Study On Molecular Mechanism Of Sex Expression Of Pistacia Chinensis Bunge

Pistacia chinensis Bunge is a pioneer tree for afforestation,and of high value as an ornament and for timber and medicine.It has also become the preferred biofuel tree in northern China in current years,because its oil content of fruit is more than 40%.However,it is dioecious and lacks good varieties,leading to waste of manpower and field,thus resulting in low and unstable yield.Rare monoecious individuals were found,to explore its biological characteristics,origin and floral development mechanism,dioecious and monoecious materials were used to explore the phenotypic characteristics of different sex types of P.chinensis in this study.Moreover,DNA molecular markers,sex stability,and inflorescence bud differentiation were studied.Additinally,the inflorescence buds of different sex types during the critical stage of sex differentiation were selected for RNA transcriptome and small RNA sequencing,and the key genes affecting sex determination were screened.Two candidate genes were selected for the sequences analyses and functional identification.The main results are as follows:1.A total of 99 monoecious Pistacia chinensis were found in Hebei and Henan province,including male and female on different branches,on the same shoots,inflorescences,and even formed bisexual forets.A detailed investigation on 23 of them showed that monoecisms were account for 15%of the local Pistacia trees.The appearance of the bisexual florets is similar to that of the male florets,but the inflorescences are significantly larger than the male inflorescences while the difference in the number of florets is not significant,so that its foret spacing is larger.Inflorescences,florel organs and other variations are diverse,such as 1-6 anthers.The blooming phenophase of the monoecious strains changed a lot among different individuals and sex types.Further pollination test proved that the male and female gametophytes were fertile,and the bisexual flowers could be self-pollinated.2.Using 24(pairs of)primers that have been used for sex-identified in the genus Pistacia and 16 pairs of specific primers designed according to specific bands,the DNA samples of the female,male,and monoecious female,male,and bisexual types were amplified.The differential bands are polymorphic results between different plants or groups,regardless of sex.The amplification bands of different sex parts of the same monoecious trees were completely identical.3.The sex expressions of grafted trees were not completely consistent with the sex types of the scions from monoecious trees,and the sex of the single shoot on monoecious trees could change within one year,indicating that monoecious type did not originate from stable bud mutation.4.Paraffin section results showed that all sex types had a bisexual phase of floral primordium in the early stage of floral development.In the first year,two types differentiated-the female organ preferred type(the stamen primordia degenerate into the second round of the tepals,finally could develop into female floret)or the male organ preferred type(the pistil primordium gradually disappeared),then male organ-preferred type could develop into male floret(no pistil primordium)or bisexual flowers(reformed pistil primordium again)in the second year.The internal development process of the development can be initially judged by external morphological characteristics and phenological period.5.The genes among different sex types are similar,but their expression patterns are usually different.The final phenotype of florets may be caused by differences in the expression levels of related genes;the female promoting and male-promoting genes inhibit each other,and their expression trend during the critical sex differentiation phase is opposite.For the bisexual floret,the male promoting genes were highly expressed in the first year,while most of the female promoting and male promoting genes are up-regulated to varying degrees in the second year.In addition,the second sexual differentiation of the bisexual floret may relate to light and cold specific genes,indicating that the formation of bisexual floret is linked to vernalization.Besides,HSP,MADS,AP2 family and other genes play important roles in sex differentiation.6.A total of 385 matures and 735 hairpins of known miRNAs were identified,and 86 matures and 109 hairpins of novel miRNA were predicted in the Small RNA sequencing of different sex type during the two critical sex differentiation stages.Also,23 miRNAs differentially expressed in sex were screened.Association analysis with the transcriptome revealed more important miRNAs and pathways regulating floral development.7.The full-length of PcHSP70-1 and PcHSP90 genes were obtained and their sequence analysis and functional verification were performed.Overexpression of PcHsp70-1 could upregulate the flowering-related genes,promoting bolting and flowering,and could maintain more boltings under drought stress.This study ruled out the hypothesis that the monoecious P.chinensis originated from bud mutations.It is speculated that the gene signature caused by environmental persistence affects sex determination.The final sex expression may change with the gene signature accordding to the environmental factors,which could be an epigenetic phenomenon in which the genotype remains unchanged while the gender changes.