slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Marker Assisted Selection (MAS) PowerPoint Presentation
Download Presentation
Marker Assisted Selection (MAS)

Loading in 2 Seconds...

play fullscreen
1 / 53

Marker Assisted Selection (MAS) - PowerPoint PPT Presentation


  • 556 Views
  • Uploaded on

Marker Assisted Selection (MAS). 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).

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Marker Assisted Selection (MAS)' - lora


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide2

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)
types of markers
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
slide4

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
slide5
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.
slide6
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.
what is mas
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)
slide9

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.
slide11

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

slide12

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

slide13

Overview of ‘marker genotyping’

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

slide14

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.
slide15

Target gene

MAS

Marker 1 (more tightly linked than 2)

1

2

markers must be tightly linked to target loci

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
slide17

-

Marker

Plant A

Plant B

Plant C

Monomorphic bands

Polymorphic bands

+

Presens of a band, ”1”

Absence of a band, ”0”

markers must be polymorphic
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!

dna extractions
DNA extractions

Mortar and pestles

Porcelain grinding plates

LEAF SAMPLING

Wheat seedling tissue sampling in Southern Queensland, Australia.

DNA EXTRACTIONS

pcr based dna markers
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

agarose gel electrophoresis
Agarose gel electrophoresis

UV transilluminator

UV light

slide22

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.

slide23

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).
slide24

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.
slide25

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.
slide26

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.

slide27

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.
slide29

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)
slide30

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.
slide31

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.
slide32

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.
slide33

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.
slide34

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.
slide36

Advantages

  • CAPS markers are co-dominant.
  • Most CAPS genotypes are easily scored and interpreted.
  • CAPS markers require only small quantities of genomic DNA.
slide37

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
slide38

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.
slide39

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.
slide40

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!
slide41

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
slide42

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.
slide43

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.
slide44

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.
slide45

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.
slide46

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.
slide47

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).
slide48

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.

factors to consider when choosing markers
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.
slide50
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
slide51
Reproducibility
    • Repeatability of findings over time and space.
  • Labor intensity and safety
    • RFLPs Vs SSRs.
  • Technical demand
    • Skills required, equipment needed

Hybridization >PCR-based

slide52
Operational costs
    • Chemicals, supplies, visualization techniques e.g. labeling, staining methods etc.
  • Development costs
    • Construction of genomic libraries and development of site specific PCR-primers for SSRs and Probes for RFLPs, Sequencing is expensive
slide53
Quantity of DNA required for analysis
    • RFLPs = 5-10µg, PCR =5-100ng per reaction
    • Important when only small tissues are available.
  • Amenability to automation
    • Increase in sample throughput
    • Manually, 100 PCR reactions > 2hrs to set up; Robot <20 min