Establishment Of Flow-Sorting System And Analysis Of Single Chromosome Genome For Erianthus Arundinaceus Chromosomes From Backcross Progenies Between Saccharum Spp. And E. Arundinaceus

The Erianthus arundinaceus is a relative plant of genus Saccharum.E.arundinaceus has many desirable agronomic traits for sugarcane breeding,such as strong resistance,broad adaptability,excellent perennial root property and a high biomass,which is favored by sugarcane breeders and has been used in cross breeding of sugarcane.However,E.arundinaceus genome research is secular stagnation.Because E.arundinaceus with breeding value is a hexaploidy plant,and the size of its genome is about 6.98 Gb,seriously hindering its full utilization in sugarcane breeding and superior genes mining.To reduce the difficulty of assembling the E.arundinaceus genome,it is one of feasible ways to reduce ploidy level or chromosome number.Therefore,single chromosome of E.arundinaceus was sorted by flow cytometry for sequencing in this study,which was hoping to solve the problem of genome assembly.First of all,the true hybrids were identified by PCR with AGRP52/53 primer pair in the progenies of BC₄ or BC₅ between Saccharum spp.and E.arundinaceus.The number of chromosomes were identified in these true hybrids using GISH,screened and saved the hybrids containing one E.arundinaceus chromosome.Then,in order to sort a large number of the target E.arundinaceus chromosomes using flow cytometry,a flow-sorting technique system was established for the E.arundinaceus chromosomes from backcross progenies between Saccharum spp.and E.arundinaceus.Finally,whole genome amplification was performed on a large number of sorted E.arundinaceus chromosomes by multiple displacement amplification（MDA）method.The third generation high-throughput sequencing technology,PacBio,was used to sequence and analyze the genome of single E.arundinaceus chromosome.The following main results were obtained through experimental analysis in this study.1.The chromosome transmission was studied in high generation backcross materials of E.arundinaceus and the clones with only one E.arundinaceus chromosome were identified and obtained.A 364-bp tandem repeat unit EaHN92 was cloned from the E.arundinaceus clone Hainan92-105 with E.arundinaceus-specific primer pair AGRP52/53,and was localized on sub-telomeric regions of all E.arundinaceus chromosomes in Hainan92-105,and was only localized on sub-telomeric regions of 7chromosomes in YCE06-61（BC₃）.True hybrid progenies between Saccharum spp.and E.arundinaceus were precisely identified using PCR with a primer pair,AGRP52/53.By GISH analysis,we found that hybrid progenies of YCE06-61 contained 1-6 E.arundinaceus chromosomes,and hybrid progenies of YCE07-71 harbored 1-5 E.arundinaceus chromosomes.According to the number distribution of E.arundinaceus chromosomes in the progeny clones,the occurrence probability of a clone with half number of paternal E.arundinaceus chromosomes was higher than others,and the occurrence probability of a clone with one E.arundinaceus chromosome was low.The fewer number of E.arundinaceus chromosome in the parent,the higher probability of clones with one E.arundinaceus chromosome in the progenies.2.A flow-sorting technique system was established for the E.arundinaceus chromosomes from backcross progenies between Saccharum spp.and E.arundinaceus.After cell cycle synchronization,the sugarcane root tips were fixed with 2%formaldehyde fixator at 4℃,then the root tips were cut in 0.1 cm.A high-quality chromosome suspension with LB01 buffer was obtained after homogenate of 0.1 cm root tips with 18000 rpm for 15 s.When we performed the genomic in situ hybridization in suspension（GISHIS）,250 ng/μL genomic probes of E.arundinaceus were added 9μL in 500μL suspension.The process of hybridization was need to keep rotated for 6 h at 37℃.The GISHIS results showed that FITC fluorescence was specifically labeled on the chromosomes of E.arundinaceus,and the hybridization signal was clear and full.However,FITC fluorescence was not observed on the chromosomes of Saccharum spp..When the samples were stained only with DAPI,the voltages of FSC,SSC and DAPI were set at 780 V,260 V and 525 V,respectively,and the suitable sorting gates were drawn to sort the sugarcane chromosomes.The results of sorting showed that they were independent chromosomes without chromosome clusters and nuclei.Therefore,the results of sugarcane cell cycle synchronization and sugarcane chromosome suspension preparation were very good,which could be used for subsequent experiments.After the samples were performed on GISHIS and re-stained with DAPI,the voltages of FSC,SSC,DAPI and FITC were set at 500 V,410 V,530 V and 450 V,respectively,and suitable sorting gates were drawn to sort FITC-labeled E.arundinaceus chromosomes.The purity of these sorted chromosome was above 97%,which could be used for sequencing research.3.E.arundinaceus chromosome 1 was sorted,sequenced,assembled and analyzed.A total of 218,100 single E.arundinaceus chromosomes from 1679-33,a backcross progeny between Saccharum spp.and E.arundinaceus,were collected using flow-sorting,with an average purity of 97.85%.A total of 29,629.2 ng DNA was obtained through whole genome amplification with MDA method.Total of 10.45 Gb data was obtained using Illumina Navo Seq 6000 sequencing with 2×150 bp mode.This short reads data was mapped to Sorghum genome using bwa software,and the results determined that the sorted chromosome was chromosome 1 of E.arundinaceus.CCS reads data of 1.24 Gb were obtained finally using PacBio CCS sequencing with 10,499 bp of N50.Using the Hi Fiasm software,the chromosome 1 genome was assembled and obtained 144.29Mb genome with 21,700 bp of contig N50.Through bioinformatics analysis,110.77 Mb repeat sequences were predicted,accounting for 76.77%of the genome.8,875 encoding genes were predicted,with a total length of 18.37 Mb,accounting for 12.73%of the genome.Among them,8,585encoding genes function were annotated,accounting for 96.73%of the total number of predicted genes.2074 non-coding RNAs with a total length of 214,720 bp were predicted,accounting for 0.142%of the genome.4.The phylogenetic relationship of E.arundinaceus and other species was studied using chromosome 1.The coding sequence（CDS）of chromosome 1 of E.arundinaceus,Sorghum bicolor,Zea mays,Oryza sativa,Saccharum spontaneum and Saccharum hybrid（R570）to construct a phylogenetic tree,it was found that E.arundinaceus was far related to Oryza sativa and Zea mays,followed by Sorghum bicolor,and E.arundinaceus was closely related to S.spontaneum and Saccharum hybrid.At the same time,analysis of distribution characteristics of gene components suggested that the phylogenetic relationship between the E.arundinaceus and the Saccharum hybrid was closer than that of other species.