Molecular Basis Of Apomixis And Grafting Chimerism In Citrus

Most citrus species produce offspring through asexual reproduction,mainly in two ways:one is producing seed through apomixis reproduction;second,agronomic measures through grafting propagation method.The phenomenon of citrus polyembryony caused by apomixis makes it difficult for most citrus to get hybrid after hybridization,which affects the breeding efficiency.But at the same time,apomixis leads to no character segregation and provides an important way to fix heterosis.Grafting,as a traditional method of asexual propagation,has been widely used to improve the resistance,quality and yield of crops in the form of scion-rootstock combination.Grafting chimera is a kind of special material produced in the process of grafting.It provides valuable materials for study of cell layer development and intercellular interactions in plant tissues.In this study,the genetic rules of polyembryony and the molecular basis of nucellar embryo formation were revealed through molecular marker verification and transcriptome data comparison.The influence of Poncirus as a citrus rootstock on scion growth,metabolite accumulation and DNA methylation was analyzed,and the DNA methylation maps for shoots and roots of Poncirus and sweet orange were generated.The cell layer origin model of different tissues and organs of citrus were constructed by analyzing two citrus graft chimeras.The main research results are as follows:1.Analysis of polyembryony candidate genes and development of associated molecular markerBased on previously located 80 Kb polyembryony candidate region,gene cloning and sequence analysis revealed that there were 11 candidate genes in this region.Association between the 11 candidate genes and embryo phenotype was tested by genotyping the SNPs in these genes.Two candidate genes（Cg4g018970 and Cg4g018910）showed the highest correlation with polyembryony.In the gene Cg4g018910,10 SNPs and a 6bp deletion were found to be highly correlated with polyembryony.The 6bp deletion was further developed into a codominant InDel marker,which was used to verify and analyze 786 citrus accessions.It was found that the 6bp deletion was in all 213 polyembryonic accessions,while no 6bp deletion was found in all 573 monoembryonic accessions.At the same time,the marker was also used to analyze the F₁ hybrid population of“HB pummelo（monoembryony）×Fairchild mandarin（polyembryony）”.Among the 124 fruit-yielding individuals,58polyembryonic progeny all had a 6bp deletion,while the other 66 monoembryonic progeny showed no 6bp deletion.The above results suggested that the 6bp deletion was highly correlated with citrus polyembryony,and the InDel marker could effectively identify citrus monoembryony and polyembryony genotype.2.Transcriptome analysis of monoembryonic and polyembryonic citrusTranscriptome data of four different tissues（leaf,ovule,fruit and seed）of monoembryonic pummelo and polyembryonic sweet orange were compared.1637and 1840 genes with specific high expression in ovules were respectively screened from pummelo and sweet orange.Among these genes,853 genes were highly expressed in both pummelo and sweet orange.GO enrichment analysis indicated that987 genes that were only highly expressed in ovules of sweet orange were enriched in almost the same gene ontologies as the group of 853 genes that were highly expressed in the ovules of both pummelo and sweet orange,suggesting that nucellar embryo and zygotic embryo experienced similar development initiation process.In order to explore the molecular basis of the initiation and development of nucellar embryo and zygotic embryo,comparative transcriptome analysis was conducted on ovules from three pairs of monoembryonic and polyembryonic citrus cultivars（pummelo-grapefruit,citron-lemon,and clementine-ponkan）at the key stage of nucellar embryo initiation.29 genes with markedly different expression levels between each pair of monoembryonic and polyembryonic cultivars were screened.Functional annotation analysis showed that some of the se genes were related to biological processes such as stress response and redox reaction.In addition,the expression of the key candidate gene CitRWP controlling polyembryony expressed significantly higher in the ovules of polyembryonic cultivars than those of monoembryonic cultivars.3.Influence of Poncirus as a rootstock on scionThe effects of Poncirus as a rootstock on the physiological indexes,metabolites and methylation levels of the scion were analyzed by setting two grafting combinations:sweet orange auto-grafting and sweet orange grafted on Poncirus.Determination of the growth index and mineral elements of the scion revealed that Poncirus had a dwarfing effect on the scion and Poncirus as a rootstock increased the content of potassium（K）and boron（B）in the scion,and reduced the content of calcium（Ca）,magnesium（Mg）,strontium（Sr）and lead（Rb）.A total of 682metabolites were detected by metabolome analysis of the scion leaves,among which47 metabolites were significantly increased in the scion leaves grafted on Poncirus,and 21 metabolites were significantly decreased.Further analysis showed that the metabolites were mainly concentrated in the flavonoid metabolic pathway,and the upstream genes of the flavonoid metabolic pathway were also up-regulated,indicating that grafting with Poncirus as a rootstock can promote the accumulation of flavonoid in scions.In addition,expression analysis of DNA methylation related genes in the scion revealed that three DME（DNA Glycosylases DEMETER）genes related to demethylation were up-regulated.The results of sulfite sequencing showed that Poncirus as rootstock resulted in 3%decrease of DNA methylation level in the scion.4.Methylation map of shoots and roots of Poncirus and sweet orangeIn order to compare the DNA methylation patterns of common rootstock s and cultivars,the bisulfite sequencing for the shoots and roots of Poncirus and sweet orange were conducted.The results showed that among shoots of Poncirus,roots of Poncirus,shoots of sweet orange and roots of sweet orange,CG methylation levels ranged from 42.75%to 49.14%,CHG methylation levels ranged from 23.78%to28.89%,and CHH methylation levels were about 7%.The DNA methylation levels of CG and CHG in Poncirus were lower than those in sweet orange.In Poncirus,CG and CHG methylation levels of roots were higher than those of shoots,while the opposite is true for that in sweet orange.In both Poncirus and sweet orange,the CHH methylation level of roots was lower than that of shoots.DNA methylation sites were unevenly distributed on chromosomes.Combined analysis of DNA methylation and transcriptome data indicated that DNA methylation could partially affect gene expression levels.5.Genetic mechanism of grafted chimeras and cell layer origins of different tissues and organs of citrusA series of InDel and SNP markers were developed to verify the cell layer origin of the two chimeras,?Zaohong?chimera and?Hongrou Taoye?chimera.Using the two chimeras,the cell layer origins of different tissues and organs of citrus were traced.Leaf and flower organs are developed from three cell layers.In fruit,the exocarp,segment wall and juice sac are developed from three cell layers,while the mesocarp and vascular bundle originate from L2/L3 cell layers.In seeds,the endopleura is derived from the three cell layers,while the episperm,cotyledon,embryo and germinated seedlings are developed from the L2/L3 cell layers.At the same time,it was found that the contribution ratio of each cell layer was different in different tissues and organs.Using the transcriptome data of?Zaohong?chimera and its two parents,215genes that activated and 155 genes that inhibited by the intercellular interaction were screened in the peel or juice sac of?Zaohong?chimera.Using transcriptome data and SNP information in gene coding region between the two parents,59 genes specifically expressed in L1 layer and 1116 genes specifically expressed in L2/L3 layer were identified.