Indiana University

Further studies on preferential segregation --M. M. Rhoades and Ellen Dempsey The mechanism responsible for preferential segregation proposed in 1952 (Rhoades, in Heterosis, Iowa St. Coll. Press) had three essential components: 1) preferential segregation of the knobbed chromatid in a knob/knobless heterozygote would occur only after a crossover in the knob-centromere region produced a heteromorphic dyad; 2) extra spindle fibers at the knob region were necessary to orient such dyads in a nonrandom fashion at anaphase I; and 3) the preferential orientation was maintained into the second meiotic division so that the knobbed chromatid was directed into the basal megaspore. Components 2 and 3 exist only in the presence of abnormal chromosome 10 (K10).

Our studies with K10-type I (Rhoades and Dempsey, Plant Genetics, Alan R. Liss, Inc., 1985) indicated that this chromosome differs from normal chromosome 10 (N10) by the possession of a mainly euchromatic internal segment of unknown origin (the differential segment) and a large heterochromatic knob, as well as by the inversion and transposition of a distal stretch of 10L. We found neocentric activity in plants with terminally deficient K10 chromosomes, which retained the differential segment of K10-type I but had lost the terminal knob and varying portions of the subterminal inverted region--i.e., neocentric activity was possible in the absence of the K10 knob. We also studied the same Df K10 plants genetically by following the female transmission of the yg2 allele, closely linked to a large terminal knob (K9l) vs. small (K9s) on the short arm of chromosome 9 in K9l yg2/K9s Yg2 heterozygotes. There was no preferential segregation of the yg2 allele, suggesting that the K10 knob was necessary for this phenomenon. While the differential segment sans the K10 knob induced neocentric activity at knobs in heterologous chromosomes, no preferential segregation of these knobs resulted. It seemed possible that the K10 knob was involved in component 3--i.e., maintenance of preferential orientation through the second meiotic division.

If this idea is correct, the K10 knob plays a role after the end of the first meiotic division. If its activity is cell-limited, segregation of the K10 knob into only one of the prophase II daughter cells would result in random segregation of heterologous knob-knobless dyads at the second division in the other daughter cell. One might at first expect to find a reduction in preferential segregation frequencies in K10/N10 plants as compared with K10/K10 homozygotes because of the higher frequency of prophase II cells without K10 in the former group. While there is a slight decrease in K10/N10 vs. K10/K10 plants, the difference is small, probably because the occurrence of a crossover in the long arm of almost every chromosome 10 bivalent would give prophase II cells both of which had one K10 chromatid. To circumvent this difficulty, we utilized trisomics of K10 N10 N10 constitution, in which the long arm of the K10 chromosome seldom undergoes crossing over with either of the N10 homologs and prophase II cells without any K10 knob should be frequent. Two crosses were made differing only in the marker genes on chromosome 10:

Cross A: K10 R-st/N10 R-nj/N10 R; K9s Yg2/K9s Yg2
    X N10 R/N10 R; K9l yg2/K9l yg2

Cross B: K10 R/N10 R-nj/N10 r; K9s Yg2/K9s Yg2
    X N10 R/N10 R; K9l yg2/K9l yg2

Progeny of the two crosses are all K9l yg2/K9s Yg2 heterozygotes. Sporocytes were collected to determine the chromosome 10 constitution and the plants were testcrossed on r/r female parents to ascertain which R alleles were present. To test preferential segregation of chromosome 9 alleles, the same plants were used as female parents in crosses with yg2 male parents. Segregation of yg2 vs. Yg2 was followed in plants of K10/N10/N10, K10/N10, and N10/N10 constitution. If our hypothesis as to the role of the K10 knob is correct, the highest frequency of preferential segregation of yg2 should be found in the K10/N10 disomics, a reduced frequency is expected in the K10/N10/N10 trisomics and a random segregation of 1:1 should occur in the N10/N10 disomics.

Table 1. Frequencies of preferential segregation and recombination found in female testcrosses of disomic and trisomic progeny of Cross A and Cross B.
% yg
Recombination percents
Cross A, 2n + 1
Cross B, 2n + 1
Cross A, 2n
Cross B, 2n
Cross A & B, 2n

Table 1 shows the frequencies of preferential segregation found with trisomic and disomic progeny of Cross A and Cross B. Similar values were observed with populations from two crosses. As expected, the highest percentages of yg2 were found in testcrosses of K10/N10 female parents, somewhat lower percents occurred in K10/N10/N10 trisomic plants and the lowest values were in the N10/N10 plants. The N10/N10 data came mainly from progeny of Cross A; these plants surprisingly showed a slight excess of yg2 when 50% yg2 was expected. The reason for the excess of yg2 is not clear. In other respects, the data are understandable and both sets (Cross A and B) are in agreement with the predicted outcome. If it is assumed that all of the daughter cells following the first meiotic division in K10/N10 plants contain a K10 chromatid and that 60% is the basic rate of preferential segregation of the K9l chromosome in such cells, the reduction to 55.4-56% yg observed in K10/N10/N10 plants would indicate that in a high proportion of the meiotic divisions (80-90%) one daughter cell receives no K10 chromosome.

Cytological counts bearing on the meiotic segregation in K10/N10/N10 plants are available (Rhoades, in Heterosis, 1952). At diakinesis, 60% of the cells possessed a trivalent configuration and in 90% of these the K10 chromosome was joined to a N10 chromosome by a chiasma in the short arm and had no chiasma in the long arm. In the 40% of cells where a bivalent + univalent situation was found for chromosome 10, the univalent was the K10 chromosome in 83% of the cells. In these cells (90% of the trivalent group and 83% of the bivalent + univalent group), the K10 chromosome would pass intact to one pole or the other since there is no opportunity for a crossover in the long arm of 10 resulting in formation of a heteromorphic chromosome 10 dyad. These data are reinforced by similar observations on two K10/N10/N10 plants from the Cross A progeny. 65.9% of 387 diakinesis cells had a trivalent association and 34.1% had a bivalent + univalent situation. Within the latter group, the K10 chromosome was the univalent in 114 (86.2%) of the cells and N10 was the univalent in only 18 (13.8%). A high percentage of resulting segregations should have no K10 chromosome at one of the poles. When daughter cells without K10 undergo the second meiotic division in megasporogenesis, no preferential orientation occurs and the genetic data indicate that the overall rate of preferential segregation is reduced. We conclude that the K10 knob must be present in the second division daughter cells to allow completion of the process resulting in preferential segregation.

Recombination was followed in the chromosome 9 bivalents in the genetic study (Table 1). Total recombination in the wx-yg2 segment of 9S did not vary greatly in disomic and trisomic plants with K10, although the K10/N10 plants from Cross A progeny had a different distribution of crossovers in the two regions studied. Thus, heteromorphic chromosome 9 dyads were present in K10/N10 disomics and K10/N10/N10 trisomics in similar frequencies. According to the hypothesis explaining preferential segregation, such dyads are required if preferential segregation is to occur. The difference in preferential segregation of yg2 observed in the two groups cannot be ascribed to a lower amount of crossing over in the trisomic 10 group. Incidentally, in the K10/N10 and K10/N10/N10 plants, recombination frequencies in both yg2-bz and bz-wx regions were higher than in the N10/N10 controls. This is consistent with the secondary role of the K10 chromosome in promoting recombination. Since crossing over occurs in prophase of the first meiotic division before segregation has taken place, no difference is expected in the frequency of recombination in the K10/N10 and K10/N10/N10 plants.

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