Studies On Chromosomal Stability Of The Synthetic Cucumis Allotetraploid Species

Polyploidy(or whole genomic duplication,WGD)has played a significant role in the evolutionary history of eukaryotic lineages.It is estimated that most flowering plants are polyploid,including many agricultural crops.Allopolyploidization(i.e.inter-specific hybridization and genome doubling)has been a driving force in plant evolution.Classic cytological and molecular cytogenetic studies have shown that experimental and natural neoallopolyploids often have meiotic irregularities,producing chromosomally variable gametes and progeny.The genomic consequences of polyploidy have been extensively studied,but the mechanisms for chromosome stability and diploidization in polyploids remain largely unknown.The Cucumisxhytivus(2n=4x=38)is an allotetraploid synthesized from interspecific hybridization between cucumber(C.sativus,2n=2x=14)and its wild relative C.hystrix(2n=2x=24)followed by chromosome doubling,and has been self-pollinated for many years.Thus,it is art ideal system for investigating speciation,evolution and stabilization of allopolyploid species that derived from distant chromosome bases.However,cytogenetic research on C.×hytivus is still lacking,there are only few studies on cytological observation.The development of comparative chromosome analysis among Cucumis species based on FISH(fluorescence in situ hybridization)provides an excellent opportunity to analyze the chromosomes of the allotetraploid C.×hytivus usjing cross-species FISH.In the present study,we used GISH(genomic in situ hybridization)and cross-species FISH to construct the karyotype of the wild parent C.hystrix.Based on this result,we distinguished the 19 pairs of homoeologoxxs chromosomes of the Cucumis allotetraploid,and investigated the distribution of repetitive sequences on C.×hytivus chromosomes.Finally,the meiotic chromosome paring and pollen fertility were analyzed to elucidate mitotic stability in relation to meiosis chromosome behavior of the synthetic Cucumis allotetraploid species.The main results are as follows:1.Karyotype construction of the wild Cucumis species C.hystrix using GISH and cross-species FISHIn this study,we performed cross-species FISH using cucumber fosmid clones;combining with GISH patterms,the 12 pairs of homologous chromosomes of C.hystrix were identified.29 cucumber fosmids that generated distinctive signals on C.hystrix mitotic metaphase chromosomes were selected from cross-species FISH.12 fosmids and genomic DNA(gDNA)of C.hystrix were used to construct a standard karyotype for C.hystrix in combination with rDNA probes.The results showed that 45S rDNA signals were located at chromosomes H08,H10 and H12,whereas 5S rDNA loci were at H10.A second karyotype was rapidly constructed using C.hystrix gDNA and a C.hystrix BAC clone(457-F11)through repeated hybridizations.2.Karyotype analysis and distribution of repetitive sequences of the allotetraploid Cucumis species C.xhytivus using GISH and cross-species FISHIn the present study,GISH and cross-species FISH were conducted using repetitive sequences and cucumber fosmid clones to identify the 19 homoeologous chromosomes of the Cucumis allotetraploid C.Xhytivus.Using a repeat-element probe mix which contains gDNA from cucumber and C.hystrix,Type Ⅲ and the BAC clone 457-F11 in two successive hybridizations allowed for routine identification of the C.×hytivus chromosomes.The karyotype of 15 C.×hytivus individuals was analyzed.Meanwhile,the distribution of four major repetitive sequences(Type Ⅰ/Ⅱ,Type Ⅲ and Type Ⅳ),45S and 5S rDNA,and the Arabidopsis telomere sequences on C.×hytivus chromosomes was investigated.The results showed that the location and signal strength of repetitive sequences on metaphase chromosomes of C.×hytivus was consistent with that reported in cucumber,and showed no significant changes.Four pairs of 45S FISH signals could be unambiguously detected on cucumber-subgenome on the allotetraploid chromosomes,whereas the weakest pair of 45S could not be detected occasionally.The three pairs of 45S loci did not show changes in signal strength and location on C hystix-subgenome.The strength of 5S signals on H10 was much stronger compared with that on C5.26 cucumber fosmids that generated distinguishing signals on C.hystrix were hybridized to mitotic metaphase chromosomes of C.×hytivus to determine the distribution pattern in the genome of the Cucumis allotetraploid.The results indicated that no aneuploids were identified in all C.×hytivus individuals that were characterized,and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid,indicating a rather stable subgenome structure.Nonetheless,small-scale recombination or transfer of chromosome segments may have occurred.3.Meiotic chromosome behavior and pollen fertility of the allotetraploid Cucumis species C.×hytivusGISH was performed using cucumber and C.hystrix genomic DNA to examine chromosome pairing between two subgenomes at different stages during meiosis Ⅰ in 60 PMCs of the Cucumis allotetraploid.The resxults showed that chromosome pairing was mostly presented as bivalents(Ⅱ),with a mean frequency of 11.21(7-14)per cell.A low frequency of trivalents(Ⅲ,average 0.97)and quadrivalents(Ⅳ,average 2.12 per cell)was observed.Unpaired univalents(I)were observed in 84.5%cells with an average of 2.7 per cell.The mean chromosome configurations for C.×hytivus were 2.7 Ⅰ+11.21 Ⅱ+0.97Ⅲ+2.12 Ⅳ.Intergenomic bivalents and multivalents were frequently observed;chromosome lagging and conglutination were also observed in some cells.Mature pollens of C.xhytivus were stained using 1%acetocarmine to calculate pollen viability.The stainability of C.xhytivus was 41.84%,thus after selfed for many generations there were still over half pollens aborted.