This lecture was a part of Plant Genetics Seminars - PGS 2017/2018 at Assiut University. These seminars organized by Dr. Ahmed Sallam, Department of Genetics, Faculty of Agriculture, Assiut University Abstract Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. This study was the first time to compare two Punica species, viz., P. protopunica and P. granatum. Three molecular markers, i.e. SRAP, TRAP, and ITAP were used to analyse twelve accessions collected from Egypt, Mexico and Yemen, along with analysis of a gene involved in anthocyanin biosynthesis (pgWD40). All markers revealed the relationship between Punica species and among P. granatum accessions. Sequence analysis of pgWD40 exhibited 98% identity between the two species, supporting earlier reports that P. protopunica could be an ancestral species of P. granatum. The information herein could be used for pomegranate genotyping and breeding programs.

1. Remarks on genetic diversity and relationship of Punica
protopunica and P. granatum assessed by molecular analyses
Associate Professor
Department of Genetics
Assiut University
mkhirshy@aun.edu.eg
29.11.2017
Dr. Muhammed Elkhirshy
2017/2018
Published in Genetic Resources and Crop Evolution

2. Remarks on Genetic Diversity and Relationship
in Punica protopunica and P. granatum Assessed
by Molecular Analyses
Dr. Muhammad Elkhirshy
Associate professor, Genetics Dept., Agriculture, Assiut University
mkhirshy@aun.edu.eg
1

3. 2

4. Introduction
Objectives
Materials & Methods
Results
Conclusion
Prospective
3
Contents

5. Introduction
Family: Punicaceae - Lythraceae
Genus: Punica
Species:
granatum L.
protopunica Balf.
Chromosome number:
2n=2x=16 (granatum)
2n=2x=14 (protopunica)
4

6. Introduction
Origin: Iran, Afghanistan and Turkmenistan to India
Secondary centre of diversification:
Albania, Montenegro and Tunisia, Morocco, Spain, Turkey, Egypt
P. protopunica is endemic to the Yemeni island of Socotra
5

7. 6
P. protopunica
P. granatum
Introduction

8. 7
P. protopunica

9. Introduction
Importance! )ٌ‫ل‬ْ‫خ‬َ‫ن‬َ‫و‬ٌ‫ة‬َ‫ه‬ِ‫ك‬‫َا‬‫ف‬‫َا‬‫م‬ِ‫ه‬‫ِي‬‫ف‬َ‫و‬ٌ‫اا‬َّ‫م‬‫ُر‬(
Anthocyanin
8

10. Introduction
Anthocyanin
Leaves
Stems
Roots
Flowers
fruits
flavonoids
9

11. Introduction
Anthocyanin
Ben-Simhon et al., 2015
10

12. Introduction
Anthocyanin
Ben-Simhon et al., 2015
11

13. Introduction
Anthocyanin
Ben-Simhon et al., 2015
MYB – bHLH – WD-40
MBW
The main regulatory genes encode proteins containing a R2R3-MYB
domain, a basic helix—loop—helix (bHLH) domain and WD40 repeats.
Combinations of the R2R3-MYB, bHLH and WD40 proteins and their
interactions determine the set of genes to be expressed.
12

14. Introduction
Genetic Diversity
Over the past decade, an increasing number of studies have focused
on the molecular characterization and genetic diversity assessment
of P. granatum by means of different molecular markers including:
AFLP – RFLP – RAPD – SSR – ISSR – SRAP.
The genetic relationship, at the molecular level, between the two
species of Punica has not been revealed previously.
13
Ercisli et al. 2011, Melgarejo et al. 2009, Singh et al. 2013, Ismail et al. 2014, Soleimani et al. 2012

15. Introduction
Genetic Diversity
SRAP: Sequence Related Amplified Polymorphism
TRAP: Target Region Amplification Polymorphism
ITAP: Intron Targeted Amplified Polymorphism
Based on PCR amplification of DNA regions related to coding
sequences, providing information to exploit polymorphisms that
are mainly associated with functional regions of the genome.
14

16. Introduction
SRAPLi and Quiros (2001)

17. 16
Introduction
TRAP
ITAP
Forward primer of a specific gene (fixed) with SRAP forward or
reverse primers.
Forward primer of 3` widely distributed conserved intron-exon
splice junction sequences with SRAP reverse primers
GACTGCGTACCTGCAAATExample
Xiong et al., 2013
Hu and Vick, 2003

18. 17
Objectives
1- To focus on the genetic relationship between the two species of Punica.
2- To assesses the genetic diversity among and within P. granatum cultivars
and landraces collected from different geographic regions of the world;
By means of:
Three molecular markers (SRAP – TRAP - ITAP)
The phylogenetic analysis of the pgWD40 gene, which is involved in
anthocyanin biosynthesis

19. 18
Materials & Methods
Plant Materials
No
Accession
name
Species
Place of
collection
Cultivation
type
Abbreviation
1 Hegazy P. granatum Egypt, Assiut Cultivar EGY-1
2 Manfalouty P. granatum Egypt, Assiut Cultivar EGY-2
3 Nab Elgamal P. granatum Egypt, Assiut Cultivar EGY-3
4 Khazemy P. granatum Yemen, Saada Cultivar YEM-1
5 Atemy P. granatum Yemen, Dhamar Landrace YEM-2
6 Melhany P. granatum Yemen, Almahwit Landrace YEM-3
7 Yucatan-1 P. granatum Mexico, Yucatan Landrace MEX-1
8 Yucatan-2 P. granatum Mexico, Yucatan Landrace MEX-2
9 Yucatan-3 P. granatum Mexico, Yucatan Landrace MEX-3
10 Yucatan-4 P. granatum Mexico, Yucatan Landrace MEX-4
11 Yucatan-5 P. granatum Mexico, Yucatan Landrace MEX-5
12 Soqatra P. protopunica Yemen, Soqatra Wild type PROT

20. 19
Materials & Methods
Total genomic DNA of the pomegranate accessions was extracted using
a modification to the Dellaporta protocol (Youssef et al. 2015)
DNA extraction
1 2 3 4 5 6 7 8 9 10 11 12

21. 20
Materials & Methods
SRAP – TRAP - ITAP
Primer SRAP (5`-3`) TRAP (5`-3`) ITAP (5`-3`)
1
Me3: TGAGTCCAAACCGGAAT
Em3: GACTGCGTACGAATTGAC
PGAF: CCTATGAGTCCCCCTAC
Em1: GACTGCGTACGAATTAAT
ITP3: GACTGCGTACCTGCAGAC
Em8: GACTGCGTACGAATTAGC
2
Me3: TGAGTCCAAACCGGAAT
Em6: GACTGCGTACGAATTGCA
PGAF: CCTATGAGTCCCCCTAC
Em2: GACTGCGTACGAATTTGC
ITP5: GACTGCGTACCTGCAGCT
Em8: GACTGCGTACGAATTAGC
3
Me3: TGAGTCCAAACCGGAAT
Em7: GACTGCGTACGAATTATG
PGAF: CCTATGAGTCCCCCTAC
Em3: GACTGCGTACGAATTGAC
ITP3: GACTGCGTACCTGCAGAC
Em5: GACTGCGTACGAATTAAC
4
Me8: TGAGTCCAAACCGGTGT
Em7: GACTGCGTACGAATTATG
PGAF: CCTATGAGTCCCCCTAC
Em4: GACTGCGTACGAATTTGA
ITP1: GACTGCGTACCTGCAAAT
Em6: GACTGCGTACGAATTGCA
5
Me2: TGAGTCCAAACCGGAGC
Em9: GACTGCGTACGAATTACG
PGAF: CCTATGAGTCCCCCTAC
PGAF: GACTGCGTACGAATTAAC
ITP1: GACTGCGTACCTGCAAAT
Em8: GACTGCGTACGAATTAGC
6
Me8: TGAGTCCAAACCGGTGT
Em9: GACTGCGTACGAATTACG
PGAF: CCTATGAGTCCCCCTAC
Em6: GACTGCGTACGAATTGCA
ITP2: GACTGCGTACCTGCATGC
Em7: GACTGCGTACGAATTATG
7
Me5: TGAGTCCAAACCGGAAG
Em2: GACTGCGTACGAATTTGC
PGAF: CCTATGAGTCCCCCTAC
Em7: GACTGCGTACGAATTATG
ITP2: GACTGCGTACCTGCATGC
Em10: GACTGCGTACGAATTTAG
8
Me6: TGAGTCCAAACCGGTAG
Em2: GACTGCGTACGAATTTGC
PGAF: CCTATGAGTCCCCCTAC
Em8: GACTGCGTACGAATTAGC
ITP4: GACTGCGTACCTGCAATG
Em3: GACTGCGTACGAATTGAC
9
Me9: TGAGTCCAAACCGGTCA
Em2: GACTGCGTACGAATTTGC
PGAF: CCTATGAGTCCCCCTAC
Em9: GACTGCGTACGAATTACG
ITP4: GACTGCGTACCTGCAATG
Em7: GACTGCGTACGAATTATG
10
Me8: TGAGTCCAAACCGGTGT
Em10: GACTGCGTACGAATTTAG
PGAF: CCTATGAGTCCCCCTAC
Em10: GACTGCGTACGAATTTAG
ITP4: GACTGCGTACCTGCAATG
Em10: GACTGCGTACGAATTTAG

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Materials & Methods
Analysis of pgWD40-repeat protein gene
A complete cDNA sequence was obtained from Punica granatum
(NCBI-GenBank: HQ199314.1)
Forward: 5’-CCTATGAGTCCCCCTAC-3’
Reverse: 5’-TAATCTCCGAACCAGCA-3’
Ben-Simhon et al. 2011
870bp
PCR products were purified using ethanol precipitation, two samples were sequenced
in forward and reverse sense at Macrogen, Inc.
1 2 3 4 5 6 7 8 9 10 11 12

23. 22
Materials & Methods
Data Analysis
EGY-1 EGY-2 YEM-1 YEM-2 MEX-1
1 1 1 1 1
1 1 1 1 1
0 0 1 1 0
1 0 1 0 0
1 0 1 0 0
0 1 1 0 1
0 1 1 0 1
0 1 0 1 1
1 0 0 1 1
1 0 0 1 1
1 1 0 0 1
1 1 1 1 0
%P
PIC
Rp
DI
Sequencher Mega
NTsys

24. 23
Results
SRAP TRAP ITAP
Molecular markers

25. 24
SRAP TRAP ITAP
Primer TNB %P PIC Rp TNB %P PIC Rp TNB %P PIC Rp
1 35 71.43 0.12 4.83 35 71.43 0.17 8.17 23 60.87 0.09 2.33
2 29 68.97 0.14 5.17 39 87.18 0.22 11.17 11 36.36 0.06 0.67
3 26 84.62 0.20 7.00 26 73.08 0.12 3.67 20 55.00 0.14 3.67
4 28 75.00 0.15 4.83 31 70.97 0.13 5.00 16 68.75 0.13 2.67
5 34 67.65 0.14 6.00 23 78.26 0.16 4.67 15 60.00 0.15 3.33
6 23 78.26 0.15 4.00 25 64.00 0.11 3.17 18 83.33 0.19 4.67
7 32 75.00 0.16 6.50 27 77.78 0.14 4.50 15 93.33 0.20 3.83
8 30 73.33 0.16 6.00 16 62.50 0.14 3.17 16 93.75 0.17 3.17
9 31 80.65 0.17 6.33 19 73.68 0.13 2.83 16 75.00 0.19 4.33
10 31 77.42 0.17 6.67 19 68.42 0.15 3.67 16 68.75 0.12 2.33
All 299 74.92 0.16 57.33 260 73.85 0.15 50.02 166 69.88 0.14 31.00
Results
A survey of total number of bands, percentage of polymorphism, polymorphism
information content and resolving power of SRAP, TRAP and ITAP molecular markers
TNB: total number of bands, %P: percentage of polymorphism, PIC: polymorphism information
content, Rp: resolving power
Molecular markers

26. 25
Results
Category
Total number of bands % of polymorphism
SRAP TRAP ITAP SRAP TRAP ITAP Average
P. protopunica vs. P. granatum 299 260 166 68.56 67.31 65.66 67.18
P. protopunica vs. EGY 275 245 158 67.27 68.57 65.82 67.22
P. protopunica vs. YEM 293 251 165 69.62 67.33 66.67 67.87
P. protopunica vs. MEX 283 246 164 67.84 66.67 65.85 66.79
Within P. granatum 214 183 122 28.04 28.96 19.67 25.56
Within Egyptian accessions 193 163 114 7.77 4.91 4.39 5.69
Within Yemeni accessions 210 174 122 15.24 17.24 13.11 15.20
Within Mexican accessions 202 169 122 20.30 21.30 18.85 20.15
EGY vs YEM 215 179 122 10.23 11.73 7.38 9.78
EGY vs MEX 208 176 125 10.10 11.93 11.20 11.08
YEM vs MEX 218 183 124 11.01 12.57 3.23 8.93
All 299 260 166 74.92 73.85 69.88 72.88
Levels of polymorphism in Punica accessions as revealed by SRAP, TRAP and ITAP analysis
Molecular markers

27. 26
Category
Unique and shared bands
SRAP TRAP ITAP Total
P. protopunica 78 74 41 193
P. protopunica – P. granatum 92 85 57 234
P. protopunica – EGY 2 0 1 3
P. protopunica – YEM 1 2 0 3
P. protopunica – MEX 3 3 1 7
P. granatum (Bulk) 125 101 68 294
Egyptian accessions (Bulk) 1 3 0 4
Yemeni accessions (Bulk) 12 9 0 21
Mexican accessions (Bulk) 4 7 1 12
EGY – YEM (Bulk) 1 5 2 8
EGY – MEX (Bulk) 1 2 0 3
YEM – MEX (Bulk) 7 7 7 21
Total 327 301 180 808
Unique SRAP, TRAP and ITAP markers characterized Punica genotypes, represented
by number of specific band per category.
Results Molecular markers

28. 27
Results
Dendrogram of genetic similarities between
Punica protopunica and P. granatum regional
bulked accessions using UPGMA cluster
analysis.
SRAP
TRAP
ITAP
Molecular markers

29. 28
Results
Dendrogram of genetic similarities among Punica granatum regional accessions
revealed by UPGMA cluster analysis.
SRAP TRAP ITAP
Molecular markers

30. 29
Results WD40-repeat protein
Alignment of 516 bases of DNA
sequences of pomegranate WD40,
different sequences are highlighted
in black background.

31. 30
Results
Phylogenetic analysis of WD40 gene. Nucleotide sequence based
phylogenetic tree of WD40 from various monocot and dicot plants
including the sequences of Punica granatum and P. protopunica
accessions.
WD40-repeat protein

32. 31
Results WD40-repeat protein

33. 32
Conclusion
This study provide for the first-time novel molecular evidence supporting the
genetic relationship between the two species of Punica.
SRAP, TRAP and ITAP genotyping, identified that the P. protopunica and P.
granatum are closely related, and revealed the close similarity among the
pomegranate accessions from different geographical regions as representatives
of P. granatum species.
The phylogenetic analysis, of WD40 gene sequence, provided further
information about evolutionary relationships between P. protopunica and the P.
granatum accessions and suggested that the former could be an ancestral
species of the latter.

34. 33
Prospective
Evaluation of Egyptian grown pomegranate cultivars for
anthocyanin content using:
Spectrophotometer determination of anthocyanin
TRAP – RFLP of MYB, bHLH and WD40-repeat protein
genes

35. 34
Prospective
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
1 2 3 4 5 6 7
Araby Assiuty Nab-gamal Wardy Manfalouty Hejazy Wonderful

36. 35
mkhirshy@aun.edu.eg