New genes (or lineage-specific genes) can facilitate functional innovations. MALE STERILITY 5 (MS5) in Brassica napus is a fertility-related new gene, which has two wild-type alleles (BnMS5 a and BnMS5 c) and two mutant alleles (BnMS5 b and BnMS5 d) that could induce male sterility. Here, we studied the history and functional evolution of MS5 homologs in plants by phylogenetic analysis and molecular genetic experiments. We identified 727 MS5 homologs and found that they define a Brassicaceae-specific gene family that has expanded partly via multiple tandem gene duplications and also probably transpositions. The MS5 in B. napus is inherited from a basic diploid ancestor of B. rapa. Molecular genetic experiments indicate that BnMS5 a and BnMS5 c are functionally distinct in B. napus and that BnMS5 d can inhibit BnMS5 a in B. napus in a dosage-dependent manner. The BnMS5 a protein can move in coordination with meiotic telomeres and interact with the nuclear envelope protein SUN1, with a possible crucial role in meiotic chromosome behavior. In summary, BnMS5 belongs to a Brassicaceae-specific new gene family, and has gained a novel function that is essential for male fertility in B. napus through neofunctionalization that has likely occurred since the origin of B. rapa.
With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.
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