and % polymorphic loci Tested for differences in frequency between the original seed population and each of the regeneration populations

This work indicated that generally, the combination of bulk sampling and increased regeneration cycles led to more frequent changes in allele frequency from those in the original populations.

Lolium multiflorum field study Morphological data gathered on each plant: --Heading date and anthesis date for each plant --Spike length --Length, width and area of the penultimate leaf from the spike measured for length --Leaf was dried and specific leaf area was calculated --Above ground biomass was rated from 1 (least) to 9 (most) In one block at each location actual biomass was measured so a linear equation between the ratings and actual biomass could be established and used to calculate dry weight values per plant.

Population Regeneration cycle BiomassLeaf lengthLeaf areaLeaf dry wt. g per plantcmcm 2 mg OriginalR BalancedR R R BulkedR R2154*22.6†12.8*55.9* R3145*22.5†12.1 †50.6 *, †, Means are different from the original population at P=0.05 and P=0.10, respectively. Mean differences between the original seed population (R0) and samples that were balanced and bulked for three regeneration cycles in three Lolium multiflorum accessions.

Is harvesting balanced samples practical in most regeneration nurseries? NO…….

Effective Population Size The effective population size (Ne) rather than the census population size (Nc) is the key parameter in genetic drift. It is the size of an ideal population that would have the same amount of random genetic drift as the actual census population. Ideally Ne/Nc=1 when each parent contributes equally to the gamete pool. In most cases Ne<Nc.

2000- Sampling Method Study Three sampling methods were used on three accessions of three species of cross-pollinated grasses grown at two locations.

Types of Sampling Bulk sampling (cut and rub): plants are harvested without regard to variation in seeds per plant. This is a major factor reducing Ne well below Nc. Inflorescence sampling: bulk sampling in which a constant number of inflorescences are harvested per plant Sampling Method Study

Counting the number of inflorescences on each plant.

Cut harvest method- each plant cut at maturity and placed in a bag. Rub harvest method- each plant visited numerous times until it was judged that all seeds had been harvest. Cut and rub seeds were weighed to obtain yield per plant. Inflorescence sampling- two heads judged to be physiologically mature were taken from each plant. Each were bagged separately in order to estimate variation in seeds per head. In addition to the 3 species in the experiment, four accession of six other species were surveyed using only this method Sampling Method Study

Relationship between the ratio of effective to census population size and increasing inflorescences sampled per plant averaged for four accessions each of six cross- pollinated grass species.

Summary Sampling a constant number of inflorescences per plant can significantly improve effective population size by reducing the variation in seeds per plant. The major benefit is derived after just a few inflorescences. The technique can be applied to regeneration of outcrossing species and self pollinators that are heterogenetic, and when field collecting germplasm.

Contact Richard Johnson if you are interested in developing an inflorescence sampling curve for your crop.

Johnson, R.C Genetic structure of regeneration populations of annual ryegrass. Crop Sci. 38: (For abstract: Johnson, R.C., V.L. Bradley and M. A. Evans Inflorescence sampling improves effective population size of grasses. Crop Sci. 44: (For PDF file: Johnson, R.C., V.L. Bradley and M. A. Evans. Effect of seed sampling method during regeneration on genetic population structure and growth of model ryegrass populations. Plant Genetic Resources Newsletter, in press. (March, 2006) For more information on these studies….

Practical Considerations

Not practical for all accessions (tangled heads, heavy shattering) We’re still refining our techniques. We record the harvest method used and how many heads we cut, on the harvest label. This data is collected each year along with seed weight and volume so we can assess how well our choice of harvest method worked. This is helping us form guidelines for species we grow routinely. Did we get enough seed from the number of heads we cut? Could we harvest fewer heads for this species?

Harvest Method Guidelines

The Good Increasing effective population size Faster harvesting than we thought Seed cleaner can work through harvested materials faster because not cleaning an overabundance of seed The Bad Lots of seed left in field- volunteers The Ugly (challenges) Time consuming- lots of planning needed, walking field during spring busy season Keeping abreast of shattering is always a challenge, but not much flexibility with I-sampling Record keeping can be a nightmare WHAT DO WE DO ABOUT ACCESSIONS WITH LOW PLANT NUMBERS?

How do you deal with: Accessions for which there are only a few seeds Accessions that have “zero” (or very low) germination but there are thousands of seeds What is the minimum population you put in your nurseries? How do you decide what is worth the extra time/money (resources)? What do you do when you have one plant of a cross-pollinated species? Minimum population size Discussion questions