Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution

Abstract

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Keywords: B chromosome; genetic drive; nondisjunction; preferential fertilization.

Fig. 1.

The maize B chromosome. (A) Root tip metaphase spread of a line possessing nine B chromosomes (red signal). The red signal identifies the ZmBs B chromosome repeat in and around the centromere with a minor representative at the distal tip of the B long arm. Green signal identifies several chromosomal features, namely, the CentC centromeric satellite, 45S ribosomal DNA repeats, and TAG microsatellite clusters. DAPI stains the chromosomes (blue). (B) Schematic view of the acrocentric maize B chromosome at pachynema of meiosis. The chromosome is divided into the B short arm (BS), B centromere (BC), proximal heterochromatin (PH), proximal euchromatin (PE), four blocks of distal heterochromatin (DH1-4), and the distal euchromatin (DE). The B-specific repeat ZmBs, CentC satellite, CRM2 retrotransposon, knob heterochromatin, and TAG microsatellite cluster are color coded along the length of the chromosome. (C) Depiction of nondisjunction of the B chromosome. The B chromosome (blue with a red centromere) is shown in the generative nucleus (G) after the first microspore division. After replication, the two chromatids proceed to the same pole at the second microspore mitosis in the vast majority of divisions. Thus, most mature pollen grains contain two sperm (S) with only one containing the B chromosomes. V: vegetative cell. (D) Depiction of preferential fertilization. For most lines of maize, the sperm with the two B chromosomes will preferentially fertilize the egg (E) as compared with the central cell (C) in the process of double fertilization. The fertilized egg develops into the next generation embryo and the fertilized central cell develops into the endosperm. The combination of nondisjunction at the second pollen mitosis and preferential fertilization comprise the drive mechanism of the B chromosome.

Fig. 2.

Repetitive sequences in the B chromosome. (A) Distribution of repetitive elements and genes along the maize B pseudomolecule. Tandem repeats are clustered mainly at the 5′ pseudomolecule end, i.e., the region close to the centromere: red, ZmBs; gold, 180-bp knob repeat; and dark green, CentC satellite. Transposable elements are evenly distributed along the B chromosome with the exception of the region occupied by tandem repeats. LTR-TEs: light violet, gypsy; dark purple, copia; and blue, unassigned. Occupancy by each class of elements (in percentage of sequence) is given per 500-kb window. For genes, the y axis represents the length of coding sequence. (B) Phylogenetic analysis based on the sequence of the reverse transcriptase domain of elements in both B and A chromosome(s). (C) Abundance analysis of elements of distinct families of LTR-TEs in A chromosomes and (D) the B chromosome sequence of maize (log10 scale). Only families with at least 10 elements were analyzed. Light violet, gypsy superfamily; dark purple, copia superfamily. (E) Estimated time since insertion of elements in the B (blue) and A (red) chromosome(s) in the 20 most abundant families. The age estimation is based on the sequence similarity of 5′ and 3′ LTRs of the respective element. Median values are marked by dots. Note similar age distribution for most families for A and B copies, indicating a shared history. Only elements of age below the 99th age percentile are visualized.

Fig. 3.

Characterization of the B chromosome centromere. (A) Snapshot of the distribution of centromere-specific repeats and CENH3 nucleosomes in the B chromosome pseudomolecule (chrB: 0 to 250,000 bp) and scaffold12456, the largest in the centromeric region. This scaffold maps between the breakpoints of the centromere misdivisions of miniB876 and isoTB-6Lc (Dataset S9). The horizontal scale marks the centromere locus in kilobases. The tracks from the Top to the Bottom are: CENH3-ChIP-seq; input; B-specific repeat ZmBs; centromeric satellite CentC; centromeric retrotransposon CRM. (B) Midpoint positions of CENH3-ChIP-seq (red) and input (blue) reads along the ZmB sequences. Two major and three minor CENH3 nucleosome positions (arrows) are indicated by alignment of the CENH3-ChIP-seq reads. Only two major CENH3 nucleosome positions are observed in the input reads. (C) Characterization of CentC satellite repeats in maize. Violin plot of the sequence identity to the CentC consensus sequence as sampled in the maize A and B chromosomes by the CENH3-ChIP-seq and input reads. **P < 0.001 (two-tailed Student’s t test). The greater difference between the violin plots of CENH3-B versus input-B compared to CENH3-A versus input-A indicates that the CentC in the B centromere is more similar to those in the A centromeres than the dispersed CentC copies in the B long arm. (D) Distribution of the midpoint positions of CENH3-ChIP-seq and input reads from the maize A and B chromosomes along the trimer of CentC satellite consensus sequence. (E and F) Distribution of the midpoint positions of CENH3-ChIP-seq and input reads from maize A and B chromosomes along the CRM1 (E) and CRM2 (F) sequence.

Fig. 4.

Region of the cis factor for nondisjunction. (A) Mini B#20 disjoins regularly itself during the second pollen mitosis. However, in the presence of complete B chromosomes, it undergoes nondisjunction as well, indicating the presence of the cis factor in the sequence of mini B#20 (41). (B) Analysis of the sequence composition of mini B#20 revealed 10 genes and a prevalence of repetitive elements in and around the B centromere. The black line corresponds to the beginning of B chromosome pseudomolecule (chrB v1.0); the gray part represents additional unordered scaffolds belonging to the sequence of mini B#20. The major components of mini B#20 are tandem arrays of ZmBs (red) and 180-bp knob repeats (gold) in the pericentromeric region, which are respectively displayed in the second and third tracks. The following tracks present the CentC satellite (dark green); LTR-TE, copia (dark purple), gypsy (light violet), unassigned (blue); helitron (light green); and TIR (pink). The final track shows the position of the genes. (C) Enlargement of the B chromosome pseudomolecule region (at position 900 to 1,300 kb) containing the border between the segment covered by ZmBs (red) and the 180-bp knob units (gold). The coverage of the region by reads originating from the B73 A chromosomal complement indicates homology of all the sequences but the ZmBs repetitive arrays, illustrating its specificity to the B chromosome. The Upper blue track shows a histogram of mapping density for reads representing 20x coverage of the B73 genome mapped to mini B#20 (with maximum of coverage 61,707). The Lower blue track shows the same data in a log scale.

Fig. 5.

Evolution of the B chromosome sequence. (A and B) Comparison of genes with homologs in the maize and S. bicolor genomes. Comparison of K_a/K_s for homologs in maize A chromosomal complement and S. bicolor (y axis) vs. K_a/K_s for homologs in the maize B chromosome and maize A chromosomal complement (A), and vs. K_a/K_s for homologs in the maize B chromosome and S. bicolor genome (B). Color corresponds to the estimated divergence time of the B gene and the A counterpart. Note the global increase of K_a/K_s when the B chromosome genes are involved (x axis), which indicates a relaxation of purifying selection. (C) Comparison of the maize B chromosome and the B73 A chromosomal complement. Track A: chromosomes and organellar DNA; magnification of the mitochondrial and chloroplastic DNA by 200x and 500x, respectively. Track B: gene density in 5-Mb windows. Track C: density of paralogs of B chromosome genes in 5-Mb windows. Track D: link between B and A paralogs with mitochondrial gene links colored in orange. (D and E) Copy number analysis in disomy or monosomy of TB-4Lb, a translocation between the B chromosome and the long arm of chromosome 4, and haploid controls using the read counts from gene regions. For chromosome 4, TB-4Lb disomy contains two copies of the long arm of chromosome 4 (4, B-4Lb) in an otherwise haploid plant and most of the B chromosome. TB-4Lb monosomy has the chromosome constitution of one copy each of 4Lb-B and chromosome 4 together with the addition of the distal tip of the B long arm. The disomy and monosomy of TB-4Lb show a two-fold higher and lower copy number variation (CNV) on 4L, respectively. No large region with CNV was found on other chromosomes that might result from homology to the portions of the B chromosome present.