1. Radiation hybrid map of the zebrafish genome
By: Garvey Xu and Ariela Khandadash

2. THE BIG PICTURE? Mapping Zebrafish Genome
Zebrafish: 1st vertebrate organism mapped genome
Mutation; valuable for the study of vertebrate development and diseases
RH mapping of the zebra fish genome is done In order to map candidate genes and increase marker density achievable/usable for positional cloning
What makes me, MEEE?
Say :
Candidate genes:
A gene that is suspected of being responsible for a particular trait or illness.
Generated hybrids extracted DNA of zebrafish
designed primers: used 1451 markers for contructing RH map , 1029 SSLP markers that were previouslky genetically mapped
Used 551 SSLP markers to anchor
“PCR products” samples form amplification were analyzed by gel electrophoresis
based on linkage info given by gel electrophoresis generated RH map using SAMapper to construct RH map
design PCR for these markers
use PCR and electrophories (if the marker is present is the pcr coming up with anything to confirm specific size the pcr that you ran than that marker is positive for that sample
if band fate then increased concetration of starting DNA then reran it to see if it would get a solid band
generated hybrids extrated DNA , design pcr primers based on the markers ( SSts & ESTS ) for those
each marker had to have a primer
based on marker (ssts , est ) already new the mrna of the zebra fish
onrun all
based on linkage on gunning on gel
put all that information to generate the RH map for them using the computer program

3. Background Information
Known:
Zebrafish-
Model organism
Previously found markers used as “anchors”
Genetics map already exists
RH based genome wide maps for mammals
e.g rat, mouse, dog and human
Advantages of RH
High Resolution
Don’t require polymorphic markers
Used existing RH panel and STS markers in order to generate anchor their RH map to their genetic map
wanted to make it easier to map genes in the future and to increase number of mapped markers

4. Unknown:
Many marker locations to be discovered
RH Efficiency-
RH previously only done on mammals
No prior chromosomal localization markers for Zebrafish
Require high LOD to avoid false linkages

5. Radiation Hybrid Mapping: Physical Mapping
RH: using X-ray breakage chromosomes to determine the distance between DNA markers as well as their order on the chromosome
fusion of irradiated zebrafish AB9 cells with Hamstercells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the
Radiated cells die (
cells that have fusion
in this case They use molecular analysis : PCR
Picture from youtube.com
My notes to read:
The method consists of fusing irradiated cultured cells of one species with cultured cells of a different species. A panel of hybrid cells is then tested for the occurrence of pairs of markers. The closer two markers are to each other, the more likely that both are present in an individual hybrid cell.
picture:

6. Radiation Hybridization Mapping: Technique
Irradiated Zebrafish cells (AB9) are fused with Hamster cells (Wg3H)
Some Zebrafish DNA hybridizes to Hamster’s
Some DNA fragments are lost
Observed which markers were retained
PCR primers amplified markers signals
Gel electrophoresis showed if markers were present
If markers show up together often = linked
Frequency of separation proportional to distance
RH Achieved:
•By Irradiating cells from a donor species causing random chromosomal breaks Zebra( AB9)
•Fuse these to a cell line from a different species Hamster(Wg3H)
Rodent cells are able to stably incorporate genetic material from fused cells
•Retained Donor cell chromosome fragments to reword: Donor-cell chromosome fragments are retained to different extents in the ensuing hybrid cells. •
•Typing a panel of RHs with PCR-based sequence-tagged sites creates an RH map in which the frequency of breakpoints between two markers is proportional to the distance between them.

7. PCR: Polymerase Chain Reaction
Reproduce/Amplify DNA
Essential 4 Ingredients:
Buffer
DNA template( DNA sample of 94 zebrafish hybrid lines)
DNA polymerase enzyme
Primers (custom made designed primes for cloned genes & ESTs using Primer3 Program)
PCR: Reproduce/Amplify DNA or RNA
Multiple PCR cycles produce many copies of DNA
1 PCR cycle (1 DNA copy)= 2 DNA copies
Essential 4 Ingredients:
Buffer
DNA template( DNA sample of 94 zebrafish hybrid lines)
DNA polymerase enzyme
Primers (custom made designed primes for cloned genes & ESTs using Primer3 Program)
PCR steps:
1: denaturing DNA: separation of the two strands of DNA (double helix)
Heat to 95° C breaks H-bonds that holds DNA strands
Add polymerase
2: Primer Annealing: cooled from 45-75° C allows primers to bind to complementary sequence in the template DNA.
3: Extension: Heat to 72° C optimal temperature for DNA polymerase to act
Say: Lab procedure: PCR on 12 plates , initial denaturing for 2 mins 35 cycles of denaturing
Annealing at 60° C
Extension at 73° C
pic:

8. Gel Electrophoresis and Scoring
Ran each marker in duplicate
Even very light signals were deemed positive
Ran a third plate if necessary
SAMapper used to construct map

9. Figure 1: Comparison of RH map of zebrafish chromosome LG1 & Genetic Map
Lod Score= probability markers are linked
Markers mapped at Lod score 6 +
Bigger # = more likely linked
Smaller #= less likely linkage
Gaps=two point lod scores of less than 6
207 novel mapped genes using their RH technique
Figure 1: LG1 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish.
For each linkage group (LG), Genetic map on the left and RH map on the right.
Only markers selected for anchoring the radiation hybrid map are indicated. The radiation hybrid map on the right shows all markers linked to at least one other marker with a lod score of 6 or more.
Distances on the genetic map are shown in cM,
distances on the radiation hybrid map in cR.
Labels in lowercase are marker name
Gaps indicate two-point lod scores of less than 6. ( no linkage)
lod score = 6 + higher it is more linked they are
Say:
CentiMorgans=recombination Frequency
Centi-Rays
Lod Score= probability markers are linked
Figure1:Comparison of RH map of Zebrafish Chromosome LG1 & Genetic Map
Bigger # = more closely linked
smaller #= less linkage
207 novel mapped genes using their RH technique
Cell hybrids are one of the most fastest method for assigning genes to chromosomes because mapping with cell hybrids doesn’t require gene polymorphism.
Polymorphism: gene is said to be polymorphic if more than one allele occupies that gene’s locus within a population.
Cell hybrids fastest method, allows for assigning genes to chromosomes
SST and EST markers that have been maps identified genes use to anchor genetic map ( uses those marks ) to the RH
used 551 of these anchor our radiation map to our genetic map this was done for LG1 and on fig 2 they show that for lg1-25
207 novel mapped genes est using their RH in fig 1

10. Fragments that are mapped & connected is indicated by the line going across
SSLP markers are what anchor RH to Genetic markers
Same sequence anchoring RH map and LM

11. Figure 2: Marker Retention Percentages
Avg. Marker retention frequency: 18.4%
Selective HPRT marker causes peak in LG14 (38.8%)
Other chromosomes range from 13% (LG7) to 21.5%(LG5)
Width: approx length of chromosome
No noticeable trends
Figure 2:
marker retention
based on distance btw the markers
The Panels average marker retention frequency 18.4%
Chromosomes (LG14) shows a peak in marker retention
average retention frequency of 38.8%.
Peak signifies the position of the HPRT marker ; used for building the panel and must be present in ever hybrid.
The average retention frequencies of the remaining chromosomes vary between 13.0% (LG7) and 21.5% (LG5). Didn’t reword
No clear trend is observable in the relative retention of centromeric and telomeric regions within a chromosome. Didn’t reword

12. Table 1: Zebrafish RH panel compared to others
High resolution despite low radiation dose reason be due to zebrafish cells more sensitive to irradiation
compare g3 map:
Combine panels can bridge gaps
Use T51 panel can bridge gaps
High resolution, low radiation dose→ due to zebrafish sensitivity
Compare with G3 map: not possible to reach complete coverage
T51 combined with lower characterized resolution panel remaining gaps can be bridged

13. Data Summary
1275 Markers linked to at least one other with a LOD of at least 6
207 of these have not been previously mapped
243 high-confidence 1000:1 bins
group of markers are very likely to be in this order
190 linkage groups (7.6 per chromosome)
82% of ESTs could be mapped
Potential Resolution is 350 kb (avg fragment size/ avg # hybrid lines markers show up in)

14. Issues with Zebrafish RH
40 (8.1%) anchor SSLPs differ from genetic map
27 genes and ESTs did not have linkage
3 genes assigned to different chromosomes than on the genetic map
2.1% of non-anchor and 0.4% anchor markers violate bin boundaries
What could be Improved?
Gaps in the RH map due to low LOD scores
Close Gaps by combining data with another panel
testing more markers would not solve this
combining T51 panel with another panel could close gaps

15. Concluding Slide Data: Discovered 207 ESTs (expressed sequence tags)
Conclude overall: Zebrafish Goodfellow T51 radiation hybrid panel useful tool for mapping Zebrafish genes
RH: Match zebrafish mutation with Candidate genes
Analyze conserved synteny between zebrafish and humans
Positional cloning of mapped mutations
Synteny= 2 or more genes are present on the same chromosome
Concluding slide

16. Further Reading
Gautier, M. and Eggen, A The construction and use of radiation hybrid maps in genomic research. Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics. 2:2.2:14.
General Information about the use of RH in research
Hitte, Christophe, Et. Al Facilitating Genome Navigation: Survey Sequencing and Dense Radiation-Hybrid Gene Mapping, Nature Reviews Genetics
review article on RH mapping