The Zebrafish Genome Map

An improved Zebrafish meiotic map

There are two overwhelming reasons to significantly improve the zebrafish meiotic map:

  • to confirm and genetically anchor the genome assembly
  • provide rapid entry to the genome sequence for Zebrafish researchers mapping mutations of interest.

Abstract

The Zebrafish genome sequence generated by the Wellcome Trust Sanger Institute has allowed the large scale discovery of a new type of genetic marker: single nucleotide polymorphisms (SNPs), with ~700,000 SNPs predicted so far. SNP technology is far advanced allowing the genotyping of thousands of SNPs and samples for the lowest per genotype costs which are still falling. The high fecundity of the Zebrafish allows the collection of huge numbers of progeny from a given cross and therefore very high resolution meiotic mapping. We believe meiotic markers are superior to Radiation Hybrid (RH) markers: because they represent the order of markers in viable fish rather possible aneuploid cells used to make the RH panel, and because they are the genetic markers researchers will use to enter the genome sequence when mapping mutations.

SSLP mapping methods

Figure 1.

Figure 1.

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Although the zebrafish is an established genetic model organism with a near complete genome sequence, the mapping of forward genetic mutations can still be slow and painful. While there is a dense map of thousands of polymorphic markers, these are predominantly SSLPs (Simple Sequence Length Polymorphism). SSLPs require researchers to carry out many PCRs and careful score them for size polymorphisms on ethidium bromide stained agarose gels or even with radioactivity on long polyacrylamide gels. Therefore SSLPs do not lend themselves to a highly parallel approach, and furthermore have a low polymorphism rate in many crosses (~30% for agarose scoring).

Typical pipeline to map one mutation:

  • PCR and score SSLP agarose scan set (400 markers) on pools of mutant and sibling DNAs,
  • From those only around 100 will be polymorphic (on average 4/chromosome) from this data we will be able to establish linkage and chromosome arm/section.
  • Identify new SSLPs on meiotic maps.
    • order primers,
    • test new markers on YOUR cross to find new polymorphisms.
  • Repeat PCRs from individuals with the new markers and score across 100s to 1000s of mutants to establish minimal genetic and therefore physical distance.
  • Repeat the process as many times as you require to refine the region.

SNPs

SNPs are the densest polymorphism in most genomes and already ~700,000 candidate SNPs have been mined from the Wellcome Trust Sanger Institutes zebrafish genome sequence with documented polymorphism rates of 65-86% in predicted polymorphic crosses (Guryev et al., 2006, Bradley et al., 2007). This is a marker density of ~1SNP/20kb, and yet is unlikely to be complete since Stickney et al (2002) found 1 SNP/145bp between C32 and SJD strains alone (extrapolated over the whole genome this is over 9 million SNPs). Furthermore another teleost, Medaka, is predicted to have over 16 million SNPs between two common strains in a genome half the size of the Zebrafish genome (Kasahara et al 2007). SNP genotyping technologies are massively parallel and commercially available with upto 1 million SNPs assayed on one chip. With so many markers available and at high density the next things is to map them, the full MGH reference cross contains DNA from 520 F2 progeny between AB and India strains, this is sex averaged cross covering 1040 meiosis providing a resolution of ~0.1cM which corresponds to ~60kb (Knapik et al., 1996). This combination of large numbers of available markers, a robust and high throughput genotyping technology and a high resolution reference cross could provide us with a meiotic map at better than BAC clone resolution.

The 3k panel

Figure 2.

Figure 2.

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We have constructed a pilot panel of 3,212 SNPs selected to be polymorphic between Tuebingen and Tupfel long fin strains and their common crosses. The SNPs were also selected to be high confidence and well spread over the genome. The panel is based on Affymetrix MIPs technology, and should facilitate rapid genetic mapping of zebrafish mutations.

Figure 3.

Figure 3.

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Using this panel we have significantly improved the marker density of the two most characterised zebrafish mapping panels (Heat Shock and MGH) and characterised the genotypes and phylogenetic relationships of nine common zebrafish strains.So far we have nearly doubled the number of confirmed zebrafish SNPs, such that now confirmed SNP markers are nearly as dense as SSLPs markers.

We are now using these markers to genetically anchor genomic contigs, and to localise unmapped mutations.

* quick link - http://q.sanger.ac.uk/dqterivl