Prof dr naglaa abdel monem mahmoud ashry an introduction to the use of molecular genetics in plant b

Contact: Dr. Naglaa A. Ashry

naglaashry@yahoo.com

An introduction to the use of molecular genetics in plant breeding

Enzymes in DNA replication

Helicase unwinds parental double helix

DNA polymerase binds nucleotides to form new strands

Binding proteins stabilize separate strands

DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases

Primase adds short primer to template strand

Ligase joins Okazaki fragments and seals other nicks in sugar25 phosphate backbone

26.72%

Fingerprinting Varietal/line identification Diversity studies Novel allele detections Marker-assisted selection Seed testing

Types and applications of molecular markers

A marker/ tag An identifier of a particular phenotype and/or genotype; its inheritance can easily be followed from generation to generation.

Types of markers 1. Morphological marker A visible character (phenotype) (flower color, seed color, height, leaf shapes).

2. Biochemical marker based on the detection of natural products (protein and/or isozymes) being produced in a specific tissue (profiles).

3. Molecular marker based on DNA polymorphism detected by DNA probes or amplified products of polymerase chain reaction.

Types of markers 4. Cytological marker Chromosomal banding patterns produced by different stains; (G banding technique produce differently stained regions on condensed chromosomes)

5. Biological Host pathogen or host parasite interaction can be used as a marker.

Biochemical markers Protein banding patterns Advantages • Inexpensive • Markers are co-dominant

Disadvantages • Many DNA variants do not result in changes in amino acid sequence • Only one set of structural genes of organisms are represented in these proteins (the coding regions) and this set may not be representative of the whole genome.

Advantages: • genic Isozymes Enzyme forms which catalyze • inexpensive • Co-dominant the same reaction but are coded by more than one • Reproducible locus • Easy to develop & assay • Relatively polymorphic Disadvantages: • limited number • biased • tissue demanding • automation is impossible

DNA based molecular markers • • • •

They are not subject to environmental influence They are unlimited in number Usually they are more objective They can be easier to analyze

Common DNA marker techniques • • • •

Restriction Fragment Length Polymorphisms (RFLPs).

Random Amplified Polymorphic DNA (RAPD).

Amplified Fragment Length Polymorphism (AFLP).

Simple Sequence Repeats (SSRs)

What is polymorphism?

Molecular markers are based on naturally occurring polymorphisms in DNA sequences (deletions, substitutions, additions or patterns) Definition: Polymorphism = The “presence of many forms” •In genetic terms “the co-existence of two or more alternative phenotypes in a population or among populations”.

•At the molecular level, polymorphism refers to the co-existence of alternative banding patterns or DNA variants

Common Procedures used for analysis of molecular markers • Restriction digestion • Gel electrophoresis • PCR

Marker visualization on agarose gel (electrophoresis)

ďƒ˜ Agarose is a polysaccharide extracted from marine alge and used as a matrix to separate DNA molecules ďƒ˜ Because DNA ia a (-) charged molecule when subjected to an electric current it will migrate towards a (+) pole

Advantages: • numerous • Co-dominant • Reproducible • representative • relatively polymorphic Disadvantages: • laborious • complex patterns • large amount DNA required • automation is difficult

Structure

Amplified Polymorphic DNA

(RAPD)

Target Sequence

= arbitrary primer (e.g. ggcattactc) High Variability: Probably due to mutations in priming sequences

PCR-based method Amplify regions between priming sites by polymerase chain reaction

Analyze PCR products by agarose gel electrophoresis.

Marker is dominant (presence/absence of band).

No prior sequence knowledge required

Random Amplified Polymorphic DNA (RAPD) Advantages:

• numerous • inexpensive • easy to develop & assay • very polymorphic Disadvantages:

• low reproducibility • anonymous • dominant

AFLP  Polymorphism based on gain or loss of restriction site, or selective bases

 Many markers generated, mostly dominant  No prior sequence knowledge required The major steps of AFLP technique * Restriction digestion of Genomic DNA * Adapters Ligation Reaction * Preselective PCR Amplification Reactions * Selective Amplification PCR Reactions

Amplified Fragment Length Polymorphism (AFLP)

Advantages: •numerous •moderately expensive •very polymorphic Disadvantages: •dominant •difficult to score •technically demanding •requires high quality and quantity of DNA

Sequence Characterized Amplified Regions (SCARs) Also called Sequence Tagged Sites (STS) RAPD or AFLP fragments correlated to the presence of a favorable allele, can be converted to a more reliable marker called a SCAR (Paran and Michelmore 1993).

* purify the DNA fragment (by cutting it out of the gel). * clone and sequence. * Design specific primers .

Cleaved Amplified Polymorphic Sequences (CAPS) *A CAPS marker represents a refinement of a STS .

Where an STS assay shows no allelic variation in amplicon size,it may still be informative if the amplicon varies in sequence between individuals. such sequence variation can be identified by treatment with a restriction enzyme after the PCR. note that the "C" in CAPS stands for "cleaved" to reflect

the need for restriction digestion to identify the polymorphism)

.

• CAPS marker needs to specify both • the primers and the specific • restriction enzyme used.

Microsatellites (Simple Sequence Repeats) ďƒ˜ Structure

Unique flanking regions

= Repeat (e.g., ga)

ďƒ˜ Number of repeats is highly variable among individuals

Design primers (

) complementary to flanking regions

Amplify repeat region by polymerase chain reaction

Analyze PCR products by agarose gel and polyacrylamide gel electrophoresis Marker is codominant

Microsatellites or Simple Sequence Repeats (SSRs)

Advantages: •numerous •Co-dominant (mostly) •reproducible (within species) •very polymorphic •automation is possible Disadvantages: •expensive to develop •Prior sequence Knowledge required •low transferability across genera

Single Nucleotide Polymorphism (SNPs) * SNPs are differences in DNA sequence of just one (or sometimes a small number of) nucleotides. ** These differences occur within a genic sequence.

**They can be associated with a change in the amino acid sequence of the gene product. ** They are very common, and are distributed throughout the genome SNP genotyping can be relatively simple, but SNP discovery generally requires extensive DNA sequencing.

Single Nucleotide Polymorphism (SNP) Advantages: • numerous • Co-dominant • easy to assay • reproducible • potentially suited for automated technology (DNA-chips) Disadvantages: • laborious detection • not universal

Other marker types New marker types are always being developed; just a few additional types are noted here, TRAP (Targeted Region Amplified Polymorphism) (Miklas et al. 2006) SRAP (Sequence Related Amplified Polymorphism, targeting open reading frames) (Li and Quiros 2001) DArT (Diversity Array Technology): http://www.diversityarrays.com

Retromarkers

There are 5 conditions that characterize a suitable molecular markers • Must be polymorphic • CO_dominant inheritance • Randomly and frequently distributed throughout the genome • Easy and cheap to detect • Reproducible

Application of molecular markers • Fingerprinting – Identification of genotypes

– Monitoring genetic diversity in breeding materials.

– Efficient management of genetic resources

• Quantitative Trait Loci mapping • Marker-Assisted Selection (MAS)

Advantages of Molecular Markers in plant breeding • They can save a lot of time in the breeding process • They may aid in discovering more information about the function of the genes of interest • They have many uses, including genetic diversity assessment, quality control (e.g. in variety development), marker-assisted breeding