Construction Of Citrus Fine Mapping Platform And Its Application In The Study Of Polyembryony And Color Bud Mutation

The researches of plant omics have rapidly developed with the advance of the omics technologies and bioinformatics methods.As one of the most important fruit crops,citrus has made constant process in improving omics platform.Based on these,the genetic mapping for the agriculturally important genes and the identification of genetic variation have become the research hotspots in the field of citrus study.In this study,the comparative genomics and population genomics analyses of the represented primitive,wild,and cultivated citrus were performed to elucidate the selection and variation during the evolution of citrus.A combination of bulk segregant analysis?BSA?on genetic population and local association analysis on natural population was used to localize the polyembryony locus.Based on the comparisons among in vitro cultured calli,we demonstrated the effects of somaclonal variation on the somatic embryogenesis ability.By the comprehensive analyses of genome,transcriptome of pummelo bud mutants,we studied the characteristics of bud mutations.The main results were as followed:1.The comparative genomics and population genomics analyses among the primitive,wild,and cultivated citrus?1?The do novo assembly and annotation of genomes for Atalantia buxifolia,Citrus ichangensis,C.medica,and C.grandis had been performed in our lab,and the genomes of sweet orange and clementine mandarin had been released in the published papers.These six citrus genomes were used to conduct comparative genomics analyses.A.buxifolia is a primitive citrus,and others are belong to the true citrus fruit trees group,among which C.ichangensis is a wild citrus,while citron,pummelo,sweet orange,and clementine mandarin are modern cultivated citrus accessions.The comparisons of genome sequences revealed that the genome sizes of the true citrus fruit trees were similar,but the genome size of atalantia was much less.Based on the presence-absence variation?PAV?analysis,the annotated genes in the uniquely presented sequences in pummelo compared to atalantia were significantly enriched in three GO terms:defense response,proteolysis,and pectate lyase activity.The unique regions in atalantia relative to pummelo had a higher TE content?54.20%?than the entire atalantia genome?43.55%?.?2?For each of the 8,551 single-copy orthologs shared by six citrus genomes,the ratio of nonsynonymous to synonymous substitutions?dN/dS?was calculated.In atalantia,12.79%of the genes showed a value greater than one,which was higher than the average value of 6.82%from other citrus.These results indicated greater sequence divergence in atalantia relative to other citrus,and might have functional associations with the biological features of atalantia.?3?The natural citrus population consisted of 108 represented atalantia,ichangensis,citron,pummelo,mandarin,and orange accessions was used for citrus diversity analysis and genetic differentiation analysis.The fixation index(F_ST)values among these citrus populations fluctuated around 0.28,which indicated moderate genetic differentiations among citrus populations.Nucleotide diversity???analysis indicated that atalantia had the highest genetic diversity and that papeda had a higher level of genetic diversity than pummelo with a dramatic reduction in genetic diversity between atalantia and papeda.Comparative population analysis suggested that genomic regions harboring energy-and reproduction-associated genes were probably under selection in cultivated citrus,for instance,the flowering time controling FPA and WOX coding WUSCHEL-related homeobox.2.The genetic mapping for citrus polyembryony and effects of in vitro culture on the somatic embryogenesis ability?1?The BSA-seq was conducted on the segregating population derived from a cross between HB pummelo?monoembryony?and Fairchild mandarin?polyembryony?,and the polyembryony locus was localized to a region spanning from 23.695 to 25.655 Mb on chromosome 4.To fine map the polyembryony locus,a local association analysis with the variant information from the protein-encoding genes in the 1.96 Mb region was performed using the genome sequencing data from the 108 aforementioned accessions,including 45 polyembryonic and 63 monoembryonic accessions,and the candidate region was narrowed to an⁸0-kb region on chromosome 4,between 24.54 and 24.62 Mb,which contains 11 genes.Consistent with this result,selection signature between monoembryonic and polyembryonic citrus on the region was identified.Furthermore,a co-dominant InDel marker in the polyembryony-related region was developed for the identification of citrus polyembryony/monoembryony.?2?Taking the in vitro cultured calli as experimental materials,comprehensive analyses were conducted to study the molecular basis of the reduced ability of somatic embryogenesis.The transcriptomic profiling showed that the stress response and the synthesis of cell wall related genes were significantly down-regulated in the long term in vitro cultured calli.There existed a clue of negative regulation of CHG DNA methylation of gene body region on the gene expression level.?3?For the comparison of long term cultivated calli to the newly induced callus,nine somatic embryogenesis related genes with differential methylation level and expression level were identified,which were probably related to the reduced ability of somatic embryogenesis.By the weighted gene co-expression network analysis?WGCNA?of 12transcriptomes from different tissues and stages of sweet orange,147 hub genes specifically expressed in the newly-induced callus were identified.Among them,there were genes involved in somatic embryogenesis under somaclonal epigenetic variation,for instance,LEA coding late embryogenesis abundant protein and KOR coding endoglucanase.3.The study of pummelo flesh color bud mutants and the genome wide association study?GWAS?for flesh color of pummelo?1?The SNP calling and genotyping analyses on six tissues of flesh color mutants from pummelo cv.“Guanxi miyou”demonstrated that the genotypes on some sites throughout a plant were not completely consistent,suggesting genomic chimerism of bud mutants.?2?The fruit of the red-flesh mutant of pummelo is rich in lycoypene,and the fruit of the orange-flesh mutant of pummelo is high in?-carotene.However,besides NCED2in the red-and orange-flesh mutants were highly expressed,the expression of carotenoid pathway genes in the red-and orange-flesh mutants relative to the wild type did not show significant difference.The significantly up-regulated genes in the red-and orange-flesh mutants were overrepresented in oxidoreductase activity,electron carrier activity,and heme binding related categories.Other up-regulated genes in orange-flesh mutant to the wild type were enriched in the GO terms of transporter activity and nutrient reservoir activity.?3?The genomic copy number of the red-and orange-flesh mutants remained stable.The number of single nucleotide variations?SNVs?with high confidence in the red-flesh mutant was 1,413 with mutation rate of 4.086×10^-6 SNV/bp.Among the genes overlapped by these SNVs,13 genes showed differential expression in the red-flesh mutant compared to the wild type.The number of SNVs with high confidence in the orange-flesh mutant was 2,019 with mutation rate of 5.839×10^-6 SNV/bp.Among the genes overlapped by these SNVs,17 genes showed differential expression in the orange-flesh mutant compared to the wild type.?4?The natural pummelo population of 191 represented pummelo accessions was used for the GWAS for the mapping of the red-flesh related loci.The significant associated loci were on the two loci on chromosome 7,one locus on chromosome 9,and one locus on chromosome 2,respectively.In summary,this study had improved the citrus genomic platform,and finished the genetic mapping for citrus polyembryony-related loci and candidate loci for fruit flesh by optimized mapping strategies,and showed a valuble approach for the genetic mapping of agriculturally important genes in the citrus study,and also provided multi-omics integrated analysis pipeline for the identification of citrus somatic variation.