RADIATION HYBRID MAP OF THE ZEBRAFISH GENOME

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RADIATION HYBRID MAP OF THE ZEBRAFISH GENOME
Tavleen Sandhu Breanne Atha Winter 2009
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Radiation Hybrid Mapping Introduction

• Old but an useful technique.
• Somatic cell genetic technique used to construct
  long range maps of mammalian chromosomes.
• Based on fusion of lethally irradiated cells of a
  species with a rodent cell line.
• Eg Zebrafish cells fused with hamster cells.
• Pieces of Zebrafish DNA either integrate into the
  hamsters chromosome or reside as a plasmid in the
  hamster cell.

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• of such radiation hybrids together form a
  panel. Eg Goodfellow T51.
• Markers like STSs, ESTs, SSLPs and cloned
  candidate genes are mapped on these RH panels.
• Which two markers mapped together?
• Others mapped by linkage to ones already mapped.
• Species mapped
• Before Human, dog, mouse and rat.
• After cow, pig, horse, baboon etc.

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DEFINITIONS
Sequence tagged sites (STSs) short (200-500 bp)
DNA sequence that occurs only once in the genome,
location and base sequence known. Expressed
sequence tags (ESTs) short sub-sequence of a
transcribed cDNA sequence. Identifies the gene
transcripts. Simple sequence length polymorphism
(SSLPs) short sequences of DNA repeated multiple
times in tandem (polymorphic).
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Advantages of RH Mapping over Genetic Mapping

• Markers need not be polymorphic. Thus increased
  of loci potentially mapped.
• Better resolution-especially in centromeric
  regions, suppression of crossing over near
  centromeres
• Distances on a RH map are determined by the
  frequency of radiation induced breaks between the
  markers. Breaks are distributed randomly,
  therefore the calc. distance along a RH map are
  proportional to physical distances.
• DNA kept in the cell lines eliminating the cost
  of animal care and breeding.

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ZEBRAFISH

• The National Institutes of Health (NIH) has been
  a leading advocate of the Zebrafish as a model
  organism for the study of vertebrate development,
  physiology, and disease.
• ZEBRAFISH as a model organism
• overall generation time of Zebrafish is
  comparable to mice
• Zebrafish embryos develop rapidly. (eggs to
  larvae in under three days).
• The embryos are large, robust, transparent and
  develop externally to the mother, facilitating
  experimental manipulation and observation.
• fluorescent markers.

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What did they do?

• Selected Goodfellow T51 RH Panel
• 94 radiation hybrids
• Tested 2125 zebrafish markers for PCR performance
• Rejected 467-no PCR product or zebrafish/hamster
  bands not distinguishable
• Used remaining markers 145 untested markers for
  PCR with radiation hybrids
• Zebrafish hamster controls

• Select RH Panel Markers
• PCR Gel Electrophoresis
• Scoring
• Map Construction

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What did they do?

• Designed primers for cloned genes and ESTs
• Ran PCR on DNA samples from 94 radiation hybrid
  lines and zebrafish and hamster controls
• Carried out gel electrophoresis on each DNA
  sample
• Ran each marker in duplicate

• Select RH Panel Markers
• PCR Gel Electrophoresis
• Scoring
• Map Construction

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What did they do?

• Scored each marker as present or not on each gel
• Discrepancy Re-score by running third plate,
  whichever occurred twice
• 352 markers failed to give bands that could be
  scored

• Select RH Panel Markers
• PCR Gel Electrophoresis
• Scoring
• Map Construction

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What did they do?

• Used 1451 markers for map construction
• 2125 original markers-467 rejected for PCR145
  untested-352 failed to give bands1451
• Include
• 1029 SSLPs-previously generated, partially
  mapped
• 551 used to anchor RH map to genetic map
• 229 cloned genes-represent most zebrafish
  published to date (1999)
• 193 ESTs-not selected by similarity to known
  genes
• SAMapper used to anaylze data and construct map

• Select RH Panel Markers
• PCR Gel Electrophoresis
• Scoring
• Map Construction

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• Figure 1
• RH map on the right. cR (polymorphic and
  non-polymorphic markers)
• Genetic map on the left. cM (polymorphic markers
  only)
• In humans, the cM is approximately equal to one
  million bp.
• 1 cR3000 in Zebrafish is approx 61,000 bp.

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Data Analysis

• 1275 markers (87.9 of those successfully scored)
  were linked to at least one other marker at a LOD
  score of 6
• 207 cloned genes and ESTs-not previously mapped
• Markers per Chromosome 29-81
• 243 high confidence 10001 bins
• Groups of markers ordered so that the odds are
  10001 against them being incorrectly ordered
• 190 Linkage Groups 7.6/chrm
• Separated by gaps with 2-point LOD scores lt 6
• Map Coverage 81.9-based on fraction of scored
  ESTs

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How Does It Compare?

• 27 genetically mapped genes ESTs have no
  linkage on RH map
• Of 241 SSLPs on both genetic and RH map
  (non-anchoring), 14 are not in the same order
• Only 5 (2.1) violate 10001 bin boundaries
• Mostly inversions between neighboring markers
• 40 (8.1) of anchor SSLPs are in a different
  order
• 2 (0.4) of which violate bin boundaries
• 3 genes (2.0) were assigned to different
  chromosomes than on the genetic map
• Small error due to statistical error of either
  mapping method

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Map Length Potential Resolution

• Eliminate markers that expand distance between
  neighboring markers by gt20cR
• Cuts on either side of marker/Experimental Error
• Map Length 27,729 cR
• Zebrafish genome size 1.7 Gb
• Potential Resolution Calculation
• 6.1 Mb/17.3350kb
• (Avg Fragment Size/ of RH lines each marker is
  represented by, on average)
• Retention Frequency radiation hybrids
  retaining a given marker/total of radiation
  hybrids test w/ the marker
• Higher retention frequencyhigher potential
  resolution

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Figure 2Marker Retention Frequency

• Avg. marker retention frequency 18.4
• LG14 highest peak with freq. 38.8 indicating
  the position of HPRT marker. (retained in every
  hybrid)
• For other chromosomes this freq. ranges between
  13.0(LG7) to 21.5(LG5).

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Conclusion

• Goodfellow T51 RH Panel-good for mapping
  zebrafish
• Avg retention Between Human G3 GeneBridge 4
  panels
• Potential Resolution Close to G3 despite lower
  irradiation (3,000 vs 10,000 rad)
• No prior knowledge of chromosomal localization of
  markers unlike human effort
• Eliminate potential false linkages
• Able to rapidly position 82 of ESTs
• Fraction will increase as more markers add to map
• 1st RH map of non-mammalian genome

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Further Reading

• Advances in Genetics by Jeffrey Hall-Chapter 3
• http//books.google.com/books?idXMqiLdem95YCprin
  tsecfrontcoverPPP1,M1
• Good technical explanations and definitions
• Facilitating Genome Navigation Survey Sequencing
  and Dense Radiation-Hybrid Gene Mapping,
  Christophe Hitte, Et. Al. Nature Reviews Genetics
  2005
• A more recent review of the use of RH mapping
  along with sequencing