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Rapid recombination mapping for high-throughput genetic screens in Drosophila.

Sapiro AL, Ihry RJ, Buhr DL, Konieczko KM, Ives SM, Engstrom AK, Wleklinski NP, Kopish KJ, Bashirullah A - G3 (Bethesda) (2013)

Bottom Line: Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster.This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing.We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222.

ABSTRACT
Mutagenesis screens are a staple of classical genetics. Chemical-induced mutations, however, are often difficult and time-consuming to identify. Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster. This method requires only two generations and a total of six crosses in vials to estimate the genetic map position of the responsible lesion with high accuracy. This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing. We have used this approach to successfully map a collection of mutations from an ethyl methanesulfonate-based mutagenesis screen on the third chromosome. We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available. By facilitating the rapid identification of mutated genes, our mapping strategy removes a primary obstacle to the widespread use of powerful chemical mutagenesis screens to understand fundamental biological phenomena.

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Use of pairs of dominant markers to map a lethal mutation on the third chromosome. An example of the mapping process illustrating the effectiveness of using pairs of dominant markers. (A) The analysis described in Figure 1 is conducted with four pairs of dominant markers (R,D, Gl,Sb, Sb,H, and H,Pr) to map a lethal mutation on the third chromosome, psg24 (indicated with a purple asterisk). Scoring the viable F2 progeny indicates that only one pair has no unmarked progeny, thus the mutation is located inside the H,Pr pair. Consistent with this interpretation, the ratio of “splits” in the R,D, Gl,Sb and Sb,H crosses point to the H,Pr region. (B) The recombinant “splits” in H,Pr are used to calculate the approximate location of psg24. The formula provides the relative distance of the mutation from the left marker. This distance is indicated by the frequency of loss of the left marker among the viable F2 progeny “splits.” In this case, the estimated genetic map position for psg24 is approximately 80 cM. This genetic location is then used to estimate a cytological location with positional information of known genes (see File S1), estimating the physical location of psg24 to around 95A. (C) We used complementation tests with deficiencies near 95A to identify the actual location of psg24. The mutation was crossed to 10 deficiencies from the DK3 collection spanning the region from 94A to 96C, and it failed to complement Df(3R)BSC619 in 94E, which is about 1 cM or approximately one deficiency away from the initial estimated physical map position. The arrow reflects the approximate reliability of the recombination analysis, where the base of the arrow (dot) represents the estimated genetic map position and the arrowhead represents the actual physical location.
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fig2: Use of pairs of dominant markers to map a lethal mutation on the third chromosome. An example of the mapping process illustrating the effectiveness of using pairs of dominant markers. (A) The analysis described in Figure 1 is conducted with four pairs of dominant markers (R,D, Gl,Sb, Sb,H, and H,Pr) to map a lethal mutation on the third chromosome, psg24 (indicated with a purple asterisk). Scoring the viable F2 progeny indicates that only one pair has no unmarked progeny, thus the mutation is located inside the H,Pr pair. Consistent with this interpretation, the ratio of “splits” in the R,D, Gl,Sb and Sb,H crosses point to the H,Pr region. (B) The recombinant “splits” in H,Pr are used to calculate the approximate location of psg24. The formula provides the relative distance of the mutation from the left marker. This distance is indicated by the frequency of loss of the left marker among the viable F2 progeny “splits.” In this case, the estimated genetic map position for psg24 is approximately 80 cM. This genetic location is then used to estimate a cytological location with positional information of known genes (see File S1), estimating the physical location of psg24 to around 95A. (C) We used complementation tests with deficiencies near 95A to identify the actual location of psg24. The mutation was crossed to 10 deficiencies from the DK3 collection spanning the region from 94A to 96C, and it failed to complement Df(3R)BSC619 in 94E, which is about 1 cM or approximately one deficiency away from the initial estimated physical map position. The arrow reflects the approximate reliability of the recombination analysis, where the base of the arrow (dot) represents the estimated genetic map position and the arrowhead represents the actual physical location.

Mentions: Recombinant F2 progeny were scored for presence of the mapping dominant markers and for absence of the balancer dominant marker (see genetic scheme in Figure 1A). Phenotypes of dominant markers used for mapping are as follows: Roughened (R), scored for rough eye; Dichaete (D), scored for extended wings and missing alulae; Glued (Gl), scored for smaller rough eyes; Stubble (Sb), scored for short and thick bristles on the notum; Hairless (H), scored for loss of the postvertical bristles in the head; and Prickly (Pr), scored for short and thin-tipped bristles on the notum (of note, H and Pr together cause a loss of bristles). For the purpose of this mapping method, the relevant F2 progeny are those that have lost one or both markers; for example, when the Sb,H pair for mapping is used, the relevant progeny to be scored are Sb,+, +,H and +,+ (the remaining progeny are not needed to calculate relative position; see Figure 2B). The balancer used was marked by Drop (TM6B,Dr), which shows a near-complete ablation of the eyes. The use of the Dr-containing balancer facilitates sorting of F2 progeny (the Dr eye phenotype is considerably stronger than those of R and Gl and thus easily identified even in their presence). Although we describe this recombination mapping method for the third chromosome, the principle of using pairs of dominant markers can be applied for mapping mutations on other chromosomes. The second chromosome has a number of good dominant markers that can be used for recombination mapping (see Supporting Information, Table S1 for examples). Moreover, even though dominant visible mutations are rare on the X chromosome, the principle of pairs of dominant markers can be applied by scoring recessive markers in male recombinant progeny.


Rapid recombination mapping for high-throughput genetic screens in Drosophila.

Sapiro AL, Ihry RJ, Buhr DL, Konieczko KM, Ives SM, Engstrom AK, Wleklinski NP, Kopish KJ, Bashirullah A - G3 (Bethesda) (2013)

Use of pairs of dominant markers to map a lethal mutation on the third chromosome. An example of the mapping process illustrating the effectiveness of using pairs of dominant markers. (A) The analysis described in Figure 1 is conducted with four pairs of dominant markers (R,D, Gl,Sb, Sb,H, and H,Pr) to map a lethal mutation on the third chromosome, psg24 (indicated with a purple asterisk). Scoring the viable F2 progeny indicates that only one pair has no unmarked progeny, thus the mutation is located inside the H,Pr pair. Consistent with this interpretation, the ratio of “splits” in the R,D, Gl,Sb and Sb,H crosses point to the H,Pr region. (B) The recombinant “splits” in H,Pr are used to calculate the approximate location of psg24. The formula provides the relative distance of the mutation from the left marker. This distance is indicated by the frequency of loss of the left marker among the viable F2 progeny “splits.” In this case, the estimated genetic map position for psg24 is approximately 80 cM. This genetic location is then used to estimate a cytological location with positional information of known genes (see File S1), estimating the physical location of psg24 to around 95A. (C) We used complementation tests with deficiencies near 95A to identify the actual location of psg24. The mutation was crossed to 10 deficiencies from the DK3 collection spanning the region from 94A to 96C, and it failed to complement Df(3R)BSC619 in 94E, which is about 1 cM or approximately one deficiency away from the initial estimated physical map position. The arrow reflects the approximate reliability of the recombination analysis, where the base of the arrow (dot) represents the estimated genetic map position and the arrowhead represents the actual physical location.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852393&req=5

fig2: Use of pairs of dominant markers to map a lethal mutation on the third chromosome. An example of the mapping process illustrating the effectiveness of using pairs of dominant markers. (A) The analysis described in Figure 1 is conducted with four pairs of dominant markers (R,D, Gl,Sb, Sb,H, and H,Pr) to map a lethal mutation on the third chromosome, psg24 (indicated with a purple asterisk). Scoring the viable F2 progeny indicates that only one pair has no unmarked progeny, thus the mutation is located inside the H,Pr pair. Consistent with this interpretation, the ratio of “splits” in the R,D, Gl,Sb and Sb,H crosses point to the H,Pr region. (B) The recombinant “splits” in H,Pr are used to calculate the approximate location of psg24. The formula provides the relative distance of the mutation from the left marker. This distance is indicated by the frequency of loss of the left marker among the viable F2 progeny “splits.” In this case, the estimated genetic map position for psg24 is approximately 80 cM. This genetic location is then used to estimate a cytological location with positional information of known genes (see File S1), estimating the physical location of psg24 to around 95A. (C) We used complementation tests with deficiencies near 95A to identify the actual location of psg24. The mutation was crossed to 10 deficiencies from the DK3 collection spanning the region from 94A to 96C, and it failed to complement Df(3R)BSC619 in 94E, which is about 1 cM or approximately one deficiency away from the initial estimated physical map position. The arrow reflects the approximate reliability of the recombination analysis, where the base of the arrow (dot) represents the estimated genetic map position and the arrowhead represents the actual physical location.
Mentions: Recombinant F2 progeny were scored for presence of the mapping dominant markers and for absence of the balancer dominant marker (see genetic scheme in Figure 1A). Phenotypes of dominant markers used for mapping are as follows: Roughened (R), scored for rough eye; Dichaete (D), scored for extended wings and missing alulae; Glued (Gl), scored for smaller rough eyes; Stubble (Sb), scored for short and thick bristles on the notum; Hairless (H), scored for loss of the postvertical bristles in the head; and Prickly (Pr), scored for short and thin-tipped bristles on the notum (of note, H and Pr together cause a loss of bristles). For the purpose of this mapping method, the relevant F2 progeny are those that have lost one or both markers; for example, when the Sb,H pair for mapping is used, the relevant progeny to be scored are Sb,+, +,H and +,+ (the remaining progeny are not needed to calculate relative position; see Figure 2B). The balancer used was marked by Drop (TM6B,Dr), which shows a near-complete ablation of the eyes. The use of the Dr-containing balancer facilitates sorting of F2 progeny (the Dr eye phenotype is considerably stronger than those of R and Gl and thus easily identified even in their presence). Although we describe this recombination mapping method for the third chromosome, the principle of using pairs of dominant markers can be applied for mapping mutations on other chromosomes. The second chromosome has a number of good dominant markers that can be used for recombination mapping (see Supporting Information, Table S1 for examples). Moreover, even though dominant visible mutations are rare on the X chromosome, the principle of pairs of dominant markers can be applied by scoring recessive markers in male recombinant progeny.

Bottom Line: Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster.This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing.We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222.

ABSTRACT
Mutagenesis screens are a staple of classical genetics. Chemical-induced mutations, however, are often difficult and time-consuming to identify. Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster. This method requires only two generations and a total of six crosses in vials to estimate the genetic map position of the responsible lesion with high accuracy. This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing. We have used this approach to successfully map a collection of mutations from an ethyl methanesulfonate-based mutagenesis screen on the third chromosome. We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available. By facilitating the rapid identification of mutated genes, our mapping strategy removes a primary obstacle to the widespread use of powerful chemical mutagenesis screens to understand fundamental biological phenomena.

Show MeSH
Related in: MedlinePlus