The Evaluation Of Apomixes Candidate Gene CitRWP In Citrus And Its Close Related Genus

As the most planted fruit trees in the world,Citrus has a huge planting area and a rich germplasm resources.Nucellar embryony in citrus is a kind of apomixis,which formed by invasion of nucellar tissues into the embryo sac,it interfers the cross breeding and has a profound impact on the genetic evolution and taxonomy in citrus.If it is introduced into crops,the process of breeding will be simplified and breeding efficiency improved.There have been many different hypotheses on the genetic regulation of apomixis in citrus,but none of them are able to lock the true regulatory sequence until the recently reported candidate gene citRWP.However,the support evidences for the judgment of citRWP as the major or only major gene is still not very sound,which still needs more research to verify.With monoembryonic sweet orange,polyembryonic Poncirus,hybrid progeny of Poncirus and other materials collected in our laboratory,the citRWP gene was analyzed from the following aspects: M/P molecular marker check at the genomic level,relative expression detection at the RNA level and cDNA cloning and sequencing at the sequence level.In addition,overexpression vectors were constructed with the sequence of cit RWP of Hamlin sweet orange to transform tobacco.We aim to draw a comprehensive map of the gene function of citRWP by the combining of these research.1.Detection of M/P molecular marker at the genomic levelThe M/P marker designed by Shimada was used to detect different embryonic genotypes.The 0.75 kb and 1.0kb fragments corresponded to monoembryonic alleles M-marker and polyembroynic alleles P-marker,respectively.In the test of M/P marker in our materials,we found the marker was not completely consistent with embryonic genotypes.Both M-marker and P-marker were found in all sweet orange embryonic types,which might indicate that the embryonic trait of sweet orange was not regulated by a single citRWP gene with MITE insertion.In the hybrid offspring of W-Murcott×Flying dragon trifoliate orange,the M/P molecular marker was consistent with the results of embryo investigation.In three Poncirus genotypes,no polyembryonic P-marker was found,it is possible that the Cit RWP might not contain the MITE insertion or a huge mutation might occur in upstream of CitRWP in Poncirus.2.Expression analysis of CitRWP in different tissues or organs of different materialsNucellar embryo occurrs in ovule,the gene expression in ovules of different embryonic varieties was analyzed.RT-PCR detection in ovule cDNA of different embryonic varieties revealed that CitRWP was expressed in all varieties.Further analysis by Quantitative Real-time PCR revealed no significant difference between monoembryonic and polyembryonic sweet oranges in detection of 2018,and same results were found in 2019 also.In addition,the expression value of monoembryonic sweet orange was much higher than polyembryonic Flying dragon trifoliate orange,which indicated that the expression of CitRWP might not directly related to embryonic phenotypes.CitRWP expression was tested in ovary(young whole fruit)of three trifoliate orange varieties,and the W-Murcott ×Flying dragon trifoliate orange hybrid offspring population.The expression was detected in a very low level in three trifoliate orange varieties.In the F1 hybrid offsprings,the expression of the CitRWP gene showed an extreme two-stage differentiation.There are 5 offsprings highly expressed and 10 offsprings lowly expressed,which are consistent with the embryonic type investigation.These results indicated somehow that the Cit RWP gene expression value has correlation with embryonic phenotype in the W-Murcott ×Flying dragon trifoliate orange hybrid offspring population.CitRWP expression was tested in organs or tissues such as young stems,young leaves,old leaves,and ovary of mono/polyembryonic sweet orange also.It was found that the expression value in different organs or tissues were significantly different due to embryonic phenotype.Among polyembryonic sweet oranges,young leaves were the most highly expressed parts,the expression value of ovules were slightly lower than that in young leaves,and other organs or tissues were similar and much lower than that in ovlues.The highest expression tissue was ovules in monoembryonic sweet orange,while the expression in young leaves tended to be consistent with other tissues.The young leaves had the highest expression indicated that CitRWP was not specifically expressed in reproductive tissue.3.cDNA clone and sequence analysis of citRWPMolecular marker detection and quantitative analysis generally detect a part of the gene sequence which meets the requirements of primer design,and the correct gene function needs the complete sequence,so CitRWP full-length gene was cloned and sequenced in this study.CitRWP cDNA from Poncirus,mono/polyembryonic sweet oranges,and monoembryonic pomelo were cloned and sequenced.Big changes in the citRWP gene were found and the changes could be divided into three types.The first is a deletion of 106 bp segment at the 406 bp position(relative to the start codon,the same hereinafter),the second is a 5 bp(-TGCAG-)insertion at the 774 bp position,and the third is the mutation in the initiation codon or termination codon.The first two mutations cause the change of CDS length to a length unsuitable for normal translation with triplet codons and no functional protein could be translated.The third type change also leads to the mistranslation of functional protein.All changes were found in Poncirus sequenced clones and only 22.2% of the sequenced clones could be normally translated.Only the 106 bp fragment deletion and 5bp(-TGCAG-)insertion were found in the sequenced clones of mono/polyembryonic sweet orange and monoembryonic pomelo,but with a higher ratio of normal ranslation.The rate of clones could be normally ranslated was 80% in polyembryonic Tarocco blood orange,and 62.5% in monoembryonic weet orange TD.The normal translation rates were 100% and 80% in monoembryonic Long’an pomelo and Wanbai pomelo,respectively.These data further indicate that citRWP is not the only major gene or only major gene that regulates apomixes in Citrus and its close related genus.4.Overexpression of citRWP in tobaccoGene overexpression research in a non-citrus genetic background is valuable for exploring whether Cit RWP requires other citrus genes to coordinate in nucellar embryony regulation,and it is also an important step for testing citrus apomixis genes whether work on fixing heterosis in other crops.The CitRWP overexpression vector was successfully constructed and transformed into tobacco(Nicotiana tabacum L.cv."W38"),and obtained 10 transgenic tobacco plants.Compared with wild-type tobacco,the growth of transgenic tobacco was not significantly changed.Because of the growth cycle,genetically modified tobacco seeds are currently not available.