1. Male reproductive investment and success in a Larix occidentalis seed orchard population Tomas Funda Faculty of Forestry, University of British Columbia, Canada tfunda@interchange.ubc.ca

2. Outline Introduction – Seed orchards – Fertility variation Materials and Methods – Western larch clonal seed orchard – Microsatellite DNA paternity analysis Results – Regression analyses – Genetic gain and diversity estimates Summary – Benefits of the simplified methods

3. Seed orchards  Artificial populations of genetically superior trees  Mass production of improved seed http://www.hupptreefarm.com/seed_orchard.htm

4. Genetic gain  Parental breeding values  Reproductive success  Pollen contamination  Number of parents  Reproductive success  Mating pattern  Co-ancestry  Inbreeding Genetic diversity © John Marshall

5. Reproductive investment vs. success Investment  Female Number/volume/weight of seed cones Number of seed per cone Proportion of filled seed  Male Number/volume of pollen buds/cones

6. Reproductive investment vs. success ??? Investment Success unknown

7. Reproductive investment vs. success ??? Investment Success unknown

8. Reproductive investment methods M 0 : Male reproductive success is assumed to be equal to parental representation (i.e., number of ramets per plus tree) Example: two parents in a seed orchard parent #1: 9 ramets parent #2: 1 ramets  then parent #1 assumed to produce 90% of successful male gametes in seed crop whereas parent #2 only 10% or

9. Reproductive investment methods M 0 : Parental representation M 1 : Male reproductive success is assumed to be proportional to crown volume or age of a particular parent

10. Reproductive investment methods M 0 : Parental representation M 1 : Crown volume or age adjustment M 2 : Visual assessment of pollen production on at least 50% of ramets (partial pollen survey) M 3 : Visual assessment of pollen production on every ramet (full pollen survey)

11. Seed orchard Species: western larch, 1 st generation Location: Vernon, British Columbia, Canada (50°14’N, 119°16’E, 480 m a.s.l.) Established: 1989 Population size: 41 parents (as of 2005)

12. Plant material  Parental population young foliage from 41 parents

13. Plant material  Parental population young foliage from 41 parents  Offspring population 37 half-sib families dormant vegetative buds from 2-year-old seedlings 3-cm-long embryos

14. Reproductive success  10 nuclear microsatellite DNA markers multilocus probability of exclusion ≅ 1

15. Reproductive success  10 nuclear microsatellite DNA markers  Paternity assignment using CERVUS 3.0.3 Maternal parentage known Unassigned offspring – contamination – insufficiently informative genotypes

16. Reproductive success  10 nuclear microsatellite DNA markers  Paternity assignment using CERVUS 3.0.3  Linear regression using SAS 9.1.3 Dependent variable – DNA-based male reproductive success (baseline) Independent variables – fecundity scores for each simplified method (M 0, M 1, M 2, or M 3 )

17. Reproductive success  10 nuclear microsatellite DNA markers  Paternity assignment using CERVUS 3.0.3  Linear regression using SAS 9.1.3 Dependent variable – DNA based male reproductive success (baseline) Independent variables – fecundity scores for each simplified method (M 0, M 1, M 2, or M 3 )  Evaluation based on R 2, RMSE, and 95% prediction intervals

18. Reproductive success  10 nuclear microsatellite DNA markers  Paternity assignment using CERVUS 3.0.3  Linear regression using SAS 9.1.3 Dependent variable – DNA based male reproductive success (baseline) Independent variables – fecundity scores for each simplified method (M 0, M 1, M 2, or M 3 )  Evaluation based on R 2, RMSE, and 95% prediction intervals  Impact on genetic gain and diversity Error? Bias?

19.  Does parental representation reflect actual male reproductive success? Questions  Is male reproductive investment assessed through a field survey a good indication of actual male reproductive success?  Does this assessment provide unbiased estimates of crops’ genetic parameters, such as gain and diversity?

20. Questions Genetic diversity  status effective number (N e ) – fertility variation – co-ancestry  Does parental representation reflect actual male reproductive success?  Is male reproductive investment assessed through a field survey a good indication of actual male reproductive success?  Does this assessment provide unbiased estimates of crops’ genetic parameters, such as gain and diversity?

21. Pedigree reconstruction  Total number of analyzed offspring: 2088  Assigned to one of the 41 candidate fathers: 1848 (88.5%)  maximum pollen contamination 11.5%  Selfing 7.6%

22. Methods’ evaluation M 0 : Male reproductive success is a function of parental representation R2R2 RMSE M0M0 0.2750.072 M1M1 0.4990.060 M2M2 0.7020.046 M3M3 0.7310.044

23. Methods’ evaluation M 1 : Male reproductive success is a function of parental representation adjusted for crown volume R2R2 RMSE M0M0 0.2750.072 M1M1 0.4990.060 M2M2 0.7020.046 M3M3 0.7310.044

24. Methods’ evaluation M 2 : Male reproductive success is a function of male fecundity scored on 50% of trees (partial pollen survey) R2R2 RMSE M0M0 0.2750.072 M1M1 0.4990.060 M2M2 0.7020.046 M3M3 0.7310.044

25. Methods’ evaluation M 3 : Male reproductive success is a function of malefecundity scored on all trees (full pollen survey) R2R2 RMSE M0M0 0.2750.072 M1M1 0.4990.060 M2M2 0.7020.046 M3M3 0.7310.044

26. Methods’ evaluation M 2 : Male reproductive success is a function of male fecundity scored on 50% of trees (partial pollen survey) R2R2 RMSE M0M0 0.2750.072 M1M1 0.4990.060 M2M2 0.7020.046 M3M3 0.7310.044 correlation = 0.97

27. Crops’ genetic parameters

28.  Does parental representation reflect actual male reproductive success? X No, it does not due to substantial male fertility variation. Questions

29.  Is male reproductive investment assessed through a field survey a good indication of actual male reproductive success?  Does parental representation reflect actual male reproductive success? X No, it does not due to substantial male fertility variation. Yes, it is.

30.  Does this assessment provide unbiased estimates of crops’ genetic parameters, such as gain and diversity? Yes, it does, at least in western larch. Questions  Is male reproductive investment assessed through a field survey a good indication of actual male reproductive success?  Does parental representation reflect actual male reproductive success? X No, it does not due to substantial male fertility variation. Yes, it is.

31. “Congruence between parental reproductive investment and success determined by DNA-based pedigree reconstruction in conifer seed orchards” Canadian Journal of Forest Research (in press)

32. Acknowledgement Co-authors: Cherdsak Liewlaksaneeyanawin Charles Chen Irena Fundova Chris Walsh Jack Woods Supervisor: Yousry A. El-Kassaby

33. Thank you for your attention

34.  Does this assessment provide unbiased estimates of crops’ genetic parameters, such as gain and diversity? Yes, it does, at least in western larch. Questions  Is male reproductive investment assessed through a field survey a good indication of actual male reproductive success?  Does parental representation reflect actual male reproductive success? X No, it does not due to substantial male fertility variation. Yes, it is.