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Genetic markers are of three distinct types:1.

Visually assessable traits (traits that can be studied morphologically like agronomic traits). 2. Biochemical markers (based on gene products like allozymes monoterpenes). 3. DNA based genetic markers ( ESTs,VNTR,SSR)

SINGAL LOCUS(Co-Dominant)
1 ) Restriction Fragment Length Polymorphism (RFLP) 2) Cleaved amplified polymorphic sequences(CAPS) 3) Simple Sequence Length Polymorphism(SSLP) oVariable number of tandem repeates (VNTR) [using Minisatellites] o Simple sequence repeats/ Simple tandem repeats (SSR/STP) [using microsatellites] 4) Sequence characterized amplified region (SCAR) 5) Single nucleotide polymorphism (SNPs) oDynamic allele-specific hybridization oDNA chips oDNA sequencing oSingle strand conformation polymorphism(SSCP)

DETECTION
Hybridization PCR PCR Hybridization or PCR PCR

PCR Hybridization Hybridization Sequencing Conformation

MULTIPLE LOCI (Dominant) 1) Amplified product length polymorphism (AFLP) 2) Random amplified polymorphic DNA (RAPD) 3) DNA amlification fingerprinting (DAF) 4) Arbitrarily primed-PCR (AP-PCR)

DETECTION PCR PCR PCR PCR PCR PCR

5) Simple sequence length polymorphism (SSLP) when multiple pairs of primer were used
6) Inter-simple sequence repeat (ISSR) 7) Single nucleotide polymorphism (SNP) -Single strand conformation polymorphism (SSCP) when used to scan for random located SNPS

Conformatio n

ASAP ASO AS-PCR AFLP AMP-PCR ASSR AP-PCR CAPS DOP-PCR Dart

Allele Specific associated primers Allele specific oligos Allele specific-polymerase chain reaction Amplified fragment length polymorphism Anchored microsatellite primed-PCR Anchored simple sequence repeats Arbitrary primed-PCR Cleaved amplified polymorphic sequence Degenerated oligonucleotide primed-PCR Diversity arrays technology

DAF EST ISSR IPCR ISTR MP-PCR MASDA RAMP RAM RAPD

DNA amplification fingerprinting Expressed sequence tags Inter-simple sequence repeats Inverse-PCR Inverse sequence tags Microsatellite primed PCR Multiplexed allele-specific diagnostic assay Random amplified microsatellite polymorphism Random amplified microsatellite Random amplified polymorphic DNA

SSLP SSR SNP RFLP SAMPL SCAR SSAP STMS

Simple sequence length polymorphism Simple sequence repeats Single nucleotide polymorphism Restriction fragment length polymorphism Selective amplification of microsatellite polymorphic loci Sequence characterized amplified regions Sequence specific amplification polymorphism Sequence tagged microsatellite

STS STR

Sequence tagged site Short tandem repeats

SPAR SSCP SSI

Single primer amplification repeats Single standard conformational polymorphism Site selected insertion PCR

SDA YNTR

Strand displacement amplification Variable number tendem repeats

(1) To locate any variation or mutation in genetic sequence. (2) To create a genetic linkage map or database. (3) To study associations between traits of great economic and biotechnological importance and their responsible genes (polygene may be). (4) To study interspecific traits and interspecific breeds(within a genus) so in result we can observe transfer of genes ,satellites from one gene pool of one species into other species, we can also form interbreed cross on basis of molecular markers assisted breeding. Usually markers are not normal genes ,mostly they are DNA sequence with no hereditable effects, instead they are indexes of genes location.

(1) Mode of inheritance of genes (I) Bi-parental nuclear inheritance(both female & male chromosomal portion(nuclear genes).
(ii) Maternal Organelle inheritance and paternal organelle maternal organelle inheritance ( Biparental Organelle inheritance)

(2)

Dominant or co-dominant markers.

Mode of gene action:-

(i) Dominant:A dominant marker allows amplification of several loci in one PCR with one sample of DNA( e.g RAPD.AFLP,SAMPLS) (ii) Co-dominant:Marker analyze one locus at a time ,more informative( RFLP, Microsatellite).because at a time a particul locus allelic variations can be distinguished.

1. Hybridization based (hybridization of DNA segments by hybridizing probes and restriction enzymes) e.g RFLP

2. PCR based techniques. two main types:(1) Arbitrary primer (without previously known sequence). (2) Known DNA sequences( which are going to be amplified). Arbitrarily Amplified DNA Markers. MAAP multiple arbitrary amplicons profiling. (Caetan anoles et al.,)(RAPD or AP-PCR or DAF , ), AFLP,ISSR These techniques are used to differentiate two individuals of a species. Site-targeted PCR techniques Develop from known DNA sequences ( e.g EST, CAPS,SSR,SCAR,STS) used to differentiate genotype of two different species.

Principle:Restriction fragment length polymorphism (RFLP) technique was developed in 1980s primarily for mapping human genome and was later applied to plants.

We can generate many different DNA fragments by digesting total DNA with specific restriction enzymes.

These fragments are relatively small in size and are co-dominant in nature i.e both strands can be assayed.
If two individuals differ by as little as a single nucleotide in the restriction site, the restriction enzyme will cut the DNA of one but not the other. Restriction fragments of different lengths are thus generated. The analysis requires a relatively complex technique that is time consuming and expensive.

The hybridization results can be visualized by 1. Autoradiography (if the probes are radioactively labeled) 2. Chemiluminesence (if non-radioactive, enzyme-link methods are used for probe labeling and detection, digoxigenin, antibodies etc).

Uses:-

Locating disease causing mutants. Further prediction of inheritance pattern. (which one would be carrier( or homozygous for disease causing allele)
An autoradiograph detecting parent (P1&P2) and homozygous and heterozygous (H ) F1 segregation

RAPD (Random Amplification of polymorphic DNA.)


RAPD also known as, AP-PCR(Arbitrarily primed PCR), DAF (DNA amplification fingerprinting)& MAAP(Multiple arbitrary amplicon profiling) DNA segment can be amplified using short oligodeoxynucleotide primer of arbitrary nucleotide sequence (amplifier) and polymerase chain reaction procedure (Khal et al., 2001 ) . RAPDs are produce by PCR using genomic DNA and arbitrary primers Taq polymerase is used to amplify DNA segment between closely spaced sequence (< 2kb) and complementary to the short random oligomers (typically 10-mers) RAPD polymorphism result from change in the primer-binding site in the DNA sequence

Segments of DNA to be amplified are arbitrary or random.

Short primers mostly 10 nucleotides or 8-12 bp are used.


Arbitrary primers should have at least 40% GC content, mostly used 50-80%(Williams et al.,) so that they can withstand annealing temperature i.e. 72 Celsius Absence of palindromic sequence. PCR products obtained are run on 1.6%-2% agarose gel stained with Ethidium Bromide or with polyacrylamide in combination with radioactive labeled nucleotide. The band obtained after running on gel shows two types of polymorphism, brighter bands show homozygous dominance and less brighter will hetrozygously dominant. so it is very difficult to distinguish or differentiate between them, so markers are known as Dominant markers, and homozygous recessive DNA fragments or loci are not amplified

In variety A there are 4 primer binding sites resulting in two RAPD products, variety B lacks one of the binding sites resulting in only one RAPD marker being produced

Template DNA

Primers point in the same direction, so amplification wont happen

Template DNA

> 2,000 bases

Primers too far apart, so amplification wont happen

Template DNA

100 - 1,500 bases

Primers are just the right distance apart, so fragment is amplified

Jatropha curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica and J. Tanjorensis

Jatropha is a genus of flowering plants in the spurge family, Euphorbiaceae. The name is derived from the Greek words , meaning "physician," and , meaning "nutrition," hence the common name physic nut. Higher classification: Euphorbiaceae Lower classifications: Jatropha curcas,

RAPD profiles of different species of Jatropha L. with primer OPL 5 (17) and IDT E 4. (814). Lane 1 & 8 J. tanjorensis; 2 & 9 J. curcas; 3 & 10J. glandulifera; 4 & 11J. gossypifolia; 5 & 12J. multifida; 6 & 13J. podagrica; 7 & 14J. integerrima; M1 Kb DNA ladder

Low Reproducibility.

This issue can be eradicate by proper handling and using efficient protocol without any contaminants

Dominant Marker effect

RAPD doesnt differentiate between Hetero and homozygous dominant alleles, no fragments will be produced from homozygous recessive allele.

Homology
Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

Difference between corresponding DNA fragment from two organisms can be detected by amplification of restricted fragments, hence polymorphism can be observed.

Principle
1.

The amplified fragment length polymorphism technique combines components of RFLP analysis with PCR technology.

2.

Total genomic DNA is digested with a pair of restriction enzymes normally a frequent and rare cutter.
Adaptors (18-24bp)of known sequence are then ligated to the DNA fragments.

3.

4.

Primer complementary to the restriction fragments.

adaptors are used to amplify the

5.

The PCR amplified fragments can then separated by gel electrophoresis and banding patterns visualized.

Eco :- (1) 5- CTC GTA GAC TGC GTA CC - 3 Eco :- (2) 5-AAT TGG TAC GCA GTC TAC-3 Mse:- (1) 5 GAC GAT GAG TCC TGA G-3 Mse:- (2) 5 TAC TCA GGA CTC AAT-3 {Whole genomic DNA + Restriction enzymes + adaptors + ligase enzyme}

now Mse 1(5-3) arrange to anneal with 5-3target strand. and Mse 2(3-5) Arrange to anneal with 3-5 target strand similarly Eco 1 with 5-3 target dna strand and Eco 2 with 3-5 strand.

Principle

Genomic DNA

For restriction digestion we use two type of cutter i.e Rare cutter (6bp) EcoRI Frequent cutter (4bp) MseI
Interstitial cohesive ends Adaptor ligation

PCR amplification using EcoRI/MseI

Principle
PCR Amplification take place either By no selective base TTCA AAGTN TTCAN By 1, 2, or 3 selective base TTCAA AAGTN 4 time reduction in 16 time reduction in 64 TTCAN amplification TTCAACG AAGTNNN TTCANNN

NAAGT.. NTTCA... AAGT.

NAAGT.. NTTCA... GAAGT.. Primer(+1) for pre-selective amplification

NNNAAGT.. NNNTTCA... TAGAAGT.. Primer(+3) for selective amplification

Case study
AFLP profiles of different species of Jatropha L. with selective amplification with primers CAT/E-AAG, (17); M-CAA/EACA(814) and M-CAA/E-ACT (15-21). Lane 1, 8 & 15 2, 9 & 16 3, 10 & 17 4, 11 & 18 5, 12 & 19 6, 13 & 20 7,14 & 21 J. Curcas J. tanjorensis; J. glandulifera; J. gossypifolia; J. multifida; J. podagrica; J. integerrima;

M1 Kb + 100 bp DNA ladder mix

Case study

Application of AFLP

Advantage

Disadvantage

CAPS
Introduction & Principle
Cleaved amplified polymorphic sequences (CAPS)

They are co-dominant genetic markers that use PCR amplification and subsequent restriction endonuclease digestion to detect [single nucleotide] polymorphisms (SNPs) or insertion/deletion (INDELs) in a region of interest (Konieczny and Ausubel 1993). When a SNP is found that distinguishes alleles underlying a desired trait, the CAPS marker becomes a functional marker and should be 100% predictive of the presence/absence of the allele and its corresponding phenotype.

Introduction & Principle


Variety A Variety B

Mutation occur EcoRI site PCR amplification and restriction digestion

Case study

Case study

The rice xa5 gene for disease resistance to Xanthomonas oryzae encodes the gamma subunit of transcription factor IIA (TFIIAgamma) . TFIIAgamma is a general eukaryotic transcription factor with no previously known role in disease resistance. xa5 is unusual in that it is recessive.

Sequencing of TFIIAgamma in resistant and susceptible isolines revealed substitutions of two nucleotides(in dominant), which results in an amino acid change from valine to glutamic acid at position 39 of the protein resulting resistant and susceptible cultivars.
Due to the change in 2 nucleotide a restriction site is created in dominant gene and hence with the help of CAPS it is used in MAS

Case study

Resistance

Susceptible

Application of CAPS

Advantage

Disadvantage

Application of CAPS

1. ISSR involves amplification of DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction (Figure9). 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, trinucleotide, tetra-nucleotide or penta-nucleotide. The primers used can be either unanchored (Meyer et al., 1993; Gupta et al., 1994;Wu et al., 1994) or more usually anchored at 3` or 5` end with 1 to 4 degenerate bases extended into the flanking sequences ( Zietkiewicz et al., 1994; Figure 9). ISSRs use longer primers (1530 mers) as compared to RAPD primers (10 mers), which permit the subsequent use of high annealing temperature leading to higher stringency. 2. The annealing temperature depends on the GC content of the primer used and ranges from 45 to 65C. The amplified products are usually 2002000 bp long and amenable to detection by both agrose and polyacrylamide gel electrophoresis. ISSRs exhibit the specificity of microsatellite markers, but need no sequence information for primer synthesis enjoying the advantage of random markers (Joshi et al., 2000). The primers are not proprietary and can be synthesized by anyone. The technique is simple, quick, and the use of radioactivity is not essential. ISSR markers usually show high polymorphism (Kojima et al., 1998)

Application of ISSR

REMAP & IRAP

Definition
Any difference in DNA sequence between two genomes, detected by polymerase chain reaction-mediated amplification of the region between a long terminal repeat of a retrotransposon and a nearby microsatellite

Principle
The dispersion, ubiquity and prevalence of retrotransposon-like elements in plant genomes can be exploited for DNA-fingerprinting Two DNA techniques based on retrotransposon like elements are introduce here IRAP (Inter-Retrotransposon Amplified Polymorphism) ,

REMAP (REtrotransposon-Microsatellite Amplified Polymorphism) (Kalendar et al., 1999).

Principle
The IRAP (Inter- Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two retrotransposons using outward facing primers annealing to their long terminal repeats (LTRs)

Retrotransposon

Principle
In REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) the DNA sequence between the LTRs and adjacent microsatellites (SSRs) are amplified using appropriate primers

Microsatellite

Retrotransposon

Application of IRAP & REMAP

SNPs

Definition
Any polymorphism between two genomes that is based on a single nucleotide exchange, small deletion or insertion

Principle
Small nucleotide polymorphism (SNP) is a new marker technology originally developed in human. SNPs are the most abundant polymorphic marker with 2-3 polymorphic sites every kilobase (Cooper et al., 1985). Originally discovered in humans, SNPs have now been developed for genotyping in plants. SNP technology is heavily dependent upon sequence data. Several methods are available for SNP detection including Automated fluorescent sequencing denaturing high-performance liquid chromatography (DHPLC, Underhill et al., 1996), DNA microarrays (Hacia and Collins, 1999), Single-strand conformational polymorphic-capillary electrophoresis (SSCP-CE, Ren, 2001) Microplate array diagonal-gel electrophoresis (MADGE, Day et al., 1998) and Matrix-assisted laser desorption /ionisation time of flight (MALDI-TOF, Griffin and Smith, 2000)

Principle

Application of SNPs

Advantage

Disadvantage