6. Molecular Markers

Question 1

What is a marker?

Answer 1

1. A marker is an allele of known genomic position
2. can be used in genetic assays to establish the localisation of other genes

Question 2

What are the characteristics of Molecular Markers?

Answer 2

1. They identify differences at the DNA sequence level
2. they are not influenced by the environment or by the developmental stage of the plant
3. their number is practically infinite
4. they are ubiquitous within each genome (both from transcribed and not- transcribed regions)
5. they are neutrals because they do not have any direct influence on the phenotype (difference from morphologic markers)
6. Relatively easy to identify (depending on the technique used)

Question 3

What are the Base techniques for molecular markers detection?

Answer 3

1. DNA Electrophoresis
2. DNA Restriction
3. DNA hybridisation
4. DNA Amplification by PCR

Explained in the slides..

Question 4

What are the main Applications of molecular markers?

Answer 4

1. Varietal identification or “fingerprinting”
2. Phylogenetic analysis
3. Genetic map construction
4. Linkage with phenotypic traits (linkage analysis)
5. Marker-Assisted Selection or MAS
6. Isolation and cloning of genes of agricultural interest

Question 5

Explain the classification of the main molecular markers?

Answer 5

• On the base of the combination of the different detection techniques

1. Restriction and Hybridisation

• RFLP = Restriction Fragment Length Polymorphisms

2. Amplification

• RAPD = Random Amplified Polymorphic DNA
• SCAR = Sequence Characterised Amplified Regions
• SSR = Simple Sequence Repeats o Microsatellites Amplification and Restriction
• CAPS = Cleaved Amplified Polymorphic Fragments

3. Restriction, Ligation and Amplification

• AFLP = Amplified Fragment Lenght Polymorphisms

4. Sequencing

• SNP = Single Nucleotide Polymorphism

Question 6

Classification of the main molecular markers

Please explain Restriction and Hybridisation?

RFLP = Restriction Fragment Length Polymorphisms

Answer 6

1. The basic technique for the detecting of RFLPs involves fragmenting a sample of DNA by a restriction enzyme
2. which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest.
3. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis and transferred to a membrane via the Southern blot procedure
4. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe
5. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis.

Question 7

What are the advantages of RFLP?

Answer 7

1. RFLPs are applicable in each species
2. They have a simple mendelian inheritance
3. The a priori knowledge of the probe sequences are not necessary
4. Reliable technique
5. Good result reproducibility among different labs
6. Illimitate number
7. Co-dominant
8. Phenotypically neutral

Question 8

What are the disadvantages of RFLP?

Answer 8

1. Complex technique
2. Needs large quantities (10 μg/lane) of highly pure DNA
3. Lab costs are medium-high
4. The radioactive method is the more widely used but there are higher lab risks because of the use of radioactive probes

Question 9

Please explain the RAPD or “Random Amplified Polymorphic DNA”?

Amplification Technique

Answer 9

RAPD Modification of the base PCR technique:

1. a single “primer” of 10 bp is used→low annealing T° (usually at 36 °C)
2. amplification is possible each time that two primer annealing sites (on the opposite DNA strands) are at amplificable distance (generally < 2,5-4 kb)
3. random amplification of some DNA fragments / reaction fragments separation in agarose gels→amplification
4. “patterns” (specific for each primer and used genotype)
5. a wide set of primers with random sequence is available and these primers can be used in any species

Question 10

What are the Advantages of RAPD markers?

Amplification technique

Answer 10

1. Technique easy and quick→name “Rapid”
2. Set of decamer “primers” with random sequence→can be used in any species
3. No information on the amplified sequence is required
4. Do not require radioisotopes
5. Needs a very low DNA amount per reaction
6. Limited cost per reaction
7. Amplification from any genome regions→Polymorphism level in fruit tree species→medium-low
8. High discrimination power

Question 11

What are the disadvantages of RAPD?

Answer 11

1. Dominant markers (it is not possible to distinguish heterozygotes and dominant homozygotes→band)
2. Limited transportability (reduced allelism→presence/ absence)
3. Amplification results are depending on:
   DNA quality and extraction method and the reagent concentrations
4. Sometimes the result reproducibility between labs is low

Question 12

What are the Reaction components for DNA amplification?

Answer 12

1. Reaction buffer (reagents that favour the reaction, i.e. Mg)
2. Nucleotides (as “bricks” for the ex-novo synthesis of new DNA)
3. Primer (flanking the specific DNA fragment to be amplified)
4. DNA polymerase (enzyme able to make the synthesis of new DNA)
5. total DNA (as template for the synthesis of new DNA)

• The enzyme used for the PCR reaction is a thermostable DNA polymerase→Taq polymerase, extracted from the bacteria Thermus acquaticus that live at high temperature

Question 13

What are the advantages of AFLP?

Answer 13

1. Technique that can be applied to all the species
2. No knowledge on DNA sequences is required
3. Illimitate number and simultaneous scoring of many loci in each analysis (About 50 fragments)
4. Degree of polymorphism→medium
5. Low amount of DNA are required (about 500 ng/digestion) Good result reproducibility between different labs

Question 14

What are the disadvantages of AFLP?

Answer 14

1. AFLPs require high quality DNA
2. Medium lab costs

Question 15

What is SNP?

Answer 15

• Single nucleotide polymorphism (SNP) is a point mutation (a change in one nucleotide) for which a short flanking sequence is known.

1. Each allele is defined by the actual nucleotide in the sequence.
2. SNPs are generated by point mutations at any place in the genome.
3. SNPs are single locus markers and mostly have two alleles in each locus
4. Their mode of inheritance is co-dominant

Question 16

What are the advantages of SNP?

Answer 16

1. Relativly easy
2. The use of SNP Chips makes it possible the analysis of thousands of SNPs well distributed in the whole genome of a species in a single experimental run

Question 17

Overview and characteristics of different DNA markers

Answer 17