Maize Genetics Cooperation Newsletter 80. 2006.

Columbia, missouri

University of Missouri

A trans-acting factor required for non-disjunction of the B chromosome is located distal to the TB-4Lb breakpoint on the B chromosome

--Lamb, JC; Han, F; Auger, DL; Birchler, JA

At the second mitotic division during pollen development, the B chromosome is not distributed to both of the daughter cells. Instead, at a high frequency one sperm cell receives both of the sister B chromatids and the other sperm gets none. The mechanism that causes this phenomenon, called non-disjunction, is not known.

In a reciprocal translocation stock involving A and B chromosomes, the chromosome with the B centromere (the B-A chromosome) is subject to non-disjunction, but not the reciprocal translocation element containing an A centromere and the distal portion of the B chromosome (the A-B chromosome) (Roman, Genetics 32:391-409, 1947). In order for the B-A chromosome to non-disjoin, an A-B chromosome must be present in the same cell, suggesting that a trans-acting factor resides near the distal tip of the B chromosome long arm (Roman, Genetics 35:132, 1950).

Translocation TB-4Lb resulted from an interchange involving the long arm of chromosome 4 and the B chromosome (Beckett, MNL 56:47, 1982). The breakpoint on the B chromosome is very near the tip of the long arm so that the B-A chromosome contains almost the entire B chromosome (Figure 1). To determine whether

Figure 1. (A) shows a mitotic chromosome spread from a TB-4La tertiary trisomic plant that is hybridized with Cent4 in red and the B chromosome specific element, ZmBs, in green. The arrow heads indicate the location of the Cent4 element and the arrow points to the centromere of the TB-4Lb chromosome. Cent4 also hybridizes to the 180 bp knob element allowing confirmation that two intact copies of chromosome 4 are present (The knob on 4L is labeled with Cent4). (B) is a mitotic chromosome spread from a euploid heterozygote TB-4Lb plant labeled with the same probes as (A). The 4Lb-B translocation chromosome is identified by the smaller size and absence of the knob on the long arm. In (C), FISH was performed on the pollen from the individual in (A) using the 180 bp knob repeat (in green) and the ZmBs element (in red). All three nuclei are labeled by the ZmBs element (seen more clearly in (C�) where only the red signal is presented) indicating that nondisjunction did not occur in the development of this pollen grain. (D) shows FISH on a pollen grain from the individual in (B). About half of the pollen from the euploid heterozygote individuals which had a ZmBs signal showed non-disjunction and about half did not consistent with expectations.

the physical location of the trans-acting factor on the B chromosome long arm was proximal or distal to the breakpoint, we tested the ability of the B-4Lb chromosome to undergo non-disjunction without the reciprocal 4Lb-B chromosome.

B-4Lb is particularly interesting because its breakpoint is distal to a small region near the tip of the long arm of B that is enriched for the B-specific sequence (Lamb et al., Chromosoma 113:337-349, 2005) (Figure 1), which is primarily present in and around the B centromere (Alfenito and Birchler, Genetics 135:589-597, 1993; Jin et al., Plant Cell, 2005). This B-specific region could be involved with the trans-acting effect on the action of the B centromere, given their similarity in sequence. If non-disjunction results from an interaction between the two sites of B specific repeats, then the B-4Lb chromosome should exhibit non-disjunction in the absence of the 4-B chromosome.

For many B-A translocations, including TB-4Lb, pollen containing the B-A chromosome and a normal chromosome 4 will not succeed in pollinating a tester line because the A chromosome segment on the B-A chromosome is present in two copies, reducing the competitive ability of the pollen (Auger and Birchler, J. Hered. 93:42-47, 2002). Because plants cannot be recovered that contain the B-4Lb chromosome from a test cross using a plant with two intact copies of chromosome 4 and a B-4Lb chromosome, we performed fluorescent in situ hybridization on pollen grains to assay directly the ability of TB-4Lb to undergo non-disjunction.

Plants that contained two intact copies of chromosome 4 and the B-4Lb chromosome (called tertiary trisomics), as well as plants containing one intact chromosome 4, one B-4Lb and one 4-B chromosome (euploid heterozygotes), were selected from progeny of a euploid heterozygote crossed as a female by a tester line. The karyotype of each plant was determined by examining mitotic chromosome spreads using a combination of FISH probes made from DNA elements specific to chromosome 4 (Cent4), centromeres (CentC), and the B chromosome (ZmBs) (Figure 1). See Kato et al. (Proc. Natl. Acad. Sci. USA 101:13554-13559, 2004) for a further description of the probes.

Mature pollen from both genotypes was fixed in 3:1 acetic acid:ethanol for 24 hours and then stored in 70% ethanol at -20�C. Pollen was rinsed in 2XSSC and then suspended in a probe mixture containing the B-specific element (ZmBs) and the 180 bp knob repeat in 2XSSC, 50% formamide and heated to 95C for 5 minutes. After heating, the pollen was incubated in the probe mixture at 37C for 24 hours. The pollen was suspended in VectaShield containing DAPI to stain the DNA, dropped onto slides, and covered with a cover slip. After waiting one hour for the DAPI to penetrate the pollen walls, the slides were examined with a fluorescent light microscope and images captured using a Magnafire CCD camera.

Over 100 well-labeled pollen grains from both the tertiary trisomic and the euploid heterozygote were examined for the presence of the B-specific probe signal in one or both sperm nuclei. The 180 bp knob probe serves as a positive control to confirm that the probe had penetrated into and hybridized to the three pollen nuclei. In the tertiary trisomic, about half of the pollen contained signals from the B-specific element and in every case both sperm nuclei contained the B-specific signal. About half of the pollen from the euploid heterozygote contained the B-specific probe and of these, half of them showed signal in both sperm. The remaining quarter of the total pollen had signal in only one of the two sperm, which is an indication of non-disjunction (Figure 1).

The pollen FISH procedure allows direct visualization of the results of B non-disjunction in the pollen grain. This allowed us to determine that the B-4Lb chromosome alone is incapable of non-disjunction. This result indicates that the trans-acting factor responsible for B non-disjunction is distal to the breakpoint of TB-4Lb and does not involve the distal ZmBs region of the B chromosome (Figure 1A). Thus, the mechanism for B non-disjunction does not involve interaction between the centromeric and distal B-specific sequences, and the trans-acting factor resides at a very distal position on the chromosome.

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