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

4. 2-D Representation of Amylopectin
Reducing end
Non-reducing end
Alpha-1,4 glucosidic linkage
Alpha-1,6 glucosidic linkage
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group.

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

6. 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

7. 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

8. 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
cDNA λ

9. 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

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

11. 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?

12. 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

13. Figure 3. SBEI RFLP’s Between Near Isogenic Lines of Pea RR and rr Can Be Observed

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

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

16. 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

17. 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.