The Developmental Process And Genetic Rule Of Multi-Ovary Trait And Its Molecular Mechanism Of Heterogeneous Cytoplasmic Suppression In Wheat

As an important food crop,wheat plays an essential role in world food security.Although wheat has obvious heterosis,large-scale effective utilization of wheat hybrids has not been achieved.One of the most significant reasons is that the propagation coefficient of hybrid is low,which leads to a high cost of seed production.This has become a crucial bottleneck problem for the efficient use of hybrid wheat.Multi-ovary wheat has the obvious advantage of increased grains per spike,thereby improving the propagation coefficient,reducing seed production cost,and potentially solving the key problem of hybrid wheat production.In a previous study of the genetic basis of the multi-ovary trait in the wheat,we found that the phenotypes of the reciprocal crosses between multi-ovary wheat DUO?and the alloplasmic line TeZhi??TZ??were different.Specifically,using DUO?as the female parent and TZ?as the male parent,the phenotype of F₁ was multi-ovary,but when TZ?was the female parent and DUO?was the male parent,the F₁ expressed the mono-ovary trait.This suggested that the expression of the multi-ovary gene was influenced by the alloplasmic cytoplasm.In order to further study the heterogeneous cytoplasm suppression of the multi-ovary gene in wheat,we first investigated the developmental process,genetic rule and cytoplasm suppression of multi-ovary.Then,we compared the DNA methylation status,transcriptome,and proteome of the reciprocal crosses between DUO?and TZ?.The main results and conclusions were as follows:1.By observing the developmental process of additional pistils,we found that the additional pistil was derived from a protrusion generated at the base of the main pistil,between the frontal stamen and lateral stamen.Its development was greatly delayed compared to the main pistil at an early stage.However,after the awn exposed stage,it developed very rapidly to a mature pistil,thereby,within the maturity time of the main pistil.Generally,the grains originating from additional pistils were smaller than the grains from the main pistil.And the seed orientation was opposite to common wheat.By studying the penetrance and germination conditions of multi-ovary wheat,we found that no matter which ovary the grain originated from,they had the same penetrance.However,the germination ability of grains generating from the main pistil was significantly higher than that of grains from additional pistils.2.To study the genetics governing DUO?under the influence of heterogeneous cytoplasm,we made the reciprocal crosses between DUO?and TZ?.By investigating the multi-ovary trait in the reciprocal crosses and the F₂,F₃,BC₁,and BC₁F₁ offspring,we found that the DUO?multi-ovary trait was controlled by a dominant gene,and that the heterogenous cytoplasm of TZ?suppressed the expression of this gene.In addition,the special heterogeneous cytoplasm could only suppress the expression of the heterozygous,but not homozygous,dominant multi-ovary gene,which resulted in the heterozygous dominant multi-ovary plant producing a mono-ovary phenotype,and the homozygous dominant plant producing the multi-ovary trait.3.In order to study the relationship between DNA methylation and the cytoplasmic suppression of the multi-ovary gene,we used methylation-sensitive amplification polymorphisms to assess the DNA methylation status of the reciprocal crosses.Genome-wide,14584 5?-CCGG-3?sites were detected and the overall methylation levels were 31.10%and30.76%in the respective crosses DUOII×TZI and TZI×DUOII.Compared with DUOII×TZI,TZI×DUOII showed 672 sites?4.61%?in which methylation–demethylation processes occurred.The demethylation process included 360 sites,and the methylation process included312 sites.The results showed that the special heterogeneous cytoplasmic significantly changed DNA methylation,and this might be associated with the cytoplasm suppression of the multi-ovary gene.4.We RNA-sequenced the entire transcriptome of 2–6 mm long young spikes,the critical stage of additional pistil primordium development,obtained from the reciprocal crosses between DUOII and TZI.A total of 600 differentially expressed genes?DEGs?was identified.Of which,330 DEGs were up-regulated,and 270 DEGs were down-regulated in TZI×DUOII.Functional annotation of these differentially expressed genes showed that the heterogeneous cytoplasmic suppression of additional pistil development mainly involved four pathways,i.e.,chloroplast metabolism,DNA replication and repair,hormone signal transduction,and trehalose-6-phosphate in the primordium development stage,which cooperated to modulate the multi-ovary gene expression under heterogeneous cytoplasmic suppression.5.We first established an optimized two-dimensional electrophoresis profile which was suitable for the study of young spikes in wheat.Then,using this profile,we compared the quantitative proteomic profiles of 2–6 mm long young spikes,the critical stage of additional pistil primordium development,obtained from the reciprocal crosses between DUOII and TZI.A total of 90 differentially expressed proteins?DEPs?were identified.Of which,18 DEPs were up-regulated,and 72 DEPs were down-regulated in TZI×DUOII.These proteins had obvious functional pathways mainly implicated in chloroplast metabolism,nuclear and cell division,plant respiration,protein metabolism,and flower development.By drawing relationships between the 90 differentially expressed proteins,we found that these proteins revealed a complex network which is associated with multi-ovary gene expression under heterogeneous cytoplasmic suppression.In addition,based on the comprehensive analysis of the results of DNA methylation,transcriptome and proteome,we proposed a molecular mechanism that was responsible for the cytoplasmic suppression of multi-ovary trait,which provided a solid foundation for further studies and utilizations of multi-ovary trait in wheat.