Molecular characterization of meiotic recombination across the 140-kb multigenic a1-sh2 interval of maize

Abstract

The 140-kb a1-sh2 interval of the maize genome contains at least four genes (a1, yz1, x1, and sh2). Partial sequence analysis of two haplotypes has revealed many single nucleotide polymorphisms and InDel polymorphisms, including several large structural polymorphisms. The physical positions of 101 meiotic recombination breakpoints are not distributed uniformly across the interval and are instead concentrated within three recombination hot spots. Two of these recombination hot spots are genic (a1 and yz1) and one is apparently nongenic. The x1 gene is not a recombination hot spot. Thus, these results suggest that not all hot spots are genes and indicate that not all genes are hot spots. Two of the 101 recombination events arose by means of either noncrossover events involving conversion tract lengths of at least 17 kb or double-crossover events. Only one recombination breakpoint mapped to the approximately 80-kb distal portion of the a1-sh2 interval that contains large amounts of repetitive DNA including retrotransposons; in this region the ratio of genetic to physical distance is less than 0.5% of the genome's average. These results establish that the retrotransposon faction of the maize genome is relatively inert recombinationally.

Figure 1

Physical and genetic characterization of the a1-sh2 interval. (A) Genetic organization of the a1-sh2 interval on chromosome 3L. (B) A restriction map of the 140-kb a1-sh2 interval includes rare cutting restriction enzyme sites (labeled vertical bars; N = NotI; P = PacI; A = AscI; S = SfiI) and EcoRI sites (unlabeled short vertical bars). The a1 and sh2 genes are not drawn to scale. The sizes of the individual cosmids that comprise a contig of the a1-sh2 interval are shown (the proximal end of this contig is located within TD2, Fig. 2). Unlabeled short vertical bars on the cosmids represent the corresponding rare cutting restriction enzyme sites indicated in the restriction map. Shaded horizontal bars (not drawn to scale) represent RFLP markers.

Figure 2

The distribution of breakpoints associated with the 101 recombinants across the 140-kb a1-sh2 interval. The positions of six RFLP markers, php10080, a1, 9-10a5, x1, 2-32a2, and sh2 are indicated relative to the proximal and distal ends of chromosome 3. Intervals I–XIII are defined by the positions of RFLPs and IDPs. Interval IV consists of 1.7 kb in the A1-LC Sh2 haplotype and 0.4 kb in the a1∷rdt sh2 haplotype. Interval V is approximately 80 bp in both haplotypes. The numbers of recombinants that map to each individual interval and the resulting ratios of genetic to physical distance (cM/Mb) are shown. Figure is not drawn to scale. E = EcoRI. B = BglII.

Figure 3

High-resolution mapping of recombination breakpoints within (A) the a1 gene (interval II), (B) the IR (interval VI), (C) the yz1 gene (interval VIII), and (D) the x1 gene (interval X). The A1-LC Sh2 haplotype (gray boxes) is positioned above the a1∷rdt sh2 haplotype (dotted boxes). Vertical bars and parentheses represent DNA sequence polymorphisms between the two haplotypes; IDPs indicated by parentheses were used to design allele-specific primers. The widths of vertical bars are proportional to the numbers of bases in a polymorphism. Polymorphisms in the region flanked by primers a1rdt3273 and a1rdt1541 (B) were confirmed to exist in the A1-LC Sh2 haplotype by sequencing the 34 recombinant alleles that mapped to the IR. The region from the A1-LC Sh2 and a1∷rdt sh2 haplotypes that are flanked by primers XL1 and x502 (D) were sequenced. Primers used in PCR and sequencing are indicated by horizontal arrows. Universal primers are indicated in italics. Primers used for RT-PCR are underlined. Allele-specific primers are positioned close to the haplotypes they amplify. The numbers of recombination breakpoints that resolved within each interval and the resulting ratios of genetic to physical distance (cM/Mb) are shown. The triangle represents the rdt transposon insertion in the a1∷rdt allele. Restriction enzyme sites used in cleaved amplified polymorphic sequence analyses are indicated.