Genetic marker (1)

Biological markers

1. Genetic Markers

2. Variation The differences that distinguish one individual from another are encoded in the individual’s genetic material, the deoxyribonucleic acid (DNA). DNA is packaged in chromosome pairs, one coming from each parent. The genes , which control a plant’s characteristics, are located on specific segments of each chromosome.

3. Marker

4. Genetic markers Genetic markers are the biological features that are determined by allelic forms of genes or genetic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene.

5. Genetic Markers represent genetic differences between individual organisms or species do not represent the target genes themselves but act as ‘ signs’ or ‘ flags located in close proximity to genes (i.e. tightly linked) may be referred to as gene ‘ tags do not affect the phenotype of the trait of interest because they are located only near or ‘linked’ to genes controlling the trait occupy specific genomic positions within chromosomes (like genes) called ‘loci ’ ( singular ‘locus’)

6. Classification of Genetic Markers C lassical markers Morphological markers , Cytological markers Biochemical markers DNA markers RFLP, AFLP, RAPD, SSR, SNP

7. Morphological markers: visible traits, such as leaf shape, flower color, pod color, seed color, seed shape, awn type and length, fruit shape, stem length Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding

8. Example of morphological markers Mendelian characters In wheat breeding, the dwarfism governed by gene Rht10 was introgressed into Taigu nuclear male-sterile wheat by backcrossing and a tight linkage was generated between Rht10 and the male-sterility gene Ta1 Then the dwarfism was used as the marker for identification and selection of the male-sterile plants in breeding populations

9. Drawbacks of morphological markers Limited in numbers many of these markers are not associated with important economic traits (e.g. yield and quality ) influenced by environmental factors or the developmental stage of the plant However, despite these limitations, morphological markers have been extremely useful to plant breeders

10. Cytological markers: In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin

11. Biochemical/protein markers: Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution

12. Drawbacks of biochemical markers Limited in numbers influenced by environmental factors or the developmental stage of the plant However, despite these limitations, biochemical markers have been extremely useful to plant breeders

13. Molecular/DNA markers DNA markers are defined as a fragment of DNA revealing mutations/variations, which can be used to detect polymorphism between different genotypes or alleles of a gene for a particular sequence of DNA in a population or gene pool. Such fragments are associated with a certain location within the genome and may be detected by means of certain molecular technology . DNA marker is a small region of DNA sequence showing polymorphism (base deletion, insertion and substitution) between different individuals

14. Defined • RFLPs are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites. RFLP assays are performed by hybridizing a chemically labelled DNA probe to a Southern blot of DNA digested with a restriction endonuclease. Restriction Fragment Length Polymorphisms (RFLPs)

15. RFLPs Restriction fragment length polymorphism Co-dominant Requires: single copy DNA probe Restriction enzyme Southern blotting DNA polymorphism

16. RAPDs Randomly amplified polymorphic DNA Based on a 10 bp single arbitrary primer Cheap, easy Insufficient reproducible maize lines; only primer 2 and 5 demonstrate polymorphism

17. 3/14/2017 RAPD – dominant marker A B

18. 3/14/2017 AFLP: Major Steps • Restriction endonuclease digestion of genomic DNA and ligation of specific adapters • Amplification of the restriction fragments by PCR using primer pairs containing common sequences of the adapter and two or three arbitrary Nucleotides • Analysis of the amplified fragments using gel electrophoresis

19. 3/14/2017 AFLPs : amplified fragment length Polymorphisms A combination of PCR and RFLP Informative fingerprints of amplified fragments

20. 3/14/2017 NIBGE Ph.D lecture AFLP-Major Steps Amplified Fragment Length Polymorphism Genomic DNA double digests with a 4-cutter ( Mse I) and a 6-cutter ( Eco R1) Ligate adapters to the Eco R1 and Mse I RE sites Primers complementary to Adapters with selective nucleotides at 3’ ends and perform PCR amplification Separate DNA fragments on high-resolution gels After detection, screen for band polymorphisms

21. 3/14/2017 AFLP: Restriction and Ligation to Adapters

22. 3/14/2017 AFLP: Pre-Selective Amplification Primer (+ 1) for pre-selective amplification

23. 3/14/2017 AFLP: Selective Amplification Primer (+ 3) for selective amplification

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25. 3/14/2017 AFLP – band polymorphisms

26. Simple Sequence Repeats (SSRs) Defined • Simple sequence repeats (SSRs) or microsatellites are tandemly repeated mono-,di-, tri-, tetra-, penta-, and hexa-nucleotide motifs. SSR length polymorphisms are caused by differences in the number of repeats. • SSR loci are “individually amplified by PCR using pairs of oligonucleotide primers specific to unique DNA sequences flanking the SSR sequence”.

27. Why Have SSRs Had Such a Large Impact on Genomics? • SSRs tend to be highly polymorphic. • SSRs are highly abundant and randomly dispersed throughout most genomes. • Most SSR markers are co-dominant and locus specific. • Genotyping throughput is high and can be automated.

28. SSR / microsatellite Isolation of DNA fragments (vectors) containing a simple sequence repeat (microsatellite), e.g. [AT] n [GC] n , [CGA]n, [GATA]n Sequencing regions flanking the SSR Designing primers for border sequences Testing in population for duplications and SSR polymorphism

29. SSR - methodolgy Genotype A Genotype A Genotype B PCR amplification with radiolabelled nucleoltide Polyacrylamide Gel Electrophoresis of PCR products and autoradiography [AT] 18 [AT] 22 [AT] 18 [AT] 22 Genotype B

30. SSRs display multiple alleles

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35. Distribution of SNP SINGLE NUCLEOTIDE POLYMORPHISM

36. Distribution of SNP SNP

37. HUMAN SNP DISTRIBUTION Most common changes Transitions: Purines to Purines Pyrimines to Pyrimidines Transversion: Purines to Pyrimidines Pyrimidines to Purinces Single-base insertions & deletion ( indel ) Distribution of SNP

38. HOW SNP ARE INDICATED Distribution of SNP

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51. Criteria for ideal DNA markers selectively neutral because they are usually located in non-coding regions of DNA High level of polymorphism unlimited in number and are not affected by environmental factors and/or the developmental stage of the plant Even distribution across the whole genome (not clustered in certain regions) Co-dominance in expression (so that heterozygotes can be distinguished from homozygotes) Clear distinct allelic features (so that the different alleles can be easily identified) Single copy and no pleiotropic effect Low cost to use (or cost-efficient marker development and genotyping) Easy assay/detection and automation High availability (un-restricted use) and suitability to be duplicated/multiplexed (so that the data can be accumulated and shared between laboratories) No detrimental effect on phenotype However, no molecular markers fulfill all these characteristics. Researchers choose the molecular marker according to their need and availability