Studies On Chromosome Evolution Of Cucumis Species Based On Oligo-Painting

The genus Cucumis contains about～66 species and is divided into two subgenera:the subgen.Melo including C.melo（2n=2x=24）,C.metuliferus（2n=2x=24）,C.anguria（2n=2x=24）,and wild relatives within the genus（2n=24,2n=48,and 2n=72）,and the subgen.Cucumis including the cucumber（C.sativus,2n=2x=14）and closely related wild species C.hystrix（2n=2x=24）.However,the evolutionary relationships and origin mechanisms underlying the distinct karyotype of Cucumis species are still poorly understood.Clarifying the origin of these species will contribute to the genetic improvement of cultivated crops and even to the de novo domestication of wile or semi-wild species with crop potential.Closely related wild species with a variety of excellent genes are an important resource for improvement in broadening the genetic basis of cucumber germplasm.Successful interspecific hybridization between C.hystrix and C.sativus var.sativus and subsequent genome doubling have created synthetic allotetraploid C.×hytivus（gocumber,2n=4x=38）.Subsequently,a series of cucumber-C.hystrix addition lines and introgression lines were created by chromosome engineering technology,which significantly broadened the genetic basis.Newly formed allopolyploids typically exhibit disturbed meiotic behavior,resulting in variation in chromosome number and structure in offspring.This meiotic challenge must be overcome in established allopolyploid,but the evolutionary mechanisms they experienced remain unclear.Synthetic allotetraploid C.×hytivus can serve as a traceable system for studying the evolutionary mechanisms of newly formed polyploid due to their clarity genetic background.The oligo-painting based on fluorescence in situ hybridization（FISH）is a powerful tool for identifying individual chromosomes,tracking chromosome behavior,visualizing chromosome structure and probing chromosome evolution.In this study,a set of oligo-painting probes based on conserved Cucumis sequences was developed to analysis the karyotype and fine-scale genome structure of eight Cucumis species from different evolutionary branches,including the Asian origin species C.melo,C.hystrix and C.sativus,and five African origin species C.metuliferus,C.dipsaceus,C.zeyheri,C.anguria and C.subsericeus.A genus-level common ancestral karyotype was reconstructed through comparative genomics.The complex karyotypic evolutionary history of these species was reconstructed from this ancestral karyotype.Secondly,the meiotic behaviors of interspecific F₁ and multiple C.×hytivus generations were traced and analyzed using oligo-painting and genomic in situ hybridization（GISH）.Meiotic biases between subgenomes and the effect on fertility were investigated.Finally,a set of species-specific oligo-painting probes was developed to construct C.hystrix standard karyotypes and to identify H-chromosomes in allotetraploids,allotriploids and alien addition lines.The main results were as follows:1.The karyotype evolutionary history of Cucumis species was reconstruction based on comparative oligo-paintingIn this study,a set of oligonucleotide-painting probes containing seven chromosomes was developed using the cucumber genomic data.The probe pool for each chromosome was divided into eight sub-pools based on physical location,for a total of 56 segmented probe pools.Seven cucumber chromosomes ware identified by multiple rounds of FISH using these probes.Syntenic analysis was performed in eight Cucumis species using whole chromosome painting probes.The structures of pachytene chromosomes from these species were visualized by 56 segmented painting probes.Using the obtained genomic structure data,a genus-level common ancestral Cucumis karyotype（ACK）containing 12 ancient chromosomes and 114 genome blocks was reconstructed.Then,we constructed a phylogenetic-based evolutionary framework.Our results show that large-scale chromosome inversions were occurred among six species with similar karyotypes（n=12）.The genome origin of C.metuliferus underwent four inversions and a centromere repositioning event.C.dipsaceus,C.zeyheri,and C.anguria experienced divergent inversion events arising from a common ancestral karyotype.C.subsericeus is an allotetraploid with subgenomic stasis.The origin of melon genome underwent 15 large-scale inversions and four centromere repositioning events and as independent branches away from the ancestor of cucumber and C.hystrix.Two melon subspecies underweant independent domestication events after species formation.C.hystrix genome originated from the common ancestor of cucumber and C.hystrix by species-specific inversions and translocations.The seven cucumber chromosomes were created by large-scale genomic reorganization that occurred in 12ancestral chromosomes.By a reconstructed evolutionary framework,we have elucidated the evolutionary trajectory and events during the karyotype origin of these Cucumis species.2.Oligo-painting and GISH reveal the subgenome dominance and meiotic harmonious evolution of allotetraploid C.×hytivusUsing the oligo-painting and GISH techniques,we traced and analyzed the meiotic pairing behavior of interspecific F₁ and allotetraploid C.×hytivus multi-generation.Homoeologous pairings in F₁ were prevalent but its frequency is low.Many autosyndesis were detected in meiosis cells（25.5%）of F₁,suggesting that the H-subgenome is more susceptible to interspecific hybridization than the C-subgenome.We observed prevalent meiotic disturbed behavior in allotetraploid C.×hytivus,such as univalent,multivalent,subgenomic pairing,asynchronous segregation and chromosome lagging.By comparison between the two subgenomes,the C-subgenome exhibits more regular meiotic behavior.H-subgenome exhibits higher univalent and chromosome lagging frequencies.These results suggest that meiotic biases between two subgenomes are the major factor causing the low fertility of allotetraploid C.×hytivus.As selfing generations increase,increased meiotic stability in the S14 generation compared with the S4 generation,including synchronous meiosis behavior,reduced univalent and chromosome lagging events.Two subgenomes display a degree of meiotic harmony,which causes a significant increase in pollen fertility and meiotic stability of C.×hytivus.Therefore,meiotic harmony evolution between subgenomes is a major subject in the stabilization of newly formed allopolyploid.3.Identification of alien chromosomes in different types plants using C.hystrix species-specific oligo-paintingUsing species-specific oligo-painting probes developed from C.hystrix genome,we established standard C.hystrix karyotype and detected three assembly errors on chromosomes H8,H9 and H10.C.hystrix-specific oligo-painting probes show very specific hybridization signals for C.hystrix-chromosomes in interspecific F₁.Then,we have used these probes to identify C.hystrix-chromosomes in allotetraploid,allotriploid,and addition lines.One aneuploid with H4 chromosome loss and one plant with homoeologous exchange between H4-C3 were detected in allotetraploid C.×hytivus,the remaining populations still maintaining a stable subgenome structure.Our results establish a species-specific oligo-painting technique,which can effectively and specifically identify alien chromosomes or segments in plant with complex ploidy and genetic backgrounds.Therefore,it can be widely applied in chromosome engineering breeding of other crops.