Study On The Genetic Basis Of Excellent F₁ Hybrids And Drought Resistance Heterosis Of Fraxinus Mandshurica×Fraxinus Rhynchophylla

Cross breeding is one of the important methods to create excellent germplasm resources by using heterosis,and it plays an important role in the breeding of improved varieties of Fraxinus mandshurica.Aiming at the problems of short growth period and easy dormancy of Fraxinus mandshurica,this study compared the differences between Fraxinus hybrid progeny F₁ and Fraxinus mandshurica female parent,Through the methods of field character investigation,physiological index determination,epigenetic regulation and differential expression analysis of key genes,the causes of F₁ Heterosis were analyzed from the perspectives of growth parameters,physiological indexes,gene expression and hormone content.Further,we will clone the differentially expressed gene Fm GA20ox2,bioinformatics analysis,construct an overexpression vector to infect Fraxinus mandshurica instantaneously,and analyze the effect of gene overexpression on the expression of related drought resistant genes.The dominant mechanism of F₁ offspring was analyzed layer by layer from phenology,growth,physiology,gene expression and apparent modification.The main contents are as follows:1.The progeny F₁ has growth advantages:compared with the female parent,the high growth period of the progeny F₁ lasts for 28 days,the defoliation period is 11 days later,the annual average height increases by 45.9%,and the annual average stem growth increases by29.8%.The research shows that the chlorophyll content,relative moisture content,SOD and pod of the progeny F₁ are significantly higher than those of the female parent,while the relative conductivity and MDA are significantly lower than those of the female parent,which provides physiological support for the growth advantage of the progeny F₁;Further studies showed that the gibberellin,cytokinin and auxin of offspring F₁ were significantly higher than those of female parents,while the contents of ethylene and abscisic acid hormones were lower,which was the hormone guarantee for the active growth of offspring F₁.The later expression of F₁ rhythm genes TOC1,LHY and CCA1 in offspring delayed the growth dormancy node time,and the lower and later expression of aging genes NAC29,ABA and Sn RK2 transformed by abscisic acid synthesis and EIN2 and EIN3 genes promoting ethylene synthesis made F₁ have growth advantages and stronger dormancy inhibition ability.The degree of methylation and deacetylation of offspring F₁ was lower,which made the growth more active and dormant later.2.The progeny F₁ has the advantage of drought resistance:in terms of growth parameters,the growth rate of progeny F₁ is higher than that of female parent under drought stress and does not stop growing;In terms of physiological indexes,the content of defensive enzymes,soluble sugar and other stress markers in F₁ offspring increased more.On the 18th day of drought,the content of chlorophyll and water content were higher than that of female parent,and the water content was 37.6%higher than that of female parent,while the relative conductivity and MDA were lower than that of female parent（84.9%and 52.0%respectively）;In terms of endogenous hormones,ethylene and abscisic acid increased rapidly,while cytokinin and gibberellin decreased less.These indexes changed in the direction of enhancing drought resistance;In terms of related gene expression,the high expression of SAG113（sense associated gene）gene in offspring F₁ under short-term drought reduced stomatal opening and enhanced drought resistance.Under drought stress,the expression of Fm GA20ox2 gene in F1progeny decreased less,172.11 times higher than that of female parent,and the gap was the largest,and the expression of rhythm gene and ethylene gene EIN2 was lower;At the apparent level,the methylation growth of offspring F₁ was less,and the above indicators of offspring F₁recovered rapidly after rehydration.In conclusion,offspring F₁ had stronger drought resistance and recovered rapidly after rehydration.3.Apparent advantage analysis of F₁ offspring:DNA,RNA methylation and HDAC（histone deacetylase）enzyme activity of F₁ offspring under non drought stress were lower than those of Fraxinus mandshurica female parent.The molecular F₁ offspring in June were0.31%,0.07%and 31242.12 U/min/mg respectively,which were 86.1%,76.8%and 92.1%of the female parent,and 78.5%,73.2%and 73.9%of the female parent in August.Combined with the results of related gene expression,physiological and biochemical indexes and growth traits,hypomethylation made the growth of offspring F₁ more active,and the high expression of rhythm genes was later,which delayed the growth dormancy node and ensured the growth advantage of offspring F₁.The DNA methylation of progeny F₁ under drought stress was lower,and the DNA methylation,RNA methylation and HDAC enzyme activity of progeny F₁ after18 days were only 83.1%of that of female parent.After 14 days of rehydration,the DNA methylation,RNA methylation and HDAC enzyme activity of progeny F₁ were 52.4%,42.9%and 73.9%of that of female parent respectively,indicating that low methylation played an important role in the formation of Heterosis of progeny F₁.Under drought stress,the expression of Fm GA20ox2 gene in F1 progeny decreased less,172.11 times higher than that of female parent.Spray 200μmol/L on Fraxinus mandshurica After 9 days of drought stress treatment,the expression of Fm GA20ox2 was 81.6 times higher than that of the control group,indicating that hypomethylation promoted the expression of Fm GA20ox2.It is speculated that the high expression of Fm GA20ox2 in offspring F₁ under long-term drought promotes gibberellin synthesis,alleviates drought and enhances dormancy inhibition through gibberellin.4.Bioinformatics analysis and transient expression of Fm GA20ox2:the differential gene Fm GA20ox2 was cloned.The total length of the gene was 1116 bp,encoding 371 amino acids,and the relative molecular weight of the protein was 41940.66 Da;Theoretical isoelectric point is 5.32;It is a stable hydrophilic protein without signal peptide and transmembrane domain.The predicted results showed that it was located in the nucleus and cytoplasm.72 hours after transient infection,the expression of Fm GA20ox2 gene was up-regulated to 87.53 times that of the control group.At the same time,the expression of ATG8e,TOC1 and SAG113 genes were significantly reduced,and the expression of SAG113 gene was reduced by 4.16 times.This indicates that Fm GA20ox2 gene is involved in the formation of F₁ Heterosis in offspring by affecting the expression of autophagy,rhythm,aging and other genes.