Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids

Abstract

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid x euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A-A and/or C-C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

Figure 1.—

Detection of autosyndesis at metaphase I using FISH on pollen mother cells from haploid Darmor-bzh. Mitotic chromosomes of B. oleracea cv. HDEM (a) and B. napus cv. Darmor-bzh (b) are probed with BAC BoB014O06 (green) and counterstained with DAPI (blue), to show that this BAC stains only C genome chromosomes. DAPI staining is then used on anther meiocytes to establish the meiotic behavior of every Darmor-bzh PMC at MI (c, e, and g) and then combined with BoB014O06 FISH signals (d, f, and h) to distinguish autosyndetic and allosyndetic associations. Red and green arrows indicate autosyndetic bivalents between A and C chromosomes, respectively. (d) Seven bivalents including one ring bivalent (four autosyndetic and three allosyndetic) and five univalents. (e) Seven bivalents (two autosyndetic and five allosyndetic) and five univalents. (f) Six bivalents (two autosyndetic and four allosyndetic) and seven univalents.

Figure 2.—

Analysis of chromosome number in the progenies of B. napus haploids. Flow cytometry was used to estimate the chromosome number for 293 haploid-derived offspring. Cytological observations at MI of meiosis on a mean number of 17 PMCs per plant were performed on 55 plants selected to have chromosome estimates ranging from 37 to 39 (open and solid bars).

Figure 3.—

Segregation of molecular markers in the progenies of B. napus haploids. This chromatid model of homeologous exchanges assumes that B. napus haploids produce restituted gametes with 19 chromosomes. When no homeologous recombination occurs during meiosis of the haploid, then all markers from the haploid parent (HP) are transmitted to its progeny (offspring A). When homeologous recombination occurs during meiosis of the haploid parent, recombinant chromatids may either (i) move to the same pole of anaphase, meaning all the markers from the HP are transmitted to its progeny (offspring B and C) despite the presence of a homeologous reciprocal translocation (HRT), or (ii) move to opposite anaphase poles, meaning some of the markers from the HP are not transmitted to its progeny while others, carried by the (partially homeologous) substituting chromosomal segment are duplicated (offspring D and E). These latter exchanges are commonly termed homeologous nonreciprocal translocations (HNTRs). r, frequency of homeologous recombination; jagged arrowhead, fertilization by male gametes (n = 19) produced by the euploid male parent.

Figure 4.—

Inheritance of the HP allele at CB10081b in the F₁ progeny of the Darmor-bzh haploid, D1. One plant lacks the Darmor-bzh HP allele (arrow).

Figure 5.—

Determination of copy number of the Darmor-bzh HP allele at CB10081b. A quantitative value for the Yudal allele peak area (x-axis) was used as a baseline to which the Darmor-bzh allele peak area (y-axis) has been compared by maximum-likelihood analyses: O, plants with no Darmor-bzh allele at the CB10081b locus (loss); +, plants with a single Darmor-bzh allele at the CB10081b locus (no duplication, no loss); Δ, plants with two Darmor-bzh alleles at the CB10081b locus (duplication).

Figure 6.—

Expected and observed segregation of the N11.N1 homeologous nonreciprocal translocation in the backcross progeny of D1H29. (A) Expected segregation of the N11.N1 HNRT assuming no further recombination event in the genetic intervals carrying the breakpoints of this HNRT. Three markers are considered: M1, CB10081a; M2, Na12C08; and M3, CB10081b. D1H29 lacks the Darmor-bzh allele at CB10081a (M1^D) and had two copies of the Darmor-bzh allele at CB10081b (M3^D). Chromatid sorting results in four groups of BC₁ offspring that can be unambiguously recognized by comparing allelic composition of Darmor-bzh and Yudal alleles at CB10081a and CB10081b (M1^Y/M3^D/M3^Y). If the cosegregation pattern of the Darmor-bzh alleles at CB10081b (M3^D) and Na12C08 (M2^D) is used, groups 1 and 3 can be identified by absence–absence (00) and presence–absence (10), respectively; however, groups 2 and 4 cannot be distinguished using these two marker alleles. (B) Observed segregation. Physical location on this plot reveals the four expected groups of BC₁ offspring (circled) from analysis of copy number ratios at CB10081a and CB10081b loci. Five outlying individuals (arrows) display abnormal allelic composition at CB10081a and/or CB10081b. Each plant is symbolized by its cosegregation pattern for two Darmor-bzh alleles at CB10081b and Na12C08 loci, respectively. The expected cosegregation pattern in the absence of recombination at the Na12C08–CB10081b interval is indicated above each of the four circles. Plants that display discrepancies between the expected cosegregation pattern (00 in group 1, 01 in group 3, and 11 in groups 2 and 4) and that observed are considered recombinant at the Na12C08–CB10081b interval. Plants in agreement with expected copy number ratios are considered nonrecombinant.

Figure 7.—

Schematic of three rearrangements detected in B. napus haploid-derived progeny. Rearrangements A and B are HNRTs that originated from homeologous exchanges between DY1a/N11 and DY1b/N1 (A, D1H29) and between DY5/N9 and DY8/N18 (B, D11H2). The origin of the third rearrangement (C, Y9H11) is unknown. The positions of breakpoints and the sizes of the lost and duplicated fragments (in centimorgans) are indicated on the parental linkage groups. Genotyping data (present, 1; absent, 0), the number of copies of the HP allele (solid bar, two copies; shaded bar, one copy; dotted bar, no HP allele), and the segregation pattern (e.g., 1:1) are indicated. Dotted lines between multilocus PCR markers indicate a syntenic relationship between homeologous regions; solid markers to the left of DY linkage groups indicate homeologous relationship between DY linkage groups but were not used in our molecular analysis.

Figure 7.—

Schematic of three rearrangements detected in B. napus haploid-derived progeny. Rearrangements A and B are HNRTs that originated from homeologous exchanges between DY1a/N11 and DY1b/N1 (A, D1H29) and between DY5/N9 and DY8/N18 (B, D11H2). The origin of the third rearrangement (C, Y9H11) is unknown. The positions of breakpoints and the sizes of the lost and duplicated fragments (in centimorgans) are indicated on the parental linkage groups. Genotyping data (present, 1; absent, 0), the number of copies of the HP allele (solid bar, two copies; shaded bar, one copy; dotted bar, no HP allele), and the segregation pattern (e.g., 1:1) are indicated. Dotted lines between multilocus PCR markers indicate a syntenic relationship between homeologous regions; solid markers to the left of DY linkage groups indicate homeologous relationship between DY linkage groups but were not used in our molecular analysis.

Figure 7.—

Schematic of three rearrangements detected in B. napus haploid-derived progeny. Rearrangements A and B are HNRTs that originated from homeologous exchanges between DY1a/N11 and DY1b/N1 (A, D1H29) and between DY5/N9 and DY8/N18 (B, D11H2). The origin of the third rearrangement (C, Y9H11) is unknown. The positions of breakpoints and the sizes of the lost and duplicated fragments (in centimorgans) are indicated on the parental linkage groups. Genotyping data (present, 1; absent, 0), the number of copies of the HP allele (solid bar, two copies; shaded bar, one copy; dotted bar, no HP allele), and the segregation pattern (e.g., 1:1) are indicated. Dotted lines between multilocus PCR markers indicate a syntenic relationship between homeologous regions; solid markers to the left of DY linkage groups indicate homeologous relationship between DY linkage groups but were not used in our molecular analysis.