Centromere function and nondisjunction are independent components of the maize B chromosome accumulation mechanism

Abstract

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.

Figure 1.

Genetic Test of Nondisjunction of 9Bic-1. Plants containing two B chromosomes, one standard chromosome 9, and one chromosome 9Bic-1, both with the dominant color marker C1, were crossed reciprocally with c1 tester plants. The left ear in (A) is from a 9Bic-1 (C1)/ 9 (C1) + 2B plant pollinated by the c1 tester. All the resulting kernels are fully colored. The right ear is from a c1 tester plant crossed with pollen from the 9Bic-1 (C1)/9 (C1) + 2B plant. When the 9Bic-1 chromosome nondisjoins, it places two copies of C1 in a single sperm and no copies in the other. Fertilization of the endosperm can occur by either sperm. Kernels with colored endosperm and colorless embryos (B), colorless endosperm and colored embryos (C), fully colored kernels (D), and mosaic endosperms with colored embryos (E), which result from chromosome 9S breakage, are present on the ear to the right. These kernel types are the expected products if 9Bic-1 undergoes nondisjunction in the presence of the normal B chromosomes.

Figure 2.

Possible Outcomes Resulting from Nondisjunction of 9Bic-1 or 7Bic-1. In this illustration, the B chromosome is depicted as a small telocentric chromosome. The magenta circles indicate tandem arrays of the B-specific element, ZmBs, that are present in and surrounding the B centromere. The larger blue chromosome with ZmBs at the tip of the short arm represents either 9Bic-1 or 7Bic-1. The green chromosome illustrates a representative A chromosome. Centromeres from A chromosomes are illustrated with a white circle. During the first pollen division (1), sister chromatids separate and move to opposite poles, producing two cells: the vegetative (v) and the generative (g). Chromosomes in the generative cell are replicated a second time. During the second pollen division (2), nondisjunction of the B chromosome occurs. 9Bic-1 may disjoin properly (3a), nondisjoin and move to the opposite pole as the B chromosomes (3b), nondisjoin and move to the same pole as the B chromosomes (3c), or break in the short arm as the active A centromeres move to opposite poles but the inactive B centromeres are adhered together. The different outcomes of the second division produce a variety of different sperm (4). Chromosome breakage produces a large fragment containing 9L, the centromere of 9, and a small portion of 9S in one sperm (s) and a small fragment from the short arm of 9Bic-1 in the other (4d). Because the small fragment does not contain an active centromere, it will be lost in the cell divisions that follow fertilization unless it acquires centromere activity (data not shown), or it becomes attached to another chromosome (5). Either of the two sperm depicted in each pollen grain can fertilize the egg (giving rise to the embryo) or the polar nuclei (giving rise to the endosperm). Kernels representing fertilization of the egg by the first sperm or the second sperm are depicted (6). The color of the embryo and endosperm depends on the presence of the C1 allele on 9Bic-1. When chromosome breakage occurs, if the breakpoint is distal to C1, the fragment containing 9L, the centromere, and C1 will enter the chromatid-type BFB cycle, producing a mosaic color pattern in the endosperm (6d). If breakage occurs proximal to C1, the chromatid entering the BFB will not produce any color, so the kernels will appear the same as in 6a.

Figure 3.

Pollen FISH Analysis of Nondisjunction. Pollen FISH was performed with the ZmBs probe (magenta) on microsporocytes at the three-cell stage and counterstained with 4′,6-diamidino-2-phenylindole (blue). Arrowheads indicate ZmBs signals from the B centromere region. The small ZmBs signal from the B chromosome tip is not indicated. The pollen in (A) was obtained from a plant with two B chromosomes. One signal is observed in the vegetative nucleus (v). Nondisjunction places two B chromosomes in one sperm (s). ZmBs signal is seen in one of the sperm and not the other. Only one ZmBs centromere signal is typically observed in the sperm, although two can occasionally be distinguished, suggesting that the sister centromeres have not separated. The pollen in (B) contains a single 9Bic-1 chromosome. In the absence of the B chromosome long arm, 9Bic-1 disjoins properly and each sperm receives a copy of the chromosome. Because the 9Bic-1 chromosome contains an inactive B centromere region, ZmBs signals are present in all three nuclei. (C) to (F) show observed outcomes for pollen containing a 9Bic-1 chromosome and an intact B chromosome. 9Bic-1 may disjoin properly (C), nondisjoin and both copies move to the same sperm as do the B chromosomes (D), or move to the opposite sperm (E). In immature pollen, pairs of sperm were occasionally observed that were connected by a long thin strand of DNA (F).

Figure 4.

Somatic Chromosome Spreads Demonstrating Nondisjunction. In all images except (B) and (D), the B-specific sequence, ZmBs, is labeled in magenta and the 180-bp knob repeat is labeled in green. Magenta arrows indicate B chromosomes, and yellow arrows indicate inactive B centromeres from 9Bic-1, 8Bic-1, or 7Bic-1. (A) to (F) Progeny of ♀ c1/c1 × 9(C1)/9Bic-1(C1) + 2B testcrosses include proper disjunction of 9Bic-1 (A), nondisjunction of 9Bic-1, and movement to the same sperm as the B chromosome followed by fertilization of the egg by a sperm without any chromosome 9 or B chromosomes (B) or by the sperm with two B chromosomes and two 9Bic-1 chromosomes (C). Progeny, in which the nondisjoined 9Bic-1 chromosomes moved to the opposite sperm as did the B chromosomes, were recovered but are not shown. Separation of the chromosome 9 centromeres of 9Bic-1 but failure to separate the inactive B centromere can lead to chromosome breakage, producing fragments containing 9L, the centromere from 9, and a portion of 9S. In (D), a spread containing a fragment is labeled with the White Cap probe (magenta), which detects the Zm CCD1 gene that is present as tandem arrays on 9L. The fluorescence intensity of the White Cap signal in FISH preparations reflects the variation in array size present among different lines (B.C. Tan and D.R. McCarty, personal communication). The 9Bic-1 long arm is strongly labeled by the White Cap probe, making it easy to distinguish from the weakly labeled chromosome 9 of the c1 tester line (white arrow). The centromere is labeled with CentC (green). The reciprocal fragment containing the centromere from 9Bic-1 and a small portion of 9S would also be produced but is usually lost since it lacks a functional centromere. However, it can be recovered if it becomes attached to an A chromosome. A chromosome containing such a translocated fragment was identified as chromosome 8 by application of a cocktail of repetitive elements that allows each chromosome to be identified (E). The cocktail includes CentC (green), 5S ribosomal genes (green), TAG microsatellite (magenta), Cent4 (magenta), TR1 (white), 1.1-kb subtelomeric repeat (magenta), subtelomeric repeat 4-12-1 (green), the 180-bp knob repeat (blue and light magenta), 45S ribosomal genes (green), and ZmBs (magenta). The assignment of chromosome 8 was confirmed by serial application of a number of chromosome-specific probes ruling out the other nine chromosomes. A second translocation chromosome was found that has an inactive B centromere on 7S (F). The spread was hybridized with a 22-kD zein B gene probe that labels 7S (white), ZmBs (magenta), and the 180-bp knob repeat (green). The inset shows an enlarged view of the translocation chromosome, including the gray value image of the 22-kD zein B gene probe. The white arrow indicates the site of the 22-kD zein B gene hybridization on the other chromosome 7 homolog. The 7Bic-1 chromosome also nondisjoins. (G) to (I) Chromosome spreads from progeny of a tester plant crossed by a pollen parent containing one copy of the 7Bic-1 chromosome and two B chromosomes include individuals in which the 7Bic-1 chromosome disjoins properly (G) or nondisjoins and can move to the same sperm as the B chromosomes (H) or to the opposite sperm (I). Also recovered but not shown were individuals with only 19 chromosomes resulting from fertilization of the polar nuclei by the sperm with two 7Bic-1 and two B chromosomes.