Pratt, D.
The preceeding research note deals with a large linkage study on male-sterile genes. The linkage screening technique used in that study may be of interest to others who are attempting to map genes controlling mature plant characters by analyzing segregations from repulsion crosses to stocks carrying seedling marker genes. The technique reduces, three fold, the number of plants which must be grown to maturity and scored for the mature plant character. It is simply to rogue the F2 at the seedling stage. Removing all plants except those homozygous for the seedling marker gene. Any linkage is indicated by the segregation for the mature plant character among those homozygotes. The following paragraphs explain the method and point out some of its advantages and defects.
A X^2 analysis of the full F2 segregation normally serves for the detection of repulsion linkage. As an example consider the F2 segregation of ms\9 versus wf, white flower:
phenotypic classes + + ms + + wf ms wf
plants observed 44 23 19 11
e, expected from 2 x 2 48.5 18.5 14.5 5.5
contingency table
|d| - 1/2, where d is the 4.0 4.0 4.0 4.0
deviation expected
(|d| - 1/2)^2 16.0 16.0 16.0 16.0
(|d| - 1/2)^/e 0.3 0.9 1.1 2.9
X^2=5.2, d.f.=1, .02 < P < .05
The large value of X^2 indicates a departure from independant assortment of
the ms\9 and wf genes. It should be noted that the wf portion of the F2,
roughly 1/4 of the total plants, contributes about 3/4 of the value of X^2.
If both genes segregated in perfect 3:1 ratios the figures would be exactly
1\4 and 3\4. This has the following basis. In such X^2 tests of two-by-two
contingency tables (used to obtain a linkage X^2 independent of the
segregation ratios of the two genes, all four classes show the same absolute
deviation from the excpected, therefore the same squared deviation. The
contribution of each class to the linkage X^2 is proportional to this d^2, but
inversely proportional to the expected, e, value for the class. The e values
of the four classes form a ratio of 9:3:3:1. Therefore, the X^2
contributions, d^2/e, form a l:3:3:9 ratio. Clearly the ms segregation in the
wf portion of the F2 provides a larger share of the X^2 and therefore, greater
efficiency in detecting linkage than the ms segregation among the non-mutant
plants. More precisely, the tester-mutant portion of the F2 has nine times
greater efficiency per plant than the non-mutant portion. The mutants are
consequently three times as efficient per plant as the full F2.These considerations suggested that efficiency of linkage screening might be improved by scoring only the fraction of F2 populations homozygous for the tester genes. This increased efficiency assumes importance with such tester genes as d, a, e, l\1, etc. which may be distinguished soon after the seedling emerges. Thus by roguing the non-dwarf plants from the F2 seedling flat -- before going thru the steps of moving the plants to the field, growing them to flowering, and scoring them for male sterility -- any linkage, of ms (or other mature plant mutant) to d could be detected with one third as many plants in the field as if the full F2 were used. It is estimated that such roguing for seedling mutants in the ms linkage studies decreased by roughly half the field space and attendant time and expense required.
In the rogued F2 families a X^2 test detects any significant reduction of the number of ms plants below the 25 percent expected with no linkage. Such a reduction might result from low viability, reduced transmission, or poor penetrance of the ms- (or other mature plant-) mutant, as well as from linkage. The roguing technique, therefore, would only prove useful for mature plant mutants showing normal Meendelian behavior. A full F2 or a testcross of F1 to the appropriate F2 recombinant should be grown to pursue all indications of linkage in the rogued F2 families. These follow-up families provide additional information for estimation of linkage values, and in cases in which the F2 has been rogued to homozygosity for two or more tester mutants, show which mutant is linked.