Cloning And Functional Study Of A Gene Related To S-type Cytoplasmic Male Sterility In Onion(Allium Cepa L.)

Onion?Allium cepa L.?belongs to the Lily family and the Allium genus,which is a biennial cross-pollinated plant.Onion has high heterosis as evidenced by the fact that the F₁hybrids exhibit a 20%-50%increase of the yield.Onion has a small flower organ and the seeding rate of a single flower is low.In addition,the growth cycle is long.Therefore,it is time-consuming and costly to select hybrids by conventional breeding methods.The discovery and successful breeding of male sterile lines has opened up a new way of utilization of onion heterosis,while the development and application of molecular marker technologies has greatly shortened the breeding period.In this study,differential expression analysis of 58open reading frames?ORFs?in the whole genome of onion mitochondria was performed.A differentially expressed fragment,orf393,was discovered,and a SCAR marker was successfully developed to promptly and accurately identify the S-type and N-type cytoplasm types.Using an Agrobacterium-mediated genetic transformation method,the function of orf393 was investigated.The main findings are as follows:1.Differential expression analysis of 58 ORFs was performed using a S-type cytoplasmic male sterile line and a maintainer line.One differentially expressed fragment,orf393,was identified,which was only expressed in the male sterile line but not in the maintainer line.By sequencing analysis,it was found that orf393 had a 36-base deletion in the maintainer line,and 29 single nucleotide polymorphisms?SNPs?were identified.The 11^thh SNP caused the mutation of CAG to TAG,leading to the early termination of translation.2.Based on the difference of the orf393 sequence in the S-type cytoplasmic male sterile line and the maintainer line of onion,a SCAR marker,namely GXX393,was developed,which could be used to identify the cytoplasmic types of onion.Using this marker,a specific band of 232 bp and 196 bp was amplified in the S-type cytoplasmic male sterile line and the maintainer line,respectively.This method was verified by using 10 combinations of known cytoplasmic types with different genetic backgrounds,which showed great agreement with the field phenotyping results of onion.By using this marker,the S-type and N-type cytoplasm can be promptly and accurately distinguished by a simple PCR.3.Quantitative PCR primers were designed based on the orf393 sequence,and the real-time fluorescent quantitative PCR was utilized to analyze the spatio-temporal expression of orf393 in the root,leaf,stem,and the buds in four developmental stages,i.e.,pollen mother cells,tetrads,mononuclear pollens and mature pollens,of the S-type cytoplasmic male sterile line and maintainer line.The results showed that orf393 was expressed in all samples of the S-type cytoplasmic male sterile line,especially those collected during the tetrads,during which,the expression of orf393 reached the maximum level,and then returned to the level similar to that in the vegetative growth period.However,no expression was detected in the maintainer line.These results indicate that orf393 is a constitutive gene and may be related to the abortion of CMS pollen.4.Using the In-Fusion Cloning method,a functional vector with mitochondrial targeting signal,pROK2-Rf1b-orf393,was successfully constructed in order to study the function of orf393.By Agrobacterium-mediated floral-dip technique,a kanamycin-resistant Arabidopsis thaliana was successfully obtained.The transgenic plants were obtained through phenotypic identification,PCR detection,Alexander's staining and anatomical observations.Compared with the wild-type plants,the transgenic plants had no pollen attached to the stigma of the pistil,and the stamens were short.The anthers were dull without cracking,and only a small amount of active pollen grains were present.The fruits were short and seeds were rarely seen.These results showed that the transgenic plants did not process normal pollen dispersal,and the fertility was greatly decreased,indicating that orf393 may be an essential gene related to the cytoplasmic male sterility of onion.