| Chinese chestnut is native to China,mainly distributed in mountainous with poor soil,and is an important ecological and economic forest tree for farmers to get rich in mountainous areas of China.As a woody grain,Chinese chestnut is the main chestnut producing country in the world.The average yield per mu was only 65 kg,but the low production capacity per unit land area is one of the key problems in the development of chestnut industry.Chinese chestnut is a dioecious plant,male and female flowers are important traits to determine the formation of yield,the ratio of catkins to female of is1:5-10,the ratio of male to female flowers is 1:2000-3000,less female flowers,more male flowers and consume tree nutrients,is the main reason for low yield.Therefore,breeding less male and more female cultivars and adjusting the male to female flower ratio is the core and key to improve the yield.In the course of the preliminary germplasm resources survey,a special material with staminate catkins completely abortive(referred to ‘Huaiqi’)was found.The ratio of catkin to female inflorescences was 1.48:1,catkins were very significantly less than common cultivars,female flowers were significantly higher than common cultivars,and only staminate catkins were defeated,while bisexual catkins were not defeated and female flowers were normal.It is a mutant material for studying male flower development.In this study,we revealed the endogenous hormone balance of staminate catkins abortive and the effect of exogenous plant growth regulators on it.And key candidate genes of staminate catkins abortion were identified,and the functions of the candidate genes were verified in this study,for explore the mechanism of staminate catkins abortion.This will lay a theoretical foundation for male reduction and female increase and yield improvement,and lay the theoretical foundation for chestnut yield formation and regulation as well as molecular improvement breeding,which have realistic guiding significance for chestnut production.The main results are as follows:1.Characteristics and determination of the critical stage of staminate catkins abortion in Chinese chestnut ‘Huaiqi’.The staminate catkins grew normally in the leaf-spreading stage,and the male flower clusters ceased to develop and withered from the top before the male flowers bloomed,and then fell off from the base.Both bisexual catkins and female flowers of on the same fruiting branch developed normally.Grafting trials for three consecutive generations showed that staminate catkins occurred at the time of abortion from May 10-15 in the Beijing,China.The average length of pure staminate catkins at abortion stage was 4.24 cm,which was 23% of the normal inflorescence length,and the ratio of male to female inflorescence was 1.48:1.The critical stage of pure staminate catkins abortion was observed in the middle of stamen primordium development by microstructure.Complete abortion and abscission of the staminate catkins occurred in the late stamen primordia,and compared with the control,the aborted staminate catkins could not develop to the anther-forming stage.2.Discovery and proof of the relationship between auxin and staminate catkins abortion.First of all,determination of the endogenous hormone balance of staminate catkins abortion of Chinese chestnut ‘Huaiqi’.Endogenous hormones showed that the contents of IAA,CTK and JA decreased significantly,and the content of IAA in aborted staminate catkins was 44.27% of that in control.and the ratio of IAA+CTK/GA in abortive staminate catkins was significantly lower than that of the control.Second,the exogenous growth regulator IAA can restore the phenotype and fertility of abortive staminate catkins.To further explore the effect of exogenous growth regulators on abortive staminate catkins,by spraying 11 different plant growth regulators,we screened that 300 mg/L IAA treatment could restore phenotype and fertility to some extent by spraying different types and concentrations of plant growth regulators,in which 63.86% of staminate catkins on fruiting branches were restored.The pollen germination rate in staminate catkins with restored phenotypes reached 42%,which maintained the same level as the pollen germination rate in bisexual catkins.At middle stamen primordium stage,the major synthetic precursor of auxin hormone indole-3-pyruvic acid(IPA)and endogenous auxin hormone were significantly higher in the staminate catkins,4.22-fold and 3.63-fold higher than in the aborted staminate catkins,respectively.In addition,the endogenous jasmonic acid content was significantly increased by 3.88-fold than the control.3.Identified the key genes associated with staminate catkins abortion.By transcriptome analysis,the differential gene analysis species of staminate catkins and restored phenotypic staminate catkins during the critical period of staminate catkins abortion,KEGG enriched in phytohormone signaling and tryptophan metabolism pathways during the middle stamen primordium stage,in which tryptophan metabolism is the main metabolic pathway for growth hormone synthesis.Combined with the changes in the content of precursors of auxin,the key genes of auxin synthesis,CmTAA1/CmTAR and Cm YUCCAs genes,were tapped to play major roles in aborted staminate catkins.In addition,it was also found Cm AUX/IAA,Cm ARF genes of auxin signaling pathway and Cm AOC1,Cm JAZ10 genes of jasmonic acid synthesis and signaling pathway would be involved in staminate catkins abortion.4.The key candidate genes were executed to validate the function.CmTAR-RNA itransiently transformed in male inflorescences on bisexual catkins of Chinese chestnut‘Huaiqi’ underwent abortion,and the CmTAR gene was significantly down-regulated in bisexual catkins abortion.It also caused a significant decrease in IPA and IAA content,and the downstream expression of auxin genes Cm YUCCAs,and auxin signal transduction genes Cm ARF to be down expression regulated.Further the expression of CmTAR gene was significantly down-regulated in the development of staminate catkins of similar staminate catkins abortive materials,supporting the involvement of CmTAR gene in regulating the abortive staminate catkins of Chinese chestnut. |
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