Molecular Cytogenetics Of Cucumis Hystrix Chakr.and Evolution Of Major Cucumis Species Based On Repetitive Sequence Analysis

Cucumis hystrix Chakr.(2n=2x=24)belongs to the subgenus Cucumis of Cucumis from the family Cucurbititaceae,is the only wild species which can be successfully hybridized with C.sativus up to now and considered to be the key species to study the origin and evolution of Cucumis species and relationship between the variation of two chromosome cardinality.It has the same progenitor with cucumber and is the important species in evolution of C.sativus.C.hystrix also contains the resistant gene(s)against downy mildew,root-knot nematodes,gummy stem blight and some other diseases,which makes it to be an important gerplasm to broaden the genetic basis of cucumber.The study of genome,in which repetitive sequences occupy a large proportion and are important to eukaryotes,is very important for deeply understanding a species.There are many different types of repetitive DNAs with different characteristics.Repetitive DNAs evolve rapidly and show variation in sequences,copy numbers and chromosomal distributions.Thus,tandem repeats have been a useful tool for chromosome identification and karyotyping analysis.However,the knowledge about the genome composition and structure of C.hystrix is very limited.In this study,we used bioinformatics and FISH to analyze the repeat composition,genome structure,chromosomal distributions and evolution of major tandem repeats of C.hystrix.At the same time,we also carried out comparactive analysis on the repetitive DNAs among C.sativus,C.hystrix,C.melo,C.anguria and C.metuliferus to study the genome structural differences of these five species and furtherly clarified their relationships.The major results were as follows:1.Analysis on the repetitive sequences of C.hystrix genomeWe found that about 19%of the C.hystrix genome is consist of repetitive sequences.Finally,eight types of repeats of LTR.Gypsy,LTR.Copia,LTR.Caulimovirus,LINE.L1,DNA.MULE.MuDR,DNA.CMC.EnSpm,rDNA and satellite have been detected.In the genome of C.hystrix,LTR retrotransposons were the most abundant repeats,which occupied 6.8%of the genome;followed by tandem repeats(including satellite and rDNA)and LINE.L1,which accounted for 4.1%and 3.115%,respectively;the content of DNA transposons were the least,accounting for only 0.233%of the genome.2.Chromosomal distribution analysis of the repetitive sequences of C hystrix genomeThe FISH results showed that the repetitive sequences of C.hystrix genome were mainly located at the end of all chromosomes.The FISH signals of Arabidopsis telomeric DNA were located at the least significant ends,while the signals of CuhyCL1?3 and CuhyCL31 were located at the subtelomeric regions of all chromosomes.The signals of 45s rDNA were located at the terminal regions of Chr.8,Chr.10 and Chr.12,the 5s rDNA loci were located at the interstitial regions of Chr.12.The signals of BAC clone 457-F11 were located at the pericentromeric regions of all chromosomes,and the FISH signals of CuhyCL7 were not only located at the pericentromeric regions of all chromosomes,but also distributed at the interstitial regions of some chromosomes.3.Homology and evolution analysis of repetitive sequences between C.hystrix and C.sativus based on comparactive FISHThe FISH signals of C.hystrix gDNA and CuhySatl?3 were located at the subtelomeric regions of all C.hystrix chromosomes,and two Cuhy5S loci were located at the interstitial regions of one single chromosome pair.All five C.hystrix probes produced signals on C.sativus chromosomes,with the signals of CuhySat1 were located at the subtelomeric regions of all chromosomes,two Cuhy5S loci were located at the interstitial regions of one single chromosome pair.While CuhySat3 showed a pericentromeric signal distribution,C.hystrix gDNA and CuhySat2 showed both subtelomeric and pericentromeric signal distributions on C.sativus chromosomes.These results revealed a high homology of repetitive sequences between C.hystrix and C.sativus genome,while the observed signal distribution variations indicated that chromosome rearrangement events,such as fusion and invension,might have occurred during the evolution.4.Evolutionary analysis of five major Cucumis species based on the comparactive analysis of genomic repetitive sequencesThe results showed that not only the repeat content of five species varied greatly,but the composition of repeat types and the genome proportions of different repeat types also varied greatly.Therefore,the evolution of Cucumis species has been companied by the genomic structure divergence.LTR retrotransposons were the most evolutionarily dynamic repeats in five species.The phylogenetic analysis of Ty3/Gypsy and Ty1/Copia families showed that C.hystrix had a close relationship with C.sativus and C.melo,but the relationship of C sativus and C.hystrix were closer,and C.anguria and C.metuliferus had a close relationship.Chromosomal distribution analysis of representative Ty1/Copia and Ty3/Gypsy retrotransposons showed that Ty1/Copia or Ty3/Gypsy retrotransposons of different species in the same subclass had different chromosomal distributions.In the same species,Ty1/Copia and Ty3/Gypsy retrotransposons showed different chromosomal distributions while the Ty3/Gypsy retrotransposons of different subclasses also showed different chromosomal distributions.These results indicated that retrotransposons not only evolved rapidly among species but also evolved rapidly within the same genome.Among the five species,the rDNA sequences were highly conserved in the gene regions,but highly polymorphism in the intergenic regions.On the chromosomes of Melo subgenus,the rDNA loci showed conserved number and location.However,there were differences in both number and location of 45s rDNA loci on the chromosomes of Cucumis subgenus,but the number and location of 5s rDNA loci were conserved.The phylogenetic analysis of five species based on the obtained ITS sequences revealed that C.hystrix had a close relationship with C.sativus and C.melo,while C.anguria and C.metuliferus had a close relationship.Therefore,we draw the following conclusions on the dimension of repetitive sequences:C.sativus is closer to C.hystrix than C.melo,and the relationship among C.hystrix,C.sativus and C.melo is closer than that among C.hystrix,C.anguria and C.metuliferus.