Bhattacharyya et al, 1990

1. The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Bhattacharyya et al, 1990

2. Storage compounds are located in the embryo of dicotyledonous seeds Seed coat Endosperm Embryo

3. Amylose (Alpha-1,4 glucosidic linkage) Amylopectin (Alpha-1,4 glucosidic linkage& Alpha-1,6 glucosidic linkage) Amylose and Amylopectin differ in their branching

4. Reducing end Non-reducing end Alpha-1,4 glucosidic linkage Alpha-1,6 glucosidic linkage 2-D Representation of Amylopectin osmotic pressure

5. Differences Between RR and rr rr RR

6. Differences Between RR and rr rr RR

7. Table 1. Composition of Mature RR and rr Pea seeds differ in many ways • Ranges refer to studies on a number of genotypes (given in paranthesis) • a, b and c indicate paired studies with near isogenic lines of RR and rr plants • % dwt indicates percent dry weight

8. Isogenic lines (identical genetic backgrounds except for R vs r) (AA bb) RR x rr (aa BB) Back Cross 1(BC1) Rr x RR. BC2 RR and Rr self self RR disgard 25% rr x RR BC Rr x RR Continue Back crossing for at least 7 generations. This will result in the genotypes of the two lines (RR vs rr) being identical except at the R locus

9. (a) Purified SBE I Figure 1. SBEI Antiserum Cross Reacts with Purified SBEI and Crude Extracts of Pea Embryos WESTERN Preimmune serum) WESTERN (Antiserum) SDS-PAGE (b) SBE I in crude embryo extracts

10. ’R’ – SBEI Pick the best answer! ***iClicker! Based on the data in the previous slide what can be concluded about the relationship between R and SBEI? • The R gene encodes the SBEI protein. B. The R gene function is required for the SBEI protein. • The R gene and SBEI are separate loci. D. The SBEI gene influences the R gene. E. All of the above.

11. Cloning of the SBEI Gene from Pea bacteriophage λ gt11– a vector for cloning and expressing in bacteria cDNAs derived from any organism. Cloning site Bacterial promoter λ P λ + cDNA λ P A cDNA from one gene λ

12. cDNA synthesis • https://www.youtube.com/watch?v=rKPJpxCW2qw

13. Cloning of the SBEI Gene from Pea Pea embryo cDNA + λ gt11 = pea embryo expression library. --each λ gt11 has a cDNA for a different gene expressed in the pea embryo. λ P cDNA λ

14. Nitrocellulose filter Screening the λ gt11 library Petri plate top view Bacteria lawn + λ gt11 library Expose to SBEI antibody and detect.

15. Cloning of the SBEI Gene from Pea Following screening of λgt11 Pea library: --7 λgt11 clones identified. --all clones contained cDNA sequence from a single gene. -- with sequence similarity to bacterial glycogen branching enzyme. Is it the SBEI gene and if so is SBEI the R gene?

16. List experiments that were done that establish a connection between R and SBEI. - - - - -

17. (b) RNA from RR and rr embryos Figure 2. RNA blot Analysis of SBE I (a) Restriction map of putative SBEI cDNA (c) RNA from different stages of development of pea embryo

18. Figure 3. SBEI RFLP’s Between Near Isogenic Lines of Pea RR and rr Can Be Observed Probe used was the entire 2.7 cDNA

19. The SBEI Gene and R Gene Cosegregate Probe = 1.3 kb EcoR1 cDNA fragment

20. Figure 5. Analysis of the SBE I Gene from RR and rr plants RR gene rr gene

21. (b) Homology of the Ips-r inverted repeats with those of other elements • Sequences of 12 bp inverted repeats and flanking 8 bp direct repeats Figure 6. Sequences of the Terminal Inverted repeats of the Insertion Element Ips-r

22. Conclusions • The R gene encodes a starch branching enzyme. • The r allele results in a wrinkled phenotype because a transposable element insertion in the SBEI gene eliminates SBEI activity causing a decrease in amylopectin.