Genomic Basis Of Citrus Domestication And Somatic Mutation

It has a long history of citrus cultivation in southern China.People has bred many well-known citrus varieties,and many wild citrus resources still grow elsewhere vigorously.Although large endeavor was made to citrus resource collection and genetic analysis for many years,the domestication and evolution history of citrus family has long been controversial due to the polyembrony in citrus plants,and worldwide spread througth clonal propagation,complex genetic background and frequent interspecific hybridization.Based on a citrus genomic platform,we developed a set of InDel markers and an online software Citrus ID for the identification of citrus unknown species.Scientific bioinformatic method was used to study the characteristics of genetic background and the domestication history of mandarin population.We identified genomic region under domestication and gene related to the low acid cultivated mandarin,and we also identified somatic variation in citrus hybrids(mainly in sweet orange)using rigorous methodology.The genomic landscape of the citrus bud mutation during clonal propagation was the first report in our study.The major results are following:1.InDel markers development based on agarose gel experiment.We develop the Indel marker by a gap alignment.The sweet orange reference genome was cut into 300 bp fragments,and compared to other citrus genomes including Clementina,pummelo,Mangshan wild mandarin.We use Blat to detect the high-quality gap alignment.The gap length was limited between 30 and 500 bp.Then Indel primer was designed and amplificated using the agarose gel experiment.We developed the InDel marker for some citrus representative species and the verification rate was over90%,and 268 high-confidence InDel markers was developed based on agarose gel experiment and we established a set of convenient and labor-saving DNA molecular markers for citrus germplasm resources identification.2.Development of online software for citrus identification-Citrus ID.By comparing the population of mandarin,pummleo,citron,Ichang papeda,kumquat and Poncirus citrus,we identified 2.1M high quality SNP which is specific to the six citrus populations,ie mandarin,pummelo,citron,Ichang papeda,poncirus orange,kumquat.We develop a method to detect the admixture pattern based on these species-specific sites,and a pipeline to analysis the genomic characteristic and genetic background.The database currently contains 489 known citrus germplasm with sequenced genomic data,including mandarins,pummelos,citrons,Ichang papedas,kumquats,poncirus oranges,sweet orange,and sour orange,lime,lemon,grapefruits and other wild citrus material.We established Citrus ID,the first online database of citrus for genetic identification.3.Genomic basis for the mandarin domestication.66 mandarins including wild mandarin,semi-domesticated mandarin and cultivated mandarin was collected around the Nanling Mountains and regions nearby.Compared with wild mandarin,citric acid content is significantly reduced while sugar content had almost no difference in cultivated mandarin.By population genomics comparison of mandarin with the four other citrus species,ie pummelo,citron,Ichang papeda and Atalantia,we identified 225 mandarin population specific genomic region called“Genomic Island”.The wild mandarin is genetically more homozygous compared to cultivated mandarin,while the cultivated mandarin has a wide range of introgression from pummelo.The percentage of pummelo introgression ranged from 1.3% to 14.1%.By phylogenetic analysis and genomic structure analysis for mandarin population,we found the Mangshan wild mandarin is a primitive and typical wild mandarin,which is different from the Citrus manshangsensis.In addition,Daoxian wild mandarin,Hezhou wild mandarin,Jiang Yong wild mandarin,which are Chinese landrace,are all true and typical mandarin varieties.The cultivated mandarin population is clutered into two independent groups,MD1 and MD2,which are located in the north and south of the Nanling Mountains respectively.6.5% and 6.8% genomic region was under selection in MD1 and MD2 cultivated mandarins,and only 37.2% of these regions was overlapped,which indicated that MD1 and MD2 was under independent domestication.PSMC model was used to analysis the change of effective size during population.The evolution history of the population show that the Daoxian wild mandarin and MD1 group experienced expansion and bottleneck;while the effective population size of the Mangshan wild mandarin and MD2 group continued to decrease.In addition,MD1 and MD2 cultivated mandarin populations have different pummelo introgression patterns on 9 chromosomes.And the wild mandarin population was detected to have genetic introgression from a kind of wild citrus(Ichang papeda).We found an aconitate hydratase(Cit ACO2)gene was in a genomic region under selection and is expressed differentially between wild and MD2 culitvated mandarin.4.Genomic landascape of somatic mutations in sweet orange genomesWe identified 4167 somatic single nucleotide variation in 30 sweet orange population with validation over 85% and 63 large genomic structure variation including33 allelic deletion or homozygous deletion,18 allelic duplication and 2 gene conversion events.We also detect 5 allelic duplication and 5 allelic deletion in other four citrus hybrid including the Satsuma mandarin,grapefruit,sour orange and lemon.The two largest somatic variation was in the genome of Fukomoko and Newhall navel orange,which were identified 10.1 MB and 12.5 MB allelic replication events,respectively,account for 3.15% and 3.9% genomic region respectively,Other sweet orange‘Jincheng’ were detected a total of 9 allelic deletion events on chromosomes 1,2,3,4,5 and 8.We also found six SNVs and an allelic deletion associated with navel oranges.We identified 262 differientially expressed gene,21 somatic SNV,an allelic deletion on chr3 and two LTR retrotransposon insertion that were highly related to acidless sweet orange.