Inbreeding drives maize centromere evolution

Abstract

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000-95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems.

Keywords: centromere drive; centromere paradox; founder effect; hemicentric inversion; linkage disequilibrium.

Fig. 1.

Functional centromeres are spatially correlated with CR2 insertions. (A) Anti–cenH3 ChIP-seq coverage at CEN5 delineates the functional centromeres of a diverse set of maize inbreds, including representatives of the blue, green, brown, orange, and red lineages, as well as inbreds IL14H, P39, and CML333 (and others in SI Appendix, Fig. S1). Only CML333 uses CEN5M exclusively. Expressed genes [fragments per kilobase of transcript per million mapped reads (FPKM) >10 in shoot apical meristem and ear; triangles] are primarily located in cenH3-free regions, whereas genes within the functional centromeres are generally not expressed (SI Appendix, Fig. S8). (B) CEN5M of the B73 reference genome is marked by CentC (green) and ancestral CR1 elements, but the functional centromere of B73 is located at CEN5L. Many more junctions (see C and D) than datable CR elements are identified owing to the incomplete assembly of the reference genome in these repeat-rich regions. (C) Ancestral elements (most of them CR1s) are clustered around CEN5M (indicated by stippled lines in A). Two clusters of ancestral elements (enclosed in stippled boxes) and other ancestral CR1 and CR2 elements associated with small upstream CentC clusters were likely relocated from CEN5M by small inversions. (D) CR2 elements shared by multiple maize inbreds inserted into their common ancestor, allowing the grouping of both inbreds and CR2 elements into colored lineages. Recently inserted lineage-specific CR2 elements identified in GSS or ChIP-seq data are found almost exclusively in neocentromeres (SI Appendix, Fig. S6). Unique CR2 insertions are those found in only one of the maize inbreds examined here. A presumed inversion (purple double-headed arrow) specific to the blue-b lines (Fig. 2) was inferred from lineage-specific CR2 elements (SI Appendix, Fig. S6). (E) Three distinct nonrecombinant CEN5 regions were identified (SI Appendix, Fig. S7) that allow phylogenetic reconstruction of CEN5 in the 28 maize inbreds examined.

Fig. 2.

Microevolution of Zea CEN5L-M. Phylogeny of domesticated maize and teosinte (TIL) inbreds reveals two major lineages that diverged 18.3 ka, the blue/orange/brown lineage (related to TIL14) and the red/green lineage (related to CML333, which acquired its CEN5L-M region, including the CentC-rich CEN5M, by recombination). The number of CR2 elements inserted into CEN5L (blue), CEN5M (green), or CEN5R (red) (SI Appendix, Fig. S6) is marked above each branch, and the shift of CR2 insertions from CEN5M to either neocentromere identifies the time interval of cenH3 movement (thick branch). Neocentromere formation in all five colored lineages postdates domestication, whether measured as maximum leaf–leaf (nodes marked with arrows), average leaf–node (indicated below neocentromere branch) or individual leaf–node distances (branch lengths given in SI Appendix, Table S3). Z. mays ssp. mexicana lines TIL08 and TIL25 (26) are intermingled with the 15 Z. mays ssp. parviglumis lines, indicating centromere movement among these subspecies. Deletions shared by multiple lines are indicated on the corresponding branch (SI Appendix, Fig. S5). Nodes with >90% bootstrap support are marked by dots. The neighbor-joining tree was constructed from HapMap2 SNP data and background calls and artificially rooted on Z. luxurians. Tree is drawn on two scales (black and gray). Ancestral CR2 insertions are in black irrespective of insertion site. Hi47, a red-B line, and Hi60, which contains 50 line-specific CR2 insertions into CEN5R (SI Appendix, Dataset S1), cannot be displayed on this tree because their CEN5L-M region was not sequenced.

Fig. 3.

Tropical inbreds contain higher amounts of CentC at their CEN10 than comparable temperate inbreds. The CentC content of CEN10 is highly variable, but in general, temperate lines (blue) contain less CentC than tropical lines (red). Nine CEN10 haplotypes for the region spanning both CentC clusters are represented by the maize inbreds examined (SI Appendix, Fig. S31), and direct comparisons between tropical and temperate inbreds sharing the same haplotype are most informative. The temperate representative of haplotype 1 (MS71) contains less CentC at CEN10 than its tropical counterpart CML247; the estimated divergence time was 2.5–5.8 ka (SI Appendix, Fig. S32). Similarly, both temperate M162W and TX303 contain less CentC than their tropical relative CML322 (estimated divergence time, 3.1–4.5 ka). CentC reduction at CEN10 of tropical TZI8 is likely related to a founder effect of this Nigerian inbred. Other temperate/tropical comparisons are less meaningful because they involve different predomestication haplotypes, but confirm the general trend. The five tropical clade A inbreds and the related KI11 contain more CentC than the ten temperate clade A lines from which they are estimated to have diverged 21.8 ka. The three tropical lines CML277, CML69, and NC358 contain greater amounts of CentC than the temperate lines MO17 and OH7B. The sole apparent exception to the rule is tropical CML103, which appears to have acquired its temperate CEN10 containing very little CentC through recombination (between the two CentC clusters) with a temperate haplotype 8 line (SI Appendix, Fig. S31). Inbreeding of temperate lines near CEN10, likely owing to a linked improvement locus related to growth in temperate environments, has led to a decrease in the amount of CentC in CEN10 of temperate lines.

Fig. 4.

Low genetic diversity near most centromeres indicates selection for centromere-linked genes. Sequence diversity along maize chromosomes was assessed by Tajima’s D, the observed minus expected site frequency spectra for maize (black) and teosinte (gray); maximum distance, the divergence date of the most distantly related maize inbreds; TIL descendants, the number of teosinte inbreds contained within the ancestral node shared by all domesticated maize lines; and number of maize lineages, the number of distinct maize lineages at 15 ka. Centromere locations determined by ChIP-seq are highlighted in green (CEN1, CEN5M, and CEN7), blue (CEN5L), and red (CEN5R). CEN1 is the region of lowest sequence diversity on chromosome 1 by all four measures. On chromosome 5, region D (yellow highlight just downstream of CEN5) has undergone a selective sweep in all 28 maize lines, but not teosinte. The low Tajima’s D in CEN5 is caused by rare recombinants arising from crossovers between CEN5 and region D that gave rise to all colored lines. For accurate representation, maize lines carrying deletions (purple lines) within and flanking region D were excluded from trees constructed from these regions (SI Appendix, Text S2). On chromosome 7, the centromere proper (green highlight) does not exhibit any of the characteristics of reduced genetic diversity seen in all other chromosomes (SI Appendix, Fig. S18); however, it is flanked by two regions with low Tajima’s D (blue arrows) that are correlated with a decrease in the number of maize lineages at 15 ka. Negative selection of the haplotypes overrepresented in these two regions at CEN7 appears to contribute to the high diversity and CentC retention rate in this centromere (SI Appendix, Text S1). The y-axis scales are linear, except in the bottom panel of each chromosome. HapMap3.1 SNP calls were used for regions lacking HapMap2 data (SI Appendix, Table S17).

Fig. 5.

Genetic diversity is reduced at most centromeres of Z. mays ssp. mays. The number of haplotypes with an estimated divergence date of >10 ka is given for each of the 10 maize centromeres and three published domestication loci known to have undergone a postdomestication sweep for each of three datasets containing 28, 51, or 895 maize lines. The 10 centromeres are divided into three groups based on the number of haplotypes represented in the 28 I dataset and the percentage of all maize lines carrying the dominant haplotype in the HapMap3 dataset (30). All three datasets show very low genetic diversity at CEN1, CEN4, and CEN6 similar to that seen at the domestication loci. The middle group of four centromeres contains two dominant haplotypes. CEN5, CEN7, and CEN10 contain five or more haplotypes, with CEN7 exhibiting the greatest diversity of haplotypes. Haplotypes are classified based on the phylogenetic trees in Fig. 2 and SI Appendix, Figs. S11, S15, S24, and S29. Data are summarized in SI Appendix, Table S18. Color code: blue, red, green, proportions of the first, second, third, etc., most abundant haplotypes in the dataset; black, haplotypes not present in dataset 28 I. Maize lines: 28 I, 28 improved inbreds, including 25 NAM lines (23), B73, Mo17, and W64A; 28 I + 23 L, dataset 28 I plus 23 landraces (all HapMap2 data); 895 HM3, 895 maize lines of the HapMap3 dataset. Domestication loci: p2, pericentromere 2 (91.5–91.6 Mb); 10 L, chromosome 10 sweep (27); D, chromosome 5 region D (9).