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

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

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

2-D Representation of Amylopectin Reducing end Non-reducing end Alpha-1,4 glucosidic linkage Alpha-1,6 glucosidic linkage osmotic pressure A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. Glycogen is a more highly branched molecue than amylopectin-otherwise they are similar molecules.

Differences Between RR and rr round wrinkled

Differences Between RR and rr round wrinkled Less sweet low sucrose Sweeter high sucrose More starch Less starch Amylopectin/amylose high Amylopectin/amylose low Lower water content Higher water content Lower lipid higher protein Higher lipid lower protein Cell size smaller lose less volume Cell size larger lose more volume Larger simple starch grains Smaller compound starch grains More legumin Less legumin Lower osmotic pressure Higher osmotic pressure SBEI No SBEI

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

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

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

’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.

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

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

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

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

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?

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

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

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

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

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

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

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.