Analysis of the chromosome-type breakage-fusion-bridge cycle
--Zheng, Y and Carlson, W

The study of dicentrics in maize was initiated by McClintock (Mo. Agric. Exp. Stn. Res. Bull. 290:1-48, 1938; Genetics, 26:234-282, 1941; Cold Spring Harbor Symp. Quant. Biol. 9:72-81, 1941; 14:13-37, 1951; PNAS 28:458-463, 1942; Carnegie Inst. Wash. Ybk. 42:148-150, 1943). She studied both chromatid- and chromosome-type dicentrics. The chromosome-type dicentrics were constructed by introducing two broken chromosomes, one from the male and one from the female, into the zygote. Fusion of the broken ends of the two chromosomes produced the dicentric. McClintock's studies showed that chromosome-type dicentrics are unstable during early plant development because they undergo the chromosome-type breakage-fusion-bridge cycle. Eventually, the dicentrics are converted to monocentrics and the cycle ceases. McClintock did not identify the time during development of dicentric stabilization.

The type of chromosome that McClintock used to produce dicentrics is referred to as duplication 9 (Dp-9). It contains a complete chromosome 9 plus a duplication of nearly all of the short arm, attached inversely to the end of the normal short arm. The duplicated region of Dp9 was combined with the B-9 chromosome of TB-9Sb through crossing over (Carlson, Corn and Corn Improvement, pp. 259-341, 1988). It should be noted that McClintock produced several duplication 9 chromosomes. The one used here is referred to as Type-I in Figure 9 of McClintock (Genetics, 26:234-282, 1941). At the first division of meiosis, the B-9-Dp9 chromosome frequently engages in foldback pairing and internal crossing over, with production of a chromatid-type dicentric B-9. This dicentric forms a single bridge at anaphase II. Following breakage of the bridge and DNA replication, the broken ends fuse and form a chromatid dicentric again. This initiates McClintock's chromatid-type breakage-fusion-bridge cycle. The cycle continues during the first pollen mitosis. However, at the second pollen mitosis, nondisjunction interrupts the cycle. Mitotic nondisjunction of the B-9 produces one sperm with the dicentric and another without it. In this process, the chromatid-type dicentric B-9 is converted into a chromosome-type dicentric. Consequently, a B-9-Dp-9 chromosome can produce chromosome type dicentrics when transferred through the male parent. This makes production of the dicentrics simpler than with McClintock's method.

In order to construct chromosome dicentrics with B-9-Dp9, kernels with a dominant C Wx phenotype were selected from crosses of 9-B(wx1) 9-B(Wx1) B-9-Dp9(C1C1) X 9(wx1) 9-B(Wx1) B-9(c1) B-9(c1). Plants grown from the seeds were classified for pollen type. Plants with all Wx pollen and 50% pollen sterility were selected. These should be 9-B(Wx1) 9-B(Wx1) B-9-Dp9. The selected plants were crossed as male parents to a tester: 9(bz1 yg2) 9(bz1 yg2) X 9-B(Wx1) 9-B(Wx1) B-9-Dp9(Bz1 Yg2 Yg2 Bz1). Fertilization of the egg by sperm containing a B-9 dicentric and of the polar cells by sperm lacking the B-9 gives the desired type. The endosperm has a recessive brown (bz) phenotype. The embryo has a B-9 dicentric and should give a variegated yellow and green (Yg/yg) phenotype.

A total of 747 brown seeds were planted in a search for chromosome dicentrics. The plant phenotypes were classified as: 194 green (26.0%), 66 yellow (8.8%), 54 dead or did not germinate (7.2%), 433 variegated green and yellow (58.0%). The variegated plants were studied in detail. Among 433 variegated plants, the primary root tips of 410 were checked for double bridges in mitotic anaphase. They were found in 364 plants. The percentage of variegated plants with double bridges was 88.8%. Next, 148 plants with double bridges were examined at weekly intervals for up to 10 weeks. A single root tip was examined each time to check double bridge configurations in 25 anaphase cells per plant. The data are summarized in Table 1. The percentage of variegated plants with double bridges in the roots declined gradually during plant development, indicating that the dicentric chromosomes were stabilized over the 10 week period in most plants. Only a few plants (6.5%) showed double bridges at week 10. From the curve in Figure 1, it appears that there is no specific time for elimination of the dicentric condition.

The findings do not distinguish between a) gradual elimination of dicentrics at different times in different sectors of a plant, or  b) elimination of dicentrics at a specific time in development for each plant, with the time varying between plants. It should also be noted that the method of dicentric B-9 stabilization has not yet been completely documented. McClintock found, with a dicentric 9, that conversion to monocentrics occurred. With the B-9 dicentric, conversion to monocentrics is found, but lagging and loss of the dicentric in anaphase may also occur. Both events "stabilize" the dicentric by eliminating double bridges.

Table 1. Percentage of variegated plants with double bridges over time. The number of variegated plants checked each week varied since the root tips of some unhealthy plants were not available each week and some plants died.

Figure 1. Percentage of variegated plants with double bridges during plant development. The data are listed in Table 1.

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