The Study On Genomics Of Origination And Evolution In Saccharum Spontaneum

Sugarcane(Saccharum spp.,Poaceae)was considered as one of the four major crops that had changed the human history,and the cultivation of sugarcane in the new world has promoted large-scale population migration worldwide and profoundly affected the human civilization and the way we live.Sugarcane belongs to Saccharum in Poaceae,and is an important sugar crop and energy crop as it affords 80% sugar and 40% ethanol in the world.Modern sugarcane cultivars are hybrids,which are mainly derived from interspecific cross between S.spontaneum and S.officinarum,resulting in the background of stress resistance in modern sugarcane cultivar contributed by S.spontaneum.Therefore,S.spontaneum is one of the two founding species for modern sugarcane cultivars breeding,and promoted the extensive commercial cultivation of sugarcane.However,mainly due to its highly complex genetic background and high level of polyploid,the Saccharum genome decoding is more challenging than that of other staple crops,consequently,the study on S.spontaneum is relatively backward and the genomics of origination and evolution in S.spontaneum,is still remain unknown.In this study,to understand the evolution and genetic basis of important biological characters and genome evolution in Saccharum,we performed whole genome sequencing and de novo assembly for natural auto-tetraploid S.spontaneum Np-X withancestral karyotype of X=10.To understand the origination and evolution of S.spontaneum,102 accessions were collected from all over the world with diverged genetic background for population genetic analysis.The main results are as follows:Deciphering S.spontaneum genome with ancestral karyotype is the basis for study of genome evolution and population genetics.To obtain the high-quality S.spontaneum Np-X genome,the Circular Consensus Sequencing(CCS)technology of Pac Bio Sequal II platform and Hi Seq X platforms were used to perform the whole genome sequencing for S.spontaneum Np-X,a total of 52 gigabases(Gb)of Pac Bio CCS long reads and 417 Gb of Illumina short reads were obtained for genome de novo assembly by using the Canu(v1.9)software.We generated an initial contig-level genome with N50 of 405 Kb and total assembled size of 2.76 Gb,accounting for 97.53% of the estimated genome size(2n=4x,～2.83 Gb)which evaluated by K-mers based genome survey.To acquire chromosome level genome for the autopolyploid,we further used the ALLHi C to scaffold the genome by integrating ～105x Hi-C pair-end reads.An high quality genome with 40 chromosomes included in 10 homolog groups were acheived and 99.12% of contigs were anchored in the genome.Among the 40 chromosomes,the intact centromeres and telomeres can be detected in 82.5%(except for Chr2 B,4C,5C,6B,6C,6D and 8A)and65%(except for Chr2 A,3B,3D,4D,5C,5D,6B,6C,6D,7A,8B,9A,10 Cand 10D)of chromosomes,respectively,and over 95% of conserved core gene set of BUSCO and CEGMA can be found in our assembly,which is much better improved than before publicated S.spontaneum genome(～94%).A total of122,945 alleles representing 35,830 protein coding genes were annotated in the S.spontaneum Np-X genome.Among them,24.1%,35.9%,30.1%and 9.9% of the genes contain 4 alleles,3 alleles,2 alleles and 1 allele,respectively.Of the annotated genes,～93.67% of genes can be found with functional information in major databases(KOG,NR,GO,KEGG etc).The characteristics of repetitive sequence were explored in S.spontaneum Np-X genome,a total of 1,560.66 Mb transposon sequences accounting for57.52% of the whole genome were annotated from the S.spontaneum Np-X genome.Among them,40.64% of transposon sequences were belong to LTR-type transposons,which can be further divided into 54 families.These results indicated that we have constructed a high quality autotetraploid S.spontaneum Np-X genome and provided a foundation for further study of evolution and population genetics in S.spontaneuum.To understand the genome evolution of Saccharum,compartive genomics were peformed for Saccharum species and its relative speices.Firstly,we analyzed the genome collinearities for S.spontaneum and its close relatives.The results confirmed that S.spontaneum Np-X with a basic chromosome X=10 was the ancestral species of S.spontaneum,andindicated that the evolution process of from the basic chromosome number with X=10 to X=8 in S.spontaneum is accompanied with the breakage and recombination of ancestral Chromosome 5 and Chromosome 8 of S.spontaneum.The detail comparsion showed that the breakages of the two ancestral chromosomes is caused by splits of centromeres which inactivated and degenerated in the reconstructed chromosomes.Secondly,we analyzed the Synonymous substitutions ratio(Ks)among the relative species including S.spontaneum Np-X,S.spontaneum AP85-441,S.officinarum,Sorghum and Miscanthus to characterize the phylogenetic status of S.spontaneum.The results indicated that Np-X vs AP85-441 was0.8 million years ago(MYA),S.spontaneum-S.officinarum was 1.6 MYA,the Saccharum vs Miscanthus was 4.0 MYA,and Saccharum vs Sorghum was 6.4 MYA,suggesting that the whole genome duplication events(WGD)occurred in the Saccharum were independent.Finally,we estimated the insertion time of LTR-type TEs in Np-X,AP85-441,S.officinarum,Miscanthus and Sorghum to clarify the evolutionary relationship of Saccharum,The results indicated that the burst of LTR-type TE were consistent to the differentiation of these species.These results provided substantial knowledge for Saccharum evolution.Sugar accumulation,stress resistance and photosynthesis were considered as the key for sugarcane breed improvement and industrial development.To investigate the genetic base of these important biologicaltrait in sugarcane,we investigated the relative gene families mainly including sugar metabolism,sugar transport,C4 photosynthesis and stress resistance constraction and expansion in Poaceae.The results indicated that the gene families involve metabolism of sugar,sugar transport,C4 photosynthesis and stress resistance were underwent expansioned in S.spontaneum.There are 6 families(SUS,INV,SWEET,SWEET,PPDK-RP,NBS)were specific found in S.spontaneum Np-X,and 10 gene families(SPS,SUT,STP,VGT,NAD-ME,C4,INT,CA,PPDK-RP,NADP-ME)expansion may be associated with the intra-specific differentiation of S.spontaneum.Given the fact that sugar transport is crucial for sugar accumulation among sugarcane,we focused on the phylogenetics and function of sugar transporter(ST)superfamily in S.spontaneum.105 ST genes were identified and clustered into 8 subfamilies in S.spontaneum.Comparative genomics revealed that tandem duplication events contributed to ST gene expansions of two subfamilies,PLT and STP,in S.spontaneum,indicating an early evolutionary step towards high sugar content in Saccharum.The analyses of expression patterns were based on four large datasets with a total of 226 samples of RNA-seq from S.spontaneum and S.officinarum.The results clearly demonstrated 50 ST genes had different spatiotemporal expression patterns in leaf tissues,10 specifically expressed STs in the stem and 10 STs responded to the diurnal rhythm.Furthermore,metabolomics analysis suggested STP7 was a sugarstarvation-induced gene,STP13 had a function in retrieving the sugar in senescent tissues.PLT11,PLT11＿T1,TMT3 and TMT4 contributed to breaking the limitations of the storage sink.SUT1,SUT1＿T1,PLT11,TMT4,p Glc T2,and VGT3 responded for different function in these two Saccharum species.This study demonstrated the evolutionary expansion and functional divergence of ST gene family and will enable the further investigation of the molecular mechanism of sugar metabolism in Saccharum.The precise regulation on gene expression could be regulated by three-dimensional(3D)structure of chromatin,however,to our knowledge,the study on 3D genomics of auto-polyploid is still a blank.To investigate the variation of 3D structure of chromosome influenced by chromosome recombination and genome evolution,we explored the 3D structure feature of S.spontaneum Np-X,S.spontaneum AP85-441,Sorghum and Rice using Hi-C data.The genomic regions were then classified into A/B compartments according to the sign of the first eigenvector.The results showed that the non-conserved spatial structure characters were existed in auto-tetraploid genome in despite of the high similar existed among homolog chromosomes which may caused by the dosage redundancy in the autopolyploid.To further explore the variations of the three-dimensional structure of reconstituted chromosomes during the evolution of S.spontaneum,we performed three-dimensional spatial structure comparisonof reconstructed chromosomes of S.spontaneum Np-X,rice,sorghum,and S.spontaneum AP85-441,The results revealed the relatively conservative three-dimensional structure were existed in corresponding homolog chromosomes 2,5,7 and 9 of S.spontaneum Np-X in rice,sorghum,and S.spontaneum AP85-441,while chromosomes 5 and 8 showed a higher level of proportion(46.79% and 66.04%)of A to B compartment switch in S.spontaneum Np-X and S.spontaneum AP85-441 compare with other chromosomes(average 34.51%),indicating that the chromosome reconstruction of S.spontaneum might induce the deactivation of reconstructed chromosome.S.spontaneum population have high level of genetic diversity with chromosome number ranged from 36 to 128.To explore the origination and population evolution history of S.spontaneum,we performed resequencing for 102 S.spontaneum accessions worldwide to study the origination and population evolution of S.spontaneum.A total of13,140,400 single nucleotide polymorphism(SNP)data sets from the S.spontaneum population were used for the population genetics analysis.The Principal Component Analysis(PCA)results showed that the S.spontaneum population can be divided into 4 Groups(Group I-IV),and Group I,II,III and IV were mainly distributed in northern India,southwest China,southeastern China,and Malay Peninsula,respectively.The population structure analysis indicated that the 4 Groups were originatedand evolved independently.To understand the evolution of subpopulation with different basic chromosome number(X=8,9 or 10),we performed align of illumine reads of these accessions population against on Np-X genome to detect the large scale genome variation.Based on the depth of mappable reads of Chr05 and Chr08 in Np-X,we discriminated that 88,9and 4 S.spontaneum accessions were X=8,X=9 and X=10,respectively.Phylogeny of population based SNPs indicated that S.spontaneum accessions with different basic chromosome number were originated independently.Furthermore,Linkage Disequilibrium(LD)analysis showed that the S.spontaneum population with X=8 existed high frequency recombination as it has higher decay rate compare with X=9 and X=10.Tajima’D analysis indicated that the S.spontaneum population with X=9 and X=10 were subjected to the natural selection,while the population with X=8 have went through population bottleneck effect.Finally,we proposed a schematic model for the population evolution of S.spontaneum with different basic chromosome number.In summary,we constructed a high quality genome with basic chromosome number of X=10 for the autopolyploid S.spontaneum Np-X,and revealed the phylogenetics,origination and evolution of S.spontaneum with different basic chromosome number.Our study also explored the genetic base of important biological trait of sugarcane,providing a foundation for sugarcane breed improvement and newknowledge for auto-polyploidy study.