Molecular Markers, their application in crop improvement
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Molecular markers such as SNPs, SSRs, RAPDs, AFLPs, and RFLPs can be used for crop improvement through applications like marker-assisted selection, linkage mapping, and trait-based selection. Molecular markers are DNA sequences that can identify specific locations in the genome and are linked to important agronomic traits. They are useful because they are selectively neutral, co-segregate with traits of interest, and follow Mendelian inheritance patterns.
Molecular Markers, their application in crop improvement
- 1. Molecular MarkersMolecular Markers Their applications in crop improvementTheir applications in crop improvement Presented By : Mrinali M. M.Sc 1st Year Dept of Seed Science and Technology
- 2. What is a Marker?What is a Marker? • Marker is an allelic difference or variation at a given locus in the DNA that can be observed at morphological, biochemical or molecular level • Molecular marker are based on naturally occurring changes or polymorphism in DNA sequence (deletion, substitution, addition, tandem repeat or duplication) • All molecular markers occupy specific genomic positions within the chromosome k/as ‘loci’ • Markers located in close proximity to desirable genes (tightly linked) are k/as ‘gene tags’ • Agriculturally important traits are governed by many genes ‘Polygenic’ (Quantitative traits) • Regions in genome containing genes associated with a particular quantitative trait are k/as Quantitative Trait Loci (QTL)
- 3. Why Molecular Marker ?Why Molecular Marker ? • Because they are selectively neutral as they are present in the non coding region of the genome. • Makers are co-segregating with the trait of interest. • They follow exactly the Mendelian pattern of inheritance. • Free from epistatic interaction or pleiotropic effect
- 4. A Perfect Molecular MarkerA Perfect Molecular Marker • Polymorphic • Co-dominant • Reproducible • Robust • Cost effective • Easy to use • High throughput • Closely linked to the trait of interest Marker Trait Marker
- 5. Level of analysis of markersLevel of analysis of markers Class of Marker Level of Analysis 1. Morphological Markers Phenotype 2. Biochemical Markers Gene Product 3. DNA markers / genetic markers DNA Sequence
- 6. 1. Morphological Markers1. Morphological Markers • Are botanical descriptors of plant which are visually or phenotypically characterized • K/as DUS descriptors or universal markers 1. D- Distinctiveness 2. U- Uniformity 3. S- Stability • Seed colour, seed shape, seed size, flower colour, growth habits, plant pigmentation
- 7. 2. Biochemical Markers2. Biochemical Markers • Are of two types: 1. Protein based 2. Enzyme based known as Isozyme • First true biochemical marker was an allelic variant of enzyme pyruvate dehydrogenase • Isoforms can be resolved by gel electrophoresis based on their size, shape and amino acid differences • Can be easily assayed and detected • They are low in polymorphism as compared to DNA markers
- 8. 3. DNA based Markers3. DNA based Markers A. On the basis of ability to discriminate between same or different species 1. Co-dominant: discriminate between homo and heterozygotes 2. Dominant: which do not discriminate between homo and heterozygotes • They can be visualized by: a. Gel electrophoresis b. Ethidium bromide or silver staining c. Radioactive or colorimetric probes
- 9. Comparison between co-dominant &Comparison between co-dominant & dominant markersdominant markers P1 P2 F1 AA aa Aa P1 P2 F1 BB bb Bb
- 10. Classification ofClassification of Molecular MarkersMolecular Markers Hybridization based markers
- 11. • RAPD :Random Amplified Polymorphic DNA • RFLP: Restriction Fragment Length Polymorphism • AFLP: Amplified Fragment Length Polymorphism • SSR :Simple Sequence Repeat • ISSR: Inter Simple Sequence Repeat • SNP : Single Nucleotide Polymorphisms • SCAR: Sequence Characterized Amplified Region • CAPS :Cleaved Amplified Polymorphic Sequence • TRAP: Target Region Amplification Polymorphism • DArT : Diversity Arrays Technology • DAF: DNA Amplification Fingerprinting • SRAP: Sequence Related Amplified Polymorphism Markers : Full FormsMarkers : Full Forms
- 12. • On the basis of principles and methods of detection Collard B.C.Y. et al , 2005. (Euphytica)
- 13. A. Hybridization based markersA. Hybridization based markers • RFLP – Restriction Fragment Length Polymorphism • Based on polymorphism arising due to chromosomal aberrations occurred in the specific regions of DNA • Are co-dominant. Can identify a unique locus as c- DNA of known function are used as probe, chromosomal positions of the specific gene are identified • Restriction enzyme can recognize and cut DNA wherever a specific short sequence occurs • Molecular probe for Southern hybridization may be radioactive or non radioactive • Size of defined restriction fragments can be compared electrophoretically
- 14. Major events in RFLP Analysis
- 15. B. PCR based markersB. PCR based markers • Reduce time, efforts and expenses • Based on use of pair of primers (reverse and forward) • Designed either on the basis of random sequences or on specific sequences flanking the DNA segment that needs to be amplified 1. Single primer used as forward and reverse primer - AP- PCR(~20nt), RAPD(~10nt), DAF (~6-8nt) (same random primer is inversed within an amplifiable distance) 2. A pair of primers used – STSs, SCARs or STARs
- 16. PrimerPrimer • Primer are short DNA sequences having free 3’-OH (~ 20bp) usually used to amplify the marker loci
- 17. i. RAPDi. RAPD • RAPD – Random Amplified Polymorphic DNA • It was the 1st PCR based marker technique and it is by far the simplest. 1st time reported by Welsh and McClelland (AP- PCR) & Williams et al. in 1990 • Short PCR primers (approx. 10 bp) are randomly selected to amplify random DNA segments throughout the genome • The resulting amplification product is generated at the region flanking a part of the 10 bp priming sites in the appropriate orientation • RAPD is a dominant marker • RAPD products are usually visualized on Agarose gels stained with ethidium bromide • Modified approach of RAPD is DNA Amplification Fingerprinting (DAF)
- 18. Major events in RAPD
- 19. ii. AFLPii. AFLP • AFLP - Amplified Fragment Length Polymorphism • Are differences in restriction fragment lengths caused by SNPs and INDELs that create /abolish restriction sites • Based on selective PCR amplification of restriction fragments from a total digest of genomic DNA • Oligonucletotide adapters (~20 bp) are ligated at the end of DNA fragments • Adapted DNA fragments are amplified by PCR
- 20. Major events in AFLP
- 21. SCARsSCARs • SCAR – Sequence Characterized Amplified Region • Based on sequence of polymorphic bands from RAPD/RFLP/AFLP linked to trait of interest • Longer primers (15-30 bp) are designed for specific amplification of particular locus • Higher in reproducibility than RAPD/RFLP • Are co-dominant
- 22. iii. Single Nucleotide Polymorphism (SNPs)iii. Single Nucleotide Polymorphism (SNPs) • SNP is a DNA sequence variation occurring when a single nucleotide (A, T, C, or G) in the genome differs between members of a species • Eg. AAGCCTA and AAGCTTA (called alleles C and T) •Can be known only after DNA sequencing of several genomes • Used in biomedical research, crop and livestock breeding programs
- 23. iv. Simple Sequence Repeats (SSRs)iv. Simple Sequence Repeats (SSRs) • Also k/as Short Tandem Repeat (STRs)/microsatellites which are repeating sequences of 2-6 nt of DNA (motifs). • Are co-dominant, abundant, dispersed throughout the genome • Occur in DNA when a pattern of two or more nucleotides are repeated which are adjacent to each other • Eg. (CATG)n in a genomic region is typically in the non-coding intron region • Of four types: EST-SSR, Genomic-SSRs, Mitochondrial-SSRs, Chloroplastic-SSRs • Expressed Sequence Tags are the expressed regions in a DNA sequence from a c-DNA clone that corresponds to m RNA. As they represent the transcribed part of genome they show higher transferability. Used for developing STSs, SSR, SNPs • Used extensively in Rice genome (www.gramene.org)
- 24. Major events in SSR Analysis
- 25. Applications of MarkerApplications of Marker 1. Confirmation of hybridity 2. Linkage Mapping 3. Marker Assisted Selection 4. Trait based Selection 5. Saturated maps 6. Orthologous gene mapping 7. Gene tagging 8. Heterosis breeding 9. Haplotype mapping 10.DNA fingerprinting for varietal identification 11.Phylogenetic and evolutionary studies • Interspecific hybridization
- 26. Confirmation of HybridityConfirmation of Hybridity • Heterozygosity of F1 can be detected
- 27. Linkage MappingLinkage Mapping • For linkage mapping we want mapping population which is immortal, universal, homozygous (true breeding type) and does not fluctuate • BC1F2, F2, DH, F2:F3, RILs, NILs
- 29. Marker Assisted Selection (MAS)Marker Assisted Selection (MAS) • MAS consists of identifying association between molecular markers and genes controlling agronomic traits (major genes) , and using these to improve plant populations • Selection is made on genotype rather than phenotype, which increases the speed and efficiency of selection • It is used for manipulating both qualitative (disease resistance) and quantitative (yield) traits • Molecular markers are used to increase the probability of identifying truly superior genotypes, by early elimination of inferior genotypes
- 30. X Back crossings Wild P1 with resistant gene Cultivated P2 with susceptible gene F1 F2 F3 F4 F5 F6 F7 Plant breeder aims to improve the resistance of a cultivated sp. Thus, he performs a cross between susceptible cultivated sp. with a wild sp. that possess the required resistance. At least 6 BC steps are necessary to get 99% of genome of recurrent parent
- 31. Collard B.C.Y. et al , 2005. (Euphytica) A robust marker is developed for a major QTL controlling disease resistance (arrow). By using this marker plant breeder may substitute large field trials and rough out many unwanted plants (75%)
- 32. Trait based selectionTrait based selection • Earliest demonstrations of power of molecular markers was provided by Beckman and Soller (1986) for indirect selection of gene in breeding program • In South Australian Barley, a single dominant resistance gene to cereal cyst nematode (Ha2) was transferred from a resistance to susceptible variety through 3 cycles of marker- assisted backcrossing using a single molecular marker
- 33. Orthologous gene mappingOrthologous gene mapping • Also known as “Comparitive mapping” • c DNA clones of one crop plant are being mapped onto the linkage maps of other crops • More saturated maps can be generated by mapping marker clones • 1st comparative maps in plants were generated by Tanksley in Tomato & potato in 1988 • Generated especially in grasses
- 34. Gene TaggingGene Tagging • Refers to mapping of genes of agricultural importance present close to known markers • A Molecular marker very closely linked to a gene can act as a ‘tag’ • Gene tagging is the pre requisite for MAS and Map based gene cloning • Eg. Cereal cyst nematode were tagged in wheat, Leaf rust resistance genes Lr9, Lr10, Lr19, Fertility restorer genes for Polima and Ogura cytoplasm in Brassica
- 35. Heterosis BreedingHeterosis Breeding • Lee et al (1989) in corn suggested that RFLP analysis provides an alternative to field testing • Since then several attempts were made to correlate heterosis with variability at molecular level • Melchinger et al (1991) analyzed 32 maize inbred lines for heterosis • Stuber et al (1992) mapped QTLs contributing to heterosis in the cross between elite maize inbred lines B73 and Mo17 • Xio et al (1995) mapped QTLs for heterosis in one of the highest yielding indica x japonica hybrids and proposed domiance as the major cause of heterosis in rice
- 36. DNA fingerprinting for varietalDNA fingerprinting for varietal identificationidentification • Smith and Smith (1992) have presented comprehensive review on need of fingerprinting of crop varieties • RAPD, microsatellite and AFLPs are the markers of choice for purpose because all are PCR based markers and does not require any prior information on nucleotides • It is useful in protection of proprietry germplasm especially the CMS lines, characterization of accessions in plant germplasm collections • Eg. Sex identification in dioecious plants by Parasnis et al (1999) using microsatellite markers (GATA)4
- 37. Phylogenetic and evolutionary studiesPhylogenetic and evolutionary studies • To study the evolutionary relationships within and between species, genera or larger taxonomic groupings • Studies are made on similarities and differences among taxa using numerous genetic markers especially RAPD and RFLP (Paterson et al, 1991) • Eg. Two lines of rice Azueena and PR 304 were classified as indicas using morphological characters while these behaved as japonicas in crossing studies
- 38. ReferencesReferences • Collard B.C.Y., Jahufer M.Z.Z., Brouwer J.B., Pang E.C.K. (2005) An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica 142: 169–196 • Xu, Y. and J.H. Crouch. (2008) Marker-assisted selection in plant breeding: from publication to practice. Crop Sci. 48: 391-407 • Gupta, P.K., Balyan, H.S., Sharma, P.C., Ramesh, B. 1996. Microsatellites in Plants: A New Class of Molecular Markers. Current Science, 70:45-54 • Dudley, J.W. 1993. Molecular Markers in Plant Improvement: Manipulation of Genes Affecting Quantitative Traits. Crop Science, 33(4): 660-668 Thank YouThank You