Gene conversion between direct noncoding repeats promotes genetic and phenotypic diversity at a regulatory locus of Zea mays (L.)

Abstract

While evolution of coding sequences has been intensively studied, diversification of noncoding regulatory regions remains poorly understood. In this study, we investigated the molecular evolution of an enhancer region located 5 kb upstream of the transcription start site of the maize pericarp color1 (p1) gene. The p1 gene encodes an R2R3 Myb-like transcription factor that regulates the flavonoid biosynthetic pathway in maize floral organs. Distinct p1 alleles exhibit organ-specific expression patterns on kernel pericarp and cob glumes. A cob glume-specific regulatory region has been identified in the distal enhancer. Further characterization of 6 single-copy p1 alleles, including P1-rr (red pericarp/red cob) and P1-rw (red pericarp and white cob), reveals 3 distinct enhancer types. Sequence variations in the enhancer are correlated with the p1 gene expression patterns in cob glume. Structural comparisons and phylogenetic analyses suggest that evolution of the enhancer region is likely driven by gene conversion between long direct noncoding repeats (approximately 6 kb in length). Given that tandem and segmental duplications are common in both animal and plant genomes, our studies suggest that recombination between noncoding duplicated sequences could play an important role in creating genetic and phenotypic variations.

Figure 1.—

(A) Phenotypes of the natural p1 alleles. Mature ear pigmentation patterns specified by the p1 alleles: P1-rw1077, P1-rwCFS302, and P1-rwCFS342 (top row from left to right) and P1-rr1088, P1-rrCFS36, and P1-rr4B2 (bottom row from left to right). All alleles are homozygous. (B) DNA gel blot analyses on the p1 simplex alleles: lane 1, P1-rw1077; lane 2, P1-rwCFS302; lane 3, P1-rwCFS342; lane 4, P1-rr4B2; lane 5, P1-rr1088; and lane 6, P1-rrCFS36. Genomic DNA was digested with SalI and hybridized with the probe, p1 genomic fragment 15. The 1.2-kb band in P1-rr4B2 is a doublet, while P1-rr1088 and P1-rrCFS36 have only one copy of the 1.2-kb fragment.

Figure 1.—

(A) Phenotypes of the natural p1 alleles. Mature ear pigmentation patterns specified by the p1 alleles: P1-rw1077, P1-rwCFS302, and P1-rwCFS342 (top row from left to right) and P1-rr1088, P1-rrCFS36, and P1-rr4B2 (bottom row from left to right). All alleles are homozygous. (B) DNA gel blot analyses on the p1 simplex alleles: lane 1, P1-rw1077; lane 2, P1-rwCFS302; lane 3, P1-rwCFS342; lane 4, P1-rr4B2; lane 5, P1-rr1088; and lane 6, P1-rrCFS36. Genomic DNA was digested with SalI and hybridized with the probe, p1 genomic fragment 15. The 1.2-kb band in P1-rr4B2 is a doublet, while P1-rr1088 and P1-rrCFS36 have only one copy of the 1.2-kb fragment.

Figure 2.—

Structural comparisons of the simplex p1 alleles. The solid boxes connected by thin lines represent exon regions. The open boxes with solid arrows indicate long direct repeats in 5′ and 3′ noncoding regions. The bent arrow represents a transcription start site (Grotewold et al. 1991). The hatched boxes represent 5′-UTR and 90-bp promoter regions conserved among the p1 alleles. The open boxes labeled with C indicate a 1.1-kb region, termed fragment C, present at the 5′ direct repeats of all p1 alleles, but absent from the 3′ direct repeats in several p1 alleles. The numbered open boxes represent the sequences identified in the text as fragment 15 and fragment 14. The enhancer-containing regions in each p1 allele are highlighted by solid bars, which range from 1.6 kb (in the P1-rr alleles) to 602 bp (in the P1-rwCFS302 and P1-rwCFS342 alleles). The cob glume-specific regulatory region is indicated as shaded boxes in the enhancer-containing regions of the P1-rr alleles (which include the 189-bp sequence from fragment 15 and the 197-bp immediate downstream sequence). The regions marked 15* indicate partial fragment 15 sequences, which contain only the first 200 bp fragment 15 sequences. The triangles located in the first copy of fragment 15 in the 5′ noncoding repeats of P1-rr4B2 and P1-rrCFS36 represent insertions of a 1.6-kb transposon-like sequence. The bracketed regions marked 1 and 2 indicate 549- and 148-bp deletions, respectively, in the noncoding repeats of P1-rwCFS342. The solid arrows represent primers used to amplify 5′ and 3′ noncoding regions of p1: 1, P1rr-18; 2, PA-B4; 3, P1rr-14; 4, PA-B6; 5, EP5-16; 6, P1rr-16; 7, P1rr-13r; 8, P1rr-30; 9, P1rr-16r; 10, P1rr-29; 11, P1rr-32; and 12, P1rr-25. Positions of the restriction site, SalI, are shown on each p1 allele (only unmethylated SalI sites that flank fragment 15 are indicated on the map).

Figure 3.—

Phylogenetic analysis of the p1 distal enhancer regions. A rooted 50% majority-rule consensus maximum-likelihood tree was generated by using a 602-bp sequence, which includes fragment 15 plus a 197-bp downstream sequence. The sequence from the maize wild relative, teosinte parviglumis, was used as the outgroup. The numbers at nodes represent bootstrap values derived from 500 replicates. Sequences from the 5′ and 3′ noncoding repeats are identified as upstream or downstream, respectively. Sequences from the same repeat are indicated in numeric order from 5′ to 3′.

Figure 4.—

Nucleotide diversity analysis of noncoding regions at the p1 locus. Open boxes with solid arrows represent the 5′ and 3′ noncoding direct repeats. The position of fragment 15 is indicated with the numbered box. The solid boxes connected with thin lines represent the exon and intron structure of the p1 locus. The triangle in the second intron indicates the 723-bp sequence that is also subject to nucleotide diversity analysis. The solid arrows represent the primers for amplification of the 723-bp region: 1, 723-5; 2, 723-3.

Figure 5.—

Sequential model for p1 evolution. The stepwise progression from p progenitor gene to the distinct p1 alleles is shown. The open boxes with solid arrows indicated the noncoding direct repeats flanking the p1 coding sequence. The numbered open boxes are the same as those shown in Figure 2. The solid boxes indicate exons of the p1 and p2 genes. The vertical arrows between the p1 and p2 regions represent retroelement insertions that separated the p1 gene from p2 (Zhang et al. 2000). In each step, the rearranged regions are highlighted by solid bars. The divergence time between the p1 alleles is indicated at the branch points.