Molecular Basis For The Formation Of Male Sterile Cytoplasm And Restorer Gene Effect In G.harknessii

Cytoplasmic male sterility is the most economical and efficient method to produce hybrids.At present,the three-line hybrid cotton used in China is based on the cytoplasmic male sterile line of G.harknessii,which performs equivalently to the artificial hybrids in yield,quality,and resistance.However,higher temperature had negative effect on the male sterile cytoplasm of G.harknessii,especially in the summer in the Yangtze River basin.In addition,the effects of restorer genes for cytoplasmic male sterility in G.harknessii are not clear,which limits the further development and utilization of three-line hybrid cotton.At present,studies on the cytoplasmic effects of G.harknessii male sterility are mainly focused on agronomic characters,yield,quality,resistance,and so on,while the effects of restorer genes are rarely reported.In this study,a set of male sterile cytoplasmic restorer lines(SH),fertile cytoplasmic restorer lines(NH)and maintainer lines(NB)created by many years of backcrossing were used as materials(SH and NH were isonuclear,NH and NB were homoplasmic).Under the environmental conditions of Anyang and Jiujiang,the metabolites and gene expression changes were analyzed in seedling stage,bud stage,boll stage leaves,and mature pollen of three materials by integrated metabolome and transcriptome sequencing.The gene-metabolite network was constructed to elucidate the molecular basis of the effects of male sterile cytoplasm and restorer genes in G.harknessii during the coordination of leaves,mature pollen,and leaf-pollen source pool at different developmental stages.The main results are as follows:1.The effect of restoring gene on leaves mainly promoted the biosynthesis of ascorbic acid and the metabolism of glycerol phospholipid,and this effect was more obvious in Jiujiang environment.In the comparison group of NH and NB,L-galactose,inositol-phosphatase and L-galactose-1 Lactone dehydrogenase in ascorbic acid biosynthesis pathway,phosphatidylethanolamine,ethanolamine kinase and ethanolamine phosphotransferase in glycerol phospholipid metabolism pathway were significantly up-regulated in NH at bud and boll stage.The effect of male sterile cytoplasm on leaves was mainly due to the inhibition of flavonoid biosynthesis.In the comparison group of SH and NH,the genes encoding chalcone synthase,anthocyanin synthase and flavonol synthase as well as quercetin and myricetin were significantly down-regulated in SH at seedling,bud and boll stage.2.The effect of restorer gene had no significant effect on the fertility of mature pollen,but the fertility restoration process of the interaction between restorer gene and male sterile cytoplasm activated the biosynthesis pathway of flavonoids and induced the accumulation of a large number of flavonoids,thus promoting the restoration of pollen fertility.The effect of sterile cytoplasmic effect on mature pollen is mainly due to the disorder of lipid metabolism,especially the metabolic balance of unsaturated fatty acids,which finally leads to the decrease of pollen fertility,and this negative effect becomes more obvious with the increase of temperature.On the basis of WGCNA,key core genes such as WRKY22,WRKY53 and ARF18 were identified,which may be involved in the regulation of pollen fertility.3.The effect of restoring gene promoted the process of glucose metabolism and transport in leaves at bud stage,and enhanced the pathways of glycolysis,citric acid cycle and pentose phosphate metabolism.In the comparison group of NH and NB,two rate-limiting enzymes,phosphofructokinase and pyruvate kinase,were significantly up-regulated.At the same time,sucrose transporter SUC4 was also significantly up-regulated.The effect of male sterile cytoplasm inhibited the process of glucose metabolism and transport in leaves at bud stage,and glycolysis,citric acid cycle and pentose phosphate metabolism pathway were all inhibited in varying degrees.In the comparison group of SH and NH,3-phosphate ribose isomerase and 5-phosphate ribose isomerase were significantly down-regulated in both environments.Sucrose transporters SUC2,SUC4 and SWEET15 were significantly down-regulated in leaves at bud stage.This study preliminarily revealed the molecular basis of the effects of male sterile cytoplasm and restoring genes in G.harknessii.Restoring genes promoted ascorbic acid biosynthesis and glycerol phospholipid metabolism in leaves,but had no significant effect on mature pollen fertility.The sterile cytoplasm inhibited the biosynthesis of flavonoids in leaves and disturbed the dynamic balance of lipid metabolism in mature pollen.The research results provide theoretical support for parent selection and combination allocation of three-line hybrid cotton,and are of great significance for the popularization and application of three-line hybrid cotton.