Revealing The Genetic Basis Of Male Sterility Based On The Evolution Of Citrus Cytonuclear Genome

Seedless is an important breeding goal for citrus fruits.Our research group used cell engineering technology to transfer the male-sterile mitochondria of G1 to several citrus varieties,obtaining cybrid offspring.Among them,the cell-engineered seedless new varieties such as G1+STY and G1+HBP have obtained plant variety rights and are considered core germplasm resources with significant industrial and research value.Cytoplasmic molecular markers are the most effective way to identify the origin of cytoplasmic genomes of cell fusion progeny.The cytoplasmic genomic molecular markers currently widely used in citrus are difficult to use and have poor stability,and the development of citrus cytoplasmic genomic molecular markers can help citrus cell fusion seedless breeding.Previous studies have proved that the male-sterile genes of G1+STY and G1+HBP are derived from G1,but the origin of the male sterility gene in G1 remains unknown.In this study,the cytoplasmic genomes of several different citrus species and varieties were assembled using re-sequencing data from citrus cell fusion progeny,their parents and natural populations.The In Del molecular markers of citrus cytoplasmic genomes were developed;the assembly of citrus mitochondrial pan-genome and population genetics analysis were completed,and the cytonuclear genomes of citrus were studied.We have completed the mitochondrial pan-genome assembly and population genetic analysis,studied the conflict of cytonuclear genomes and analyzed the origin of cytoplasmic male sterility in citrus;identified the key periods of flower organ development and performed transcriptome analysis by observing the paraffin sections of flower organs of G1+STY and Shatian pummelo at several developmental stages,and studied the male sterility of G1+STY.The key genes of cytoplasmic male sterility were identified and transcriptome analysis was performed to investigate the male sterility phenotypes and related nuclear gene functions of G1+STY.The main findings were as follows:1.Citrus cytoplasmic genome assembly,comparison and In Del molecular marker developmentUsing resequencing data,12 citrus mitochondrial genomes and 4 chloroplast genomes were assembled and annotated in this study using long and short reads hybrid assembly method.A comparative study of citrus cytoplasmic genomes was performed in combination with two published mitochondrial and three chloroplast genomes.The results showed that citrus mitochondrial genomes showed extensive recombination,with recombination sites mostly in intergenic regions and great chance in the recombination process;mandarin mitochondrial recombination was more frequent than pummelo,but frequent recombination of mitochondrial genomes had minimal effect on conserved genes.Citrus chloroplast genes are more conserved,with no large structural variation(length > 50 bp)or gene number differences,and a weak expansion of the Inverted repeat region,with most of the intergenomic loci also located in the intergenic region.Molecular marker primers were designed at interspecies-specific In Del sites in the cytoplasmic genome.In mitochondria,62 samples were screened by agarose gel electrophoresis,and five pairs of mitochondrial In Del molecular markers were developed and validated;in chloroplasts,47 samples were screened by capillary electrophoresis,and five pairs of chloroplast In Del molecular markers were developed and validated.The above primers are easy to use and stable.2.Analysis of nuclear and cytoplasmic genome evolution and CMS gene origin in citrusThe 14 citrus mitochondrial genomes collected by assembly were used to construct a mitochondrial pan-genome of the genus Citrus,and to mine 184 citrus species for information on nuclear,mitochondrial and chloroplast genomic variation.Population genetic analysis showed that there was extensive cytonuclear discordance during citrus evolution.At the level of differentiation,hybridization events and evolutionary rate differences led to inconsistent evolutionary trees of cytonuclear genomes,and some citrus mitochondrial genomes had mitochondrial heterogeneity(paternal leakage)leading to differences in mitochondrial and chloroplast phylogenetic trees;at the level of domestication,orange domestication events had different effects on nuclear and mitochondrial genomes.At the level of domestication,the orange nuclear genome experienced two domestication events,but the two events originated from the same type of wild orange,while the diversity of the domesticated orange nuclear genome became larger and the mitochondrial diversity decreased,and the diversity conflict of the cytonuclear genome was the genetic basis of male sterility in the satsuma mandarin line;at the level of hybridization,in the process of citrus evolution,the orange could not be used as a parent to cross with the grapefruit,in addition to polyembryony,the existence of the mandarin mitochondrial and pummelo nuclear genes In addition to polyembryony,the lack of affinity between orange mitochondria and grapefruit nuclear genes is also a reason.Based on the above analysis,this study concluded that the Cytoplasmic male sterility(CMS)gene and Restorer factor(Rf)gene have always existed in wild mandarin,but in the history of mandarin domestication and hybridization,the Rf gene of satsuma mandarin was gradually replaced by rf(or Rf was lost),which finally This led to abnormal male sterility by cytonuclear interaction.In addition,based on cytonuclear interaction selection,a total of 168 nuclear genes interacting with cytoplasm were screened in this study using Genome wide association study of citrus natural hybrid population.3.Phenotypic observation and identification of genes related to male sterility of G1+STYThe fruit of G1+STY was seedless,with deformed floral organs,stunted development of sepals and petals,early decay due to long-term exposure of stigma,and significantly lower filament number and pollen vigor than those of Shatian pummelo.Paraffin sections revealed that most anthers of G1+STY were abnormally developed,the anther walls were abnormally thickened and the intra-anther walls could not form cellulose bands and undergo secondary lignification thickening normally at the later stage of development,and could not enter into programmed cell death,and finally the anther walls could not dehiscence normally,and even if there was partially viable pollen,it could not be dispersed.The transcriptome analysis of four floral developmental periods of G1+STY and STY screened for differential male sterility relative genes and metabolic pathways.The comparison and expression analysis of mitochondrial genomic chimeric ORFs unearthed two chimeric ORFs(orf374,orf384)as cytoplasmic male sterility gene candidates,and the analysis of cytonuclear reciprocal genes screened three PPR genes(Fh3g18750,Fh4g20550,Fh7g08550)as possible fertility recovery genes.