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Aberrant Meiotic Prophase I Leads to Genic Male Sterility in the Novel TE5A Mutant of Brassica napus

View Article: PubMed Central - PubMed

ABSTRACT

Genic male sterility (GMS) has already been extensively utilized for hybrid rapeseed production. TE5A is a novel thermo-sensitive dominant GMS line in Brassica napus, however, its mechanisms of GMS remain largely unclear. Histological and Transmission electron microscopy (TEM) analyses of anthers showed that the male gamete development of TE5A was arrested at meiosis prophase I. EdU uptake of S-phase meiocytes revealed that the TE5A mutant could accomplish DNA replication, however, chromosomal and fluorescence in situ hybridization (FISH) analyses of TE5A showed that homologous chromosomes could not pair, synapse, condense and form bivalents. We then analyzed the transcriptome differences between young floral buds of sterile plants and its near-isogenic fertile plants through RNA-Seq. A total of 3,841 differentially expressed genes (DEGs) were obtained, some of which were associated with homologous chromosome behavior and cell cycle control during meiosis. Dynamic expression changes of selected candidate DEGs were then analyzed at different anther developmental stages. The present study not only demonstrated that the TE5A mutant had defects in meiotic prophase I via detailed cytological analysis, but also provided a global insight into GMS-associated DEGs and elucidated the mechanisms of GMS in TE5A through RNA-Seq.

No MeSH data available.


FISH analysis of meiosis in both the wild-type and the TE5A mutant.Meiotic chromosomes probed with 45S rDNA (green). Chromosomes were stained with DAPI (blue). (A,E), at leptotene stage, 12 to 14 45S rDNA signals were observed in the wild-type (A) and in the TE5A mutant (E), respectively; (B,F), at pachytene stage, six paired signals (half of the total number) were detected in the wild type (B), whereas 12 to 14 45S rDNA signals were still observed in the mutant (F,C,D,G) signals in half were observed in dyads (C) and tetrads (D) of the wild-type, in contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were still observed in the mutant (G). Scale bars = 5 μm.
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f5: FISH analysis of meiosis in both the wild-type and the TE5A mutant.Meiotic chromosomes probed with 45S rDNA (green). Chromosomes were stained with DAPI (blue). (A,E), at leptotene stage, 12 to 14 45S rDNA signals were observed in the wild-type (A) and in the TE5A mutant (E), respectively; (B,F), at pachytene stage, six paired signals (half of the total number) were detected in the wild type (B), whereas 12 to 14 45S rDNA signals were still observed in the mutant (F,C,D,G) signals in half were observed in dyads (C) and tetrads (D) of the wild-type, in contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were still observed in the mutant (G). Scale bars = 5 μm.

Mentions: Further confirmation of the failure of pairing in the TE5A mutant was achieved via fluorescence in situ hybridization (FISH) using 45S rDNA as probe to determine the pairing status of the homologous chromosomes in PMCs during meiosis I (Fig. 5). In Brassica napus, there are 12 to 14 repeated 45S rDNA loci on somatic chromosomes of wild-type plants. Therefore, 12 to 14 signals should be observed in unpaired chromosomes and only six or seven in paired/synapsed chromosomes in meiosis. In both wild-type plants and the TE5A mutant, 12 to 14 45S rDNA signals were observed at leptotene (Fig. 5A,E). During early pachytene, paired signals (six signals) were detected in the wild-type (Fig. 5B), whereas 12 to 14 45S rDNA signals were also observed, thereby suggesting that a defective pairing of homologous chromosomes in the mutant (Fig. 5F). After that, the number of chromosomes was halved in the dyads (Fig. 5C) and tetrads (Fig. 5D) of the wild-type. In contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were observed at the corresponding stage in the TE5A mutant with a significantly higher number of chromosomes (Fig. 5G). These results confirmed that pairing did not occur in TE5A mutant, and the arrest of prophase I might be caused by defective pairing.


Aberrant Meiotic Prophase I Leads to Genic Male Sterility in the Novel TE5A Mutant of Brassica napus
FISH analysis of meiosis in both the wild-type and the TE5A mutant.Meiotic chromosomes probed with 45S rDNA (green). Chromosomes were stained with DAPI (blue). (A,E), at leptotene stage, 12 to 14 45S rDNA signals were observed in the wild-type (A) and in the TE5A mutant (E), respectively; (B,F), at pachytene stage, six paired signals (half of the total number) were detected in the wild type (B), whereas 12 to 14 45S rDNA signals were still observed in the mutant (F,C,D,G) signals in half were observed in dyads (C) and tetrads (D) of the wild-type, in contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were still observed in the mutant (G). Scale bars = 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037387&req=5

f5: FISH analysis of meiosis in both the wild-type and the TE5A mutant.Meiotic chromosomes probed with 45S rDNA (green). Chromosomes were stained with DAPI (blue). (A,E), at leptotene stage, 12 to 14 45S rDNA signals were observed in the wild-type (A) and in the TE5A mutant (E), respectively; (B,F), at pachytene stage, six paired signals (half of the total number) were detected in the wild type (B), whereas 12 to 14 45S rDNA signals were still observed in the mutant (F,C,D,G) signals in half were observed in dyads (C) and tetrads (D) of the wild-type, in contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were still observed in the mutant (G). Scale bars = 5 μm.
Mentions: Further confirmation of the failure of pairing in the TE5A mutant was achieved via fluorescence in situ hybridization (FISH) using 45S rDNA as probe to determine the pairing status of the homologous chromosomes in PMCs during meiosis I (Fig. 5). In Brassica napus, there are 12 to 14 repeated 45S rDNA loci on somatic chromosomes of wild-type plants. Therefore, 12 to 14 signals should be observed in unpaired chromosomes and only six or seven in paired/synapsed chromosomes in meiosis. In both wild-type plants and the TE5A mutant, 12 to 14 45S rDNA signals were observed at leptotene (Fig. 5A,E). During early pachytene, paired signals (six signals) were detected in the wild-type (Fig. 5B), whereas 12 to 14 45S rDNA signals were also observed, thereby suggesting that a defective pairing of homologous chromosomes in the mutant (Fig. 5F). After that, the number of chromosomes was halved in the dyads (Fig. 5C) and tetrads (Fig. 5D) of the wild-type. In contrast, the number of 45S rDNA FISH foci was not halved, and unpaired 45S rDNA FISH foci were observed at the corresponding stage in the TE5A mutant with a significantly higher number of chromosomes (Fig. 5G). These results confirmed that pairing did not occur in TE5A mutant, and the arrest of prophase I might be caused by defective pairing.

View Article: PubMed Central - PubMed

ABSTRACT

Genic male sterility (GMS) has already been extensively utilized for hybrid rapeseed production. TE5A is a novel thermo-sensitive dominant GMS line in Brassica napus, however, its mechanisms of GMS remain largely unclear. Histological and Transmission electron microscopy (TEM) analyses of anthers showed that the male gamete development of TE5A was arrested at meiosis prophase I. EdU uptake of S-phase meiocytes revealed that the TE5A mutant could accomplish DNA replication, however, chromosomal and fluorescence in situ hybridization (FISH) analyses of TE5A showed that homologous chromosomes could not pair, synapse, condense and form bivalents. We then analyzed the transcriptome differences between young floral buds of sterile plants and its near-isogenic fertile plants through RNA-Seq. A total of 3,841 differentially expressed genes (DEGs) were obtained, some of which were associated with homologous chromosome behavior and cell cycle control during meiosis. Dynamic expression changes of selected candidate DEGs were then analyzed at different anther developmental stages. The present study not only demonstrated that the TE5A mutant had defects in meiotic prophase I via detailed cytological analysis, but also provided a global insight into GMS-associated DEGs and elucidated the mechanisms of GMS in TE5A through RNA-Seq.

No MeSH data available.