Academic journal article Genetics

Predicting the Size of the Progeny Mapping Population Required to Positionally Clone a Gene

Academic journal article Genetics

Predicting the Size of the Progeny Mapping Population Required to Positionally Clone a Gene

Article excerpt

ABSTRACT

A key frustration during positional gene cloning (map-based cloning) is that the size of the progeny mapping population is difficult to predict, because the meiotic recombination frequency varies along chromosomes. We describe a detailed methodology to improve this prediction using rice (Oryza sativa L.) as a model system. We derived and/or validated, then fine-tuned, equations that estimate the mapping population size by comparing these theoretical estimates to 41 successful positional cloning attempts. We then used each validated equation to test whether neighborhood meiotic recombination frequencies extracted from a reference RFLP map can help researchers predict the mapping population size. We developed a meiotic recombination frequency map (MRFM) for ~1400 marker intervals in rice and anchored each published allele onto an interval on this map. We show that neighborhood recombination frequencies (R-map, >280-kb segments) extracted from the MRFM, in conjunction with the validated formulas, better predicted the mapping population size than the genome-wide average recombination frequency (R-avg), with improved results whether the recombination frequency was calculated as genes/cM or kb/cM. Our results offer a detailed road map for better predicting mapping population size in diverse eukaryotes, but useful predictions will require robust recombination frequency maps based on sampling more progeny.

A limited number of forward genetics techniques exist to isolate an allele that underlies a mutant or polymorphic phenotype and that require no prior knowledge of the gene product. These include protocols to isolate host DNA flanking insertional mutagens (e.g., transposons) (BALLINGER and BENZER 1989; RAIZADA 2003) and positional gene cloning techniques (BOTSTEIN et al. 1980; PATERSON et al. 1988; TANKSLEY et al. 1995) that permit the discovery of alleles created by chemicalmutagens, radiation, or natural genetic variation. Positional gene cloning is feasible when the following conditions are met: (1) two parents exist that differ in a trait of interest; (2) the parents can be distinguished at the chromosome level by polymorphic DNA markers (e.g., RFLP); and (3) in a population of progeny, the underlying gene can be mapped relative to nearby DNAsegmentsthathavepreviouslybeencloned (BOTSTEIN et al. 1980; TANKSLEY et al. 1995). Unfortunately, positional gene cloning suffers from unpredictability in terms of the number of post-meiotic progeny that a researcher can expect to genotype to narrow a candidate chromosomal region to a small number of candidate genes (DINKA and RAIZADA 2006). For example, in rice (ORYZA sativa L.), only 1160 gametes were genotyped to narrow the Pi36(t) allele to a resolution of 17 kb (LIU et al. 2005), whereas 18,944 gametes were genotyped to map the Bph15 allele to a lower resolution of 47 kb (YANG et al. 2004). During fine mapping, the physical distance between a known physical location on a chromosome( i.e., themolecularmarker)andthe target allele is inferred by the frequency ofmeiotic recombinants that can break cosegregation of the phenotype encoded by the target allele with physically anchored molecular markers (BOTSTEIN et al. 1980; PATERSON et al. 1988). Ideally, a gene hunt ends once a molecular marker is found that always cosegregates with the target phenotype in a large population of genotyped and phenotyped F2 (or post-F2) progeny. Therefore, the frequency of meiotic recombination in the vicinity of the target locus (defined as R = kilobase/cM), along with the local density of molecular markers, determines the size of the mapping population. We are interested in helping researchers predict mapping population size. As initial analysis assigns a target allele to a 1-5-cM map interval, the goal of this study is to determine whether the recombination frequencyat this interval size,obtainedfrom a high-density molecular marker map, can be used to predict the number of progeny required for subsequent sub-centimorgan mapping in combination with userfriendly mathematical formulas. …

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