Haplotype structure strongly affects recombination in a maize genetic interval polymorphic for Helitron and retrotransposon insertions

Abstract

We have asked here how the remarkable variation in maize haplotype structure affects recombination. We compared recombination across a genetic interval of 9S in 2 highly dissimilar heterozygotes that shared 1 parent. The genetic interval in the common haplotype is approximately 100 kb long and contains 6 genes interspersed with gene-fragment-bearing Helitrons and retrotransposons that, together, comprise 70% of its length. In one heterozygote, most intergenic insertions are homozygous, although polymorphic, enabling us to determine whether any recombination junctions fall within them. In the other, most intergenic insertions are hemizygous and, thus, incapable of homologous recombination. Our analysis of the frequency and distribution of recombination in the interval revealed that: (i) Most junctions were circumscribed to the gene space, where they showed a highly nonuniform distribution. In both heterozygotes, more than half of the junctions fell in the stc1 gene, making it a clear recombination hotspot in the region. However, the genetic size of stc1 was 2-fold lower when flanked by a hemizygous 25-kb retrotransposon cluster. (ii) No junctions fell in the hypro1 gene in either heterozygote, making it a genic recombination coldspot. (iii) No recombination occurred within the gene fragments borne on Helitrons nor within retrotransposons, so neither insertion class contributes to the interval's genetic length. (iv) Unexpectedly, several junctions fell in an intergenic region not shared by all 3 haplotypes. (v) In general, the ability of a sequence to recombine correlated inversely with its methylation status. Our results show that haplotypic structural variability strongly affects the frequency and distribution of recombination events in maize.

Fig. 1.

Recovery of recombination products in the bz1-Ac7077 and Ac7077-sh1 intervals. The cartoon depicts the parental (Left) and recombinant (Right) phenotypes. The contrasting kernel phenotypes are: Bz, purple; bz-m, spotted; and bz, bronze; Wx, nonwaxy and wx, waxy, which stain dark and light, respectively, with an I-KI solution, as illustrated at the top right of each kernel (although no staining is necessary when scoring bz Sh seed); Sh, plump; and sh, shrunken. Recombination in the bz-m2(D1)-Ac7077 interval gives rise to wx bz Sh and Wx Bz sh reciprocal recombinants. Recombination in the Ac7077-sh1 interval gives rise to wx bz-m Sh and Wx Bz sh reciprocal recombinants. The physical distance between Ac7077 and bz in B73 is from ref. ; that between sh and Ac7077 is from the maize genome sequencing project (www.maizesequence.org). These will vary from inbred to inbred and are shown for relative purposes only. Table 1 shows the seed populations screened for the Ac7077/W22 and Ac7077/B73 heterozygotes and the number of recombinants isolated in each class.

Fig. 2.

Distribution of recombination junctions in the Ds2(D1)-Ac7077 genetic interval among Sh bz recombinants from bz-m2(D1) Ac7077/Bz-W22 + (Upper) and bz-m2(D1) Ac7077/Bz-B73 + (Lower) heterozygotes. Each haplotype is identified by the name of the genetic line, followed by the physical size of the interval under study, in parentheses. The 9S centromere (proximal side) is to the left; the 9S telomere (distal side), to the right. Genes are shown as pentagons pointing in the direction of transcription; exons are in bronze, and introns in yellow. There are 7 genes in the genetic interval: bz1, stc1, rpl35A, tac6058, hypro1, znf, and tac7077. The same symbolism is used for gene fragments carried by Helitrons (Hels), which are represented as bidirectional arrows below the line for each haplotype. The vacant sites for HelA and HelB in B73 are provided as reference points and marked with short vertical strokes. Dashed lines represent deletions. Retrotransposons are indicated by solid triangles of different colors. DNA transposons, including Ds2(D1) and Ac7077, are indicated by open triangles of red color. Small insertions are indicated in light blue color. Only the genes have been drawn to scale. For analysis, the interval was subdivided into a series of roughly corresponding segments defined by the same polymorphic sites, where possible, or by polymorphisms located close to each other in each heterozygote. The locations of the different polymorphisms are numbered according to the bz-m2(D1) Ac7077 sequence, starting with the NotI site in the first exon of bz1 as position 1. The nature of the polymorphisms at each location (SNPs or indels) is identified in Tables S1 and S2. The number of recombination junctions in each subinterval is shown above (W22) and below (B73) the unique haplotype in each heterozygote. See Results for additional details.

Fig. 3.

Summary of C-methylation analysis in B73 (A) and Ac7077 (B) haplotypes. The results from methylation-sensitive restriction endonucleases are represented with a series of geometric symbols, whose meaning is described in the box at the top right of the figure. The probes used to hybridize the blots are shown as bars beneath each haplotype. The C-methylation status at the 3′ end of HelA in Ac7077 was determined by bisulfite sequencing. Unmethylated sites are represented as open stars and methylated sites, as filled stars. The C-methylation status of W22 sites between Ds2(D1) and HelB was essentially identical to Ac7077.