| Sugarcane(Saccharum spp.)is the primary sugar and biofuel feedstock crop,providing 80% of the world’s sugar and 40% of ethanol.The genus Saccharum typically includes six species,namely four cultivars,i.e.,Saccharum officinarum,Saccharum sinense,Saccharum barberi,Saccharum edule,and two wild species,i.e.,Saccharum robustum and S.spontaneum.Among the six species,S.spontaneum is the most important wild germplasm resources in sugarcane breeding.S.spontaneum species provides excellent agronomic trait genes such as disease resistance,stress resistance,lodging resistant,clumps and adaptability for sugarcane cultivars.At present,the main sugarcane cultivars in the world all contain the blood relationship of S.spontaneum.However,Saccharum spontaneum shows the highest level of genetic diversity in the Saccharum genus,with nearly 40 genotypes,whose chromosome numbers range from 2n=40 to128.The large and complex genomes seriously hinder the study of chromosomal structure and evolution in S.spontaneum species,and restricted the efficient utilization of S.spontaneum in sugarcane chromosome engineering breeding.To this end,our study developed a series of efficient cytological tools based on oligonucleotide-based fluorescence in situ hybridization(oligo-FISH)technique,and investigated the chromosomal structure and evolution of Saccharum spontaneum at the molecular cytological level,which laid a foundation for efficient utilization of S.spontaneum in sugarcane chromosome engineering breeding.The main results are as follows:1.Development of barcode chromosome identification markers and characterization of a Saccharum spontaneum with a basic chromosome number of x = 10We developed the first set of oligonucleotide(oligo)-based barcode probes based on the S.spontaneum genome(x = 8),which can be used to simultaneously distinguish each of the 8 chromosomes of octaploid S.spontaneum SES208(2n = 8x = 64)through fluorescence in situ hybridization(FISH).By comparative FISH assay,we confirmed the chromosomal rearrangements of S.spontaneum(x = 8)and S.officinarum(2n = 8x = 80),the main contributors of modern sugarcane cultivars.In addition,we examined a S.spontaneum accession,Np-X,with 2n = 40 chromosomes,and we found that it was a tetraploid with the unusual basic chromosome number of x = 10.Assays at the cytological and DNA levels demonstrated its close relationship with S.spontaneum with basic chromosome number x = 8(the most common accessions in S.spontaneum),confirming its S.spontaneum identity.Population genetic structure and phylogenetic relationship analyses between Np-X and 64 S.spontaneum accessions revealed that Np-X belongs to the ancient PanMalaysia group,indicating a close relationship to S.spontaneum with basic chromosome number of x = 8.This finding of a tetraploid S.spontaneum with basic chromosome number of x = 10 suggested a parallel evolution path of genomes and polyploid series in S.spontaneum with different basic chromosome numbers.2.Development of whole chromosome painting markers and dissection of sugarcane genome structure and evolutionThe first whole painting probe that can cover an entire S.spontaneum chromosome 2(CP2)was developed through bioinformatic pipeline.This probe can help us to deeply conducted molecular cytogenetic studies of sugarcane at the chromosome level.Then,fluorescence in situ hybridization(FISH)using this CP2 probe revealed six types of ploidies from twenty S.spontaneum clones,including 6x,8x,10 x,11x,12 x,and13x clones.The finding of S.spontaneum clones with uneven of ploid suggested that certain S.spontaneum clones come from hybridization rather than autopolyploidization.It renews our knowledge that S.spontaneum is derived from autopolyploidization.In addition,we have developed a dual-color FISH technique combining chromosome painting and S.spontaneum-specific probe that can be used to accurately identify chromosome or fragments from either S.spontaneum or Saccharum officinarum in sugarcane modern cultivars.We revealed unexpected high level of interspecific recombination from introgressive S.spontaneum chromosomes(>50.0%)in cultivars ROC22 and ZZ1,indicating frequent chromosome exchange in cultivars.Intriguingly,we observed interspecific recombination recurring among either homoeologous or nonhomoeologous chromosomes in sugarcane cultivars.3.Development of flexible chromosome painting markers and detection of meiotic chromosome behavior and structure in S.spontaneumTo deeply analyze the meiotic chromosome behavior and structural variation in S.spontaneum,we also developed the longest chromosome 1and the shortest chromosome 7 and 8 painting oligo probes of S.spontaneum except for chromosome 2 painting probes.Among them,chromosome 7 and 8 are flexible chromosome painting probes consisting of 10 chromosome segments.Then,we conducted meiotic chromosome behavior and structure studies in polyploid S.spontaneum species using four chromosome-specific painting probes.A representative S.spontaneum ancestral style(x=10,tetraploid)and two predominant cytotype(x=8,octaploid and decaploid)were selected for cytological analysis.The results showed that S.spontaneum has evolved into a diploidization state with predominantly bivalent pairing(approximately 89.7-100%)in meiosis,and conforms to a disomic inheritance pattern.By comparative FISH assays using flexible chromosome painting probes,we demonstrated that no intrachromosomal structures variations at the cytological level were identified among S.spontaneum ancestral style and two predominant cytotype,which suggested that the chromosomal synteny in S.spontaneum may has been remarkably maintained after nearly 5.27 MYA of divergence.Taken together,this study developed a series of efficient sugarcane chromosome-specific cytological markers,and elucidated the chromosome structure and evolution as well as meiotic chromosome behavior characteristics of S.spontaneum species at the molecular cytological level.This lays the foundation for the efficient use of S.spontaneum species in sugarcane distant cross breeding and chromosome engineering breeding in the future. |
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