Methods for characterization of animal genomes (RFLP, RAPD, SNP, STR, QTL ) Submitted by: Dr. Vijayata

Restriction Fragment Length Polymorphism (RFLP) RFLP is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of particular Restriction Endonucleases, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

The similarity of the patterns generated can be used to differentiate species (and even strains) from one another. This technique is mainly based on the special class of enzyme i.e. Restriction Endonucleases. They have their origin in the DNA rearrangements that occur due to evolutionary processes, point mutations within the restriction enzyme recognition site sequences, insertions or deletions within the fragments, and unequal crossing over (Schlotterer & Tautz, 1992). Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

This is not always the case with genomic DNA molecules because some restriction sites are polymorphic, existing as two alleles, one allele displaying the correct sequence for the restriction site and therefore being cut when the DNA is treated with the enzyme, and the second allele having a sequence alteration so the restriction site is no longer recognized. The result of the sequence alteration is that the two adjacent restriction fragments remain linked together after treatment with the enzyme, leading to a length polymorphism. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

The DNA molecule on the left has a polymorphic restriction site (marked with the asterisk) that is not present in the molecule on the right. The RFLP is revealed after treatment with the restriction enzyme because one of the molecules is cut into four fragments whereas the other is cut into three fragments. A restriction fragment length polymorphism (RFLP) Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

In order to score an RFLP, it is necessary to determine the size of just one or two individual restriction fragments against a background of many irrelevant fragments. Size fractionation is achieved by gel electrophoresis and, after transfer to a membrane by Southern blotting; fragments of interest are identified by hybridization with radioactive labeled probe. Different sizes or lengths of restriction fragments are typically produced when different individuals are tested. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

Southern hybridization, using a probe that spans the polymorphic restriction site, provides one way of visualizing the RFLP, but nowadays PCR is more frequently used. The primers for the PCR are designed so that they anneal either side of the polymorphic site, and the RFLP is typed by treating the amplified fragment with the restriction enzyme and then running a sample in an agarose gel. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

(A) RFLPs can be scored by Southern hybridization. The DNA is digested with the appropriate restriction enzyme and separated in an agarose gel. The smear of restriction fragments is transferred to a nylon membrane and probed with a piece of DNA that spans the polymorphic restriction site. If the site is absent then a single restriction fragment is detected (lane 2); if the site is present then two fragments are detected (lane 3). Two methods for scoring an RFLP : Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

(B) The RFLP can also be typed by PCR, using primers that anneal either side of the polymorphic restriction site. After the PCR, the products are treated with the appropriate restriction enzyme and then analyzed by agarose gel electrophoresis. If the site is absent then one band is seen on the agarose gel; if the site is present then two bands are seen. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

Applications: RFLPs can be applied in diversity and phylogenetic studies ranging from individuals within populations or species, to closely related species. RFLPs have been widely used in gene mapping studies because of their high genomic abundance due to the ample availability of different restriction enzymes and random distribution throughout the genome (Neale & Williams 1991). RFLP markers were used for the first time in the construction of genetic maps by Botstein et al Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

Random Amplified Polymorphic DNA (RAPD) PCR based technology. RAPD is a DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence (Williams et al., 1990; Welsh and McClelland, 1990). Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermo cyclic amplification. On an average, each primer directs amplification of several discrete loci in the genome, making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

Amplified products are separated on agarose gel ( %) and visualised by ethidium bromide staining. The use of a single oligonucleotide promotes the generation of several discrete DNA products and these are considered to originate from different genetic loci. Polymorphisms result from mutations or rearrangements either at or between the primer binding sites and are detected as the presence or absence of a particular RAPD band. This means that RAPDs are dominant markers and, therefore, can not be used to identify heterozygotes. Random amplified polymorphic DNA (RAPD) markers and its applications N. Senthil Kumar1 and G. Gurusubramanian2, 2011 Sci Vis 11 (3),

The standard RAPD utilises short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Welsh and McClelland independently developed a similar methodology using primers about 15 nucleotides long and different amplification and electrophoretic conditions from RAPD and called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique. Random amplified polymorphic DNA (RAPD) markers and its applications N. Senthil Kumar1 and G. Gurusubramanian2, 2011 Sci Vis 11 (3),

PCR amplification with primers shorter than 10 nucleotides [DNA amplification finger- printing (DAF)] has also been used to produce more complex DNA fingerprinting profiles. Although these approaches are different with respect to the length of the random primers, amplification conditions and visualization methods, they all differ from the standard PCR condition in that only a single oligonucleotide of random sequence is employed and no prior knowledge of the genome subjected to analysis is required. Random amplified polymorphic DNA (RAPD) markers and its applications N. Senthil Kumar1 and G. Gurusubramanian2, 2011 Sci Vis 11 (3),

Applications: The application of RAPDs and their related modified markers in variability analysis and individual-specific genotyping has largely been carried out, but is less popular due to problems such as poor reproducibility faint or fuzzy products, and difficulty in scoring bands, which lead to inappropriate inferences. RAPDs have been used for many purposes, ranging from studies at the individual level (e.g. genetic identity) to studies involving closely related species. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

RAPDs have also been applied in gene mapping studies to fill gaps not covered by other markers (Williams et al. 1990, Hadrys et al. 1992). Variants of the RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), which uses longer arbitrary primers than RAPDs, and DNA Amplification Fingerprinting (DAF) that uses shorter, 5–8 bp primers to generate a larger number of fragments. Multiple Arbitrary Amplicon Profiling (MAAP) is the collective term for techniques using single arbitrary primers. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

Microsatellites / Short tandem repeats (STRs) The term microsatellites was coined by Litt & Lutty (1989)and it also known as Simple Sequence Repeats (SSRs). Microsatellites, or short tandem repeats/simple sequence repeats (STRs/SSRs), are polymorphic loci present in DNA that consist of repeating units of one to six base pairs in length (CACACACACACACACA) (Litt and Lutty, 1989; Tautz, 1989). A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010 Int. J. Mol. Sci. 2010, 11, ; doi: /ijms

The repeated sequence is often simple, consisting of two, three or four nucleotides (di-, tri- and tetra- nucleotide repeats) and can be repeated many times. Microsatellites can be amplified for identification by PCR using the unique sequences of flanking regions as primers. A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010 Int. J. Mol. Sci. 2010, 11, ; doi: /ijms

The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated portion. Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length of microsatellites. The PCR products are separated either by slab gel electrophoresis or capillary gel electrophoresis in an automated sequencer. A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010 Int. J. Mol. Sci. 2010, 11, ; doi: /ijms

The number of repeats can be determined by carrying out a PCR using primers that anneal either side of the STR, and then examining the size of the resulting product by agarose or polyacrylamide gel electrophoresis. GENE CLONING AND DNA ANALYSIS An Introduction T.A. BROWN, Faculty of Life Sciences University of Manchester Manchester, Sixth Edition Part II The Applications of Gene Cloning and DNA Analysis in Research P180

Another approach using SSRs for the detection of polymorphism, called inter simple sequence repeat (ISSR), involves PCR amplification of genomic DNA between two microsatellite loci using a single primer composed of a microsatellite sequence anchored at the 3’ or 5’ end with one or few arbitrary, often degenerate nucleotides. These primers target simple sequence repeats that are abundant throughout the eukaryotic genome and do not require prior knowledge of DNA sequence for primer design. Biotechnology & Genetic Engineering Reviews Volume 25, Editor: Stephen E. Harding, First published 2008

Microsatellites have proved to be versatile molecular markers, particularly for population analysis, but they are not without limitations. With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process. A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010 Int. J. Mol. Sci. 2010, 11, ; doi: /ijms

Applications: In general, microsatellites show a high level of polymorphism. As a consequence, they are very informative markers that can be used for many population genetics studies, ranging from the individual level (e.g. clone and strain identification) to that of closely related species (distinguish closely related genotypes; because of their high degree of variability). Microsatellites are also considered ideal markers in gene mapping studies. Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

Single Nucleotide Polymorphism (SNP) Single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide (A, T, G or C) differs among members of a species. (Single nucleotide variations in genome sequence of individuals of a population are known as SNPs.) A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010 Int. J. Mol. Sci. 2010, 11, ; doi: /ijms

They are bi-allelic markers, indicating a specific polymorphism in two alleles only of a population. Most SNPs (about two of every three SNPs), involve the replacement of cytosine (C) with thymine (T). These are positions in a genome where some individuals have one nucleotide (e.g. a G) and others have a different nucleotide (e.g. a C) Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

SNPs represent one of the more interesting approach in genotypization, because they are abundant in the genome, genetically stable and amenable to high-throughput automated analysis (Vignal et al., 2002). SNP genotyping technologies have two components - a method for determining the type of base present at a given SNP locus, and a method for reporting the presence of the allele(s) (signal detection). MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p , 2009

SNP detection is more rapid because it is based on oligonucleotide hybridization analysis. An oligonucleotide is a short single-stranded DNA molecule, usually less than 50 nucleotides in length, that is synthesized in the test tube. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

An oligonucleotide will hybridize with another DNA molecule only if the oligonucleotide forms a completely base-paired structure with the second molecule. If there is a single mismatch - a single position within the oligonucleotide that does not form a base pair - then hybridization does not occur. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

Oligonucleotide hybridization can therefore discriminate between the two alleles of an SNP. Various screening strategies have been devised ( Mir and Southern, 2000), including DNA chip technology and solution hybridization techniques. A DNA chip is a wafer of glass or silicon, 2.0 cm 2 or less in area, carrying many different oligonucleotides in a high- density array. The DNA to be tested is labeled with a fluorescent marker and pipetted onto the surface of the chip. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

Hybridization is detected by examining the chip with a fluorescence microscope, the positions at which the fluorescent signal is emitted indicating which oligonucleotides have hybridized with the test DNA. Many SNPs can therefore be scored in a single experiment (Gerhold et al., 1999). Solution hybridization techniques are carried out in the wells of a microtiter tray, each well containing a different oligonucleotide, and use a detection system that can discriminate between unhybridized single-stranded DNA and the double-stranded product that results when an oligonucleotide hybridizes to the test DNA. Genomes, 2 nd edition Terence A BROWN Chapter 5. Mapping Genomes, DNA markers for genetic mapping. Oxford: Wiley-Liss; 2002.ISBN-10:

SNP have become the preferred markers in genetic disease studies for various livestock species, as researches direct their attention towards functional genomics (White et al., 2001). SNPs are becoming especially important as markers because they are very stable, i.e. have very low mutation rates and can be amplified with PCR for testing. One disadvantage of these markers is the lower informational content compared with that of a highly polymorphic microsatellite, but it can be compensated by the use of a higher number of markers (Werner et al. 2002, 2004). MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p , 2009

Quantitative trait loci (QTLs) The regions within genomes that contain genes associated with a particular quantitative trait are known as quantitative trait loci (QTLs). The identification of QTLs based only on conventional phenotypic evaluation is not possible. A major breakthrough in the characterization of quantitative traits that created opportunities to select for QTLs was initiated by the development of DNA (molecular) markers in the 1980s. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

QTL mapping The process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with traits is known as QTL mapping (also ‘genetic,’ ‘gene’ or ‘genome’ mapping). The most important use for linkage maps is to identify chromosomal locations containing genes and QTLs associated with traits of interest; such maps may then be referred to as ‘QTL’ (or ‘genetic’) maps. ‘QTL mapping’ is based on the principle that genes and markers segregate via chromosome recombination (called crossing-over) during meiosis (i.e. sexual reproduction), thus allowing their analysis in the progeny (Paterson, 1996a). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

QTL analysis is based on the principle of detecting an association between phenotype and the genotype of markers. Markers are used to partition the mapping population into different genotypic groups based on the presence or absence of a particular marker locus and to determine whether significant differences exist between groups with respect to the trait being measured. A significant difference between phenotypic means of the groups (either 2 or 3), depending on the marker system and type of population, indicates that the marker locus being used to partition the mapping population is linked to a QTL controlling the trait. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

Three widely-used methods for detecting QTLs are single- marker analysis, simple interval mapping and composite interval mapping (Liu, 1998; Tanksley, 1993). Single-marker analysis (single-point analysis) is the simplest method for detecting QTLs associated with single markers. The statistical methods used for single-marker analysis include t-tests, analysis of variance (ANOVA) and linear regression. This method does not require a complete linkage map and can be performed with basic statistical software programs. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

However, the major disadvantage with this method is that the further a QTL is from a marker, the less likely it will be detected. This is because recombination may occur between the marker and the QTL. This causes the magnitude of the effect of a QTL to be underestimated (Tanksley, 1993). The use of a large number of segregating DNA markers covering the entire genome may minimize both problems (Tanksley, 1993). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

The simple interval mapping (SIM) method makes use of linkage maps and analyses intervals between adjacent pairs of linked markers along chromosomes simultaneously, instead of analyzing single markers (Lander & Botstein, 1989). The use of linked markers for analysis compensates for recombination between the markers and the QTL, and is considered statistically more powerful compared to single- point analysis (Lander & Botstein, 1989; Liu, 1998). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

Composite interval mapping (CIM) has become popular for mapping QTLs. This method combines interval mapping with linear regression and includes additional genetic markers in the statistical model in addition to an adjacent pair of linked markers for interval mapping (Jansen, 1993; Jansen & Stam, 1994). The main advantage of CIM is that it is more precise and effective at mapping QTLs compared to single-point analysis and interval mapping, especially when linked QTLs are involved. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts, B.C.Y. Collard1,4, ∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1, Springer 142: 169–196.

Type of markersAdvantagesDisadvantages Restriction Fragment Length Polymorphism (RFLP) -High genomic abundance -Co-dominant markers -Highly reproducible -Good genome coverage -Can be used across species -No sequence information -Needed for map based cloning -Need large amount of good quality DNA -Laborious (compared to RAPD) -Difficult to automate -Need radioactive labeling -Cloning and characterization of probe are required Randomly Amplified Polymorphic DNA (RAPD) -High genomic abundance -Good genome coverage -No sequence information -Ideal for automation -Less amount of DNA -No radioactive labeling -Relatively faster -No probe or primer information -Dominant markers -Not reproducible -Can not be used across species -Not very well-tested Simple Sequence Repeat (SSR) -High genomic abundance -Highly reproducible -Fairly good genome coverage -High polymorphism -No radioactive labeling -Easy to automate -Multiple alleles -Can not be used across species -Need sequence information -Not well-tested Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B. K. Pandey2 Plant Omics Journal 2(4): (2009) ISSN:

FeatureRFLPRAPDSTR/SSR Amount of DNA required HighLow Maximum theoretical number of possible loci in analysis Limited by the restriction site (nucleotide) Polymorphism Limited by the size of genome, and by nucleotide polymorphism Limited by the size of genome and number of simple repeats in a Genome DominanceCodominantDominantCodominant Genomic abundance High Medium Locus specificityYesNo Reproducibility High to very high Low to medium Medium to high Type of probes/primers Low copy genomic DNA or cDNA clones Usually 10 bp random nucleotides Specific repeat DNA sequence MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p , 2009 Comparison of commonly used genetic markers (Mburu and Hanotte, 2005)

FeatureRFLPRAPDSTR/SSR Degree of Polymorphism HighLowMedium Type of polymorphism Single base changes, insertion, deletion Single base changes, insertion, deletion Changes in length of repeats Potential for studying adaptive genetic Variation Limited Transferability Across related species Across generaWithin species Within genus or species Ease of developmentDifficultEasyDifficult Technical requirement HighLowMedium Major applicationPhysical mappingGene taggingGenetic diversity MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva Biotechnology in Animal Husbandry 25 (5-6), p , 2009

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