Marker Assisted Selection (MAS)

1. Marker Assisted Selection (MAS)

2. DNA sources • Genomic DNA from chromosomes: (Fragments because usually too large to clone directly) • cDNA (complementary DNA): derived by action of reverse transcriptase from (usually) mRNA template • chemically synthesized DNA molecules (oligonucleotides)

3. Types of markers • Morphological markers • Seed color e.g. Kernel color in maize • Function based e.g. Plant height associated with salt tolerance in rice

4. Limitations • Most phenotypic markers are undesirable in the final product (Yellow color in maize). • Dominance of the markers: homozygotes/ heterozygotes not distinguishable • Sometimes dependent on the environment for expression e.g. Height of plants

5. Molecular markers • Non-DNA such as isozyme markers: Restricted due limited number of enzyme systems available. • DNA based markers: Markers based on the differences in the DNA profiles of individuals.

6. Some molecular markers are pieces of DNA that have no know function or impact on plant performance (Linked Markers): • Detected via mapping. • Linked markers are near the gene of interest and are not part of the DNA of the gene. • Other markers may involve the gene of interest itself (Direct Markers): • Based on part of the gene of interest. • Hard to get but great once you have it.

7. What is MAS? • Concept of using molecular markers particularly DNA based to detect and track presence of gene transfer in breeding programs • MAS works on the principle of linkage dis-equilibrium where markers that are tightly linked to target genes segregate together in a non random manner (due linkage)

8. Advantages of MAS

9. Improvement of response to selection (Rs) • Assays require small amount of tissue, therefore no destructive sampling. • Use of codominant markers allows accurate identification of individuals for scoring without ambiguity • Multiple sampling for various QTLs is possible from same DNA prep • Can assay for traits before they are expressed, e.g. before flowering • Time saving.

11. CONVENTIONAL PLANT BREEDING P1 P2 x Donor Recipient F1 large populations consisting of thousands of plants F2 PHENOTYPIC SELECTION Phosphorus deficiency plot Salinity screening in phytotron Bacterial blight screening Glasshouse trials Field trials

12. MARKER-ASSISTED SELECTION (MAS) MARKER-ASSISTED BREEDING P1 x P2 Susceptible Resistant F1 large populations consisting of thousands of plants F2 Method whereby phenotypic selection is based on DNA markers

13. Overview of ‘marker genotyping’ (1) LEAF TISSUE SAMPLING (2) DNA EXTRACTION (3) PCR (4) GEL ELECTROPHORESIS (5) MARKER ANALYSIS

14. Requirements for a useful molecular marker • Molecular markers must be tightly linked to a target gene. The linkage must be really tight such that the presence of the marker will reliably predict the presence of the target gene. • The marker should be able to predict the presence of the target gene in most if not all genetic backgrounds.

15. Target gene MAS Marker 1 (more tightly linked than 2) 1 2

16. RELIABILITY FOR SELECTION Marker A Using marker A only: 1 – rA = ~95% QTL 5 cM Marker B Marker A Using markers A and B: 1 - 2 rArB = ~99.5% QTL 5 cM 5 cM Markers must be tightly-linked to target loci! • Ideally markers should be <5 cM from a gene or QTL • Using a pair of flanking markers can greatly improve reliability but increases time and cost

17. - Marker Plant A Plant B Plant C Monomorphic bands Polymorphic bands + Presens of a band, ”1” Absence of a band, ”0”

18. Markers must be polymorphic RM84 RM296 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 P1 P2 P1 P2 Not polymorphic Polymorphic!

19. DNA extractions Mortar and pestles Porcelain grinding plates LEAF SAMPLING Wheat seedling tissue sampling in Southern Queensland, Australia. DNA EXTRACTIONS

20. PCR-based DNA markers • Generated by using Polymerase Chain Reaction • Preferred markers due to technical simplicity and cost PCR Buffer + MgCl2 + dNTPS + Taq + Primers + DNA template PCR THERMAL CYCLING GEL ELECTROPHORESIS Agarose or Acrylamide gels

21. Agarose gel electrophoresis UV transilluminator UV light

22. A woman is uncertain which of two men is the father of her child. DNA typing is carried out on blood from the child (C), the mother (M), and each of the two males (A and B), using probes for a highly polymorphic DNA marker on two different chromosomes (“locus 1” and “locus 2”). The result is shown in the accompanying diagram. Can either male be excluded as the possible father? Explain your reasoning.

23. DNA replication in natural systems requires: • A source of the nucleotides adenine (A), cytosine (C), thymine (T), and guanine (G). • The DNA polymerase (DNA synthesis enzyme). • A short RNA molecule (primer). • A DNA strand to be copied. • Proper reaction conditions (pH, temperature).

24. Is there any differences between PCR and mechanisms of the natural replication system? • DNA primers are used instead of the RNA primer found in the natural system. • Magnesium ions that play a role in DNA replication are added to the reaction mixture. • A DNA polymerase enzyme that can withstand high temperatures, such as Taq, is used. • A reaction buffer is used to establish the correct conditions for the DNA polymerase to work.

25. Conditions under which MAS is valuable • Low heritability traits • Traits too expensive to score: Soybean Cyst Nematode (SCN) resistance. Young (1999) • Recessive genes: Pyramiding of dominant and recessive genes conferring resistance to important crop diseases which would otherwise be very difficult • Multiple genes (Quantitative traits): QTLs underlying phenotypic and physiological traits can be traced using markers. Although QTL mapping is tedious, markers once identified can be used fast and accurately to detect the QTLs of interest.

26. Quarantine:No need to grow plants to screen for viral diseases that can not be visually detected, and small tissues can be used for DNA typing.

27. Limitations of MAS • Cost of equipment, reagents and personnel. • Data collected in the field is assumed to be normally distributed, but usually is not. • Integration of the DNA information into existing systems is difficult. • Linkage drag. As the marker distance from the target gene increases, more of the donor DNA is retained in the desired background resulting in need for more backcrosses.

28. Types of DNA based Markers

29. Hybridization based markers • Restriction Fragment Length Polymorphisms (RFLPs) where differences in the number and size of fragments is analyzed • Polymerase Chain Reaction (PCR) based • Randomly Amplified Polymorphic DNAs (RAPDs), Single Sequence Repeats (SSRs). • Amplified Fragment Length Polymorphic DNA (AFLPs) • Other variants such as SCAR, CAPS, SSCP e.t.c. • Sequence based markers • Expressed Sequence Tags (ESTs), • Single Nucleotide Polymorphism (SNPs)

30. Restriction Fragment Length Polymorphisms (RFLPs) • The first type of DNA markers that were used for genetic mapping were RFLPs. • For instance a given restriction site may be present in one line and not in the other.

31. Procedure • For detecting RFLPs involves the fragmentation of genomic DNA by a Restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs. • The resulting DNA fragments are then separated by length in agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure. • Hybridizationof the membrane to a labeled DNA probe then determines the size of the fragments which are complementary to the probe.

32. An RFLP occurs when the size of a detected fragment varies between individuals. • Each fragment size is considered an allele, and can be used in genetic analysis.

33. RFLP markers have several advantages: • They are co-dominant and unaffected by the environment. • Any source DNA can be used for the analysis. • Many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations. • A main disadvantage is that: • RFLP mapping necessitates relatively large amounts of DNA.

34. Cleaved Amplified Polymorphic Sequences (CAPS) • The principle of CAPS markers is very similar to that of RFLP markers. • The main difference is that PCR is used instead of DNA blot hybridisation to detect a restriction site polymorphism. • A genomic DNA region is amplified by PCR using specific primers and those amplified fragments are then digested with a diagnostic restriction enzyme to reveal the polymorphism. • RFLP probes can be anonymous clones, CAPS markers require sequence information to design the specific PCR primers.

36. Advantages • CAPS markers are co-dominant. • Most CAPS genotypes are easily scored and interpreted. • CAPS markers require only small quantities of genomic DNA.

37. Random Amplified Polymorphic DNA (RAPD) • RAPD markers are another type of PCR-based markers that have been used for genetic mapping. • This approach is based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. • The oligonucleotide (around 10-bp long) is used for PCR at low annealing temperatures. • When the oligonucleotide hybridises to both DNA strands at sites within an appropriate distance from each other, the

38. DNA region delimited by these two sites will be amplified. • Small nucleotide changes (polymorphism) at one of the two sites may prevent hybridisation of the oligonucleotide and hence also prevent DNA amplification.

39. Typically a RAPD primer will amplify a given fragment from line A and not from line B. • It will thus be impossible to distinguish an homozygous individual AA from an heterozygous individual AB. • In other words, RAPDs are dominant markers and are thus less efficient than co-dominant markers in extracting information from a given F2 population. • Another limitation of RAPD markers is that because of the low annealing temperatures used, the amplification of a given polymorphic band seems to be highly sensitive to PCR conditions and hence less consistently reproducible in different laboratories.

40. Advantages: • Random distribution throughout the genome • The requirement for small amount of DNA (5-20 ng) • Easy and quick to assay • The efficiency to generate a large number of markers • Cost-effectiveness!

41. Limitations • Dominant nature (heterozygous individuals can not be separated from dominant homozygous) • Sensitivity to changes in reaction conditions, which affects the reproducibility of banding patterns • The results are not easily reproducible between laboratories

42. Amplified Fragment Length Polymorphism (AFLP) • AFLP TM is a patented technology developed by KeyGene, Wageningen, The Netherlands. • In this procedure, the genomic DNA is digested by two different restriction enzymes, a rare cutter and a frequent cutter. • Double-stranded adapters are then ligated to the ends of the restriction fragments. • The fragments are then amplified by PCR using primers that correspond to the adapter and restriction site sequences.

43. These primers have additional nucleotides at the 3' ends extending into the restriction fragments, in order to limit the number of fragments that will be amplified. • The AFLP products are detected by labelling one of the two primers, and the labelled DNA fragments are separated by electrophoresis in denaturing polyacrylamide gels (similar to sequencing gels). • Typically, 50 to 100 amplification products are detected in a single lane. Polymorphic bands can be identified by comparing the amplification products derived from two lines. • Like RAPDs, AFLPs are typically dominant markers.

44. The procedure of AFLP technique is divided into three steps: • Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments. • Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences. • Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

45. Simple Sequence Repeats (SSR): Microsatellites • Regions of genome where a short (1-4 base) motif is repeated many times (can be repeated 10 to 100 times) • These microsatellite repeat sequences are usually polymorphic in different lines because of variations in the number of repeat units. • These polymorphisms are called SSR, and can be conveniently used as co-dominant genetic markers. • As compared to CAPS markers, SSR offer the additional advantage that they do not involve the use of restriction endonucleases and thus avoid the problems associated with partial digestions.

46. One common example of a microsatellite is a (CA) repeat. • CA nucleotide repeats are very frequent in human and other genomes, and present every few thousand base pairs. • Microsatellites developed for particular species can often be applied to closely related species, but the percentage of loci that successfully amplify may decrease with increasing genetic distance.

47. Single Nucleotide Polymorphisms (SNPs) Definition of SNP = Single Nucleotide Polymorphism: a single base difference in DNA sequence among individuals. • (SNP, pronounced snip), is a DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual).

48. DNA strand 1 differs from DNA strand 2 at a single base-pair location (a C/T polymorphism). In this case we say that there are two alleles : C and T.

49. Factors to consider when choosing markers • Abundance • Dependent on frequency at which the marker sites occur through out the genome. • Level of polymorphism • Determined by rate of mutations in a loci e.g. charges in a protein, number of repeats in a core sequence in microsatellite, substitutions, deletions, insertions etc.

50. Locus specificity • Homology Vs Non homology of bands need to be considered. • Codominance/dominance • Allows differentiation of homozygotes and heterozygotes and therefore determination of genotypes and allele frequencies at a loci