Burnham(1962) listed 7 methods to determine the heterogametic sex.
- Cytological identification of heteromorphic pair of sex chromosomes. Newer techniques include banding.
- Criss-cross type of sex linked inheritance helps in identification of heterogametic sex by segregation of a trait in that sex in the F2 generation.
- Reciprocal cross between dioecious and monoecious species results in all female progeny when the dioecious parent is female while monoecious female parent crossed with dioecious male parent gives half female and half male progeny, suggesting that male is heterogametic.
- Quantity of pollen resulting unequal sex ratio suggests male is heterogametic. For example, in hemp, sparse pollination gives more males, while in Melandrium, sparse pollination gave excess females, suggesting male sex is heterogametic in both cases. If female is heterogametic, sparse pollination between rare homozygous male and homozygous female in dioecious plant, Asparagus, give all males. This suggested that males were heterogametic, Pp, and females were homogametic, pp. No sex chromosomes could be identified in this case, but the results could be explained by assuming pXpX(female) and pXY(male), so that selfed pXY(M) will give 1 pXpX(F): 2pXY(M):1YY(M), of which YY males crossed to pXpX(F) will give all pXY males. Similar results were reported in Cannabis sativa, Spinacia oleracea, Thallictrum and Mercurialis.
- Reciprocal crosses between diploid homogametic and autotetraploid heterogametic indicate the heterogametic sex. For example, if male is heterogametic XX(F) x XXYY(M) give 1 XXX(F): 4XXY(M): 1XYY(M), i.e., 1 female and 5 male. If female is heterogametic sex, then it gives 1 male: 5 female. Such tests in Spinacia oleracea and in Melandrium album proved that male is the heterogametic sex. Janick and Stevenson found the ratio to vary from 2:1 to 5:1.
- Sex ratios among progeny of trisomics for chromosome 1 give 2F:1M if male is heterogametic and also suggesting that sex determining genes is located in chromosome 1.