DNA Markers: Frequently Asked Questions | Molecular Breeding | Plant Breeding

Everything you need to know about DNA markers !

Q. 1. What is marker character?

Ans. Any property of an individual showing heritable variation is referred to as a character. It includes morphological, physiological and biochemical properties in plants. Plant characters are of two types viz. qualitative (governed by one or few genes) and quantitative (governed by several genes). Those characters which can be easily identified are referred to as marker characters.

Q. 2. What are types of markers?

Ans. Markers are of three types, viz.:

i. Morphological markers,

ii. Biochemical markers and

iii. DNA markers.

These are defined below:

i. Morphological Markers:

These are related to shape, size, colour and surface of various plant parts. Such characters are used for varietal identification.

ii. Biochemical Markers:

Such markers are related to variations in protein and amino acid banding patterns. Gel electrophoretic studies are used for identification of biochemical markers.

iii. DNA Markers:

DNA markers are related to variations in DNA fragments generated by restriction endonuclease enzymes. DNA markers are also known as molecular markers.

Q. 3. What are features of an ideal DNA marker?

Ans. A DNA marker refers to a unique sequence of nucleotides found on a strand of DNA. The DNA markers are also known as genetic markers.

The main features or characteristics of an ideal marker are given below:

i. Polymorphic:

It means that the markers tend to look slightly different in different individuals. This presents variability in the population which is essential for a geneticist or plant breeder to select superior genotypes.

ii. Co-Dominant:

An ideal DNA marker should be co-dominant. Co-dominance refers to absence of intra-locus interaction. It helps in identification of heterozygotes from the homozygotes.

iii. Non-Epistatic:

It refers to absence of inter locus interaction. It helps in predicting the genotype of an individual from its phenotype.

iv. Multi Allelic:

It refers to presence of more than two alleles at a locus. It is useful in getting more variability or polymorphism for a character.

v. Neutral:

It means that the allelic substitutions at the marker locus do not alter the phenotype of an individual. Almost all molecular markers have this property.

vi. Insensitive to Environment:

It means the molecular marker should not be affected by environmental conditions. This property is found in almost all DNA markers. It helps in predicting genotype from the phenotype of an individual.

Q. 4. What are differences between morphological and DNA markers?

Ans. The DNA markers differ from morphological markers in several ways (Table 25.1).

Q. 5. What are the limitations of biochemical markers?

Ans. There are some limitations of biochemical markers (protein markers) as given below:

i. The biochemical markers are detected through isozyme analysis. Isozyme technique can detect only limited number of loci. For example it can detect 30-40 isozyme markers (protein markers) in segregating populations of rice or maize.

ii. Secondly, all the enzymes are not present or active in all the organs of an individual.

The DNA markers, on the other hand, are numerous in number and uniformly present in all organs and stage of an individual.

Thus main differences between DNA markers and protein markers are given In Table 25.2:

Q. 6. List various types of DNA markers.

Ans. There are several types of DNA markers.

The list of DNA markers which are commonly used in plant breeding is given below:

i. Restriction Fragment Length Polymorphisms (RFLPs)

ii. Amplified Fragment Length Polymorphisms (AFLPs)

iii. Random Amplified Polymorphic DNA (RAPD)

iv. Cleaved Amplified Polymorphic Sequence (CAPS)

v. Simple Sequence Repeats (SSRs)

vi. Single Strand Conformational Polymorphisms (SSCPs)

vii. Single Nucleotide Polymorphisms (SNPs)

viii. Hetero-Duplex Analysis (HA)

ix. Expressed Sequence Tags (ESTs)

x. Sequence Tagged Sites (STSs).

Q. 7. What are restriction fragment length polymorphisms (RFLPs)?

Ans. RFLPs refer to variations found within a species in the length of DNA fragment generated by specific endonuclease. RFLPs are useful as molecular markers. RFLPs are the first type of DNA markers developed to distinguish individuals at the DNA level. RFLP technique was developed before the discovery of PCR.

The RFLP technique consists of following three main steps:

i. Cutting genomic DNA with specific restriction enzymes.

ii. Separating different sized DNA fragments on electrophoresis gels (agarose), and

iii. Hybridizing a labelled, radioactive DNA probe to complementary DNA fragment on the gel in order to visualize them (using X-ray film exposure).

Q. 8. What are advantages of RFLPs?

Ans. RFLP technique has several advantages.

Important advantages of this technique are presented below:

i. It is a simple and cheaper technique of DNA sequencing.

ii. It does not require special instrumentation,

iii. The majority of RFLP markers are co-dominant and highly locus specific.

iv. RFLP markers are powerful tools for comparative and synteny mapping.

v. Other markers such as CAPS or INDEL can be developed from RFLP probe sequences.

vi. Numerous samples can be simultaneously screened by this technique using different RFLP probes.

vii. RFLP genotypes for single or low copy number genes can be easily interpreted and scored.

Q. 9. What are disadvantages of RFLPs?

Ans. Disadvantages of RFLPs are given below:

i. Developing sets of RFLP probes and markers is labour intensive.

ii. This technique requires large amount of high quality DNA.

iii. The multiplex ratio is low (typically one per gel).

iv. The genotyping through put is low (difficult to automate).

v. Mostly genotyping is done using radioactive methods, despite availability of non- radioactive methods.

vi. RFLP finger prints for multi-gene families are often complex and difficult to score.

vii. RFLP probes must be maintained physically and are thus a hassle to share between laboratories.

Q. 10. What are uses of RFLPs?

Ans. RFLPs can be used in a variety of ways for various purposes.

Important uses of RFLPs are given below:

i. RFLPs can be used in determining paternity cases.

ii. RFLPs are also used in criminal cases to determine the source of DNA sample.

iii. RFLPs can be used to determine the disease status of an individual.

iv. In plant breeding and genetics RFLPs are used for gene mapping, germplasm characterization and marker assisted selection.

v. It is also used for diagnosis of pathogen in plants even if it is in latent stage.

Q. 11. What are amplified fragment length polymorphisms (AFLPs)?

Ans. AFLPs are differences in restriction fragment length caused by SNPs (single nucleotide Polymorphism or INDELs (insertion-deletion) that create or abolish restriction endonuclease recognition sites. AFLP assay are performed by selectively amplifying a pool of restriction fragments using PCR. The RFLP technique was originally known as selective restrictive fragment amplification (SRFA). It was developed by Zabeau and Vas in 1993.

The AFLP protocol consists of the following five main steps:

i. Digestion of DNA with two different restrictions, enzymes (generally a rare and a frequent cutter).

ii. Ligation of double stranded adapters to the end of restriction fragments.

iii. Optional DNA-pre-amplification of ligated products directed by primers complementary to adapter and restriction site sequences.

iv. DNA amplification of subsets of restriction fragments using selective AFLP primers and labelling of amplified products.

v. Finally the labelled reaction products are separated by electrophoresis (using denaturing poly acrylamide gels) and exposed to X-ray films to visualise the AFLP fingerprint (if radioactive) or by running out the product on an automated DNA sequencer.

Q. 12. What are advantages of AFLPs?

Ans. Advantages of AFLPs are as follows:

i. This technique provides very high multiplex ratio and genotyping through put.

ii. This technique can be applied to virtually any organism with no formal marker development.

iii. The results are highly reproducible across laboratories.

iv. No special instrumentation is needed for performing AFLP assays except for co-dominant scoring.

v. The startup cost of AFLP is moderately low.

vi. AFLP assay can be performed using very small DNA samples.

Q. 13. What are disadvantages of AFLPs?

Ans. Disadvantages of AFLPs are given below:

i. High quality DNA is needed to ensure complete restriction enzyme digestion.

ii. The homology of a restriction fragment cannot be unequivocally ascertained across genotypes or mapping populations.

iii. It is difficult to develop locus specific markers from individual fragments.

iv. It generally involves radioactive methods. Though non-radioactive methods are available but they are rarely used.

v. In species with large genomes such as barley and sunflower, AFLP markers often densely cluster in telomeric regions.

vi. The maximum polymorphic information content for any bi-allelic marker is 0.5.

vii. Proprietary technology is needed to score heterozygotes and ++ homozygotes,

Q. 14. What are uses of AFLPs?

Ans. This technique has been widely used in the construction of genetic maps containing high densities of DNA marker. In plant breeding and genetics AFLP markers are used in varietal identification, characterization of germplasm, gene tagging and marker assisted selection.

Q. 15. What is random amplified polymorphic DNA (RAPD)?

Ans. RAPD refers to polymorphism (variations) found within a species in the randomly amplified fragments of DNA generated by restriction endonuclease enzyme. RAPDs are PCR based molecular (DNA) markers. RAPD marker assays are performed using single DNA primer of arbitrary sequence. This technique was proposed by Williams et.al. 1990, the RAPD markers are dominant. RAPD polymorphisms are produced by the presence or absence of PCR amplicons.

Q. 16. What are advantages of RAPDs?

Ans. Advantages of RAPDs are as follows:

i. This technique is simple and quick as compared to RFLPs.

ii. RAPD primers are readily available being universal.

iii. It can be performed with any species using universal primers.

iv. It provides more polymorphisms than RFLPs.

v. It does not need special equipment. Only PCR is required.

vi. The startup cost is low.

vii. RAPD marker assays can be performed using very small DNA samples (5-25 ng per individual).

viii. RAPD markers are universal, can be commercially purchased and are easily shared between laboratories.

ix. RAPD technique permits development of locus specific co-dominant. PCR based markers from RAPD markers.

x. Large number of bands are produced per primer.

Q. 17. What are Disadvantages of RAPDs?

Ans. Disadvantages of RAPDs are as follows:

i. The detection of polymorphism is limited as in case of RFLPs.

ii. This technique only detects dominant markers,

iii. The reproducibility of results across laboratories is low or inconsistent.

iv It is not feasible for use in marker assisted breeding program.

v. The homology of fragments across genotypes cannot be ascertained without mapping.

Q. 18. What are uses of RAPDs?

Ans. A segregating population is used to detect polymorphism.

RAPD makers are useful in following ways:

i. For assessment of genetic variation in breeding populations, germplasm and different species of a cross.

ii. To study phylogenetic relationship among species and subspecies.

iii. For gene tagging and construction of linkage maps.

iv. For varietal identification.

v. For DNA fingerprinting of cultivars and germplasm.

RAPD markers have played a very important role in selection of genotypes with desired characteristics and also in the production of species specific, genome-specific and chromosome specific markers.

Q. 19. What is cleaved amplified polymorphic sequence (CAPS)?

Ans. It refers to polymorphism (variations) that are found in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus specific oligonucleotides primers.

CAPs assays consist of following main steps:

i. Digesting locus specific PCR amplicons with one or more specific enzymes.

ii. Separating the digested PCR amplicons with one or more specific enzymes.

iii. Exposure to X-ray films to visualise CAPS fragments.

The modification CAPS has been proposed by some workers, called d CAPS (derived CAPS) which is based on SNPs. It is PCR based technique.

Q. 20. What are advantages of CAPS?

Ans. Advantages of CAPS are as follows:

i. It is a simple PCR assay. The markers are developed from DNA sequences of previously mapped RFLP markers.

ii. The most CAPS markers are co-dominant and locus specific.

iii. It provides moderately high genotyping through put.

iv. No special equipment is needed to perform manual CAPS marker assays.

v. The CAPS marker assays can be performed using semi-automated methods e.g. florescent assays on DNA sequencer.

vi. The startup cost is low for manual assay methods.

vii. The CAPS assays can be performed using very small DNA samples (50- 100 ng per individual).

viii. The most CAPS genotypes are easily scored and interpreted.

ix. The CAPS markers are easily shared between laboratories.

Q. 21. What are disadvantages of CAPS?

Ans. Disadvantages of CAPS are as follows:

i. Other methods better than CAPS such as SNPS assays are now available for screening locus specific DNA fragments for polymorphism.

ii. Scoring and interpretation of multi-gene families is sometimes difficult by this technique,

iii. A variety of restriction enzymes have to be tested to find polymorphism.

Q. 22. What are simple sequence repeats (SSRs)?

Ans. SSRs also called microsatellites are randomly repeated mono, di, tri, tetra, penta, and hexo nucleotide motifs. The SSR length polymorphisms are caused by differences in the number of repeats.

The SSR loci are individually amplified by PCR using pairs of oligonucleotide primers specific to unique DNA sequences flanking the SSR sequence. Jeffreys (1985) showed that some restriction fragment length polymorphisms are caused by VNTRs (Variable Number of Tandem Repeats). These are known as mini-satellites and have similarity to larger satellite DNA repeats.

Q. 23. What are advantages of SSRs?

Ans. Advantages of SSRs are as follows:

i. The SSR markers tend to be highly polymorphic.

ii. Most SSR markers are co-dominant and locus specific.

iii. This is a simple PCR based technique.

iv. SSRs technique does not require special equipments. However, sometimes DNA sequencer is required.

v. The startup cost is low for manual assay methods.

vi. It provides high genotyping throughput.

vii. The SSR assays can be performed using small DNA samples (100 ng per individual),

viii. The SSR DNA markers can be easily shared between Laboratories,

ix. The SSR markers can be multiplexed using PCR.

Q. 24. What are disadvantages of SSRs?

Ans. Disadvantages of SSRs are given below:

i. The development of SSRs is labour intensive.

ii. The cost of developing SSR markers is very high.

iii. The SSR markers are specific to plant species.

iv. The startup cost is high for automated method of SSR assays.

v. The development of PCR multiplexer is difficult and expensive. Some markers may not multiplex.

Microsatellites or simple sequence repeats (SSRs), simple sequence length polymorphisms (SSL.Ps), short tandem repeats (STRs), simple sequence motifs (SSMs), sequence target microsatellites (STMs) is a class of repetitive sequence which are widely distributed in all eukaryotic genomes. They consist of arrays of tandemly repeated short nucleotide motifs of 1-4 bases, and are called mono, di, tri or tetra-nucleotide repeats respectively.

Q. 25. What are uses of SSRs?

Ans. The use of microsatellites or genetic markers has been proposed for genetic mapping of eukaryotes.

Q. 26. What are single strand conformational polymorphisms (SSCPs)?

Ans. SSCPs refer to DNA polymorphism produced by differential folding of single stranded DNA harboring mutations. The conformation of folded DNA molecule is produced by intra-molecular interaction and thus is a function of DNA sequence. The SSCP marker assays are performed using heat denatured DNA on non-denaturing DNA sequence gels. Special gels (e.g. mutative enhancement gels) have been developed to enhance the recovery of single strand conformational polymorphisms caused by INDELs, SNPs or SSRs.

Q. 27. What are advantages of SSCPs?

Ans. Advantages of SSCPs as follows:

i. It is a simple PCR based assay.

ii. Many SSCPs markers are multi allelic and highly polymorphic.

iii. Most SSCPs are co-dominant and locus specific.

iv. SSCP assay does not require special equipment.

v. The startup cost is low.

vi. The SSCP marker assays can be performed using very small DNA samples (10-50 ng per individual).

vii. The SSCP markers can be easily shared between Laboratories.

viii. The SSCP gels can be silver stained (non-radioactivity).

ix. The complexity of PCR products can be assessed and individual fragments can be isolated and sequences.

Q. 28. What are disadvantages of SSCPs?

Ans. Disadvantages of SSCPs are as follows:

i. The development of SSCP markers is labour intensive.

ii. The development cost of SSCP markers is high.

iii. The SSCP marker analysis cannot be automated.

Q. 29. What are single nucleotide polymorphisms (SNPs)?

Ans. The variations (differences) which are found at a single nucleotide position are referred to as single nucleotide polymorphisms or SNPs. This type of DNA polymorphism results due to substitution, deletion or insertion. It occurs approximately every 1.3 Kb. Most polymorphisms of this type have only two alleles and also called biallelic loci. The SNP is the most common class of DNA polymorphism which found both in natural lines and after induced mutagenesis.

Main features of SNPs are presented below:

i. SNP markers are highly polymorphic and mostly biallelic.

ii. SNP markers provide ultra-high thorough put for genotyping.

iii. SNP markers are locus specific.

iv. SNP markers are excellent long term investment.

v. SNPs result due to substitution, deletion or insertion.

vi. SNP markers can be used to pinpoint functional polymorphisms.

vii. Small amount of DNA is required for SNP markers.

viii. Several SNP assay techniques have been developed.

Q. 30. What are advantages of SNPs?

Ans. Advantages of SNPs are as follows:

i. SNPs are useful in gene mapping.

ii. SNPs help in detection of mutations at molecular level.

iii. SNPs are useful in positional cloning of a mutant locus.

iv. SNPs are useful in identification of disease causing genes.

Q. 31. What are disadvantages of SNPs?

Ans. Disadvantages of SNPs are as follows:

i. Most SNPs are biallelic and thus less informative than SSRs.

ii. Multiplexting is not possible for all loci.

iii. Some SNP assay techniques are costly.

iv. In preparing genetic maps, three times more SNP markers are needed than SSR markers.

v. Development of SNP markers is labour oriented.

Q. 32. What are uses of SNPs?

Ans. Uses of SNPs are given below:

i. SNPs are being used in generating genetic maps. SNPs have been used in preparing human genetic maps.

ii. In plants, SNPs have not been extensively used so far.

Q. 33. What is hetero-duplex Analysis (HA)?

Ans. In refers to DNA polymorphisms produced by separating homo-duplex from hetero-duplex. DNA using non-denaturing gel electrophoresis or partially denaturing high performance liquid chromatography, the hetero-duplexes are produced due to single base mismatches between genotypes. The presence of hetero-duplexes indicates presence of DNA polymorphism.

Q. 34. What are advantages of HA?

Ans. Advantages of HA are given below:

i. It is powerful technique for the discovery of single nucleotide polymorphism (SNP).

ii. Very small quantity of DNA samples (10-50 ng per individual) is required to perform hetero-duplex analysis.

iii. Most of the hetero-duplex markers are co-dominant and locus specific.

iv. The hetero-duplex analysis can be performed using automated high performance liquid chromatography.

v. The HA markers can be easily shared between laboratories.

Q. 35. What are disadvantages of HA?

Ans. Disadvantages of HA are as follows:

i. The hetero-duplex analysis requires special equipment.

ii. One protocol may not be sufficient for hetero-duplex analysis of different targets using HPLC.

Q. 36. What are uses of HA?

Ans. Uses of HA are as follows:

i. Hetero-duplex analysis has been mostly used in human genetics to screen disease genes for DNA polymorphism.

ii. In plant breeding, it can be used for detection of pathogens which are in latent stage and thus will be useful in selection of disease free plants.

iii. It is useful in the discovery of Single Nucleotide Polymorphism.

Q. 37. What are Expressed Sequence tags (ESTs)?

Ans. Expressed Sequence Tags (ESTs) are small pieces of DNA and their location on the chromosome and sequence are known. The term Expressed, Sequence Tags was first used by Venter and his colleagues in 1991.

Main points related to ESTs are given below:

i. ESTs are short DNA sequences, usually 200-500 nucleotides long.

ii. ESTs are a type of Sequenced Tagged Site (STS).

iii. ESTs are created from CDNA, a single strand of DNA which has been copied from an mRNA molecule. In other words, an EST is a strand of DNA consisting only of the exons. Exon is the base pair sequence of a gene that is expressed.

Q. 38. What are advantages of ESTs?

Ans. Advantages on ESTs are as follows:

i. ESTs are useful in discovering new genes particularly genes that are involved in genetic diseases.

ii. It is a rapid and inexpensive technique of locating a gene.

iii. ESTs provide information about gene expression. In other words, they can be used for tissue specific gene expression.

iv. ESTs generate a large number of sequences quickly.

v. Through ESTs more information can be obtained from QTL mapping.

vi. ESTs can be used to locate disease causing genes.

Q. 39. What are limitations of ESTs?

Ans. Limitations of ESTs are given below:

i. ESTs have lack of primer specificity.

ii. Development of ESTs is time consuming and labour oriented.

iii. The error is higher than other conventional approaches.

iv. It is difficult to obtain large transcripts (>.6 kb).

Q. 40. What are applications of ESTS?

Ans. Uses of ESTs are as follows:

i. ESTs are commonly used to map genes of known function. In other words, they can be used for constructing genome maps.

ii. ESTs are used for phylogenetic studies.

iii. ESTs can be used to produce DNA arrays, a powerful tool for the analysis of the whole expression pattern of a tissue.

Q. 41. What is Sequence Tagged Sites (STSs)?

Ans. It genomics, a sequence tagged site (STSs) is a short DNA sequence that has a single copy in a genome and whose location and base sequence are known.

Main points related to STSs are given below:

i. STSs are usually 200-500 base pair long.

ii. STS occurs only once in the genome. In other words, STSs have single copy in a genome.

iii. STSs are detected by Polymerase Chain Reaction (PCR) in the presence of all other genomic sequences.

iv. STSs are derived from CDNAs,

Q. 42. What are advantages of STS markers?

Ans. Advantages of STSs are as follows:

i. STSs serve as landmarks in developing physical map of a genome. In other words, they are useful in physical mapping of genes.

ii. STSs permit use of data from different Laboratories for gene mapping.

iii. It is rapid and more specific technique than DNA hybridization techniques.

iv. It can be automated resulting in performing large number of tests.

v. It is highly sensitive means has highly degree of accuracy.

Q. 43. What are limitations of STS?

Ans. Limitations of STS are as follows:

i. Development of STS is a difficult task.

ii. It is a time consuming and labour oriented technique.

iii. Development of STS required high technical skill.

Q. 44. What are applications of STS?

Ans. Applications of STS are given in below:

i. STS is the most powerful physical mapping technique.

ii. It can be used to identify any locus on the chromosome.

iii. STSs are used as standard markers to find out gene in any region of the genome.

iv. It is used for constructing detailed maps of large genomes.

Q. 45. What are applications of DNA markers in crop improvement?

Ans. Molecular markers or DNA markers have several useful applications in crop improvement programmes. As stated above, now several types of DNA markers are available. The choice of a type of DNA marker depends on the user’s objectives.

In plant breeding, molecular markers are useful in four principal ways, viz.:

i. Assessment of genetic diversity.

ii. Gene mapping

iii. Marker assisted selection, and

iv. In crop evolution.