| Seedless citrus has become one of the breeding goals due to its high edible ratio and convenient for fresh consumption and processing.Hongjiangcheng sweet orange?Gailiangcheng?is a landrace with excellent exterior quality and edible quality but contains many seeds.The seedless mutant of Hongjiangche ng sweet orange?MT?produced from the wild type Hongjiangcheng sweet orange?WT?via cobalt-60 radiation mutation showing stable seedless character,is an ideal material in revealing citrus seedless mechanism.In this study,firstly,the primary metabolit es were profiled using GC-MS.For morphological and physiological analysis,the large-scale pollination experiment and plant microscopic techniques were carried out to investigate the floral organ development,male and female fertility,self-compatibility,parthenocarpy and zygotic embryos development in the MT.Finally,the molecular mechanism study was primarily studied through genome-wide re-sequencing and RNA-Seq.The main results were shown as follows:1. Compared with the WT,the contents of proline,valine,d-?-?-fructofuranose and d-?-?-fructopyranose in MT increased significantly?P<0.05?,whereas the contents of quinic acid,malic acid,sucrose and d-?-?-fructose in MT decreased significantly?P<0.05?.2. For the morphological and physiological aspects,the floral organs in MT were fully developed and morphologically normal.However,the most of mature pollens of MT were distorted and shrunken along with the significantly lowered fertility than WT?P<0.01?.Furthermore,the ovule number of MT was also normal.Through large-scale pollination experiment,we discovered the self-compatibility during pollen tube germination in situ and the absence of natural parthenocarpy.Notably,paraffin sectioning indicted that the zygotic embryos development ceased at the fourth week after pollination in MT,which was one of the physiological causes of seed abortion in MT.Thus,it implied that the seedless MT was related to the blocked nucellar embryos development.3. Genome-wide re-sequencing profiels and transcriptome sequencing revealed that:689434 specific single nucleotide polymorphisms?SNP?and 62430 specific insertion and deletion?In Del?were found in the MT genome.RNA-Seq indicated that a total of815 differentially expressed genes?DEGs?were identified in immature embryos at the4 th week after pollination.According to these DEGs and q RT-PCR results,three candidate genes were selected which might be related to embryonic development.The Arabidopsis homologous gene of Cs4g10930 and orange 1.1t02243 belonging to LEA family genes were late embryogenesis abundant,while that of Cs5g21900 encoding EP3chitinase is related to somatic embryogenesis.4. Sequence analysis on the promoter and CDS of above candidate genes showed no difference between WT and MT.Thus these genes might be regulated by other regulators and thus lead to seed abortion by affecting embryo development.Furthermore,14transcription factors?TFs?were predicted which could potentially bind to the promoters of Cs4g10930 and orange 1.1t02243,including ERF,b HLH,WRKY,MYB and MADS box family genes,which were closely related to plant growth and development.Notably,Cs7g02340 was the only differentially expressed MADS box gene.Our study revealed that the morphological and physiological causes of seed abortion in MT was pollen abortion,zygotic embryo abortion at early stage and blocked nucellar embryos development.Also,we found the normal functional embryo sac,the self-compatibility and the inability of natural parthenocarpy in MT.For molecular aspect,several candidate genes were selected,which might be related to embryo and seed abortion.The findings in this study could serve as reference and guidance for the mechanism research and breeding of seedless citrus. |
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