1. Experiment Summary Statements 1) The mutagen applied to the truebreeding seed induced phenotype variations in the soybeans. 2)The mutagen induced mutations (changes) in the genetic makeup of the plant which were passed on to offspring, and thus the unique phenotypes were inherited. 3)The genetic makeup of the M2 plants was not always the same. Some were homozygous, others heterozygous. 4) The mutations were random. Dr. Specht did not observe the indeterminate mutant he was hoping for, but observed many other random mutations instead.

2. What is a Monohybrid Cross? A monohybrid cross is a cross between two plants that differ by a single trait (mono). Monohybrid crosses are done to allow plant breeders to follow the inheritance of the single trait more easily. Monohybrid crosses were used on the dwarf and soybean lesion mutants to learn about the inheritance of these mutant traits. Dwarf Mutant Soybean Lesion Mutant Soybean

3. What is a reciprocal cross? A cross is made between plants with two different phenotypes using one phenotype as the male and the other phenotype as the female. In a reciprocal cross, a second cross is made switching the phenotypes of the male and female.

4. X Reciprocal Cross X M3 plants homozygous for the dwarf mutant traits were crossed to homozygous normal plants and the F1 seed harvested and bagged. DD dd DD dd Pollendonor Pollendonor Cross 1: Pollen donor = Mutant Cross 2: Pollen donor = Normal

5. X Crosses were also made for soybean lesion mutants. GG gg GG gg PollendonorPollendonor Cross 1: Pollen donor = Mutant Cross 2: Pollen donor = Normal X Reciprocal Cross

6. Why do a reciprocal cross? Sometimes traits are passed on only maternally (through the female) or paternally (throught the male). If this is true for a particular trait, the offspring of the two crosses in a reciprocal cross will have different phenotypes. This allows plant breeders to determine if a trait is passed on in this way.

7. Making a Cross Stamen and Pollen = male part Stigma and style = female part

8. Making a Cross Soybeans are self-pollinating. Therefore, cross pollination must be done manually. View soybean crossing video

9. Making a Cross The anthers are removed from female flower to prevent self pollination.

10. Making a Cross Pollen taken from anther of another flower and dusted onto stigma of female flower.

11. Soybean flowers

12. Soybean anther Soybean flowers

13. Cross pollinated flowers are labeled. Pods will form containing seed from the cross.

14. Dwarf Cross - F1 Seed The F1 seed was planted keeping the reciprocal cross seed in a separate plot. PlantSeedPlantSeed Pollen donor = Mutant Pollen donor = Normal

15. Dwarf Cross - F1 Plants The F1 plant phenotypes were observed, and the plants allowed to self-pollinate to produce F2 seed. Pollen donor = Mutant Pollen donor = Normal

16. All of the F2 seed was harvested with seeds from each individual plant placed in a separate labeled envelope. Pollen donor = Mutant Pollen donor = Normal Dwarf Cross - F1 Plants

17. The F2 seed was planted in progeny rows. Again the reciprocal cross seed was planted in a separate plot. Pollen donor = Mutant Pollen donor = Normal Dwarf Cross – F2 Seed

18. The F2 plant phenotypes were observed and recorded, and the plants allowed to self-pollinate to produce F3 seed. Pollen donor = Mutant Pollen donor = Normal Dwarf Cross – F2 Plants

19. All of the F3 seed was harvested from each plant and placed in separate envelopes labeled with the phenotype of the parent plant (Dwarf or Normal). Normal DWARF DWARF DWARF Pollen donor = Mutant Pollen donor = Normal Dwarf Cross – F2 Plants

20. DWARF Normal Normal DWARF Normal Normal Dwarf Cross – F3 Seed DWARF DWARF Normal Normal Normal Normal The F3 seed was planted in progeny rows. The reciprocal cross seed was planted in the same plot.

21. The F3 seed was planted in progeny rows. Dwarf Cross – F3 Seed DWARF Normal Normal DWARF Normal Normal DWARF DWARF Normal Normal Normal Normal

22. Soybean Disease Lesion Mutant At the same time, these steps were taken with the soybean disease lesion mutants. Lesion Mutant Soybean

23. Soybean Disease Lesion Mutant Data

24. Summary of Results 1)All F1 plants had the normal phenotype 2)In both the first cross and reciprocal cross, - Mutant phenotype not apparent in F1s - Mutant phenotype not apparent in F1s - Mutant phenotype did reappear in F2s - Mutant phenotype did reappear in F2s - About ¼ of F2 plants had mutant - About ¼ of F2 plants had mutant phenotype of dwarf or disease lesion. phenotype of dwarf or disease lesion.

25. Summary of Results 3) Two types of plants had the normal phenotype the normal phenotype in the F2. About 1/3 of in the F2. About 1/3 of the F2 normals were the F2 normals were trubreeding while 2/3 had mixed progeny rows. trubreeding while 2/3 had mixed progeny rows. 4) Mutant F2 plants were truebreeding and all of truebreeding and all of their F3 progeny were their F3 progeny were mutants. mutants. F3 rows = 1:2:1 F2

26. Explaining the Results Using the Principle of Segregation 1)The variation in the dwarf and lesion traits was controlled by genes. Genes store the information that controls the by genes. Genes store the information that controls the expression of traits. Alternative versions of genes called expression of traits. Alternative versions of genes called alleles allow for the expression of different traits. alleles allow for the expression of different traits. 2)Genes are found in pairs in somatic cells that make up the plant. the plant. 3)Paired genes separate during gamete formation and one gene from each pair goes into a gamete. gene from each pair goes into a gamete. 4)Gametes fuse at random during sexual reproduction producing the next generation seed. producing the next generation seed.

27. Explaining the Results Using the Principle of Segregation Dr. Specht now had to use the data to figure out what was happening on the gene level. We will use the data to answer some questions and see if we can arrive at the same conclusions he did.

28. Explaining the Results Using the Principle of Segregation The Principle of Segregation explains what Dr. Specht observed with his plants in the field. He can test the validity of the principle in explaining his results. We will use the data to answer some questions and see if we can arrive at the same conclusions he did.

29. QUESTION? Looking at the data for the soybean lesion mutant, which trait was dominant? Normal trait Soybean lesion mutant trait

30. ANSWER Since there are only normal and no lesion phenotypes in the F1, it can be assumed normal is dominant and masked the expression of the mutant trait (recessive). The normal trait is dominant.

31. QUESTION? AA X Aa aa X Aa What are the genotypes of the original parents for the soybean lesion trait? Use A to represent the dominant allele and a to represent the recessive allele. AA X aa

32. ANSWER The genotype of one parent is AA and other parent is aa. The experiment states that a cross was made between a truebreeding normal plant and a truebreeding soybean lesion mutant plant. Truebreeding means both parents are homozygous for the trait. Therefore, neither parent could be genotype Aa. We have determined that normal is dominant. Therefore its genotype would be AA. That means the lesion-type is recessive, genotype aa. The original cross was: AA x aa

33. QUESTION? AA Aa What is(are) the genotype(s) of the F1 generation for the soybean lesion trait? Use A to represent the dominant allele and a to represent the recessive allele. aa AA, Aa, & aa

34. ANSWER The genotype of all the F1 offspring is Aa Aa (Normal) Aa (Normal) Aa (Normal) Aa (Normal ) Normal parent alleles Lesion parent alleles AA a a The AA parent can contribute only gametes with A alleles. The aa parent can contribute only gametes with a alleles. Therefore, the F1 progeny will all have Aa genotypes.

35. QUESTION? 1 AA : 2 Aa : 1 aa What is(are) the genotype(s) of the F2 generation for the soybean lesion trait And in what ratios do they appear? Use A to represent the dominant allele and a to represent the recessive allele. 1 AA : 1 Aa : 1 aa 1 AA : 1 aa 1 Aa

36. ANSWER The genotypes and ratios of the F2 offspring are 1 AA : 2 Aa : 1 aa Normal F1 parent alleles Aa A a Since the F1 plant (Aa) is selfed, each parent contributes both an A and a allele. The gametes for the Punnett square would be 50% A and 50% a. F2 genotypic ratios are 1 AA : 2 Aa : 1 aa AA Aa aa

37. QUESTION? 2 Normal : 1 Lesion What are the phenotypes of the F2 generation for the soybean lesion trait And in what ratios do they appear? 1 Normal : 1 Lesion 3 Normal : 1 Lesion

38. ANSWER The phenotypes and ratios of the F2 offspring are 3 Normal : 1 Lesion. Normal F1 parent alleles Aa A a The A allele, which codes for the normal trait, is dominant. Therefore, the only way to get the lesion phenotype is with the aa genotype. The ¼ AA offspring and the ½ Aa offspring will both have the normal phenotype. AA (Normal) Aa (Normal) Aa (Normal) aa (Lesion)

39. QUESTION? 1 AA : 2 Aa Of the F2 generation progeny with a normal phenotype, what is the ratio of homozygous to heterozygous plants for the soybean lesion trait? 1 AA : 1 Aa 1 AA : 3 Aa

40. ANSWER The ratio of homozygous to heterozygous F2 normal plants is 1 Homozygous : 2 Heterozygous. Normal F1 parent alleles Aa A a We do not even look at the lesion offspring when figuring this ratio. Of the 3 normal offspring, 1 has the AA genotype (homozygous). And 2 have the Aa genotype (heterozygous). AA (Normal) Aa (Normal) Aa (Normal) aa (Lesion)

41. *BONUS QUESTION?* Self pollinate the plants and plant them in progeny rows to observe the offspring. If you were a plant breeder, what experiment might you perform to determine which of the F2 normal phenotype plants were homozygous and which were heterozygous? Cross pollinate the plants and plant them in progeny rows to observe the offspring.

42. ANSWER Self pollinating the plants would give the results below. If lesion plants were observed in the progeny rows, you could determine that the selfed parent had to pass on an a allele and was heterozygous. AaA a AA (Normal) Aa (Normal) Aa (Normal) aa (Lesion) Self pollinate the plants and plant them in progeny rows to observe the offspring. AAA A AA (Normal) AA (Normal) AA (Normal) AA (Normal)

43. Summary A monohybrid cross is used to study the inheritance of a single gene. A reciprocal cross is used to determine if the trait is sex-linked. Soybeans must be cross-pollinated by hand. The results of the soybean lesion cross indicate that the trait is recessive. The results observed in progeny of the experiment follow the Principle of Segregation.

44. Summary of Results 1)All F1 plants had the normal phenotype 2)In both the first cross and reciprocal cross, - Mutant phenotype not apparent in F1s - Mutant phenotype not apparent in F1s - Mutant phenotype did reappear in F2s - Mutant phenotype did reappear in F2s - About ¼ of F2 plants had mutant - About ¼ of F2 plants had mutant phenotype of dwarf or disease lesion. phenotype of dwarf or disease lesion.

45. Summary of Results 3) Two types of plants had the normal phenotype the normal phenotype in the F2. About 1/3 of in the F2. About 1/3 of the F2 normals were the F2 normals were trubreeding while 2/3 had mixed progeny rows. trubreeding while 2/3 had mixed progeny rows. 4) Mutant F2 plants were truebreeding and all of truebreeding and all of their F3 progeny were their F3 progeny were mutants. mutants. F3 rows = 1:2:1 F2

46. Explaining the Results Using the Principle of Segregation 1)The variation in the dwarf and lesion traits was controlled by genes. Genes store the information that controls the by genes. Genes store the information that controls the expression of traits. Alternative versions of genes called expression of traits. Alternative versions of genes called alleles allow for the expression of different traits. alleles allow for the expression of different traits. 2)Genes are found in pairs in somatic cells that make up the plant. the plant. 3)Paired genes separate during gamete formation and one gene from each pair goes into a gamete. gene from each pair goes into a gamete. 4)Gametes fuse at random during sexual reproduction producing the next generation seed. producing the next generation seed.