Experiment 02Mendelian Transmission GeneticsIndependent Assortment
Mendelian Monohybrid & Dihybrid Crosses: Segregation & Independent Assortment in Drosophila
Lab Duration: 3-4 Weeks (P → F1 → F2)
🎯 Aim: To conduct a reciprocal dihybrid genetic cross between homozygous wild-type flies (grey body, long wings) and double mutant homozygous flies (ebony body, vestigial wings) in Drosophila melanogaster, score filial generations (F1 and F2), and verify Mendel’s Law of Segregation and Law of Independent Assortment ($9:3:3:1$ phenotypic ratio).
🔬 Principle & Theoretical Background
Gregor Mendel’s Second Law—the Law of Independent Assortment—states that alleles of two or more different genes assort independently of one another into gametes during gametogenesis, provided the gene loci reside on non-homologous chromosomes (syntenic loci separated by $>50,text{cM}$) or on completely separate autosomes.
In Drosophila melanogaster, body color and wing shape provide classical autosomal phenotypic markers:
- Body Color Gene (Chromosome 3, locus 70.7 cM): The wild-type allele ($e^+$ or $G$) produces normal tan/grey body pigmentation and is completely dominant over the recessive mutant allele ($e$ or $g$), which produces an intensely dark ebony/black body and dark wing veins.
- Wing Shape Gene (Chromosome 2, locus 67.0 cM): The wild-type allele ($vg^+$ or $L$) produces full-length, straight functional wings and is dominant over the recessive mutant allele ($vg$ or $l$), which produces severely reduced, rudimentary, non-functional “vestigial” wings due to apoptosis in the wing imaginal disc during pupation.
Because the ebony locus resides on Chromosome 3 and the vestigial locus resides on Chromosome 2, they represent unlinked autosomal genes on non-homologous chromosomes that assort with absolute independence during anaphase I of meiosis:
Parental Cross (P): GG LL (Wild-type ♂) × gg ll (Ebony, Vestigial ♀) → Gametes: GL × glFilial 1 (F1): 100% Gg Ll (Dihybrid Wild-type: Grey body, Long wings)F1 Intercross (F1 × F1): Gg Ll × Gg Ll → F2 Gametes: 1/4 GL, 1/4 Gl, 1/4 gL, 1/4 glExpected F2 Phenotypes: 9/16 Wild-type : 3/16 Grey Vestigial : 3/16 Ebony Long : 1/16 Ebony Vestigial
📋 Step-by-Step Experimental Protocol
- Setting up Parental Cross (Week 1): Collect 5 virgin female flies from an ebony, vestigial mutant stock ($gg,ll$) and 5 healthy young male flies from a pure-breeding wild-type stock ($GG,LL$). Anesthetize, confirm purity, and transfer both into a fresh culture bottle labeled “P: ♀ ebony,vestigial × ♂ wild-type”.
- Parental Removal (Day 7-8): After 7 days, inspect the vial for developing third-instar larvae crawling on the glass walls. Anesthetize and remove all parental flies to prevent back-crossing with emerging F1 offspring.
- Scoring F1 Generation (Week 2): Upon F1 adult eclosion (days 10-12), anesthetize the flies and score their body color and wing shape. Verify that $100%$ of F1 progeny display wild-type grey bodies and long wings ($Gg,Ll$).
- Setting up F2 Intercross (Week 2): Select 5 pairs of healthy F1 males and F1 females and place them into a fresh culture bottle labeled “F1 × F1 Intercross”. (Note: Virgin collection is not strictly required for F1 sibling intercrosses).
- Clearing F1 Adults (Day 20): Remove all F1 parents 7 days after establishing the intercross.
- Scoring F2 Generation (Weeks 3 & 4): Score F2 progeny daily for 10 consecutive days following the initial eclosion. Sort flies under the stereomicroscope into four distinct phenotypic classes:
- Class 1: Wild-type (Grey body, Long wings)
- Class 2: Recombinant 1 (Grey body, Vestigial wings)
- Class 3: Recombinant 2 (Ebony body, Long wings)
- Class 4: Double mutant (Ebony body, Vestigial wings)
📊 Representative F2 Data Table
| Phenotypic Class | Genotype(s) | Observed Count (O) | Expected Ratio | Expected Count (E) | (O – E) |
|---|---|---|---|---|---|
| Wild-type (Grey, Long) | $G_ L_$ | $284$ | $9/16$ ($56.25%$) | $270.0$ | $+14.0$ |
| Grey body, Vestigial wings | $G_ ll$ | $92$ | $3/16$ ($18.75%$) | $90.0$ | $+2.0$ |
| Ebony body, Long wings | $gg L_$ | $86$ | $3/16$ ($18.75%$) | $90.0$ | $-4.0$ |
| Ebony body, Vestigial wings | $gg ll$ | $18$ | $1/16$ ($6.25%$) | $30.0$ | $-12.0$ |
| Total Sample Size (N) | — | $480$ | $16/16$ ($100%$) | $480.0$ | $0.0$ |
