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 .

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).

Fig ( 1 ) : Color variation representing a morphological marker.

·        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.

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.  

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:

1. Hybridization-based (non-PCR) techniques

2. Arbitrarily-primed PCR techniques

3. Sequence targeted an d single locus PCR

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).

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.

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.

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

Fig (2 – a ) : The different steps of RFLP analysis.

Fig (2 - b) : Polymorphism among 13 different accessions.

Arbitrarily-primed PCR techniques

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.

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 ).

2- DNA Amplification Fingerprinting (DAF) in which the products are separated on polyacrylamide gels.

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.

1-Selective Restriction Fragment Amplification (SRFA).

2-Amplified Fragment Length Polymorphism (AFLP).

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 ).

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

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.

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.

Fig ( 5 ) : SSR analysis in 20 different lines.

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 ) . 

SSR has the advantages :

strengths

•Highly abundant & evenly distributed in the genome

•Highly polymorphic

•Codominant

•Rapidly typed

•Easy to automate

Weaknesses

•Prior sequence Knowledge

•Difficult & time consuming

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 ) .