Development Of Citrus Model Material And Phylogenetic Study Of Genus Fortunella

Citrus is one of the most widely cultivated and economically important fruit crops.However,the characteristics,such as long juvenility,polyembryony,highly heterozygosity and high stem severely limited the gene function and genetic researches on this taxon.Hongkong kumquat?Fortunella hindsii?,a citrus relative characterized as short juvenility and dwarf,belongs to Fortunella genus under the“true citrus fruit tree”group.Recently,our group discovered the monoembryonic Hongkong kumquat?Mini-Citrus?in wild,which showed an unprecedented potential to be the experimental material for citrus.Thus,in present study,aiming to develop a dwarf,early flowering,crossing-friendly?monoembryony?and highly homozygous“model citrus”for scientific purposes,we breed homozygous lines of Mini-Citrus via selfing,investigated the agronomic traits of Mini-Citrus,and construct Mini-Citrus‘PN02'×F.crassifolia‘Huapi'F₁ and F₂ populations.We sequenced and assembly the genome of Hongkong kumquat by using the“pure line”accession‘S3y-45',explored the gene co-expression network via RNA-seq,and provided genetic clews for the flowering features of Hongkong kumquat.Besides,based on molecular markers and chloroplast sequences,we evaluated the genetic diversity,population structure and phylogeny of genus Fortunella.By using population analysis,we further gave a new insight into the origin of cultivated kumquat and answered the previous controversial questions about the classification around this genus.1. Breeding and agronomic evaluationA total of 118 2nd generation selfing lines?S₂?and 28 3st generation?S₃?were bred.A series of highly homozygous accessions with homozygosity higher than 90%?estimated by27 n SSR markers?were selected.Among them,‘S3y-45'is the most homozygous accession with a genomic heterozygosity of 0.62%.Under cultivation environment,about 70%of Mini-Citrus seedings blossomed in their first year with a juvenility of eight months.The monoembryony rate of Mini-Citrus is higher than 90%.The tree of Mini-Citrus showed a very dwarf size as 15.29cm,25.76cm and 35.99cm height for 1,2 and 3 years old.A series of selfing lines were selected for further experimental purposes by a criterion of fruit-bearing number on one-year old seeding;among them,‘S2f-179'is the most outstanding one with a homozygosity of 77.7%and an average fruit-bearing number of5.90.A total of 222 F₁ and 713 F₂accessions of‘PN02'בHuapi'were bred.2. Genome sequencing and related analyses‘S3y-45'was sequenced by using Pacbio,Illumina and10X genomics platforms.A genome assembly with 373.6 Mb of size,2.21 Mb of contig-N50 and 5.16 Mb of scaffold-N50 was obtained?F.hindsii genome V1.0?.A total of 32,257 protein-coding genes were identified,12,360 of them have orthologs in the 9 Aurantioideae genomes,and 986 were species-specific to Mini-Citrus.A phylogenetic tree constructed base on low-copy genes showed a close relationship between Mini-Citrus and Citrus species,pummelo,citron,sweet orange and mandarin;among them,Mini-Citrus genetically close to mandarin?estimated differentiation time was about 5.32 million years ago?but far from Poncirus.A total of 27 co-expression modules were identified by using WGCNA method based on RNA-seq data of 13 tissues collected at different phases during Hongkong kumquat development.31 of 86 key floral genes showed differential expression in Hongkong kumquat,and 18 of them involved in floral induction step.A total of 19 Fh SPLs were identified and 2 more SPL3/4/5 orthologs?Fh SPL7/9?were detected in Hongkong kumquat than in sweet orange.Realtime quantitative PCR results showed the expression of Fh SPL7/9 were significantly upregulated in adult leaf,stem and bud meristem tissues,which were highly positive correlated with key floral induction gene SOC1?r=0.94?but negative with flowering repressor mi R156a?r=-0.91?.3. Phylogeny of genus FortunellaThe collected Fortunella germplasms showed a high nuclear diversity as 4.34 of Na,2.27of Ne,0.49 of Ho,He and u He based on 46 n SSRs,but showed a low cytoplasmic diversity as 0.693 of Hd,0.00073 of Pi and 13 of Nh based on 5 chloroplast loci.Both n SSR and chloroplast phylogenetic trees indicate an independent phylogeny of genus Fortunella.PCo A and population structure analysis indicate a“Cultivated?F.margarita,F.japonica and F.crassifolia?–Wild?Hongkong kumquat?”genetic structure in genus Fortunella.For further population-genomic analysis,15 cultivated kumquat and 15 wild Hongkong kumquat samples were selected based on above genetic distance and their sampling sites.Genomic PCA and population structure analysis verified the“Cultivated-Wild”genetic structure of Fortunella again.Intriguingly,the substructure of cultivated kumquat indicates F.japonica rather than F.crassifolia has an admixture genetic background.The Fst index between each two cultivated species is higher than 0.25,indicating their authorized species status.The SNP diversity of cultivated kumquat population?Pi=0.12,Theta=0.10?is significantly lower than that of Hongkong kumquat?Pi=0.23,Theta=0.26?.The Tajima's D,Fu&Li's D*of Fu&Li's F*statistics of both populations denied neutrality test,indicating that directional selections involved in their evolutions.Genomic linkage disequilibrium analysis showed a higher strength of linkage,a slower disequilibrium decay and a longer decay distance of cultivated kumquat than those of Hongkong kumquat,indicating cultivated kumquat underwent a more forcefully directional selection.Population dynamic analysis revealed that cultivated kumquat experienced the genetic bottleneck?70-120 mya?associated with the quaternary glaciation in front of Hongkong kumquat?30-60 mya?;after that,cultivated kumquat experienced another specific population explosion during 1-2 mya,which might be associated with the human activity.