1. The American Chestnut Tree

2. During today’s lesson students will be able to: 1. Explain how the backcross breeding method works and is helping to develop disease-resistant populations of new trees. 2. Perform a ‘mock’ genetic backcross. Student Objectives Sunday, October 11, 2015

3. Warm-Up Complete the following Punnett Square. In this situation you are crossing 2 pink flowers. In the flowers, the colors are co-dominant and the traits will blend. R- Red Flower r – White Flower R r R f r 1. Describe the phenotype of the offspring. 2. What are the genotypes of the offspring?

4. It’s in the genes!!! An Introduction to the Backcross Breeding Method Explore the following slides and think about the following as you go… 1.What methods have been used as attempts to save the American Chestnut? 2.Which traits are “desirable” in a Chestnut tree? 3.What is a Back-Cross?

5. Early Attempts Attempts to save the chestnut include isolating diseased chestnuts, attacking the blight fungus, trying to mutate disease-resistant trees, and breeding for resistance to stop the blight. These efforts include…

6. Introducing a virus that would kill the fungus (carried by wasps)- unsuccesful. Cutting isolation strips to prevent the spread of the fungus- unsuccessful.

7. Looking for natural resistance in wild trees- to date there are existing unaffected trees, but it is unclear whether they are disease resistant or just lucky. Actual American Chestnut Tree from Mt. Airy, MD Natural Resistance

8. Scientists have attempted using a weaker strain of the fungus (that does not kill the tree) to build resistance in chestnuts. This works much like a vaccination would…This method is still being studied. Hypovirulence

9. Attempting to cause a mutation that will give chestnuts resistance. Irradiating chestnuts in the hopes that mutations might result in blight resistance was an outgrowth of President Eisenhower’s Atoms for Peace Program. It was suggested by Dr. Singleton From the University of Virginia who had had some success irradiating corn. To date, has not proven its effectiveness, but data is still being collected that may show positive results. Irradiation

10. Many trees in the American Chestnut Research Area on Sugarloaf Mountain are grown from irradiated nuts. Dr. Dennis Fulbright at Michigan State University came to Maryland’s Sugarloaf Mountain in the mid- 1990’s and began research to determine the effects of irradiation on blight resistance. He continues to grow, inoculate, and test seedlings from Sugarloaf at experimental orchards in Michigan. The 300+ trees growing on Sugarloaf Mountain for 40 years all have blight cankers, but are still growing and produced 4500 nuts in the fall of 2003. Irradiation

11. So, Let’s Summarize What the Genetic Problem Is…. - How can we create a tree that is blight resistant and but still has “American Chestnut” qualities?

12. The Players American Chinese

13. Chinese Chestnut Leaf Stem Nut High Resistance From Blight

14. American Chestnut Leaf Stem Nut Low / No Resistance From Blight

15. Difference in Seeds American Chestnut produces a sweet but small nut Chinese chestnut produces a large but generally tasteless nut

16. Overall Tree Comparison Chinese Chestnut American Chestnut 

17. And the Answer to the Genetic Problem Is….

18. http://www.patacf.org/bc_pgm.htm Backcrossing

19. What is a Back-Cross? -You take two different trees (Chinese and American) and cross them. -Then you keep crossing the offspring with the American Variety

20. Resistant Asian X Susceptible American Partially Resistant X American again

21. How Do You Perform a Genetic Cross in Chestnut Trees? 1.Select trees to be crossed 2. Take pollen from one tree and use it to fertilize the ovary of the second tree. Male (catkin) Female (bur) www.patacf.org/ chap_act.htm

22. 3. Harvest the nuts and plant the hybrid trees. www.me-acf.org/Tree%20%20Urchin/ Urchin_v6_n1.html

23. Backcross Diagram Observe the diagram on the next slide and your own diagrams for a better understanding of how the backcross method works.

24. THE BACKCROSS METHOD (3) American chestnut with resistance equal to that of the Chinese parent. See print version

25. For three generations, the resulting offspring is backcrossed with a pure American to get a Backcross 3 (BC3) –BC3 is intercrossed with other BC3 trees –American gene content at each generation: F1 = 1/2 BC1 = 3/4 BC2 = 7/8 BC3 = 15/16 BC3F2 = 15/16 BC3F3 = 15/16

26. Three backcrosses are done to replace Chinese genes with American genes – but NOT to replace the genes that confer blight resistance.

27. 1 out of the 4 will have 1 copy of both resistant genes The process is repeated until a final cross of 2 trees with partial resistance yields 1 having 2 copies of both resistant genes making it fully resistant

28. Resulting Generations F1Backcross 1 Backcross 2

29. Testing and Selection at Each Generation… The backcross breeding strategy was developed by Dr. Charles Burnham in 1982. An essential element of the strategy includes a selection process at each generation.

30. Testing and Selection at Each Generation… When the seedlings from each generation reach about 5 years of age, they are inoculated with a known strain of the blight and their reactions are observed. Only the trees that appear to have some blight resistance are allowed to grow fruit and breed to make the next backcross generation.

31. Testing and Selection at Each Generation… The progeny of the blight resistant trees that emerge from inoculating the third backcross generation are allowed to intercross with each other. The intercross progeny will have inherited blight resistance from both parents and will be the basis for blight-resistant trees.

32. Evidence of Success There have been many trees developed that, so far, show an increased resistance to blight. They are vigorous trees and have been used in back-cross pollination for other generations of trees.

33. Evaluate: Complete the “Bean There Done That” activity to demonstrate your ability to perform a ‘mock’ backcross.Bean There Done That

34. Section Overview Since the spread of the chestnut blight and the realization that it was not going to go away, efforts have been employed to slow it down or eliminate it. These attempts have ranged from attempted isolation of the fungus, to irradiation of nuts, to looking for natural or induced resistance in mature trees. Most recent attempts have focused on backcrossing, a technique that crosses the susceptible American chestnuts to resistant Chinese varieties, and then crosses these hybrids back to American trees for three generations. The result is a tree that is 15/16 American, with American chestnut morphology and Chinese resistance. This portion of the Chestnut Unit focuses on these efforts, with a PowerPoint presentation on past labors and the current backcross efforts. It also includes a worksheet for students to determine the percentage of American traits trees should show at each level of backcross. Finally, as students collect data on the trees they have planted, they will try to trace the resistance (or lack of it) in each tree to the parents. Print version