 | Dr Hanaiya El-Itriby, Director, Agricultural Genetic Engineering Research Institute AGERI, ARC, Egypt. | |
 | Prof.Dr Richard Jones Senior Research & Lecturer Scholar, School of Forestry& Environmental Studies, Yale Univ., USA | |
 | Dr Sami S. Adawy Senior Researcher, AGERI, ARC, Egypt. | |
 | Dr Michael Baum |
Genetic Diversity and Germplasm Conservation Using Molecular and Genomic Techniques
Dr Hanaiya El-Itriby,
Director,
Agricultural Genetic Engineering Research Institute (AGERI)
Genetic diversity: is generally defined as the amount of genotypic variability in a population, or: the number of different alleles per loci and the proportion of loci with more than one allele in a species or population. |
Knowledge about germplasm diversity and genetic relationships among breeding materials are highly valuable tools in crop improvement strategies. A number of methods are currently available for analysis of genetic diversity in germplasm accessions, breeding lines, and populations. These methods have relied mainly on the availability of genetic markers. |
A Genetic marker |
Represents variation at a particular site on the genome which is heritable, easy to assay and can be followed over generations. Therefore ,Genetic markers are of great value in genetic research and practical breeding programs, since they reflect the variability the genetic variation among individuals . |
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There are three |
types of genetic markers: |
· Morphological markers |
Mutations in genes with visible consequence, have been used in genetic studies since early in the twentieth century for example color, shape etc ( Fig 1). |
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Fig ( 1 ) : Color variation representing a morphological marker. |
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· Biochemical markers |
The development of elecrophotic techniques in the 1960 offered a promising alternative to morphological markers by providing biochemical or isozyme markers. |
In order to maintain, and increase germplasm collections efficiently and effectively, amore objective appraisal of genetic diversity and relationships among accession can be provided from isozyme data and total protein banding patterns. The advantages of using isozymes as a tool in measuring genetic variability is that allozymes generally exhibit Mendelian inheritance , co-dominant expression allowing heterozygotes to be distinguished from homozygotes and complete penetrance . |
Molecular markers |
In recent years , the field of molecular biology has provided different tools suitable for rapid and detailed genetic analysis. The most fundamental of these tools are “ DNA as molecular markers “. Molecular markers |
reflect heritable differences in homologous DNA sequences among individuals. |
They may be due to: |
Base pair changes, |
rearrangements (translocation or inversion), |
insertions or deletions. |
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The development of molecular markers holds many promises to the plant breeders and geneticists in different areas ; such as varietal identification or fingerprinting ,estimation of relatedness between different genotypes discernment of evolutionary relationships, and introgression of Mendelian traits into a population . Marker based selection, however ,is the area where molecular markers could have the greatest impact in plant breeding. Moreover , genetic maps based on molecular markers are playing on increased important role in genetic studies and plant breeding programs. |
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Nowadays, a wide array of different molecular techniques are used to detect polymorphism at the DNA level. |
Most Molecular Markers fall into 3 basic categories: |
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Hybridization based (non-PCR) techniques |
Restriction Fragment Length Polymorphism (RFLP) analysis ( |
Botstein et al, 1980 ). RFLP’s result from variation in the bases within a restriction enzyme recognition sites, insertion , or deletion within a restriction fragment or rearrangement of DNA fig ( 2-a , 2-b ). |
RFLP analysis is used for genetic analyses where the number of samples is moderately low (<300-400). |
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Strengths |
•All alleles are seen simultaneously. |
•The DNA polymorphisms act as co-dominant genetic markers. |
•No prior information on DNA sequence is needed. |
•Relatively simple technique. |
•Easily reproducible. |
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Weaknesses |
•Has relatively low throughput |
•High labor and materials costs. |
•Requires relatively large amounts of high quality DNA |
•Gel based and thus not easily automatable. |
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Applications of RFLP’s : |
· Several applications for RFLP’s have been proposed and are being investigated . the major ones are the following . |
· Mapping of qualitative traits (single genetic factors and translocation breakpoint ). |
Determination of genetic diversity and relationships in the germplasm base mapping of quantitative traits loci ( complex genetic factors involving several genes). |
Hybridization-based fingerprinting |
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Fig (2 – a ) : The different steps of RFLP analysis. |
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Fig (2 - b) : Polymorphism among 13 different accessions. |
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Arbitrarily-primed PCR techniques |
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Development of the polymerase chain reaction (PCR) removed the necessity for probe hybridization steps. |
A common feature of these techniques is the lack of requirement for sequence information from the genome under investigation. |
The range of different approaches in this category differ in the length and sequence of the primers used, the stringency of the PCR conditions and the method of fragment separation and detection. |
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This includes: |
1- Random Amplified Polymorphic DNA (RAPD) analysis ( Williams et al. 1990, and Welsh and McClelland , 1990 ). Are genetic markers resulting from PCR amplification of genomic DNA segments between closely spaced sequence that are recognized by and complementary to random primers ( usually 10 mers ) of arbitrary nucleotide sequence. |
RAPD polymorphism results from changes in the primer-binding site in the DNA sequence.The typical reaction consists of a simple buffer, the four dinucleotide triphosphates ( dATP , dCTP , dGTP , |
dTTP ) , a single primer , DNA polymerase and a few nanograms of DNA from the organism under study. Following 4-5 hours of amplification, the products are separated on gels by electrophoresis. |
In the presence of ethidium bromide the resulting DNA pattern is visualized under ultra violet light the gel is simply stained for DNA and photographed as shown in |
figures ( 3 ). |
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2- DNA Amplification Fingerprinting (DAF) in which the products are separated on polyacrylamide gels. |
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Fig ( 3 ) : RAPD profiles of the 13 varieties amplified with RAPD primers. |
Strengths |
•Non-radioactive detection |
•No prior DNA sequence information |
•Universal primers work in any genome |
•Very small amounts of genomic DNA |
•Experimental simplicity |
•No need for expensive equipment |
•Automation |
Weaknesses |
•Reproducibility of RAPD profiles. |
•Dominant markers |
•Underestimation of genetic distances |
In a second subgroup, primers used are semi-arbitrary in that they are based upon restriction enzyme sites or sequences interspersed in the genome such as repetitive elements, transposable elements and microsatellites. |
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1-Selective Restriction Fragment Amplification (SRFA). |
2-Amplified Fragment Length Polymorphism (AFLP). |
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Principles of AFLP . |
AFLP technology is a DNA fingerprinting technique that combines both of these strategies. It is based on the selective amplification of a subset of genomic restriction fragments using PCR. DNA is digested with restriction endnuclease, and double-strand DNA adaptors are ligatedto the ends of DNA fragments to generate template DNA for amplification. Thus the sequence of the adaptors and the adjacent restriction site serve as primer binding site for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction fragments are added to the 3’ ends of PCR primers such that only a subset of the restriction fragments are recognized. Only restriction fragments in which the nucleotides flanking the restriction site match the selective nucleotides will be amplified. The subset of amplified fragments are then analyzed by denaturing polyacrylamide gel electrophoresis to generate the fingerprint Fig ( 4 ). |
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Fig ( 4 ) : AFLP analysis, using different primer combinations. |
Strengths |
•PCR-based |
•Requires minimal amounts of DNA |
•Automatable |
•Robust, Reliable & reproducible |
•No prior sequence knowledge |
•High marker density |
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Sequence targeted and single locus PCR |
A series of very short (2-10), tandemly arranged, highly variable DNA sequences dispersed throughout the genome. |
If SSR loci are cloned and sequenced, primers to the flanking regions can be designed to produce a sequence-tagged microsatellite site (STMS), or SSR marker. |
Microisatellite , know as (SSRs) or short tandem repeats (STRs) are DNA stretched composed of simple motifs 2 to 6 base pairs in length. In plants early studies demonstrated that loci with microsatellites such as (AT)n , (CA)n , ( AAT)n are multi-allelic and somatically stable. This makes microsatellites good candidates for genetic markers in plants ( Akkaya , et al , 1992 ). |
The use of DNA markers in specially important for species with low levels of polymorphism such as wheat. |
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SSRs are highly attractive markers because each primer pair (typically) identifies a single locus. |
SSR loci may have many alleles because of their high mutability. |
Minisatellites are generally very difficult to clone by virtue of their size but if they can be isolated with sufficient flanking sequence for primer design, they provide single locus markers similar to STMS. |
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Fig ( 5 ) : SSR analysis in 20 different lines. |
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For SSR analysis , DNA amplification will be carried out using SSR primer pairs derived from published sequence . The products of the microsatellites based PCR will be detected by electrophoresis on 2 % ethidium bromide stained agarose gels. However, microsatellite alleles may vary in length by only few base pairs fig ( 5 ) . |
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SSR has the advantages : |
strengths |
•Highly abundant & evenly distributed in the genome |
•Highly polymorphic |
•Codominant |
•Rapidly typed |
•Easy to automate |
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Weaknesses |
•Prior sequence Knowledge |
•Difficult & time consuming |
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DATA ANALYSIS |
The banding pattern generated by different molecular markers analyses will be compared to determine the genetic distance among the studied genotypes , the amplified fragments will be scored as present (1) or absent (0). The genetic similarity coefficient (GC ) between two genotypes will be estimated according to Dice coefficients ( sneath and sokal , 1973) as show in figures ( 6 ) . |
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Fig ( 6 ) : Represent band scoring and analysis of data. |
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Fig ( 7 ) : Genetic Similarity matrix calculated according to Jaccard’s coefficient based on marker data. |
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The similarity matrix will be used in the cluster analysis . A computer program based on the unweigthed pair-Group method using arithmetic average ( UPGMA) will be used to develop adendrogram revealing the genetic relationships among the different genotypes fig ( 7 & 8 ). |
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Fig ( 8 ) : Dendrogram constructed with UPGMA cluster analysis of marker data showing the genetic relationships among the different samples. |
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Genomics |
One of the immediate applications of genomics and it’s advanced technology is: To assess the genetic diversity in germplasm collections. |
To identify superior alleles at loci of interest by comparing trait variation and molecular polymorphisms. |
By combing through both germplasm banks and the multitude of plant genome resources, such as cDNA libraries and collections of ESTs and SSRs, to identify useful gene loci, and then moving these genes into crop improvement programs. |
Using comparative genomics, to identify genes for similar traits in related species, and ultimately to build consensus gene maps of various crop species. |
Finally, to develop an informatics platform to store and analyze the data generated. |
To this end new techniques are used some of which: |
Expressed Sequence Tags (ESTs) |
Diversity Arrays™ Technology (DArT) (Kilian,2002) |
ESTs ( Expressed Sequence Tags ) |
Single-pass sequencing reads from randomly selected cDNA clones |
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Steps for EST’s |
•cDNA libraries (containing many of the expressed genes of an organism). • pick cDNA clones randomly. • rapidly determine some of the sequence of nucleotides from the end of each clone. • These ESTs could then be compared to all known sequences using a program called BLAST. |
An exact match to a sequenced gene means that the gene encoding that EST is already known. |
If the match was close but not exact one could conclude that the EST is derived from a gene with a function similar to that of the known gene. |
The EST sequences with their putative identification are then deposited in the GenBank and the clones from which they were derived are kept in a freezer for later use. |
Overview of the EST sequencing process |
Clones are picked from petrie dishes into microtitre plates, and archived for later use. All subsequent manipulations (PCR, clean up and sequencing) are carried out in microtitre plates to yield medium-throughput fig ( 9 ) . |
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Fig ( 9 ) : Steps of EST sequencing process. |
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DArT |
Diversity Arrays™ Technology is a novel genotyping method developed originally using plant genomes as models. |
It provides for low cost, high throughput, sequence-independent genotyping fig ( 10 ) . |
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Genotyping Egyptian Date palm Cultivars Using RAPD, ISSR, AFLP Markers and Estimation of Genetic Stability among Tissue Culture Derived Plants
Sami S. Adawy1, Ebtissam H.A. Hussein 2, Dina El-Khishin 1, Mahmoud, M. Sak er3 , Amina A.Mohamed1 and Hanaiya, A. El-Itriby1
1) Agricultural Genetic Engineering Research Institute (AGERI), ARC, Giza, Egypt.
2) Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt.
3) Plant Cell and Tissue Culture Dept., Genetic Engineering and Biotechnology Division, National
Research Center, Cairo, Egypt.
Abstract
Date palm (Phoenix dactylifera L.) is one of the most important fruit crops in North Africa including Egypt and in Middle East. Egypt lies in the fruit largest producer among Arab countries. However, little is currently known about the molecular characterization of date palm cultivars. Determination of genetic variability and proper cultivar identification in date palm would be of major importance in improvement programs and in germplasm characterization and conservation to control genetic erosion. In an attempt to determine a molecular fingerprint characterizing each of the Egyptian date palm cultivars, three types of PCR based markers, i.e., RAPD, ISSR and AFLPs were applied on two sets of five cultivars cultivated in two different regions, Delta and Upper Egypt .Intravarietal variations were investigated using ten random decamer primers on seven to ten individual trees representing each of the five cultivars of the two sets. All the tested primers exhibited intravarietal polymorphism among the Delta set, while the Upper Egypt set revealed negligible intravarietal polymorphism. To asses the genetic polymorphism and to develop fingerprint for each of these cultivars RAPD,ISSR and AFLP analysis were conducted on bulked DNA samples composed of the DNA of the different trees representing each cultivar .Fingerprinting of the Delta cultivars (Zaghloul, Samany, Hayani, Siwi and Amhat) was conducted using 8 ISSR and 6 AFLP primer /primers combinations. This revealed a total of 53 and 433 amplicons, respectively and a level of polymorphism of 64.1% and 53.81%, respectively. DNA profiling of the five Upper Egypt cultivars (Bertmoda, Gandila, Malikaby, Shameia and Sakkoty) was carried out using 41 RAPD, 19 ISSR and 28 AFLP primer/primers combinations, thus exhibiting 259, 159 and 1135 amplicons representing a level of polymorphism of 18.9%, 34.6% and 41.6%, respectively. The genetic similarity matrices were estimated for the two sets and used to develop dendrograms revealing the genetic relationships. Moreover, unique markers characterizing each cultivar were identified. Furthermore, the genetic stability of tissue culture derived plants from cultivars Zaghloul (Delta) Bertmoda, Gandila, and Sakkoty (Upper Egypt) were studied using RAPD and AFLPs. The DNA profiles exhibited non significant polymorphism indicating the true to type nature of these plants.
Introduction
Dates (Phoenix dactylifera L.) (2n=2x=36) are dioecious, perennial, monocotyledon fruit trees that belong to the family of Arecaceae (Barrow, 1998). Dates is the major fruit crop of arid climate region in countries of North Africa and Middle East. It represents a source of income to Oases inhabitants and creates favorable conditions for improving secondary crop culture like barley, alfaalfa and cloves as forage. The average number of date palm trees in the Arab countries is estimated to be about 62 million trees of which 7.5 million trees are cultivated in Egypt ( FAO, 1996 There are 3 main types of dates based on fruit moisture content i.e., soft, semi-dry, and dry cultivars. Little is known currently about the molecular characterization of date palm varieties .RAPDa PCR based technique established by Welsh and Mc Clelland (1990) and Williams et al.,(1990), is based on the use of a single short usually 10-mer primer of arbitrary sequence. ISSR s are semi –arbitrary markers amplified by using a single primer composed of a microsatellite repeated sequences (Nagaoka and Ogihara,1997,Hussein et al.,2003). AFLP is a method constitutes a powerful and reliable molecular technique widely reported to analyze the genetic diversity among higher plants (Vos et al., 1995). It has also been reliably used for genome mapping and examination of genetic diversity between closely related species and populations (Testolin et al., 2001). AFLP markers were also used in date palm breeding program for potential mapping population (El-Kharbotly et al., 1998). The objectives of this study were, (1) to produce a catalogue revealing the DNA profiling of the Egyptian Date palm cultivars,(2).to investigate the genetic variation at the intra and intervarietal levels in Egyptian date palm,(3) to assess the genetic relationships among the Egyptian date palm cultivars,(4) to identify unique markers characterizing each cultivar and (5) to evaluate the genetic stability of tissue culture derived plantlets. This will have its impact in germplasm collection preservation, date palm breeding and improvement programs.
Materials and Methods
Plant material
Plant material was obtained from the Egyptian Ministry of Agriculture experiment station at Aswan governorate. The date palm cultivars studied were five, i.e., Bertmoda, Gandila, Shameia, Malikaby and Sakkoty. Other five Delta Egypt date palm cultivars i.e. Zaghloul, Samany, Hayany, Siwi and Amhat collected from different locations in Egypt.
DNA extraction
Total DNA was extracted from young leaves following the method described by Porebski et al. (1997). DNA quantitation was performed by comparison of (Gibco BRL) used as standards 1 Kb and 1Kb plus DNA Ladder.
RAPD analysis
RAPD was performed using 41 and 15 decamer oligonucleotide primers as described by Williams et al., (1990) with few modifications in Upper and delta Egypt date palm, respectively.. PCR reactions were carried out in 25 ul volumes containing 25 ng genomic DNA, 20 pmoles primer, 200 uM of each dNTP, 2 mM MgCl2, 1x PCR buffer and 0.4 ul Ampli Taq Polymerase (RTS Taq DNA polymerase). Amplification was performed in a Perkin Elmer Cetus Thermal cycler 9600 with the following program: 94°C for 5 min, then 94°C for 1 min, 36°C for 1 min and 72 °C for 90 sec for 40 cycles. A final extension cycle was performed at 72°C for 7 min. The PCR products were electrophoresed on 1.4% ethidium bromide- stained agarose gels and visualized with a UV transilluminator.
ISSR analysis
Fingerprinting using ISSR was carried out as described by Adawy et al. (2002a and 2004) and Hussein et al. (2003). Nineteen and seven oligonucleotides composed of defined, short tandem repeat sequences with or without anchor and representing different micro satellites have been used as genetic primers in PCR amplifications in Upper and Delta Egypt date palm cultivars. PCR was performed in 25 ml reaction volume containing 1x PCR buffer, 2 mM MgCl2, 200uM of each dNTP, 1 mM primer, 25ng genomic DNA and 1 unit hot start Taq DNA polymerase (Qiagen). Amplification reactions were subjected to a thermocycling profile composed of an initial hot start and denaturation step at 94°C for 10 min, followed by 40 cycles of 1 min 94°C, 1 min at (40°C to 46°C), 2 min at 72°C and a final extension step of 10 min at 72°C. The PCR products were separated on 2% agarose gels and /or 8% polyacrylamide gels, stained with ethidium bromide and photographed.
AFLP analysis
AFLP method was carried out as described by Vos et al., (1995) using the GIBCO BRL AFLP analysis system1 and the AFLP starter primer Kits (Cat No. 10544-013 and 10483-014, respectively). Primers with 3 selective nucleotides comprising 8 Mse 1 primers (CAA/CAC/ CAG/CAT /CTA/ CTC/ CTG/CTT) and 7 EcoR1 primers (AAC/ACA/ACC/ACG/ACT/AGC/AGG) for a total of 28 primer combinations were employed in Upper Egypt date palm while 6 primer combinations were employed in delta region. To determine the size of the AFLP fragments (100-2000bp range),100bp DNA ladder from GIBCO BRL was used.
Data analysis
Only distinct reproducible, well-resolved fragments were scored as present (1) or absent (0). The genetic similarity between different pairs of cultivars was estimated according to Dice coefficient (Sneath and Sokal, 1973). A dendrogram was constructed for each type of molecular markers using the unweighted pair group arithmetic average (UPGMA) method.
Results and Discussion
Due to the dioceous nature of date palm trees, intra-varietal variations are expected. Therefore, a preliminary study was carried out to investigate intra and inter-varietal variations among the ten date palm cultivars using the selected primers in RAPD reactions with sixteen samples from each cultivar.
Polymorphism Among date palm Genotypes as revealed by molecular markers:Intravarietal polymorphism
Intravarietal polymorphism has been investigated among 5 date palm cultivars from the Delta region and 5 other cultivars from Upper Egypt, using RAPD analysis.
To estimate the intravarietal polymorphism, DNA samples from seven individual trees representing each of the five cultivars (Malikaby, Sakkoty, Gandila, Shameia and Bertmoda) were subjected to RAPD analysis using 12 primers. The amplification profiles of the seven samples of each cultivar were monomorphic and none of the studied primers revealed intravarietal polymorphism. While analysis of date palm cultivars from Delta Egypt (Samany, Siwi, Zaghloul, Amhat and Hayany exhibited Intravarietal polymorphism among the Delta set. Intravarietal polymorphism was detected among individuals within the same cultivars from the 16 DNA samples examined in each cultivar. Ten samples were chosen which gave consistant and reproducible results with the fifteen primers (Fig 1). This suggests that the date palm cultivars from Upper Egypt are genetically more homogenous than those from Delta Egypt.
Fig (1) RAPD profiles for individual date palm samples from delta (A) Samany, (B) Hayany, amplified by OPB12 primer. M refers to Lambda Hind III digest, Phi x174 Hae11 digest.
Intervarietal polymorphism
To investigate intervarietal polymorphism among the five Upper Egypt date palm cultivars, RAPD, ISSR and AFLP analysis have been conducted on bulked DNA samples composed of 7-10 trees representing each cultivar.
RAPD analysis
The five bulked Upper Egypt samples were assayed using 41 RAPD primers, while the Delta samples were investigated using 10 RAPD primers. All the tested primers generated reproducible and easily scorable RAPD profiles. These primers produced multiple band profiles with a number of amplified DNA fragments ranging from 2 to 13 of the Upper Egypt cultivars. Fingerprinting revealed a total number of 259 unambiguous DNA fragments with an average of 6.30 fragment /primer. The number of polymorphic bands ranged from 0 to 5 per primer with an average of 1.2 polymorphic bands per primer. The total number of polymorphic amplicons produced by the 41 primers was 49, thus, representing a level of polymorphism of 18.92% (Table1) .The genetic similarity between cultivars was assessed on the basis of the Dice similarity coefficient and complemented with the UPGMA cluster analysis. Pair wise comparisons of RAPD profiles were resulted in a similarity matrix. The dendrogram illustrating the genetic relationships among the five Upper Egypt date palm cultivars. In this context, Isabel et al. (1993) and Letouze`et al,(1998) reported that RAPD technique can be routinely applied for varietal identification and gives reproducible results. Moreover, Saker and Moursy (1998) used the RAPD technology to evaluate the genetic polymorphism among the same cultivars and found that only six of the ten studied primers revealed polymorphism. While, Letouze et al.(1998)differentiated 13 different date palm cultivars using 5 decamer primers which provided 14 polymorphic markers.
Table 1- Level of polymorphism among Upper Egypt date palm cultivars detected by RAPD primers.
Primers | Total No. Primers | Total No. of amplicons | Polymorphic amplicons | Polymorphism % |
Total | 41 | 259 | 49 | 18..9 |
Average | 6.3 | 1.2 | ||
ISSR analysis
The ISSR analysis was performed on the bulked DNA samples representing the five Upper Egypt cultivars using 19 ISSR primers composed of short tandem repeat sequences with or without anchor . Fig (2) illustrating the ISSR profile of Upper Egypt date palm . A total of 159 amplicons were generated by the tested primers with an average number of 8.4 amplicons/primer. Primer A8 exhibited the highest number of fragments (16 amplicons), while primers Amic 8 revealed the least number (3 amplicons). The total number of polymorphic bands was 55 with an average of 2.9 polymorphic amplicons per primer. This represents a level of polymorphism of 34.6% Table(2).
Table 2- Level of polymorphism among Upper Egypt date palm cultivars detected by ISSR analysis.
No. of Primers | Total No. of bands | Polymorphic amplicons | % of poly-morphism |
19 | 159 | 55 | 34.59 |
Average | 8.4 | 2.9 |
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The results of the present study revealed that the polymorphism detected by the ISSR assay, although it was lower than that detected among Delta Egyptian date palm cultivars, however, it was higher than the polymorphism detected by the RAPD assay 34.6% The number of polymorphic markers varied among the different primers. Primer A8 generated 8 polymorphic bands out of 16, while primers A6, A7 and Amic8 yielded only monomorphic amplicons.
The total number of unique ISSR markers in Upper Egypt was 31.The cultivar Malikaby was characterized by 3 positive and 2 negative markers. Gandila was distinguished by 2 positive and 3 negative markers. Sakkoty was characterized by 3 positive and 4 negative markers. Shameia revealed one positive and 7 unique negative markers. While Bertmoda exhibited 2 positive and 4 unique negative markers. The genetic distance estimates based on ISSRs ranged from 80.2% to 89.0%. The lowest similarity value was between Malikaby and Shameia. While, the highest value was between Shameia and Bertmoda. A dendrogram constructed by cluster analysis using ISSR based genetic distance is presented in Fig (3). The overall tree topology suggested a rather weak grouping association except for the cultivars Shameia and Bertmoda which clustered together.
The bulked samples of the 5 Delta Egypt date palm cultivars were analyzed using 7 Inter simple sequence repeat (ISSR) primers . These included five anchored primers at the 5` end with 3 base pairs and 2 primers composed of non- anchored repetitive sequences. The 7 ISSR studied primers produced good reproducible and scorable patterns and the amplification profiles were screened for the presence of polymorphisms among the 5 date palm cultivars. a total of 53 fragments were generated by the 7 primers, with primer Amic 1 yielding the highest number of products (11 amplicons) and primer A mic7 the least (4 amplicons).The number of polymorphic markers also varied between primers, with primer Mic3 generating only polymorphic bands (100% polymorphism) and primer Amic1 revealing 10 polymorphic bands out of 11 and Amic7 yielding only monomorphic bands. Similarly, Cekic et al. (2001) used 10 ISSR primers for screening of polymorphism between two closely related forms of F. vesca and stated that the number of polymorphic bands varied between primers. Among the seven studied ISSR primers ,six revealed unique markers characterizing each of the four cultivars. The total number of unique ISSR markers was 24 in Delta Egypt date palm cultivars .
Fig (2). ISSR profiles of the five date palm cultivars Malikaby (1), Sakkoty (2), Shameia (3), Gandila (4) and Bertmoda (5) as detected by different ISSR primers. A9 (a), A10 (b), .M refers to DNA standards.
AFLP Analysis
The technique of AFLP required initial optimization to identify primer combinations that yield reproducible and discernible patterns .Therefore, 40 primer combinations were tested with the five bulked DNA samples representing the five Upper Egypt date palm cultivars. Among the tested primer combinations, 28 EcoRI/MseI selective combinations yielded reproducible and discernible profiles. The rest of primer combinations did not yield any amplification. The 28 primer combinations produced 1135 bands, 41.59% being polymorphic across cultivars. The average number of polymorphic bands was 16.86 per AFLP primer combination (Table3). The size of the AFLP amplified fragments ranged from 2600 bp to 50 bp .The primer combination (5/5) showed the highest percentage of polymorphic bands (81.15%), while the lowest polymorphism (16.95%) was revealed by primer combination (8/1). The number of amplified bands per primer combination ranged between 17 and 69. Genetic relationships among the five date palm cultivars based on the AFLP data polymorphism using Dice coefficients of similarity are shown in Fig(3). The dendrogram confirmed that the cultivar Bertmoda does not cluster with any other cultivar tested and is easily distinguishable, while the cultivars Gandila and Malikaby were the most genetically similar among the studied cultivars, with Shameia and Sakkoty next. Moreover, eight primer combinations were tested with the five bulked DNA samples representing the five Delta Egypt date palm cultivars. Among the eight tested primer combinations, six EcoR1/Mse1 selective
Table (3): Level of polymorphism among Upper Egypt date palm cultivars detected by 28 AFLP primer combinations.
No. Primer combination | Total number of amplicons | Polymorphic amplicons | Polymorphism% |
28 | 1135 | 472 |
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Average | 40.5 | 16.9 | 41.59 |
primer combinations yielded discernible reproducible profiles. The 6 Primer combinations produced 433 bands,53.8% being polymorphic across cultivars. The average number of polymorphic bands was 29.1 per AFLP primer combination Table (4).The average number of bands per primer combination was 54.1. In this respect, similar findings were reported by Han et al., (2000) studying tea species, they found that the number of amplified AFLP bands per assay ranged from 32 to 150 with a mean of 84.7 and an average of 10.5 polymorphic bands per primer combination. Le Febvre et al. (2001) analyzed 47 pepper inbred lines with 10 AFLP primer combinations and revealed 863 selectively amplified fragments of which 378 were polymorphic (34.8%). Matthes et al. (2001) used ten AFLP primer combinations with oil palm and reported that the average number of bands per primer combination was 82 which is in agreement with our results. The number of distinguishable bands detected after selective amplification varied among the different primer combinations.
Table (4): Level of polymorphism among Delta Egypt date palm cultivars primer combinations.
Primer combination | Total number of amplicons | Polymorphic amplicons | Polymorphism% |
6 | 433 | 233 | 53.8 |
| 54.1 | 29.1 |
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The highest number of amplicons (106) was exhibited by the primer combination (4.4), whereas the lowest number was 33 as revealed by primer combination (6.6) (Table 5). In this concern, Goulao et al. (2001) reported a range of 34 to 66 fragments per primer pair in apple cultivars.The level of polymorphism ranged from 42.42% to 59.02% in primer combinations 6.6 and 7.7, respectively. Similarly, Cervera et al., (1998) applied the AFLP technique to characterize 67 different grapevine accessions. They obtained an average of 100 amplified fragments per primer combination, of which 49% were polymorphic. Moreover, Hussein et al., (2002) reported that the level of polymorphism revealed by six AFLP primer combinations on cotton ranged from 38% to 65%.
The genetic similarity estimates among the five Delta cultivars ranged from 64.4% to 76.7%. This revealed moderate levels of genetic similarity among the studied cultivars. The highest genetic similarity (76.7%) was between Siwi and Hayani. This was followed by 75.2% between Samany and Amhat, while the lowest genetic similarity (64.4%) was detected between Zaghloul and Samany. These genetic relationships were reflected on the dendrogram which represents the graphical illustration of the genetic distances among the five date palm cultivars (Fig.2). The dendrogram confirmed that the cultivars Siwi and Hayani were the most genetically similar among the studied cultivars, with Amhat and Samany next, while Zaghloul was the most distinct cultivar. Fig (4) illustrating the AFLP profiles of the five upper and delta Egypt date palm cultivars as detected by some different primer combinations.
Table (5):Unique positive and negative AFLP markersfor the five Upper Egypt cultivars.
Primer combination | Malikaby | Gandila | Sakkoty | Shameia | Bertmoda | |||||
UPM | UNM | UPM | UNM | UPM | UNM | UPM | UNM | UPM | UNM | |
Total UPM & UNM | 11 | 15 | 22 | 24 | 6 | 16 | 11 | 21 | 8 | 57 |
Grand total | 26 | 46 | 22 | 32 | 65 | |||||
Cultivar identification by unique AFLP markers
As shown in Table (5), the total number of unique bands across the five Upper Egypt cultivars was 58 positive (UPM) and 133 negative markers (UNM) in the different cultivars .The number of UPM ranged from 6 to 22 and the number of UNM ranged from 15 to 57 in the different cultivars.
Table (6), permitted the distinction of AFLP analysis among the five studied Delta date palm cultivars and the characterization of each cultivar by specific unique markers. A total of 78 positive and 48 negative markers were identified by the six AFLP primer combinations. The total number of unique markers per genotype ranged from 13 to 51. The cultivar Zaghloul was characterized by the highest number of unique positive markers (48), in addition to 3 unique negative markers. Siwi was identified by 9 positive and 7 negative markers. Hayany exhibited a total of 23 unique markers among which 10 were positive. Samani also exhibited a total of 23 unique markers, seven of which were positive while the other 16 were negative. While, Amhat revealed the lowest number of unique markers (13) with 4 positive and 9 negative markers.
Table ( 6) : Unique positive and negative AFLP markers for the five Delta Egypt date palm cultivars.
Primer combination | Siwi | Hayany | Amhat | Samany | Zaghloul | |||||
UPM | UNM | UPM | UNM | UPM | UNM | UPM | UNM | UPM | UNM | |
Total UPM UNM | 9 | 7 | 10 | 13 | 4 | 9 | 7 | 16 | 48 | 3 |
Grand total | 16 | 23 | 13 | 23 | 51 | |||||
Fig (4 - A), AFLP analysis in date palm, using different primer combinations. No. 7.7 and 8.8 .
Combined RAPD, ISSR and AFLP data
The three applied marker techniques (RAPD ISSR and AFLP ) amplify different parts of the genome. This was partially reflected on the topology of the phylogenetic trees drawn from the data of the three assays. Therefore, to obtain more balanced values for genetic similarity among cultivars and an equilibrated dendrogram representation of the relationships among the two sets of studied date palm cultivars, the data of RAPD, ISSR and AFLP analysis were combined (Fig 3).
A summary of the effectiveness of the different markers is given in Table (7). AFLP were the most effective in that all primer pairs tested detected polymorphism. AFLP showed 16.9 as average polymorphism per primer combination compared with 2.9 and 1.2 in ISSR and RAPD, respectively. While the percentage of polymorphism detected was 41.6% in AFLP compared to 34.6% and 18.9% in ISSR and RAPD, respectively.The dendrogram constructed on the basis of the combined data from RAPD, ISSR and AFLP analyses showed the same grouping pattern as generated by AFLP (Fig.3) and thus confirming that AFLP is the most effective. The use of RAPD and ISSR and AFLP techniques on the date palm genome enabled us to generate many polymorphic markers ensuring a good coverage of the genome.
RAPD ISSR
Fig (3): Dendrogram of 5 date palm cultivars based on similarity index by RAPD, ISSR, AFLP and combined data.
Table (7): Effectiveness of RAPD, ISSR and AFLP markers in detecting Polymorphism in data palm cultivars.
| RAPD | ISSR | AFLP |
Total band detected | 259 | 159 | 1135 |
Polymorphism detected | 49 | 55 | 472 |
% of total | 18.9 | 34.6 | 41.6 |
No. of primers used | 41 | 19 | 28 |
Average polymorphisms per primer/primer pair | 1.2 | 2.9 | 16.9 |
Fig (4-B). AFLP profiles of the five date palm cultivars Malikaby, Gandila, Sakkoty, Shameia and Bertmoda as detected by primer combinations (A) 1/3, (B) 1/8, (C) 7/2, (D) 4/1 and (E) 3/7. M.DNA molecular weight standard (100 bp ladder).
Genetic stability of somatic embryo- derived plantlets
The tissue culture overcomes the problem of date palm cultivation with traditional methods which prevent rapid crop improvement of the date palm trees. However, the tissue culture protocol should maintain the genotype of the original cultivar, i.e . we must prove that tissue culture – derived plants are true to type This problem could be avoided if an accurate identification method was utilized at an early stage of propagation. To investigate the genetic stability (true to type) of the clones derived from date palm tissue culture , ten seedlings derived from in vitro culture of the cultivar Zaghloul (Delta) Bertmoda,Gandila and Sakkoty ( Upper Egypt) was assayed using RAPD and AFLP. The DNA profiles of the tissue culture derived plants exhibited non significant polymorphism indicating the true to type of these plants
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