This document summarizes a study assessing genetic diversity in 25 spinach accessions using SDS-PAGE and morphological analysis. Morphological analysis found variation in seed shape, color, stem color, and leaf tips. SDS-PAGE identified 27 total protein bands, with 20 being polymorphic. Three accessions from different locations (20541 from Peshawar, 19902 from AVRDC, and 20180 from Lahore) showed significant genetic differences based on presence of distinct protein bands. While genetic differences were linked to morphological variations, no clear linkage was found between diversity and geographic origin of the accessions. The study concluded spinach accessions show genetic diversity that could benefit future breeding programs.

1. Presented By: MADIHA
RASHID

3. SPINACH (Spinacia oleracea)
 Family – Chenopodiaceae
NUTRITIONAL IMPORTANCE
 Source of Mg, Mn, Fe, Ca, K, Cu, P, Zn, Se
 Vitamins, protein, antioxidants, fatty acids,
carotenoids, flavonoids & apocyanin

4.  Whole plant – for remedy of urinary
calculi
 Leaves – bowel, lung inflammation,
febrile affliction & cooling
 Leaf extract – powerful water soluble
natural antioxidants

5.  Past – agronomic, morphological and
physiological traits – used to characterize
varieties
 Influence of environmental factors or presence
of multiple alleles
 Field testing & evaluation – difficult & time
taking
 Now – biochemical markers to assess the

6.  SDS-PAGE more simple, valid,
practically reliable and extensively
used
 Seed storage proteins – synthesized
in developing seeds
 Important for seed germination &
seedling growth
 high stability
 independence of ecological
conditions
 used to determine taxonomic and

7.  considerable progress in genetic improvement
of Spinacia oleracea L.
 but its genetic variability at molecular level has
not been investigated
OBJECTIVE
 assessment of genetic diversity of 25 Spinach
accessions using SDS-PAGE technology &
molecular markers

8. MORPHOLOGICAL
STUDY
 Seeds – IABGR-
NARC
 Grown in pots
 Temperature – 10-
39οC
 pH – 7
 Humidity – 42-67 %
 Loamy soil

9.  Seed Shape
 Seed Color
 Seed Weight (10
seeds)
 Germination Rate
 Plant Height
 Stem
(Presence/Absence)
 Stem Length
 Number of
Leaves/Plant
 Petiole Length
 Leaf Tips
 Leaf Base
 Leaf Length
 Leaf Width

10.  Urea Buffer - 0.5 M tris HCl pH 8.0, 5
M urea, 0.2 % SDS & 1 % 2-
mercaptoethanol in dist. water +
PMSF (protease inhibitor) +
Bromophenol blue
 Ground seeds + extraction buffer
 Boiling – 5 min
 Centrifuge – 15000 rpm
 Supernatant collected

11.  12 % Resolving Gel – 30 % acrylamide, 1.5 M
tris pH 8.8, 10 % SDS, 10 % APS & TEMED in
distilled water
 Resolving gel in gel plates
 n-butanol poured on it
 Stacking gel – 30 % acrylamide, 1 M tris pH
6.8, 10 % SDS, 10 % APS & TEMED in
distilled water

12.  Reducing buffer was prepared by
adding tris, glycine & SDS in distilled
water
 Protein extracts & marker in wells
 Reducing buffer & power supply
 Staining solution – methanol, glacial
acetic acid, distilled water &
Commassie Brilliant Blue
(50:10:40:0.3)
 De-staining solution – methanol,
glacial acetic acid & distilled water

14. RESULTS
MORPHOLOGICAL STUDY
 Seed shape – irregular seeds with 4-5 ends
 Prickly seeds with 2-6 spines
 Seed color – yellowish, greenish, light & dark
brown, blackish brown, brown & yellowish
brown
 Stem color – purplish pink & green
 Leaf tips – obtuse & acute

18.  protein bands – 12.6 to 91.2 kDa
 Total bands – 27
 20 – polymorphic
 4 – monomorphic
 Zone A – 9 lightly stained bands
 Zone B – 7 dark & lightly stained
bands
 Zone C – 11 medium & lightly stained
bands

20. GEL ZONES
 Zone A – 53.7 to 91.2 kDa
 5 polymorphic,1
monomorphic
 Zone B – 29.5 to 50.1 kDa
 4 polymorphic, 3
monomorphic
 Zone C – 12.6 to 28.2 kDa

21. RESULTS
 number of band variation in
accessions -10–18
 79.4, 66.1 & 63.1 kDa – in all
accessions
 53.7, 35.5, 33.1 & 29.5 kDa – in
20541
 From Peshawar
 20541, 19902 & 20180 had significant
genetic differences
 91.2, 85.1, 69.2, 53.7, 50.1, 36.3,

22. RESULTS
 20541 (Peshawar) had 53.7, 33.1 &
29.5 kDa
 19902 (AVRDC) had 69.2, 25.7 &
14.1
 20180 (Lahore) had 28.2 kDa

24. DISCUSSION
 Use of morphological and seed
storage protein characters – better
way to explore genetic diversity of
plant genotypes
 Variation in morphological characters
is based on genetic variation
 Seed storage proteins – not affected
by environmental fluctuations
 used to characterize the taxonomic
and evolutionary features of different
crops

25. Protein Profiles vs
Geography
 polymorphism in seed proteins is linked with
the geographical regions
 No linkage was found in case of spinach
 e.g., 20180 & 20188 from Lahore
 20540 & 20541 from Peshawar
 Accessions from local origin had less
variation
 SDS-PAGE – a better technique for genetic

27. CONCLUSION
 Significant differences exist in
spinach accessions
 Also some minor differences exist
 Genetic differences are directly linked
with morphological differences
 No linkage with geographical
locations was found in case of
spinach accessions

28. SUGGESTION
 Minor differences found can be studied by
advanced molecular techniques including
2D electrophoresis & DNA markers
 These variations might have certain
concealed capabilities which can be
preserved for future plant breeding
programs