Sterile Characteristics And Identification Of Candidate Genes In A Thermo-sensitive Genic Male Sterile Line 05ms Of Eggplant

Eggplant（Solanum melongena L.）is one of the most important vegetable crops in the world,which has the largest planting area in China.Eggplant has a strong heterosis,and male sterility is one of the important ways to utilize heterosis,saving time and labor,Improve seed quality and purity.Male sterility is one of the interesting research topics in biology field.However,compared with other vegetable crops,in eggplant the male sterility has limited studies and the mechanism of abortion is still unclear.Therefore,discovering the excellent male sterility resources of eggplant and exploring its abortion mechanism could improve the utilization efficiency of male sterility,accelerate breeding process,and provide theoretical guidance for hybrid utilization and molecular assisted breeding.In this study,the spontaneous thermo-sensitive genic male sterile（TGMS）line 05ms and its homologous fertile line S63 were used as the experimental materials.The anther of 05ms is male sterile in low temperature,but it is fertile in high temperature.A systematic study of 05ms was conducted in the several aspects of cytology.physiology,transcriptome and genome during the period of sterility and fertility.We analyzed the anther abortion characteristics,revealed the temperature response mechanism of fertility transformation,analyzed the main regulatory pathways of differentially expressed genes,defined the candidate intervals of thermo-sensitive male sterility.and identif ied the candidate genes of thermo-sensitive male sterility.The results will help to clarify the male sterility abortion mechanism,and provide the new germplasm and method for the utilization of male sterility in eggplant,and also provide important references for the utilization of thermo-sensitive male sterility in Solanaceae crops.The main results are listed as followed.1.The PIHCH staining method was used to observe the pollen mother cells of 05ms and S63 during the sterile period,showing that the chromosomes of pollen mother cells of 05ms were abnormal from the meiosis stage.Such as scattered arrangement,adhesion and unequal distribution,and eventually it could not form normal tetrad.The number of middle layer cells in anther wall of 05ms was increased and their arrangement was disordered.In 05ms the tapetum cells were vacuolated and delayed dissolution.finally only fragments were left in the pollen sac.And the tetrads of 05ms were surrounded by callose and glowed strongly,finally could not form microspores.However,both pollen mother cells and anther wall cells in S63 could develop normally and eventually form mature pollen grains.2.The results of physiological indexes showed that during the sterile period,the content of chlorophyll a,net photosynthetic rate,soluble sugar and soluble protein in 05ms leaves were significantly lower than in S63,while the content of ABA was significantly higher than in S63.In all four stages ABA content in flower buds in 05ms was significantly higher than in S63,IAA content was significantly lower than in S63 except pollen stage,and in microspore and pollen stages soluble sugar and soluble protein content in 05ms were significantly lower than in S63.In the fertile period,ABA and soluble sugar contents in flower buds of 05ms were significantly lower than of S63 in all stages,while soluble protein content was significantly higher than S63 in all stages,and in microsporocyte and meiosis stages IAA content was significantly higher than of S63.Comparison of the flower buds of 05ms in the sterile and fertile periods showed that in the four stages ABA and ZR contents were significantly higher in the sterile period than in the fertile period,while IAA contents were low in the sterile period except microspore stage.3.RNA-seq results showed that expressions of most genes in 05ms were down-regulated in the sterile period.Differentially expressed genes（DEGs）were mainly enriched into pathways of "plant hormone signal transduction","starch and sucrose metabolism","carbon metabolism" and "phenylpropane biosynthesis".The families with the most DEGs in transcription factors were MYB,bHLH,AP2-EREBP and Trihelix.Two candidate genes related to thermo-sensitive male sterility expressed differently between 05ms and S63:the gene 1AA4（Sme2.5₀5719.1_g00012.1）in "plant hormone signal transduction" pathway was high expression under low temperature（104.32）,but low expression under high temperature（54.39）;the expression of 4CLL1（Sme2.5₀0368.1_g00010.1）in the "phenylpropane biosynthesis" pathway was low expression under low temperature（1.46）,but high expression under high temperature（648.21）.4.The DNA pools of sterile and fertile were constructed with the F2 segregation population crossing between 05ms and S63,and the BSA-seq analysis was performed.The 30 candidate regions related to thermo-sensitive male sterility were detected,and the 26 candidate SNP and InDel polymorphisms related to thermo-sensitive male sterility were selected.Three candidate genes associated with thermo-sensitive male sterility were found by combining results of BAS-seq and RNA-seq.The gene sme2.5₀1314.1_g00006.1 encoding small heat shock protein（sHSP）,with the InDel A/-in the 1st intron,was highly expressed at low temperature and has low expression at high temperature in both 05ms and S63.The gene sme2.5 057559.1_g00003.1 encoding GPAT.with SNP mutation C>T at the downstream region,was highly expressed at low temperature and has low expression at high temperature in 05ms,while the expression level in S63 was exactly opposite.The gene sme2.5 100565.1_g00002.1 encoding CK1,with the InDel-/ACA at the downstream region,was highly expressed at low temperature and has low expression at high temperature in 05ms,the expression level in S63 had no significant difference,but it was approximately 7 times higher than that in 05ms.Through cell observation,substance content determination,RNA-seq and BSA-seq,we analyzed the anther abortion characteristics of 05ms under sterile condition.The temperature response mechanism of fertility conversion was revealed,and the main regulatory pathways of DEGs were analyzed.Furthermore,candidate TGMS genes were identified in this study.These findings provide a theoretical basis for revealing the mechanism of thermo-sensitive genic male sterility and promoting the utilization of heterosis in eggplant.