Aluminum tolerance in maize is associated with higher MATE1 gene copy number

Abstract

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world's potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

Fig. 1.

Localization of MATE1 in maize lines L53 and Al237 using FISH. Homologous metaphase chromosomes from root meristem cells are aligned as pairs. (A) L53, Al-sensitive, one copy of MATE1. (B) Al237, Al-tolerant, three copies of MATE1. A large arrowhead indicates the location of MATE1 on 6S; small arrowheads indicate minor signals. (C) Multiple MATE1 FISH signals are observed on Al237 chromosome 6S. Multicolor image and grayscale version of the respective red channel are shown. The arrowheads indicate regions with two MATE1 signals per chromatid. The MATE1 probe is labeled with Texas Red. Green fluorescein isothiocyanate-labeled probes include centromere C repeat sequences (found on all chromosomes but copy number highly variable), a TAG microsatellite repeat (chromosome arms 1L, 2S, 2L, and 4S), and the ribosomal gene cluster (6S at the nucleolar organizing region). Blue DAPI counterstain was used to visualize heterochromatic 180-bp knob regions (line-specific locations). The second karyotype shown in each panel is the grayscale version of the MATE1 probe (red) channel in the respective multicolor karyotype. Adjustment of individual color channels was performed on original images. (Scale bars, 3 μm.)

Fig. 2.

MATE1 CNV in Al237×L53 RILs correlates with aluminum tolerance. (A) MATE1 copy number in the RIL population derived from a cross between Al237 (Al-tolerant, three copies of MATE1) and L53 (Al-sensitive, one copy of MATE1), determined by qPCR. RILs are arranged based on copy number in ascending order (from one copy to three copies). The red bars denote single-copy RILs (n = 57); the blue bars denote RILs with three copies of MATE1 (n = 53). Error bars indicate 1 SD. (B) One-way ANOVA of RRG (standard phenotypic index of Al tolerance) by MATE1 copy number in the Al237×L53 RIL population. Mean diamonds represent 95% confidence intervals, and midbars in diamonds represent the group mean. The gray line represents the overall mean.

Fig. 3.

MATE1 expression in the Al237×L53 RIL population correlates with gene copy number. (A) MATE1 relative expression in root tips quantified via qPCR from maize seedlings grown under control conditions (Left), or treated with 39 µM Al³⁺ activity (Right) for 24 h. In each graph, maize lines are arranged based on MATE1 relative expression in ascending order. The bars are color-coded based on MATE1 copy number: the red bars denote one-copy RILs, and the blue bars denote three-copy RILs. The gray bars denote the parents of the population, Al237 (Al-tolerant, three copies of MATE1) and L53 (Al-sensitive, one copy of MATE1). Samples were calibrated against L53 (relative expression = 1) within each dataset. Error bars indicate 1 SD. (B) One-way ANOVA of MATE1 relative expression by MATE1 copy number in the Al237×L53 RIL population, in control and Al stress conditions. Mean diamonds represent 95% confidence intervals, and the midbars in diamonds represent the group mean. The gray line represents the overall mean.

Fig. 4.

A major aluminum tolerance QTL and eQTL for MATE1 expression colocalize with MATE1. A GLM analysis identified GBS-generated SNP markers that are associated with Al tolerance (expressed as RRG; red circles, y axis on Left) and with MATE1 expression (open triangles: expression under control conditions; solid triangles: expression under Al treatment; y axis on Right). The level of statistical association for each polymorphism is expressed as –Log P. x axis: physical position on maize chromosome 6. Inset is a magnification of a 40-Mb window on the top of chromosome 6. A gray bar indicates the location of the MATE1 locus.

Fig. 5.

Genomic organization of the MATE1 locus shows three tandemly arrayed gene copies. Diagram showing Al237 genomic BAC ZMMCBa0006o22. The triplicated regions are indicated by brackets. The three copies of MATE1 are indicated by solid red boxes. The dashed boxes of different colors indicate different families of retrotransposons (annotated using http://maizetedb.org/~maize). The unique Gypsy retrotransposon insertion upstream of MATE1-2 is indicated by a solid orange box. Except for MATE1-1, -2, and -3, no other genes are predicted within the BAC sequence according to MAKER (45). The scale bar is in kilobase pairs.

Fig. 6.

Maize inbred lines carrying three MATE1 copies also show high MATE1 expression. (A) Distribution of Al tolerance (i.e., RRG) in maize diversity panel. The Al tolerance of lines in which MATE1 expression was quantified are indicated (L = L53; B = B73; IL = Il677a; Al = Al237; C = C100-6). The number in parenthesis indicates MATE1 copy number. (B) MATE1 relative expression quantified via qPCR in root tips of maize inbred lines from a survey for MATE1 CNV. Seedlings were grown under control conditions (Upper), or treated with 39 µM Al³⁺ activity (Lower) for 24 h. The red bars denote lines with one MATE1 copy (L53 and B73, Al-sensitive), and the blue bars denote three-copy lines (Al237, C100-6, and IL677a, Al-tolerant). Error bars indicate 1 SD.