Primary Establishment Of Comparative Chromosomal Mapping And Chromosomal Evolution Analysis In The Major Cucumis

The genus Cucumis contains 52 species,including two economically significant crops,cucumber(C.sativus L.,2n=14)and melon(C.melo L.,2n=24),as well as other important wild species,such as sour cucumber(C.hystrix Chakr.,2n=24),C.metuliferus(2n=24)and C.anguria(2n=24).The abundant gene resources in genus Cucumis has provided valuable gene pool for crop improvement.Elucidation of evolutionary relationship among species is of great significance for genetic improvement of Cucumis crops.Genome sequences are available in only two Cucumis species,cucumber and melon.Molecular information of other species is still limited,thus making it difficult for studies in evolutionary relationship among Cucumis species using molecular biological information.Comparative chromosomal mapping based on fluorescence in situ hybridization(FISH)can viualize chromosome rearrangements among closely related species and reveal evolutionary relationships,which is of great importance to Cucumis species considering their relatively weak genetic studies.In recent years,comparative chromosomal mapping among Cucumis species is still in initial stage,with most studies focoused on comparision between cucumber and melon.Comparative evolutionary research in wild Cucumis species is rarely seen.In this study,firstly karyotype analysis of two cucumber variants was carried out using genomic in situ hybridization(GISH).Secondly,we analyzed the relationship between tandem repetitive sequences(TRS)and chromosomal structure in 5 Cucumis species(C.sativus,C.melo,C.hystrix,C.metuliferous and C.anguria)and revealed TRS homology of the 5 studied species.Finally,single-copy gene-based chromosome painting(ScgCP)technique was developed to further investigate evolutionary relationship among species,which was then used to construct comparative chromosomal maps between cucumber and melon to provide cytological evidence of their evolutionary relationships.The main results are as follows:1.Rapid Karyotype Analysis of Cucumber Varieties Based on Genomic in situ HybridizationUsing genomic in situ hybridization(GISH),metaphase chromosome analysis was performed for two variants[cultivated cucumber(Cucumis sativus var sativus)and wild cucumber(Cucumis sativus var hardwickii)]of cucumber(C.sativus L.,2n=2x=14)to quickly establish the chromosome karyotype of cucumber varieties.Genomic DNA from cultivated cucumber was used as probes and hybridized onto the metaphase chromosomes of cultivated cucumber itself and wild cucumber using genomic in situ hybridization technique.Individual chromosome of two variants was analyzed based on the GISH banding,and 45 rDNA signals as well.The results showed that cucumber genomic DNA probes exhibited distinguishing signal patterns on the chromosomes of two variants,and the signal distribution and intensity substantially showed distinct characteristics across seven chromosomes of two variants.Each chromosome was characterized unequivocally according to the signal patterns,and a karyotype for each cucumber variant was constructed.Compared with the cucumber repetitive sequences,the GISH patterns were found to locate mainly in TRS blocks of cucumber chromosomes.2.Evolutionary relationships among major Cucumis species revealed by comparative cytogenetic mappingDistribution and divergence of TRS could be elucidated by comparative cytogenetic maps,thus reveal the evolutionary relationships among Cucumis species.Using self-gDNA and TRS of 5 Cucumis species as probes,we carried out GISH and FISH experiment to analyze chromosomal structure and TRS divergence of the 5 species.Results have shown that the majority of TRS was positioned at the pericentromeric heterochromatin region in C.melo and C.anguria,while preferential distribution of TRS at the subtelomeric regions were found in C.sativus,C.hystrix and C.metuliferus.Further,comparative GISH(cGISH)using gDNA of C.sativus and C.hystrix as probes revealed the high homology of TRS.Repeats Type ?/? and Type ? simultaneously produced bright signals at the subtelomeric regions of C.sativus and C.hystrix,which confirmed the high TRS homology,as well as a closer relationship between these two species than other Cucumis species.The obvious signals around pericentromeric heterochromatin regions detected by C.melo gDNA in different species suggested that some conserved TRS existed in these regions.The distribution of gene density along chromosomes was analyzed based on the genome drafts of C.sativus and C.melo.Results showed that the regions with lower gene density have bright GISH signals,and the higher gene density appeared at the regions with very weak GISH signals.In conclusion,the cytogenetic mapping of TRS and cGISH revealed the distinct differentiation of repetitive sequences during species formation.3.The development of single-copy gene-based chromosome painting in cucumber and its application for chromosome analysis in CucumisChromosome painting based on fluorescence in situ hybridization(FISH)has played an important role in chromosome identification and research into chromosome rearrangements,diagnosis of chromosome abnormalities and evolution in human and animal species.However,it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes.In the present work,a chromosome painting method for single-copy gene pools in C.sativus was successfully developed.Gene probes with sizes above 2 kb were detected consistently.A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes.This single-copy gene-based chromosome painting(ScgCP)technique was performed by PCR amplification,purification,pooling,labeling and hybridization onto chromosome spreads.Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes.Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C.sativus.Three mis-aligned regions on chromosome 4 were identified by the painting patterns.A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus.A comparative chromosome map of this region was constructed between cucumber and melon.