These newer techniques include the following and may be used for animals also : (i) banding technique, which permits linear differentiation of chromosomes so that even if sex chromosomes are similar in gross morphology, these may be distinguished by banding (Q banding, C banding, G banding, R banding, etc.); (ii) in situ hybridization, for which DNA sequences have been isolated, which are specific for sex chromosome, e.g. Bkm (Banded Krait = Bungarus fasciatus, minor sat-DNA). The Bkm sequence was first isolated by Dr. Lalji Singh (presently working at CCMB, Hyderabad). This sequence when used as a probe for in situ hybridization, help in the identification of sex chromosomes, and consequently of the heterogametic sex. Cytological identification of XO type of heterogametic sex is also possible through the detection of a difference in chromosome number in two sexes, the heterogametic sex having one chromosome less than the other sex.
Genetic linkage showing criss-cross or sex linked inheritance. Criss-cross type of inheritance also helps in identification of heterogametic sex by segregation of a trait in that sex in F2 generation. For instance, in Lychnis alba, a cross, broad leaved ♀ x narrow leaved ♂, gives all broad leaved Ft plants (both ♀ and ♂), which in F2 give all females broad leaved, but part of the males broad leaved and a part narrow leaved (broad leaves is a dominant character). Such segregation in male sex suggests that male is the heterogametic sex with XY, female being XX. Several other characters in Lychnis showed a similar segregation. Sex linkage has also been reported in plant species like hemp, papaya and Rumex acetosa, thus helping in the identification of heterogametic sex.
Crosses between dioecious and monoecious species. In reciprocal crosses between dioecious and monoecious species, the sex of the dioecious parent species, which gives segregation in the progeny, represents the heterogametic sex. For instance in Bryonia, B. dioica ♀ (dioecious) x B. alba ♂(monoecious) gives all female, while B. alba ♀ (monoecious) x B. dioica ♂(dioecious) gives 1 ♀ : 1 ♂, suggesting that ♂is heterogametic. Crosses between B. multiflora (monoecious) and B. dioica (dioecious) gave similar results. Similar experiments were also conducted in Amaranthus and Acnida.
Competition among pollen (sparse vs excess pollen). If sex ratios in the progenies from sparse vs excess pollen differ, it suggests that ♂ sex is heterogametic. For instance, in hemp (Cannabis sativa), sparse pollination gave excess males, while in Melandrium, sparse pollination gave excess females, suggesting that male sex is heterogametic in both the cases. If female is heterogametic, sparse pollination should give male a-nd females in equal proportions.
Self pollination in exceptional bisexual flowers of dioecious species. In a dioecious plant species of Asparagus, few seed set on a rare ♂ plant gave a ratio 3 ♂: 1 ♀; l/3rd of ♂from this population were homozygous giving all males in crosses with a female. This suggested that males were heterogametic Pp and females were homogametiepp and the sex may be monogenically controlled. No sex chromosomes could be identified in this case, although the results could also be explained by assuming XX (♀) and XY (♂), so that selfed XY (♂)will give 1 XX (♀) : 2 XY (♂) : 1 YY (♂), of which l/3rd males i.e. YY when crossed to XX (♀) will give all XY males. Similar results were reported for Cannabis sativa, Spinacia oleracea, Thallictrum and Mercurialis.
Crosses of diploids (2x) with autotetraploids (4jc). Reciprocal crosses between diploids and autotetraploids also give information about the heterogametic sex. For instance, if male is XY and female XX, then two desired crosses will be XXXX (♀) x XY (♂)and XX (♀) x XXYY (♂), the former giving XXX (♀) and XXY (♂)in equal proportion and the latter giving 1 XXX (♀) : 4 XXY (♂): 1XYY (♂)= 1 (♀) : 5 (♂). This ratio should become 5 (♀) : 1 (♂), if female is the heterogametic sex. Such tests when conducted in Spinacia oleracea and in Melandrium album proved that male is the heterogametic sex.
In the above experiments, among XXYY individuals, a high proportion of XY gametes may results due to preferential pairing and the 5 : 1 ratio may deviate in favour of heterogametic sex. Contrary to this, Janick and Stevenson (1955) found the ratio to vary from 2 : 1 to 5 : 1 suggesting excess of segregation of X chromosomes to one pole and Y chromosomes to the other pole. . 5
Sex ratios among progeny of trisomics. Particularly when sex is monogenically controlled, trisomic analysis may also suggest if male is heterogametic. For instance, in Spinacia oleracea (2« = 12), when 2x female is crossed with all the six trisomics as male, then 1 (♀) : 1 (♂) ratio was obtained in all cases except when trisomic for chromosome 1 (Aaa)was used, which gave 2 ♀ (aa): 1 ♂ (Aa)ratio suggesting that ♂ is heterogametic and that sex determining gene is located on chromosome 1. Contrary to this if ♀ were heterogametic, in all the six trisomics used as male, same sex ratio should be available in the progeny.
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