1.
RAPD Markers <ul><li>There are other problems with RAPD markers associated with reliability </li></ul><ul><li>Because small changes in any variable can change the result, they are unstable as markers </li></ul><ul><li>RAPD markers need to be converted to stable PCR markers. </li></ul><ul><li>How? </li></ul>

2.
RAPD Markers <ul><li>The polymorphic RAPD marker band is isolated from the gel </li></ul><ul><li>It is used a template and re-PCRed </li></ul><ul><li>The new PCR product is cloned and sequenced </li></ul><ul><li>Once the sequence is determined, new longer and specific primers can be designed </li></ul>

3.
SCAR Markers <ul><li>The new primers are then used in PCR reactions </li></ul><ul><li>Because they are longer and specific, the annealing temperature can be increased </li></ul><ul><li>This increases reliability and transferability </li></ul><ul><li>New markers are Sequenced Characterized Amplified Region Markers </li></ul>

4.
AFLP Markers <ul><li>Most complex of marker technologies </li></ul><ul><li>Involves cleavage of DNA with two different enzymes </li></ul><ul><li>Involves ligation of specific linker pairs to the digested DNA </li></ul><ul><li>Subsets of the DNA are then amplified by PCR </li></ul>

5.
AFLP Markers <ul><li>The PCR products are then separated on acrylamide gel </li></ul><ul><li>128 linker combinations are readily available </li></ul><ul><li>Therefore 128 subsets can be amplified </li></ul><ul><li>Patented technology </li></ul>

8.
AFLP Markers <ul><li>Technically demanding </li></ul><ul><li>Reliable and stable </li></ul><ul><li>Moderate cost </li></ul><ul><li>Need to use different kits adapted to the size of the genome being analyzed. </li></ul><ul><li>Like RAPD markers need to be converted to quick and easy PCR based marker </li></ul>

9.
Marker Assisted Selection (MAS) <ul><li>Breeding for specific traits in plants and animals is expensive and time consuming </li></ul><ul><li>The progeny often need to reach maturity before a determination of the success of the cross can be made </li></ul><ul><li>The greater the complexity of the trait, the more time and effort needed to achieve a desirable result. </li></ul>

10.
MAS <ul><li>The goal to MAS is to reduce the time needed to determine if the progeny have trait </li></ul><ul><li>The second goal is to reduce costs associated with screening for traits </li></ul><ul><li>If you can detect the distinguishing trait at the DNA level you can identify positive selection very early. </li></ul>

11.
Developing a Marker <ul><li>Best marker is DNA sequence responsible for phenotype i.e. gene </li></ul><ul><li>If you know the gene responsible and has been isolated, compare sequence of wildtype and mutant DNA </li></ul><ul><li>Develop specific primers to gene that will distinguish the two forms </li></ul>

12.
Developing a Marker <ul><li>If gene is unknown, screen contrasting populations </li></ul><ul><li>Use populations rather than individuals </li></ul><ul><li>Need to “blend” genetic differences between individual other than trait of interest </li></ul>

13.
Developing Markers <ul><li>Cross individual differing in trait you wish to develop a marker </li></ul><ul><li>Collect progeny and self or polycross the progeny </li></ul><ul><li>Collect and select the F2 generation for the trait you are interested in </li></ul><ul><li>Select 5 - 10 individuals in the F2 showing each trait </li></ul>

14.
Developing Markers <ul><li>Extract DNA from selected F2s </li></ul><ul><li>Pool equal amounts of DNA from each individual into two samples - one for each trait </li></ul><ul><li>Screen pooled or “bulked” DNA with what method of marker method you wish to use </li></ul><ul><li>Method is called “Bulked Segregant Analysis” </li></ul>

15.
Marker Development <ul><li>Other methods to develop population for markers exist but are more expensive and slower to develop </li></ul><ul><li>Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent </li></ul><ul><li>What is the advantage to markers in breeding? </li></ul>

16.
Reducing Costs via MAS <ul><li>Example disease resistance </li></ul><ul><ul><li>10000 plants </li></ul></ul><ul><li>Greenhouse space or field plots </li></ul><ul><ul><li>$5000 - $10000 </li></ul></ul><ul><li>Time 4 months (salary) </li></ul><ul><ul><li>$10 - $15000 </li></ul></ul><ul><ul><li>total cost = $15 - $25,000 </li></ul></ul>

17.
Reducing Costs via MAS <ul><li>PCR-based testing @ $5 sample </li></ul><ul><li>$50,000 - costs more? </li></ul><ul><li>Analysis of trait not easily phenotyped </li></ul><ul><li>eg: Cadmium in Durum wheat </li></ul><ul><li>10000 plants need to reach maturity </li></ul><ul><li>Cadmium accumulates in seed </li></ul>

18.
Reducing costs via MAS <ul><li>$15 - 25 growing costs + analysis </li></ul><ul><li>Atomic Absorption @ $15 per sample </li></ul><ul><li>$150,000 + growing costs </li></ul><ul><li>PCR analysis still $50000 </li></ul><ul><li>Savings in time and money increase as more traits are analyzed </li></ul><ul><li>Many biochemical tests cost >$50 sample </li></ul>

19.
Marker Assisted Breeding <ul><li>MAS allows for gene pyramiding - incorporation of multiple genes for a trait </li></ul><ul><li>Prevents development of biological resistance to a gene </li></ul><ul><li>Reduces space requirements - dispose of unwanted plants and animal early </li></ul>