Student Objectives Monday, April 10, 2017 After today’s lesson, you will be able to: Understand how the backcross breeding method works and is helping to develop disease-resistant populations of new trees. Perform a ‘mock’ genetic backcross. Homework: -Complete the bug backcross activity questions.

Middle School Curriculum The Chestnut Project Carroll County Public Schools Middle School Curriculum November 2005 Next slide http://www.acf.org/

Complete the Punnet Square F- Average sized firefly f – Mini sized firefly F f F f f Describe the phenotype of the offspring: What are the genotypes of the offsping?

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

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…

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

Natural Resistance 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

Hypovirulence 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.

Attempting to cause a mutation that will give chestnuts resistance. Irradiation 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 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.

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?

The Players American Chinese

High Resistance From Blight Chinese Chestnut Leaf Stem Nut High Resistance From Blight

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

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

And the Answer to the Genetic Problem Is….

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

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

Resistant Asian X Susceptible American Partially Resistant American again

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

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

Punnett Squares Possible outcomes of cross between an American (AA)* and a Chinese (CC)tree. Chinese X American = F1 A C AC *A and C represent the full complement of genes contained by and American and Chinese tree, rather than individual genes.

Probabilities Genotypic Ratio: -100% AC Phenotypic Ratio: -50% American Traits 50% Chinese Traits

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

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

BC3 is intercrossed with other BC3 trees 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

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

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

Resulting Generations F1 Backcross 1 Backcross 2

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.

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.

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.

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.

Question Why are the diagrams in this Powerpoint more descriptive than a Punnett Square? What information does the Punnett Square lack. What does the Punnett Square give as the probability for the cross between a Chinese Chestnut and an American Chestnut throughout the breeding?

Extension Activity: Complete the “Genetic Backcross” activity. Be sure to answer the questions in complete sentences.

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