Studies On The Physiological,Biochemical And Molecular Mechanisms Of The Sterility Characteristics Of Watermelon Genic Male Sterile Line Se18

As an allogamous plant,watermelon（Citrullus lanatus（Thunb.）Matsum.&Nakai）possesses significant heterosis.Therefore,to prepare hybrid seeds with male sterile lines is an important way to take advantage of the watermelon heterosis.The GMS line‘Se18’is a nuclear genetic male sterile material discovered by our research group,whose sterile traits are very stable.In this study,the watermelon nuclear male sterile line‘Se18’was adopted for observation and comparison of its cytological characteristics during the anther abortion period.And by comparing the changes in physiological and biochemical indicators such as antioxidant enzyme activity,material metabolism,and endogenous hormone content during the development of male flower buds of sterile plants and fertile plants,the physiological and biochemical characteristics of male sterility were discussed.In combination with BSA（Bulked Segregant Analysis）and RNA-Seq technology,a transcriptome sequencing on male flower buds of sterile and fertile plants was performed.A systematic analysis was conducted for the biological functions of differentially expressed genes（DEGs）.A preliminary prediction was made for candidate areas of male sterility genes and functional annotation was performed for genes within the candidate areas,so as lay a foundation for exploration of the molecular mechanism for occurrence of male sterility of watermelon.The critical findings of the study are as follows:1.Observed the development process of sterile and fertile anthers with an optical microscope,and discovered that the sterile anther abortion period occurred at the end of meiosis of microspore mother cells.The reason for abortion may lie in the abnormal development of the tapetum cells,which causes the microspore mother cell to undergo only nuclear division without cytoplasmic division,failing to form a tetrad,resulting in abnormal meiosis of the microspore mother cell,and eventually leading to male sterility.2.The determination of antioxidant enzyme activity,antioxidant substance content and active oxygen content at different developmental stages of sterile and fertile male flower buds of watermelon reveled that the activity of superoxide dismutase（SOD）of sterile male flower buds at both mono binuclear microspore stage and pollen grain mature stage was significantly higher than that of the fertile male flower buds at the corresponding stage.The activity of peroxidase（POD）at different stages was significantly higher than that of the fertile male flower buds at the corresponding stages;the content of superoxide anion（O₂^-·）,hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）was also always higher than that of the fertile male flow buds.Compared with the variation tendency of the fertile male flow buds,the variation of activity of catalase（CAT）,glutathione reductase（GR）and ascorbate peroxidase（APX）as well as the content of ascorbic acid（AsA）and glutathione（GSH）shows a downward trend.Foregoing results indicate that the activity variation of antioxidant enzyme,reduction of the content of antioxidant substances and rising of active oxygen content are possibly related to anther abortion.3.Determination of the content of substances in different developmental stages of sterile and fertile male buds of watermelon revealed that the soluble protein in sterile male flower buds at different developmental stages was significantly lower than that of fertile male flower buds.With the growth and development of flower buds,the variation of free proline content of sterile male flower buds showed a downward trend,while the free proline content of fertile male flower buds quickly accumulated,which was significantly higher than that of sterile male flower buds.This indicates that the lack of soluble protein and the reduction of free proline content may be related to anther abortion.4.The enzyme-linked immunoassay was adopted for determination of the dynamic changes of auxin（IAA）,abscisic acid（ABA）,gibberellin（GA₃）,zeatin riboside（ZR）,jasmonic acid（JA）,brassinolide（BR）and isopentenyl adenine nucleoside（IPA）in watermelon plant leaves and flower buds at different developmental stages,which revealed that the endogenous hormone content of sterile plants and fertile plants had different trends of change,and the content was significantly different.No matter whether it is leaves or flower buds,the content of IAA,ZR and BR in the sterile lines is in shortage,while the content of ABA and JA is higher than that of fertile plants.Meanwhile,the balance between the hormones is also broken.It shows that the abnormal content of endogenous hormones and the imbalance of the ratio of each hormone may be related to anther abortion.5.We performed transcriptome sequencing for fertile and sterile male flower buds with Illumina Hi Seq^TM 2500 platform and obtained a total of 43.27 Gb high quality data.In total,2507 differentially expressed genes（DEGs）were identified,including 593 up-regulated and1914 down-regulated expressions.Among the differentially expressed genes,2443 genes were annotated.Gene ontology analysis results showed that these differentially expressed genes involved in processes such as signal transduction,biological regulation,reproduction and fertility,substance metabolism,transcription factor and protein synthesis.In KEGG analysis,a total of 451 DEGs were assigned to 95 different biochemical pathways.The analysis results showed that metabolic pathways such as nitrogen metabolism,alanine,aspartic acid and glutamate metabolism,arginine and proline metabolism were significantly enriched.The results of this study provide a reference for elucidating the molecular mechanism of watermelon nuclear male sterility and the identification of regulatory genes.6.Sterile and fertile male flower buds were selected to construct mixed pools separately with method of BSR-Seq（Bulked segregant RNA-Seq）.Through ED algorithm and correlation analysis,the sterile genes with total length of 10.71 Mb were initially located in two regions of Chr5 and Chr9 chromosomes.Among 112 differentially expressed genes,20genes were up-regulated and 92 genes were down-regulated.The GO analysis results showed that the genes in candidate regions involved in processes such as signal transmission,substance metabolism,transcription factors and protein synthesis.In the KEGG analysis,14genes in the candidate regions were mapped to 19 different pathways.The analysis results showed that the metabolic pathways such as diterpenoid biosynthesis,carbon fixation of photosynthetic organisms,arginine and proline metabolism,and pyruvate metabolism were significantly enriched.In the COG analysis,36 genes were mapped to 16 functional categories,and the analysis results showed that the genes in the candidate regions involved in functions such as signaling mechanisms,carbohydrate transport and metabolism,amino acid transport and metabolism.The above research results has laid the foundation for the next step of fine mapping of watermelon male sterility related genes.