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Up the top of the gel:

- Large fragments


Down the bottom of the gel:

- Smaller fragments


What are we scoring?

- The presence or absence of bands
- We don't know if this is hetero- or homo- zygous


How do you map things with AFLPs?

- Maximum signal is obtained through maximum divergence
- Reduction of the noise, to ensure that A is homozygous and isogenic


F1 will be hybrids between A and B. This can result in achiasmatic and chiasmatic individuals:

- Achiasmatic: No crossing over in meiosis in females (in butterflies)
- Chiasmatic: Crossing over in meiosis can happen in males (in butterflies)


Identical segregation patterns indicate:

- A linkage group!
- A linkage disequilibrium barcode can define the gene controlling the phenotype to the linkage group
- We still don't know how far away they are from each other


How can you determine which are recombinants?

- Offspring with one band from the father (10kb) and one from the mother (either 100kb or 200kb)


Combining genetic and physical data via common markers:

- Identify an interval surrounding your gene, and flank with two markers. You don't know how big the interval is or how many genes are within this region
- We must connect the genetic map to the physical map. How?!


Genomic library:

- A genetic library covering the whole genome with markers along it


How do we close the gap between the twp=o markers?

- Identify overlapping genomic clones, by attempting to walk toward the other marker
- Find the overlapping sequences is a challenge
- Walking in the correct direction is another challenge



- The overlapping clones covering a chromosomal region


Physical map:

- A set of contigs that covers the entire genome


Making a Bacterial Artificial Chromosome (BAC) library:

- Isolate high molecular weight DNA
- Random shearing or partial restriction digest
- Select 100-150 kb fragments
- Clone into a vector
- Pick random colonies
- Develop a colony array
- And screen this array


Marker conversion applies to all kinds of DNA markers and uses this method:

- Cut an AFLP band from the gel
- Re-amplify using PCR
- Sequence the product and sequence the tagged site (STS)
- The PCR product can be used as a DNA probe to screen gDNA library, or the DNA sequence and its genomic location can be compared to other species


Overlapping clones must share something!

- In order for them to be informative


Chromosome walking to see if you are walking in the correct direction

- Develop new markers based on BAC sequence and see how far they are from the target locus, to make sure you are walking in the correct direction
- Sequence the ends and design new primers to amplify the parents of the mapping cross (there must be a polymorphism in order to design assays to genotype progeny to allow linkage analysis)


Chromosome walking, identifying the next clone:

- PCR amplify BAC end or an internal region, hybridise the amplicon to BAC library
- Identify overlapping BAC clones and repeat the process


Minimum tile path:

- A library of clones is generated, but many of them are redundant as they overlap so much.
- The minimum tile path is the minimum number of clones needed in order to cover the entire genome
- Each tile can be shot gun sequenced to create 1kb sequences
- Can convert the BAC clone to in situ hybridising probes, so you can combine the physical map to the genetic map