DIA-based proteome profiling with PRM verification reveals the involvement of ER-associated protein processing in pollen abortion in Ogura CMS cabbage

Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

Identifying Cytoplasmic Male Sterile Types of Chinese Cabbage(Brassica campestris L.)by Molecular Markers

Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identified by molecular marker in the study, in order to better use molecular marker to conduct the assisted breeding in the future. Genomic DNA was isolated from Chinese cabbage by CTAB method. The design of two pairs of specific primers was performed on conserved flanking region of orf138 gene in the GenBank. PCR was performed with genomic DNA of the nine Chinese cabbage materials. The bands were sequenced. The homologous comparison was conducted in NCBI, and finally, the type of sterile cytoplasm was determined. The results showed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs of specific primers PUPIl and PIII/PIV, while the other five materials did not obtain the relative bands. The result was consistent with the field sterility identification. And then four molecular markers of Chinese cabbage Ogura cytoplasmic male sterility （CMS） were obtained. After conducting a homologous comparative analysis with BLAST in GenBank, it was found that the homologous degree was 100% in specific segments of tbe tbree sterility materials （L1-CI, L3-CI and L3- F1 ） and Ogu orf138 gene （GenBank accession No. ： HQ149728） of the reported broccoli Ogu CMS. The homologous degree of L1-F1 was 99% with a variation point. The type of cytoplasmic male sterility of the other five materials needed further research. Four materials of the nine were identified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.