Cloning mitochondrial gene atp9 and utilization molecular marker associated with male sterile cytoplasm in kenaf

【Objective】The present study was conducted to clone the coding sequence(CDS) region of mitochondrial atp9 gene of kenaf and analyze its evaluation relationships with other crops in order to lay the foundation for atp9 gene function verification. A molecular marker MM556 associated with male sterile cytoplasm, which was developed based on gene atp9, was used for detecting the fertility of kenaf germplasm resources in order to provide references for detecting kenaf male sterile cytoplasm germplasm resources. 【Method】The CDS region was cloned from kenaf cytoplasmic male sterility(CMS) line P3 A and its maintainer line P3 B by using homological cloning method, followed by sequence alignment between P3 A and P3 B. The phylogenetic tree of gene atp9 was analyzed amongst different crops. Meanwhile, a specific molecular marker MM556 was used for screening male sterile cytoplasm materials from 84 accessions of kenaf germplasm resources with unknown cytoplasmic fertility. 【Result 】The CDS region of atp9 was 339 bp in length for P3 A and P3 B,and only four base pairs' difference was found in P3 A and P3 B. CDS region was contained 112 deduced amino acids.Phylogenetic analysis results showed that, atp9 gene of kenaf had the closest relationship with grape being 72 of node approval rating, followed by rape and Arabidopsis, and the farthest one was leek. Ten varieties viz., UG93-2ms-1, UG93-2ms-2, UG93-2ms-3, UG93-2ms-4, UG93-2-22, KN250, KN250-1, KN142, ZB90 and 09-7 containing male sterile cytoplasm were identified from 84 germplasm resources of kenaf by using molecular marker MM556; out of which, 4 varieties viz., UG93-2ms-1, UG93-2ms-2, UG93-2ms-3, UG93-2ms-4 were derivative strain from wild-abortive type mutation of kenaf variety UG93. The results of molecular detection and field fertility verification of Hibisius radjatus showed that, molecular marker MM556 was also suitable for fertility validation of tetraploid Hibisius radjatus as well. 【Conclusion】The obtained CDS region of atp9 gene and screened kenaf germplasm resources containing male sterile cytoplasm could be used for further researches on revealing CMS molecular mechanism of kenaf. A molecular marker MM556 developed based on atp9 could be used for detecting male sterile cytoplasm of kenaf.