Chromosome inheritance and meiotic stability in allopolyploid Brassica napus

Abstract

Homoeologous recombination, aneuploidy, and other genetic changes are common in resynthesized allopolyploid Brassica napus. In contrast, the chromosomes of cultivars have long been considered to be meiotically stable. To gain a better understanding of the underlying mechanisms leading to stabilization in the allopolyploid, the behavior of chromosomes during meiosis can be compared by unambiguous chromosome identification between resynthesized and natural B. napus. Compared with natural B. napus, resynthesized lines show high rates of nonhomologous centromere association, homoeologous recombination leading to translocation, homoeologous chromosome replacement, and association and breakage of 45S rDNA loci. In both natural and resynthesized B. napus, we observed low rates of univalents, A-C bivalents, and early sister chromatid separations. Reciprocal homoeologous chromosome exchanges and double reductions were photographed for the first time in meiotic telophase I. Meiotic errors were non-uniformly distributed across the genome in resynthesized B. napus, and in particular homoeologs sharing synteny along their entire length exhibited multivalents at diakinesis and polysomic inheritance at telophase I. Natural B. napus appeared to resolve meiotic errors mainly by suppressing homoeologous pairing, resolving nonhomologous centromere associations and 45S rDNA associations before diakinesis, and reducing homoeologous cross-overs.

Keywords: allopolyploid; double reduction; homoeologous recombination; meiosis error; polysomic inheritance.

Figure 1

Homoeologous pairing and nonhomologous centromere association in pachytene of resynthesized and natural cultivar Brassica napus. (A) Two different homoeologous A–C tetravalents identified according to strength of green signals are indicated with green and white arrows, respectively. (B) Tetravalents were not detected in Stellar, and the same homoeologous sets paired in (A) are unpaired in this example [compare to green and white arrows in (A)]. (C) A gray scale image of the photo shown in (A). (D) An enlargement of a homoeologous pair shown in (A, C). Note that homologous chromosomes are not completely synapsed and pairing partner switches are observed, and that several areas show association of homoeologous chromosomes A2 and C2 (arrow) since it contains red signals from BAC clone KBrH092N24. (E) Nonhomologous centromere associations in resynthesized B. napus detected with a probe mixture containing centromere probes. When centromere probes (CentBr1 and CentBr2) were applied to pachynema in resynthesized B. napus, few, large foci were detected. (F) Nonhomologous centromere association in B. napus Stellar. When centromere probes (CentBr1 and CentBr2) were applied to pachynema in Stellar, few, large foci were detected (bright foci). Scale bar = 10 μm.

Figure 2

Nonhomologous associations detected during diakinesis in resynthesized and natural Brassica napus. (A) The average number of nonhomologous associations in resynthesized and natural B. napus indicated by bar height, and error bars represent standard deviations. Resynthesized lines were analyzed in the S₁ and S₁₁ generations (blue and red bars, respectively), and natural B. napus Stellar and DH12075 are shown with green and purple bars, respectively. The average number of nonhomologous associations across all chromosomes is summarized at the far right of the graph. (B) Homoeologous associations were analyzed from the data set presented in (A). Bar height represents the average percentage of homoeologous associations, and error bars represent standard deviations.

Figure 3

Nonhomologous chromosome association during diakinesis of Brassica napus. Hybridizations were first performed using probe mixture 1 (panels A and C) and probe mixture 2 (panels B and D) allowing unambiguous identification of all parental chromosomes (see Materials and methods). (A) Analysis of resynthesized B. napus revealed bivalents among homologs (e.g. A10, C4, C9), associations among homoeologs (green arrows; i.e. A2–C2, A3–C3), and association of 45S rDNA locus-containing chromosomes, which connect by a huge white signal from 45S rDNA probe (magenta arrow). (B) The same cell as in (A) reprobed with mixture 2 shows association of nonhomologous centromeres (white arrows). (C) Analysis of Stellar revealed normal association among homologs (e.g. A2, A4, C8, etc.), association among homoeologs (i.e. A1–C1 and A5–C4; green arrows), association among 45S rDNA locus-containing chromosomes (A6 and A9) and 5S containing chromosomes (A3 and C4, magenta arrowhead). (D) The same cell as in (C) reprobed with mixture 2 revealed nonhomologous centromere association by white CentBr1 signals (white arrows). Note: Each pair of homologs is denoted with a capital letter for the parental genome and the chromosome number. Scale bar = 10 μm.

Figure 4

Homoeologous reciprocal exchanges, homoeologous chromosome replacement, early sister chromatid segregation, and 45S rDNA association detected at telophase I of resynthesized Brassica napus. Hybridizations were first performed using probe mixture 1 (panels A and C), and reprobed with mixture 2 (panels B and D) allowing unambiguous identification of all the parental chromosomes at telophase I. (A) In these telophase I cells, homoeologous reciprocal translocations between A2 and C2 were identified (green and red arrows). Chromosome A2 and C2 are homoeologous chromosomes. Normal A2 containing two sister chromatids had two red signals from BAC KBrH092N24 on long arms and weak green signal on short arm, and normal C2 had green signals on both arms. Note that one A2 (green arrow) lost one red signal on one chromatid, while one C2 (red arrow) gained one red signal on one chromatid. A4 homologs were unpaired (denoted with A4) and one homolog migrated to the leftmost pole and the other underwent early sister chromatid separation (denoted with green arrowheads and lower case a4). 45S rDNA association was detected between chromosomes A5 and A1 in the leftmost pole. (B) Same cells of A were re-hybridized using probes mixture 2, in which red signal from C-genome specific probe showed chromosomes from C-genome, and white and green signals from CentBr1 and CentBr2, respectively, showed the constitution of centromeres. (C) In this example, both sets of A2 and C2 homoeologs underwent reciprocal exchanges and migrated to opposite poles (green and red arrows). Again, note that the chromatids on these two sets of chromosomes are heteromorphic for red signals. Homoeologous chromosome replacement occurred between A10 and C9 (green text), and one C9 chromosomes lost green signal and one A10 gained green signal on long arm, indicating homoeologous pairing and exchange before telophase I. Homologous chromosome nondisjunction resulted in two C7 chromosomes migrating to one daughter cell (yellow text). 45S rDNA association was detected between chromosomes A6 and C8 in the rightmost pole. (D) Same cells of C were re-hybridized using probes mixture 2 to use for identification chromosomes. Scale bar = 10 μm.

Figure 5

Homoeologous chromosome replacement and early sister chromatid separation Brassica napus cultivar Darmor. These hybridization results were obtained with probe mixture 1 (see Materials and methods). The same cells after hybridization with probe mixture 2 did not show here. (A) Homoeologous replacement among A2 and C2 homoeologs (white and green arrows, respectively). (B) Early sister chromatid separation of chromosome A6 (labeled with lowercase letter and white arrows). Scale bars = 10 μm.

Figure 6

Chromosome breakage and 45S rDNA translocation in resynthesized Brassica napus. (A) 5S and partial 45S rDNA loss on one chromatid of A1 (red arrowhead) translocation to one chromatid of A2 (full size red arrow). Inset shows the A1 chromosome that experienced the loss with the Cy5 channel (white) removed. (B) Chromosome breakage at the centromere position of C7 (red arrowheads). Inset left (L) shows zoom in of C7 and inset right (R) shows the breakage at centromere position (white signal and red arrowhead) using probe mixture 2. Scale bars = 10 μm.