TRIHYBRID CROSSES
Forked-line method: For three or more unlinked events, Punnett squares, which do a complete genotypic
analysis, tend to become quite cumbersome. With three unlinked genes, each parent can produce 8 different
types of gametes, which generates 64 possible genotypic combinations in the Punnett Square. For four unlinked
markers, there are 16 possible gametes from each parent and 256 cells in the complete Punnett square! It is
therefore more effective to use the forked-line method when working with 3 or more traits. The forked-line
method is based on possible alternatives using fractions, rather than all possible fertilization events. The forkedline can be used to figure out phenotypic probability, as shown below, or to figure out genotypic probability.
Each branch point represents the expected distribution of phenotypes or genotypes for a particular trait based on
the parental genotypes. Multiplying the expected fractions at each of the branches across the diagram provides
an easy calculation of the expected frequency of any particular phenotypic or genotypic combination. The
example below is for phenotypic distribution when the parents are heterozygous at three unlinked traits in peas.
3/4 Full
= 27/64 YRF phenotype
/
3/4 Round
/ \
/
1/4 constricted = 9/64 YRf phenotype
/
3/4 Yellow
/ \
/
\
3/4 Full
= 9/64 YrF phenotype
/
\ /
/
1/4 Wrinkled
/
\
/
1/4 constricted = 3/64 Yrf phenotype
/
YyRrFf x YyRrFf
\
\
3/4 Full
= 9/64 yRF phenotype
\
/
\
3/4 Round
\
/ \
\
/
1/4 constricted = 3/64 yRf phenotype
\ /
1/4 green
\
\
3/4 Full
= 3/64 yrF phenotype
\ /
1/4 wrinkled
\
1/4 constricted = 1/64 yrf phenotype
27:9:9:9:3:3:3:1 ratio: As can be seen in the forked line diagram above, a trihybrid cross yields a phenotypic
ratio of 27:9:9:9:3:3:3:1. This reflects the phenotypes generated by the 64 genotypic combinations resulting
from 8 different male gametes fertilizing 8 different female gametes.