Types and applications of molecular markers

Types and applications of molecular markers

Contact: Dr. Naglaa A. Ashry

naglaashry@yahoo.com

SAX in 1923 first reported association of a simply inherited genetic marker with a quantitative trait in plants when he observed segregation of seed size associated with segregation for a seed coat color marker in beans (Phaseolus vulgaris L. )

A marker (sometimes called a tag) is: identifier of a particular phenotype and/or genotype; its inheritance can easily be followed from generation to generation.

Types of markers 1. Morphological marker based on visible characters (phenotypic expression) e.g.. • flower color, seed color, height, leaf shapes.

2. Biochemical marker based on detection of natural enzymes (isozymes) being • produced, each individual variety has its own isozyme variability (profiles) which can be detected by electrophoresis on a starch gel.

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

Types of markers 4. Cytological marker the chromosomal banding produced by different stains; • for example,G banding (technique used in cytogenetics to produce differently stained regions on condensed chromosomes).

5. Biological Different pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites.

•

Biochemical markers 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 and this set may not be representative of the whole genome.

Isozymes Enzyme forms which catalyse the same reaction but are coded by more than one locus

Advantages: genic • inexpensive • codominant • reproducible • easy to develop & assay • relatively polymorphic •

Disadvantages: • limited number • biased • only nonsynonymous variation • tissue demanding • automation is impossible

Advantages of DNA molecular markers • They are not subject to environmental influence • They are unlimited in number • They are usually more objective • They can be easier to analyze • They may be less expensive than some types of markers (especially when they can be done in high-throughput)

DNA marker technologies • Restriction Fragment Length Polymorphisms (RFLPs).

• Random Amplified Polymorphic DNA (RAPD).

• Amplified Fragment Length Polymorphism (AFLP).

• Simple Sequence Repeats (SSRs)

polymorphism Molecular markers are based on naturally occurring polymorphisms in DNA sequences base pair (deletions, substitutions, additions or patterns) Definition: The word polymorphism technically means ‘presence of many forms •In genetic terms, it refers to the coexistence of two or more alternative phenotypes in a population or among populations.

• In general, these diverse phenotypes are caused by alternative alleles of one gene/locus •At the molecular level, polymorphism refers to the coexistence of alternative banding patterns or DNA variants

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

Theoretical basis of agarose gel electrophoresis

ďƒ˜ Agarose is a polysaccharide from marine alage that is used in 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

Pouring an agarose gel 1

2

3

4

5

6

7

8

9

Restriction Enzymes  Hundreds of restriction enzymes have been identified.

Most recognize and cut palindromic  sequences Many leave staggered (sticky) ends  by choosing correct enzymes can cut DNA  very precisely Important for molecular biologists  because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence

PCR Steps

RFLP steps

Restriction Fragment Length Polymorphism (RFLP) Advantages:

Disadvantages:

• • • • •

• laborious • complex patterns • large amount DNA

numerous codominant reproducible representative relatively polymorphic

required • automation is difficult

Random Amplified Polymorphic DNA, AP-PCR

Structure

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

RAPD analysis RAPD PCR reactions 20 ng of genomic DNA 25 pmoles primer 2 mM dNTPs 2 mM MgCl2 2.25 unit Taq polymerase (Fermentas) 1X buffer (NH4)2 SO4.

Total volume 25 ul

RAPD PCR program

Random Amplified Polymorphic DNA (RAPD) Advantages:

Disadvantages:

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

• low reproducibility • anonymous • dominant

AFLP analysis (A) Restriction digestion of Genomic DNA (B) Adapters Ligation Reaction (C) PreselectivePCRAmplificationReactions (D) Selective Amplification PCR Reactions

Amplified Fragment Length Polymorphism  Polymorphism based on gain or loss of restriction site, or selective bases  Many markers generated, mostly dominant  No prior sequence knowledge required

PCR reaction of preamplification Component Diluted ligation mix Pre-amp.primer mix 10 x PCR buffer Taq DNA polymerase Total volume

Amount / ul 5 ul 40 ul 5 ul 1 ul (5 unit / ul) 51

PCR program of preamplification

20 Cycles

94 C 30 sec

56 C 1min

72 C 1min

4 C 

Selective PCR reaction Two mixes were prepared Mix 1

EcoRI primer MseI primer with dNTP’s

0.5 ul 4.5 ul

Mix 2 Diluted pre-amp products (1:50) PCR buffer Taq DNA polymerase (5u/ul) Deionized distilled water Total volume

5 ul 2 ul 0.5 ul 7.5 ul 20 ul

Selective PCR program 94 C

23 Cycles

13 Cycles 65 C

30 sec

72 C

1min 30 sec

*

94 C

30 sec

72 C 56 C 30 sec

1min

4 C



AFLP profiles of the six maize inbred lines using primer combinations A: E- AGG+M-CAC and B: E-ACT+ M-CTC. 1: Gm 2, 2: Gm 7, 3: Gm 18, 4: Gm 30, 5: Sd 7 and 6: Sd 63. M: DNA molecular weight marker (50 bp ladder, Sigma).

Amplified Fragment Length Polymorphism (AFLP)

Advantages: •numerous •moderately expensive •very polymorphic

Disadvantages: •anonymous •dominant •difficult to score •technically demanding •requires high quality and quantity of DNA

SCARs If a RAPD or an AFLP fragment appears to be correlated with the presence of a favourable allele at an important gene, it can be converted to a more reliable marker called a Sequence Characterized Amplified Region (Paran and Michelmore 1993) (or simply a Sequence-tagged site, STS).

The idea is to first purify the DNA fragment (by cutting it out of the gel), then clone it. The DNA sequence of the clone will allow for specific primers to be designed.

Obviously this is not doable on a large scale, but it is a useful way of exploiting individual RAPD or AFLP fragments in MAB.

Cleaved Amplified Polymorphism Sequences (CAPS)

A CAPS marker represents a refinement of a STS marker. Where an STS assay shows no allelic variation in amplicon size,it may still be informative if the amplicon varies in sequence between individuals.

If such sequence variation can be identified by treatment with a restriction enzyme after the PCR, the STS becomes a CAPs marker (note that the "C" in CAPS stands for "cleaved" to reflect the need for restriction digestion to identify the polymorphism).

Since each restriction enzyme has its unique recognition site, a 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

SSR analysis PCR reaction 50 ng genomic DNA 50 ng of each forward and reverse SSR primers 2.5 mM MgCl2 0.4 mM of each dNTP 0.3 units of Ampli Taq Gold The PCR reactions were performed in 25 ul volume

PCR program 30 Cycles

10 Cycles 94 C 10min

94 C 1min

72 C 94 C

65 C * 1min

72 C 72 C 55 C

1.5 min 1min

* 1min

1.5 min 7min 4 C



SSR profiles of the six maize inbred lines using primers MZE. RACTB and MAG.E01 on 3% agarose gel and primers Phi053 and MZE.ADH2N on 8% polyacrylamide gel. 1: Gm 2, 2: Gm 7, 3: Gm 18, 4: Gm 30, 5: Sd 7 and 6: Sd 63. M: DNA molecular weight marker (100 bp ladder, Amersham).

Microsatellites or Simple Sequence Repeats (SSRs)

Advantages: •numerous •codominant (mostly) •reproducible (within species) •very polymorphic •automation is possible

Disadvantages:

•expensive to develop •Prior sequence Knowledge required •low transferability across genera

Single Nucleotide Polymorphism (SNP) SNPs (pronounced “snips�) are differences in DNA sequence of just one (or sometimes a small number of) nucleotides. Where these differences occur within a genic sequence, they are more often than not phenotypically neutral, but sometimes 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. Although not as yet not widely used in MAB

Single Nucleotide Polymorphism (SNP) Advantages: • numerous • codominant • 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, with references for further information: 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

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 Locus mapping • Marker-Assisted Selection (MAS)

The role of molecular markers in plant breeding

Genetic similarity (GS) matrix as revealed by combined data Lines

Gemmeiza 2

Gemmeiza 7

Gemmeiza 18

Gemmeiza 30

Sids 7

Gemmeiza 2

100.0

Gemmeiza 7

65.3

100.0

Gemmeiza 18

65.9

69.2

100.0

Gemmeiza 30

58.6

63

63.1

100.0

Sids 7

58.2

69.1

60

66.7

100.0

Sids 63

57.7

61.1

58.3

62.1

66.4

Sids 63

100.0

Genetic relationship as revealed by the combined data of RAPD, SSR and AFLP. .

Dendrogram for the six white maize inbred lines constructed from RAPD, SSR and AFLP combined data using UPGMA and similarity matrix computed according to Dice coefficient.

Molecular markers as predictors of combining ability and hybrid performance Simple correlation coefficients between genetic distances (GDs) and specific genetic distances (SGDs) as revealed by DNA markers (AFLP, RAPD, SSR) and their combined data, specific combining ability (SCA), mid-parents heterosis (MPH), high-parent heterosis (HPH) and grain yield combined for all locations during 2003 season.

Parameter

Grain yield

SCA

MPH

HPH

0.84** SCA MPH

0.08

0.53*

HPH

0.08

0.51*

0.92**

AFLP

-0.20 (0.09)

-0.07 (0.19)

0.24 (0.16)

0.26 (0.19)

RAPD

-0.18 (-0.09)

-0.18 (-0.11)

-0.13 (-0.06)

-0.18 (-0.13)

SSR

-0.14 (-0.26)

-0.28 (-0.37)

-0.06 (-0.19)

-0.09 (-0.20)

Com

-0.18 (0.002)

-0.04 (0.06)

0.13 (0.07)

0.12 (0.07)

GD (SGD)

Molecular markers as predictors of combining ability and hybrid performance Simple correlation coefficients between genetic distances (GDs) and specific genetic distances (SGDs) as revealed by DNA markers (AFLP, RAPD, SSR) and their combined data, specific combining ability (SCA), mid-parents heterosis (MPH), high-parent heterosis (HPH) and grain yield combined for all locations during 2003 season.

Parameter

Grain yield

SCA

MPH

HPH

0.84** SCA MPH

0.08

0.53*

HPH

0.08

0.51*

0.92**

AFLP

-0.20 (0.09)

-0.07 (0.19)

0.24 (0.16)

0.26 (0.19)

RAPD

-0.18 (-0.09)

-0.18 (-0.11)

-0.13 (-0.06)

-0.18 (-0.13)

SSR

-0.14 (-0.26)

-0.28 (-0.37)

-0.06 (-0.19)

-0.09 (-0.20)

Com

-0.18 (0.002)

-0.04 (0.06)

0.13 (0.07)

0.12 (0.07)

GD (SGD)

For studying the correlation between genetic distances and hybrid performance a good coverage of the genome is essential this could be attained either through the use of a sufficient number of primer combinations in AFLP technology or through combining the data of several types of markers. This will provide a good genome coverage leading to accurate estimate of genetic distances which in turns formulate an accurate relationship between hybrid performance and genetic distances.

Moreover, increase sample size will improve the efficiency of utilization of genetic distances as predictors for hybrid performance and aid in planning the most productive crosses in the hybrid breeding program.

Conclusion

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 gene of interest • They have many uses, including genetic diversity assessment, quality control (e.g. in variety development), marker-assisted breeding (the focus of this module) and others