Screening of target genes and localization analysis for ovule abortion in the female sterile line of Pinus tabuliformis Carr.

Female gametophyte development, which is the necessary life process for the formation of mature female gametophytes (FG) and the alternation of generations, is a precondition of sexual reproduction. However, the present research on female sterility is mainly focused on angiosperms, and investigations of gymnosperms are inadequate. Gymnosperms retain several ancestral traits inherited from spore-bearing plants, while concurrently exhibiting derived characteristics that are shared with angiosperms. Free nuclear mitosis (FNM) is the key step of FG formation in gymnosperms. Our research group previously observed that in a natural female sterile line (No. 28) of Pinus tabuliformis, the development of FG was stopped during mitosis. To identify key regulators involved in free nuclear division of the female gametophyte, we combined miRNA sequencing and RNA sequencing technologies, constructing 18 mRNA libraries and 18 small RNA libraries. By comparing the expression differences of miRNAs and their target genes between the fertile line (FL) and sterile line (SL) at different ovule developmental stages, and integrating functional analysis of target genes, a molecular regulatory network associated with ovule abortion was established. Significant differential expression of target genes involved in signal transduction, energy metabolism, cell division, and protein modification were detected between FL and SL, and these results were validated by real-time quantitative PCR. In addition, cryosectioning combined with fluorescence in situ hybridization (FISH) revealed that the PtDNAJ gene was highly expressed in FL, predominantly localized around diploid tissues, but barely expressed in the FG of SL. This research provided a new insight for the exploration the reproductive development of gymnosperms and provided a theoretical basis for understanding the developmental biology of woody plants.