Assignment Work On

Molecular Markers

GP-509 (Biotechnology for Crop Improvement )

Course Teacher :Dr.K.K.TiwariAssistant professor ,C.P.College of Agriculture,S.D.A.U. , Sardar Krushinagar

Submitted by :Satyendra singh04-agrma-01157-2013M.Sc. (Agri.) GPBC.P. College of Agriculture,S.D.A.U. , Sardar Krushinagar

MarkerMarker can be any sign or character which are considered to be associated with a particular trait morphologically and/or genetically. Criteria for desirable markers 1. High level of genetic polymorphism.2. Co- dominance (so that heterozygotes can be distinguished from homozygotes).3. Clear distinct allele features (so that different alleles can be identified easily).4. Even distribution on the entire genome.5. Neutral selection (without pleiotropic effect).6. Easy detection (so that the whole process can be automated).7. Low cost of marker development and genotyping.8. High duplicability (so that the data can be accumulated and shared between laboratories).

Types of MarkersGenetic markers fall into one of the three broad classes: 1. Morphological Markers2. Biochemical Marker3. Molecular Markers

1. Morphological MarkersThese are based on visually assessable traits (morphological and agronomic traits).Morphological markers represent genetic polymorphisms which are visible as differences in appearance. Pink colour of mango related to sweetness. Spotted guava is considered good in quality. Morphological markers are usually mapped by classical two- or three-point linkage tests.

AdvantagesLimitations

-Readily available-Less expensive-Skilled labour and laboratory not required.-Limited in number -Narrow in diversity-We cannot combine 3-4 markers-Unknown genomic information

2. Biochemical Marker (Isozyme): Tanksley and Orton 1983These are based on gene product. Two or more enzymes that catalyse the same reaction but are encoded by different genes are called isozymes. Isozymes originate through amino acid alterations, which cause changes in net charge, conformation of the enzyme molecules and their electrophoretic mobility. This change in electric charge and conformation can be detected by gel electrophoresis. Applications:Allozymes can be applied in many population genetics studies, including measurements of out crossing rates, population structure and population divergence.Allozymes are particularly useful at the level of conspecific populations and closely related species, and are therefore useful to study diversity in crops and their relatives.Allozymes have been used, in concert with other markers, for fingerprinting purposes, and diversity studies, to study interspecific relationships, the mode of genetic inheritance, and allelic frequencies in germplasm collections over serial increase cycles in germplasm banks, and to identify parents in hybrids.AdvantagesLimitations

-Useful for evolutionary studies-Isolation lot easier than that of DNA-Can be used across species-No radioactive labelling-No need for sequence information-Tedious in handling-Limited in polymorphism-Expensive (each system is unique)-Have to know the location of the tissue -Influenced by environment-stage specific

3. Molecular Markers These are relying on a DNA assay.Molecular markers are constant landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome, and transmitted by the standard laws of inheritance from one generation to the next.

DNA markers are the best candidates for efficient evaluation and selection of plant material. Since DNA markers segregate as single genes and they are not affected by the environment.The uses of molecular markers are based on the naturally occurring DNA polymorphism.Genetic polymorphism is defined as the simultaneous occurrence of a trait in the same population of two discontinuous variants or genotypes.Basis of Genetic polymorphism1. Unequal recombination2. Replication slippage3. Single nucleotide polymorphism4. INDEL

Use of genetic markers in plant breeding1. Gaining a better understanding of breeding materials and breeding system. Molecular markers can be used to characterize germplasm, develop linkage maps, and identify heterotic patterns.2. Rapid introgression of simply inherited traits.Introgression of genes into another genetic background involves several rounds of tedious backcrosses. When the source of desirable genes is a wild species, linkage drag becomes a problem. Using markers and QTL analysis we can reduce linkage drag.3. Early generation testing.Breeding for compositional traits, such as high lysine and high tryptophan genes in maize, can be advanced with early detection and selection of desirable segregants.4. Unconventional problem solving.The recessive linkage drag was removed by using DNA markers flanking the introgression to pre-select for individuals that were recombinant in the vicinity of the gene. Breeding Lettuce resistant to aphid Nasonovia ribisnigi from a wild lettuce Lactuca virosa by repeated backcross assisted by molecular markers. The life span of new cultivars can be extended through the technique of gene pyramiding (i.e., transferring multiple disease resistance genes into one genotype) for breeding disease resistant cultivars. Marker assisted backcross can be used to achieve this rapidly, especially for genes with indistinguishable phenotypes.5. Plant cultivar identification. Molecular markers are effective in cultivar identification for protecting proprietary rights as well as authenticating plant cultivars

Classification of molecular markersa) Method of analysis (Hybridization-based or PCR based markers).b) Mode of gene action (Dominant or co-dominant markers).c) Mode of transmission (biparental nuclear inheritance, maternal nuclear inheritance, maternal organelle inheritance, or paternal organelle inheritance).

DNA markers and related major molecular techniquesSouthern blot-based markers / hybridisation based markersRestriction fragment length polymorphism (RFLP)Single strand conformation polymorphic RFLP (SSCP-RFLP)Denaturing gradient gel electrophoresis RFLP (DGGE-RFLP)PCR-based markersRandomly amplified polymorphic DNA (RAPD)Sequence tagged site (STS)Sequence characterized amplified region (SCAR)Random primer-PCR (RP-PCR)Arbitrary primer-PCR (AP-PCR)Oligo primer-PCR (OP-PCR)Single strand conformation polymorphism-PCR (SSCP-PCR)Small oligo DNA analysis (SODA)DNA amplification fingerprinting (DAF)Amplified fragment length polymorphism (AFLP)Sequence-related amplified polymorphism (SRAP)Target region amplified polymorphism (TRAP)Insertion/deletion polymorphism (Indel)Repeat sequence-based markersSatellite DNA (repeat unit containing several hundred to thousand base pairs (bp) )Microsatellite DNA (repeat unit containing 25 bp)Minisatellite DNA (repeat unit containing more than 5 bp)Simple sequence repeat (SSR) or simple sequence length polymorphism (SSLP)Short repeat sequence (SRS)Tandem repeat sequence (TRS)mRNA-based markersDifferential display (DD)Reverse transcription PCR (RT-PCR)Differential display reverse transcription PCR (DDRT-PCR)Representational difference analysis (RDA)Expression sequence tags (EST)Sequence target sites (STS)Serial analysis of gene expression (SAGE)Single nucleotide polymorphism-based markersSingle nucleotide polymorphism (SNP

Classification of markersS.No.Name of the TechniqueDiscoverer

A.Biochemical markersAllozymesTanksley and Orton 1983; Kephart 1990; May 1992

B.Molecular markers

i) Non-PCR2 based techniquesRestriction Fragment Length Polymorphisms (RFLP)Botstein et al. 1980; Neale and Williams 1991

Minisatellites or Variable Number of Tandem Repeats (VNTR)Jeffreys et al.. 1985

ii) PCR-based techniques

DNA sequencingMulti-copy DNA, Internal Transcribed Spacer regions of nuclear ribosomal genes (ITS)Takaiwa et al. 1985; Dillon et al. 2001

Single-copy DNA, including both introns and exonsSanger et al. 1977; Clegg 1993a

Sequence-Tagged Sites (STS)Microsatellites, Simple Sequence Repeat (SSR), Short Tandem Repeat (STR), Sequence Tagged Microsatellite (STMS) or Simple Sequence Length Polymorphism (SSLP)Litt and Lutty (1989),Hearne et al. 1992; Morgante and Olivieri 1993; Jarne and Lagoda 1996

Amplified Sequence Length Polymorphism (ASLP)Maughan et al. 1995

Sequence Characterized Amplified Region (SCAR)Michelmore et al. (1991); Martin et al. (1991); Paran and Michelmore 1993

Cleaved Amplified Polymorphic Sequence (CAPS)Akopyanz et al. 1992; Konieczny and Ausubel 1993

Single-Strand Conformation Polymorphism (SSCP)Hayashi 1992

Denaturing Gradient Gel Electrophoresis (DGGE)Riedel et al. 1990

Thermal Gradient Gel Electrophoresis (TGGE)Riesner et al. 1989

Heteroduplex Analysis (HDA)Perez et al. 1999; Schneider et al. 1999

Denaturing High Performance LiquidChromatography (DHPLC)Hauser et al. 1998; Steinmetz et al. 2000; Kota et al. 2001

Multiple Arbitrary Amplicon Profiling (MAAP) Caetano-Anolles 1994; Caetano-Anolles et al. 1992

Random Amplified Polymorphic DNA (RAPD)Williams et al. 1990; Hadrys et al. 1992

DNA Amplification Fingerprinting (DAF)Caetano-Anolles et al. 1991

Arbitrarily Primed Polymerase Chain Reaction (AP-PCR)Welsh and McClelland 1990; Williams et al. 1990

Inter-Simple Sequence Repeat (ISSR)Zietkiewicz et al. 1994; Godwin et al. 1997

Single Primer Amplification Reaction (SPAR)Staub et al. 1996

Directed Amplification of Minisatellites DNA (DAMD)Heath et al. 1993; Somers and Demmon 2002

Amplified Fragment Length Polymorphism (AFLP)Vos et al. 1995

Selectively Amplified Microsatellite Polymorphic Loci (SAMPL)Witsenboer et al. 1997

Molecular basis of major DNA markers. Parts AE show different ways in which DNA markers (listed below each diagram) can be generated. The cross in part A indicates that mutation has eliminated the priming site. Abbreviations: as defined in Table ; VNTR, variable number of tandem repeat; CAPS, a DNA marker generated by specific primer PCR combined with RFLP; ISSR, inter simple sequence repeat.Hybridization-based molecular markersDNA-based marker systems can be described as hybridization-based makers when the DNA profiles are visualized by hybridizing fragments to labelled probes.It include: I. Restriction fragment length polymorphisms (RFLPs)

Restriction fragment length polymorphisms (RFLPs): Botstein et al. 1980The restriction fragment length polymorphisms (RFLPs) marker technology is the first generation of DNA markers and one of the best for plant genome mapping.Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms are differentiated by analysis of patterns derived from cleavage of their DNA. When two different organisms are digested with similar Restriction Endonucleases, it cut within genome at a particular sequence in both the organisms. The similarity of the patterns generated can be used to differentiate species from one another.RFLP is the most widely used hybridization-based molecular marker. RFLP markers were first used in 1975 to identify DNA sequence polymorphisms for genetic mapping of a temperature-sensitive mutation of adeno-virus serotypes (Grodzicker et al., 1975). It was then used for human genome mapping (Botstein et al., 1980), and later adopted for plant genomes (Helentjaris et al., 1986; Weber and Helentjaris, 1989).Procedure for RFLP(i) DNA isolation a significant amount of DNA must be isolated from the sample and purified to a fairly stringent degree as contaminants can often interfere with the restriction enzyme and inhibit its ability to digest the DNA.(ii) Restriction Digest - Restriction enzyme is added to purified genomic DNA under buffered conditions. The enzyme cuts at recognition sites throughout the genome and leaves behind hundreds of thousands of fragments.(iii) Gel electrophoresis The digest is run on a gel and when visualized appears a smear because of the large number of fragments.(iv) Southern blotting-transfer to nitrocellulose or nylon membrane filter(v) Probe visualization Because of the large number of fragments, probes must be constructed to visualize more specific bands in the digest. These probes consist of radio labelled oligonucleotide sequences which will anneal to the fragment sequences so that that they may be visualized on photographic paper using a technique called autoradiography.(vi) Analysis- Number of RFLP loci can be analysed after autoradiography.

RFLP workflow from DNA extraction to radio-autograph.Modified from Xu and Zhu (1994).

AdvantagesLimitations

Easy to score Sequence used as probe need not be known Transferable across populationsHigh genomic abundanceHighly reproducibleGood genome coverageCan be used across speciesNeeded for map based cloning requires the presence of high quantity and quality of DNA depends on the development of specific probe libraries Technique is not amenable for automation. Level of polymorphism is low, and few loci are detected per assay. Time consuming, laborious, and expensive Usually requires radioactively labelled probes.

PCR-based molecular markersPCR-based molecular markers operate through a PCR process i.e. directly amplifying a specific short segment of DNA without the use of a cloning method. An attractive feature of a PCR-based marker system is that only a minute amount of DNA is needed for a task and yields higher throughput than RFLPPCR-based techniques are of two types depending on the primers used for amplification:1) Arbitrary primed PCR techniques that developed without prior sequence information. e.g., AP-PCR, DAF, RAPD, AFLP, ISSR

2) Site-targeted PCR techniques that developed from known DNA sequences. e.g., EST, CAPS, SSR, SCAR, STS

Arbitrarily Amplified DNA MarkersRAPD (random amplified polymorphic DNA), AP-PCR (arbitrarily primed PCR) and DAF (DNA amplification fingerprinting) are collectively termed as multiple arbitrary amplicon profiling (MAAP; Caetano-Annolles, 1994).These three techniques were the first to amplify DNA fragments from any species without prior sequences information.The key innovation of RAPD, AP-PCR and DAF is the use of a single arbitrary oligonucleotide primer (10mer primer) to amplify template DNA without prior knowledge of the target sequence.

The difference among MAAP techniques include: Modifications in amplification profiles by changing primer sequence and length, annealing temperature The number of PCR cycles used in a reaction The thermo-stable DNA polymerase used Enzymatic digestion of template DNA or amplification products and Alternative methods of fragment separation and staining.

Random amplified polymorphic DNA (RAPD): Williams et al. 1990 RAPD is a PCR-based marker system in which the total genomic DNA is amplified using a single short (10mer) random primer. It differs from traditional PCR analysis in that it does not require specific knowledge of the DNA sequence of the target organism.

Since it is a dominant marker, polymorphisms can be detected by simply presence or absence band on gel.

The RAPD protocol usually uses a 10 bp arbitrary primer at constant low annealing temperature (generally 34 37oC).

Criteria for RADP primers giver by William et. al.(1990) :I. A minimum of 40% GC content (50 - 80% GC content is generally used), and II. The absence of palindromic sequence (A base sequence that reads exactly the same from right to left as from left to right).

Because G-C bond consists of three hydrogen bridges and the A-T bond of only two, a primer-DNA hybrid with less than 50% GC will probably not withstand the 72oC temperature at which DNA elongation takes place by DNA polymerase. The resulting PCR products are generally resolved on 1.5- 2.0% agarose gels and stained with ethidium bromide (EtBr).

AdvantagesLimitations

High genomic abundance Good genome coverage No sequence information Ideal for automation Less amount of DNA (poor DNA acceptable) No radioactive labelling Relatively faster No probe or primer information Dominant markers Not reproducible Cannot be used across species Not very well-tested Difficult to analyse Low transferability

AFLP (amplified fragment length polymorphism): Vos et al. 1995AFLP technique combines the power of RFLP with the flexibility of PCR-based technology by ligating primer recognition sequences (adaptors) to the restricted DNA.The key feature of AFLP is its capacity for genome representation: The simultaneous screening of representative DNA regions distributed randomly throughout the genome. AFLP markers can be generated for DNA of any organism without initial investment in primer/probe development and sequence analysis.Both good quality and partially degraded DNA can be used for digestion but the DNA should be free of restriction enzyme and PCR inhibitors

Procedure for AFLP(i) DNA is cut with two specific restriction enzymes, one frequent cutter (3 bp recognition site) and one rare cutter (6 bp recognition site).(ii) Oligonucleotide adapters are ligated to the ends of each fragment. One end with a complimentary sequence for the rare cutter and the other with the complimentary sequence for the frequent cutter. This way only fragments which have been cut by the frequent cutter and rare cutter will be amplified(iii) Primers are designed from the known sequence of the adapter, plus 1-3 selective nucleotides which extend into the fragment sequence. Sequences not matching these selective nucleotides in the primer will not be amplified.(iv) PCR performed

(v) Visualized on agarose gels with ethidium bromide OR on denaturing polyacrylamide gels with autoradiography AgNO3 staining OR automatic DNA sequencers.

Polyacrylamide gel electrophoresis (PAGE) provides maximum resolution of AFLP banding patterns to the level of single-nucleotide length differences, whereas fragment length differences of less than ten nucleotides are difficult to score on agarose gels(2%-2.5% gel).

AdvantagesLimitations

Highly reliable and reproducible.DNA sequence information not required.Information-rich due to its ability to analyse a large number of polymorphic loci simultaneously (effective multiplex ratio) with a single primer combination on a single gel

Co-migrating AFLP amplification products are mostly homologous and locus specific

more number of steps are required. Requires template DNA free of inhibitor compounds that interferes with the restriction enzyme. This technique requires the use of polyacrylamide gel in combination with AgNO3 staining, radioactivity, or fluorescent methods of detection, which will be more expensive and laborious than agarose gels. It involves additional cost to purchase both restriction and ligation enzymes as well as adapters.AFLP loci are dominant, hence does not differentiate dominant homozygotes from heterozygotes. This reduces the accuracy of AFLP markers in population genetic analysis, genetic mapping, and marker assisted selection.

ISSR (inter-simple sequence repeat): Zietkiewicz et al. 1994

ISSR involves amplification of DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction.The technique uses microsatellites as primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly inter simple sequence repeats of different sizes.The microsatellite repeats used as primers for ISSRs can be di-nucleotide, tri-nucleotide, tetra-nucleotide or penta-nucleotide. The primers used can be either unanchored or more usually anchored at 3` or 5` end with 1 to 4 degenerate bases extended into the flanking sequences.ISSR uses longer primers (1530 mer) as compared to RAPD primers (10 mer), which permit the subsequent use of high annealing temperature leading to higher stringency.The annealing temperature depends on the GC content of the primer used and ranges from 45 to 65oC. The amplified products are usually 2002000 bp long and amenable to detection by both agarose and polyacrylamide gel electrophoresis.The technique is simple, quick, and the use of radioactivity is not essential. ISSR markers usually show high polymorphism.

Detection of ISSR

Polyacrylamide gel electrophoresis (PAGE) in combination with radioactivity was shown to be most sensitive, followed by PAGE with AgNO3 staining and then agarose gel (2%-2.5% gel) with EtBr system of detection.

AdvantagesLimitations

Show high polymorphism Simple QuickUse of radioactivity is not essential

Reproducibility,Dominant inheritance Homology of co- migrating amplification products

Site-targeted PCR techniques

Microsatellites: Litt and Lutty (1989Microsatellites, also known as, Simple Sequence Repeat SSRs, are tandemly repeated units of short nucleotide motifs that are 16 bp long. Di-, tri- and tetra-nucleotide repeats such as (CA)n, (AAT)n and (GATA)n are widely distributed throughout the genomes of plants and animals.Presence of high level of allelic variation, make them to utilise as genetic markers.Microsatellite sequences are especially suited to distinguish closely related genotypes; because of their high degree of variability, these are, favoured in population studies and for the identification of closely related cultivars.

PCR fragments are usually separated on polyacrylamide gels in combination with AgNO3 staining, autoradiography or fluorescent detection systems. Agarose gels (usually 3%) with EtBr can also used when differences in allele size among samples is larger than 10 bp.

SSR markers are characterized by their hypervariability, reproducibility, codominant nature, locus specificity and random dispersion throughout most genomes. SSRs are reported to be more variable than RFLPs or RAPDs.

Origin of SSR The predominant mutation mechanism in microsatellite tracts is slipped-strand mispairing. When slipped-strand mispairing occurs within a microsatellite array during DNA synthesis, it can result in the gain or loss of one, or more, repeat units depending on whether the newly synthesized DNA chain loops out or the template chain loops out, respectively. The relative propensity for either chain to loop out seems to depend in part on the sequences making up the array, and in part on whether the event occurs on the leading (continuous DNA synthesis) or lagging (discontinuous DNA synthesis) strand . SSR allelic differences are the results of variable numbers of repeat units within the microsatellite structure. The repeated sequence may be, consisted of two, three or four nucleotides (di-, tri-, and tetra-, nucleotide repeats, respectively).Method of SSR genotyping Four methods:SSRs on agarose gel: These assays can usually distinguish alleles which differ by 24 bp or more.

PAGE based: to separate radio-labelled or silver-stained PCR products by denaturing or non- denaturing PAGE using ethidium bromide or SYBR staining.

Semi-automated SSR : genotyping can be carried out by assaying fluorescently labelled PCR products for length variants on an automated DNA sequencer.

Drawback of fluorescent SSR genotyping is the cost of end-labelling primers with the necessary fluorophores. e.g. 6-carboxy-fluorescine (FAM), hexachloro- 6-carboxy-flurescine (HEX) or tetra chloro- 6-carboxy-fluorescine (TET).

SSRs assayed on polyacrylamide gels typically show characteristic stuttering. Stutter bands are artefacts produced by DNA polymerase slippage. Stutters are multiple near-identical ladders of PCR products which are one or two nucleotides shorter or longer than the full length product Stuttering reduces the resolution between alleles such that 2- or possibly 4-bp differences between alleles cannot be sharply or unequivocally distinguished on polyacrylamide gels.

AdvantagesLimitations

High genomic abundanceHighly reproducibleFairly good genome coverageHigh polymorphismNo radioactive labellingEasy to automateMultiple allelesCannot be used across speciesNeed sequence informationNot well-tested High mutation ratePrimer preparation is time consuming

CAPS (cleaved amplified polymorphic sequence): Akopyanz et al. 1992CAPS is a combination of the PCR and RFLP, and it was originally named PCR-RFLP (Maeda et al., 1990). The technique involves amplification of a target DNA through PCR, followed by digesting with restriction enzyme.CAPS markers rely on differences in restriction enzyme digestion patterns of PCR fragments caused by nucleotide polymorphism between samples.Critical steps in the CAPS marker approach include DNA extraction, PCR conditions, and the number or distribution of polymorphic sites.The ability of CAPS to detect DNA polymorphism is not as high as SSRs and AFLPs because Nucleotide changes affecting restriction sites are essential for the detection of DNA polymorphism by CAPS. The development of CAPS markers is only possible where mutations disrupt or create a restriction enzyme recognition site.

A technique known as derived-CAPS (dCAPS) is developed that can eliminate the problems related with CAPS markers by generating mismatches in a PCR primer, which are subsequently used to create a polymorphism based on the target mutation.

SCAR (sequence characterized amplified region): Michelmore et al. (1991) A SCAR marker is a genomic DNA fragment that is identified by PCR amplification using a pair of specific oligonucleotide primers of 15-30 pb size.By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs.SCARs are derived by cloning and sequencing the two ends of RAPD markers that appeared to be diagnostic for specific purposes (e.g., a RAPD band present in disease resistant lines but absent in susceptible lines).

AdvantagesLimitations

Quick Easy to useHigh reproducibility Locus-specific.Due to the use of PCR, only low quantities of template DNA are required (10100 ng per reaction).

need for sequence data to design the PCR primers

STS (sequence tagged site): Olsen et al. (1989)

STS was first developed by Olsen et al. (1989) as DNA landmarks in the physical mapping of the human genome, and later adopted in plants.STS is a short, unique sequence whose exact sequence is found nowhere else in the genome. Two or more clones containing the same STS must overlap and the overlap must include STS.

STS markers are co-dominant, highly reproducible, suitable for high throughput and automation, and technically simple for use.

AdvantagesLimitations

Useful in preparing contig mapsNo radioactive labellingFairly good genome coverageHighly reproducibleCan use filters many timesLaboriousCannot detect mutations out of the target sitesNeed sequence informationCloning and characterization of probe are Required

SNP (single nucleotide polymorphism): Tautz and Renz (1984) A single nucleotide polymorphism SNP is an individual nucleotide base difference between two DNA sequences. SNPs can be categorized according to nucleotide substitution as: Transitions (C/T or G/A) or Transversions (C/G, A/T, C/A or T/G).A single base variation in cDNA (mRNA) is considered as SNPs and therefore SNPs provide the ultimate form of molecular marker.

For a variation to be considered a SNP, it must occur in at least 1% of the population. Two of every three SNPs involve the replacement of cytosine (C) with thymine (T).

SNPs may fall within coding sequences of genes, non-coding regions of genes or in the inter-genic regions between genes at different frequencies in different chromosome regions.

SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced due to redundancy in the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed synonymous, while if a different polypeptide sequence is produced they are non-synonymous. SNPs that are not in protein coding regions may still have consequences for gene splicing, transcription factor binding or the sequence of non-coding RNA.

Discovery of SNPThree general categories are possible for discovery of SNP:1. In-vitro discovery, where new sequence data is generated;2. In-silico methods that rely on the analysis of available sequence data; and 3. Indirect discovery, where the base sequence of the polymorphism remains unknown.A convenient method for detecting SNPs is RFLP (SNP-RFLP) or by using the CAPS marker technique. i.e. cut with a restriction enzyme and analyse the sequence data stored in the major databases and identify SNPs.Software approach for SNP discovery SNP discovery by alignment of sequence traces obtained from direct sequencing of genomic PCR products. It is not always possible to distinguish between sequence artefacts and true polymorphism, when two peaks are present at one position. Box1: top sequence homozygote AA, middle sequence heterozygote AG, bottom sequence homozygote GG. Box 2: The polymorphism detection software has considered the top and bottom sequences as heterozygote CT and the middle one as homozygote CC. Clonal sequence removes many of such ambiguities, since any double peak is a sequence artefact.

Method of SNP genotyping Sobrino et al. (2005) classify majority of SNP genotyping assays to one of four groups based on the molecular mechanisms:A. Allele-specific hybridization, B. Primer extension,C. Oligonucleotide ligationD. Invasive cleavage

(A) Hybridization with allelic specific oligonucleotides (ASO): Two ASO probes are hybridized with the target DNA that contains the SNP. Under optimized conditions, only the perfectly matched probe-target hybrids are stable.

(B) Primer extension reactions: Mini sequencing (B1) and allelic-specific extension (B2). (B1) mini sequencing: a primer anneals to its target DNA immediately upstream to the SNP and is extended with a single nucleotide complementary to the polymorphic base. (B2) allelic-specific extension: the 3 end of the primers is complementary to each allele of the SNP. When there is a perfect match the primer is extended.

(C) Oligo-nucleotide ligation assay (OLA): Two allelic-specific probes and one common ligation probe are required per SNP. The common ligation probe hybridized adjacent to the allelic specific probe. When there is a perfect match of the allelic-specific probe, the ligase joins both allelic-specific and common probes.

(D) Invasive cleavage: The oligonucleotides required called invader probe and allelic-specific probes, anneal to the target DNA with an overlap of one nucleotide. When the allelic-specific probe is complementary to the polymorphic base, overlaps the 3 end of the invader oligonucleotide, forming the structure that is recognized and cleaved by the Flap endonuclease, releasing the 5 arm of the allelic-specific probe.

Chemistry, demultiplexing, detection options in SNP genotyping

AdvantagesLimitations

Low mutation rateEasy to automate Cross-study comparison easy SNP

Time consuming Expensive Low information content of a single Ascertainment bias

DArT (diversity arrays technology)

Diversity array technology (DArT) is a novel type of DNA marker which employs a microarray hybridization-based technique developed by CAMBIA (http://www.diversity arrays.com) that enables the simultaneous genotyping of several hundred polymorphic loci spread over the genome.DArT can be used to construct medium-density genetic linkage maps in species of various genome sizes.

Schematic representation of DArT.

(A) Generation of diversity panels: Genomic DNAs of specimens to be studied are pooled together. The DNA is cut with a chosen restriction enzyme and ligated to adapters. The genome complexity is reduced in this case by PCR using primers with selective overhangs. The fragments from representations are cloned. Cloned inserts are amplified using vector-specific primers purified and arrayed onto a solid support.

(B) Contrasting two samples using DArT: Two genomic samples are converted to representations using the same methods as in (A). Each representation is labeled with a green or red fluorescent dye, mixed and hybridized to the diversity panel. The ratio of green: red signal intensity is measured at each array feature. Significant differences in the signal ratio indicate array elements (and the relevant fragment of the genome) for which the two samples differ. (Jaccoud et al., 2001).

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DArT markers are biallelic and behave in a dominant (present versus absent) or codominant (two doses versus one dose versus absent) manner. DArT detects single-base changes as well as indels.AdvantagesLimitations

does not need prior sequence information high throughput, quick, and highly reproducible method.Cost effective, tenfold lower than SSR markersGenetic scope of analysis is defined by the user and easily expandableNot covered by exclusive patent rights, but on the contrary open-sourceDArT is a microarray-based technique that involves several steps, so it demands an extensive investment both in laboratory facility and skilled manpower. Marker dominance Being a novel technique, it is not in use on large scale.

SUMMERY Links between the signal generation and detection: Vignal et al., 2002

Methods used to detect products generated by the allele-specific reactions are: 1: PCR-RFLP2: Oligonucleotide ligation assay (OLA)3: Good Assay4: Minisequencing techniques5: single stranded conformation polymorphism (SSCP) 6: denaturing high performance liquid chromatography (DHPLC)7: Pyrosequencing8: SNP it9: exonuclease detection (Taqman)10: Invader Assay, 11: Microarray or DNA chips

Comparison of the five most widely used DNA markers in plants

RFLPSSRRAPDAFLPISSR

Genomic abundancehighmediumvery highvery highmedium

Part of genome surveyedlow copy coding regionswhole genomewhole genomewhole genomewhole genome

Amount of DNA requiredhighlowlowmediumlow

Type of polymorphismsingle base changes,changes in length of repeatssingle base changes,single base changes,single base changes,

insertion, deletioninsertion, deletioninsertion, deletioninsertion, deletion

Level of polymorphismmediumhighhighvery highhigh

Effective multiplex ratiolowmediummediumhighmedium

Marker indexlowmediummediumhighmedium

InheritanceCo-dominantCo-dominantdominantdominantdominant

Detection of allelesyesyesnonono

Ease of uselabour intensiveeasyeasydifficult initiallyeasy

Automationlowhighmediummediummedium

Reproducibility (reliability)highhighintermediatehighmedium to high

Type of probes/primersLow copy genomic DNA or cDNA clone specific repeat DNA sequenceUsually 16 bp random nucleotidespecific sequencespecific repeat DNA

sequence

Cloning and/or sequencingyesyesnonono

Radioactive detectionusually yesnonoyes/nono

Development/start-up costshighhighlowmediummedium

Utility for genetic mappingspecies specificspecies specificcross specificcross specificcross specific

Proprietary rights statusNoNo (some are licensed)licensedlicensedno