Protocol for 2-generation Mapping, Balancing, and Homozygosing using single individuals (by CTH 2003).

1.  Male y w ?/Y; ?/?; ?/? x Female w/w; CyO/Gla Bc; TM2/TM6B

2.  F1:  If there are any white-eyed females, there are no inserts on the X-chromosome.  If all males are white-eyed and all females are red-eyed, then there are only inserts on the X chromosome.  If all females are red-eyed but only some males are red-eyed, there is an X-linked insert but there is an insert on another chromosome that was hemizygous.  Be careful here because all red-eyed progeny just means at least one insert was homozygous.  Also be careful to have strong statistical support here because only the lack of an X-linked insert can be conclusively rejected by a single fly.  If you have enough lines, it will be easiest if you just throw those out that contain inserts on the X.

Collect only red-eyed virgin females and red-eyed males with the appropriate markers to set up the crosses in #3.

3.  Perform one of the following crosses with red-eyed single flies (or both; I just do as many as I get since single-fly matings often fail):

            Male w/Y; ?/Gla Bc; ?/TM2 x Female w/y w ?; ?/CyO; ?/TM6B

            or

            Male w/Y; ?/Gla Bc; ?/TM6B x Female w/y w ?; ?/CyO; ?/TM2

If there are multiple eye colors, try to match them because these may be due to different inserts.

4.  Here are the most common results and how to interpret and act on them.  While I have just said “red-eyed,” the color will vary from pale peach to dark red.  Multiple colors that cannot be explained solely be the number of copies of an insert on the chromosome you think you have might indicate additional inserts, and you should consider other possibilities.

            a.  If there is an insert on only the 2nd chromosome, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

white-eyed

red-eyed

red-eyed

Gla Bc; ?

white-eyed

white-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed (homozygous)

red-eyed (homozygous)

?; ?

red-eyed

red-eyed

red-eyed (homozygous)

red-eyed (homozygous)

Bascially,  CyO/Gla Bc should be white-eyed, and everything else will be red-eyed (3rd chromosome and sex should not matter).  Note that if the insert has created a lethal allele, the homozygous classes will be missing; this would further suggest the insert was on the 2nd chromosome.  You should note whether the allele is homozygous viable.

Ideally, recover the bottom right corner flies (red-eyed homozygous ?/?; ?/?) to create the stock.  Red-eyed ?/CyO; ?/? would also be great.  These 2 genotypes could be mixed if there is a shortage of virgin females or males.  If necessary, ?/TM6B or ?/TM2 flies could be recovered, provided they are not mixed with one another as this will decrease the efficiency of selection.  Eventually, natural selection will remove the balancers from the stock, unless they are balancing a lethal allele.  Do not recover any flies with Gla Bc; it is not a true balancer chromosome.

            b.  If there is an insert on only the 3rd chromosome, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

white-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed (homozygous)

red-eyed

red-eyed (homozygous)

?; TM2

white-eyed

red-eyed

white-eyed

red-eyed

?; ?

red-eyed

red-eyed (homozygous)

red-eyed

red-eyed (homozygous)

Bascially,  TM2/TM6B should be white-eyed, and everything else will be red-eyed (2nd chromosome and sex should not matter).  Note that if the insert has created a lethal allele, the homozygous classes will be missing; this would further suggest the insert was on the 3rd chromosome.  You should note whether the allele is homozygous viable.

Ideally, recover the bottom right corner flies (red-eyed homozygous ?/?; ?/?) to create the stock.  Red-eyed ?/?; ?/TM6B would also be great (TM6B is a better balancer than TM2).  These 2 genotypes could be mixed if there is a shortage of virgin females or males.  If necessary, ?/CyO flies could be recovered.  Eventually, natural selection will remove the balancers from the stock, unless they are balancing a lethal allele.  Do not recover any flies with Gla Bc; it is not a true balancer chromosome.

            c.  If there is an insert on both the 2nd and 3rd chromosomes but not on the X or the 4th, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

red-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed (homozygous for both inserts)

Basically, only CyO/Gla Bc; TM2/TM6B will be white-eyed.  Again, missing classes might indicate a homozygous lethal allele, and this should be noted.

Ideally, recover the bottom right corner flies (red-eyed homozygous ?/?; ?/?) to create the stock.  Alternatively, balancing either insert of CyO or TM6B would also work.  Recovering a single-insert stock here is impossible, but one could separate the double-balanced stock later.

            d.  If there is an insert on the X chromosome of the female in addition to an insert on the 2nd chromosome of both the male and female, the results should look like this where mixed means both white-eyed and red-eyed flies are included in this class (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

mixed

mixed

red-eyed

red-eyed

Gla Bc; ?

mixed

mixed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

Whether you want to chase these depends on how badly you need more inserts.

            e.  If there is an insert on the X chromosome of the female in addition to an insert on the 3rd chromosome of both the male and female, the results should look like this where mixed means both white-eyed and red-eyed flies are included in this class (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

mixed

red-eyed

mixed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

mixed

red-eyed

mixed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

Whether you want to chase these depends on how badly you need more inserts.

            f.  If there is an insert on the X chromosome of the female in addition to an insert on both the 2nd and 3rd chromosome of both the male and female, the results should look like this where mixed means both white-eyed and red-eyed flies are included in this class (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

mixed

red-eyed

red-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

Whether you want to chase these depends on how badly you need more inserts.

5.  Since the male and female in the cross in #3 do not necessarily contain the same inserts, there are a few other possibilities shown below.  These should be rare.

            a.  If the male insert is on the 2nd and the female insert is on the 3rd, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

white-eyed

red-eyed

Gla Bc; ?

white-eyed

red-eyed

white-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

Balanced stocks of the 2nd chromosome insert can be recovered here by taking red-eyed ?/CyO; ?/TM6B flies.  Balanced stocks of the 3rd chromosome insert cannot be recovered immediately without including the 2nd chromosome insert because Gla Bc is not a balancer.

            b.  If the male insert is on the 3rd and the female insert is on the 2nd, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

white-eyed

red-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

white-eyed

white-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

Balanced stocks of the 3rd chromosome insert can be recovered here by taking red-eyed ?/CyO; ?/TM6B flies.  Balanced stocks of the 2nd chromosome insert cannot be recovered immediately without including the 3rd chromosome insert because Gla Bc is not a balancer.

            c.  If the male has an insert on the 2nd and 3rd chromosome and the female has only an insert of the 2nd, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

white-eyed

red-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

You could recover a balanced stock of the insert on the 2nd with ?/CyO; ?/TM2.  If the 2nd chromosome inserts are the same, you could also recover ?/?; ?/TM2.  You cannot recover the 3rd chromosome insert immediately without including the 2nd chromosome because Gla Bc is not a balancer.

            d.  If the male has an insert only on the 2nd chromosome and the female has an insert on both the 2nd and 3rd chromosomes, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

red-eyed

red-eyed

Gla Bc; ?

white-eyed

red-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

You could recover a balanced stock of the insert on the 2nd with ?/CyO; ?/TM6B or ?/?; ?/TM6B if the 2nd chromosome inserts are the same.  You cannot recover the 3rd chromosome insert immediately without including the 2nd chromosome because Gla Bc is not a balancer.

            e.  If the male has an insert on the 2nd and 3rd chromosome and the female has only an insert of the 3rd, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

white-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

red-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

You cannot recover a single-insert balanced stock in a single cross.

            f.  If the female has an insert on the 2nd and 3rd chromosome and the male has only an insert of the 3rd, the progeny should look like this (note that TM6B and TM2 could be reversed, depending on which of the above crosses was performed):

CyO; TM6B

CyO; ?

?; TM6B

?; ?

Gla Bc; TM2

white-eyed

red-eyed

red-eyed

red-eyed

Gla Bc; ?

red-eyed

red-eyed

red-eyed

red-eyed

?; TM2

white-eyed

red-eyed

red-eyed

red-eyed

?; ?

red-eyed

red-eyed

red-eyed

red-eyed

You could recover ?/CyO; ?/TM2 as a balanced stock of the 3rd chromosome insert.  ?/Cyo; ?/? would work if the 3rd chromosome inserts are the same.

            g.  Variants on #5a-f are also possible if the female also contains an insert on the X.  Then, the white-eyed classes would actually be mixed.  Recovering balanced stocks would be impossible in one step.

            h.  I have also not discussed the possibility of multiple inserts on the same chromosome.  For the most part, these crosses will not differentiate between multiple and single inserts.  If multiple inserts end up in repulsion or in different flies in the balanced stock, they will eventually break up by recombination, and the stock will no longer be balanced because it will leave behind y w chromosomes that could allow white eyes.

DEFINITIONS, SCORING, FLY GENETICS, ETC.

1.  Recombination does not occur in Drosophila males.

2.  Balancers are chromosomes that contain multiple inversions, a visible dominant marker, and a recessive lethal.  This suppresses viable crossovers on that chromosome.

3.  w is a recessive mutant for the white gene; white-eyed phenotype.

4.  w+ is a dominant wild type (or transgene) allele of the white gene; red-eyed phenotype.

5.   y is a recessive mutant for the yellow gene; yellow body, bristles, cuticle, etc.

6.   GlaBc is a 2nd chromosome that is homozygous lethal and has 2 easily scored dominant markers:  Glazed or Glassy eyes and narrowed eyes and small black dots all over the cuticle.  This chromosome does not carry inversions and is not a true balancer, so it should be treated with caution when in a female and unbalanced.  Gla is actually a wg allele.

7.  CyO is a 2nd chromosome balancer with a curly wings (Cy) marker.

8.  TM2 is a 3rd chromosome balancer that is marked by a tiny bristle or set of bristles on the anterior, proximal portion of the haltere.

9.   TM6B is a 3rd chromosome balancer that is marked by extra humeral bristles (on shoulder).

10.  I have used ? to symbolize a chromosome that may or may not contain a w+ insertion.
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