| 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 species.C.anguria(West Indian gherkin,2n=24)is one of important cultivated species.C.anguria is rich in vitamins and minerals in gherkin fruits and carries broad-spectrum resistance to multiplex biotic and abiotic stress,such as powdery mildew,fusarium wilt and meloidogyn incognita.C.anguria provide a valuable gene pool for crop improvement of Cucumis crops.The karyotype analysis can provide important information for the evolutionary relationship of species in taxa.The evolutionary relationship is an important theoretical basis for further breeding utilization.Fluorescence in situ hybridization is a powerful tool for karyotype analysis.Based on the homologous relationship between related species,cross-species fluorescence in situ hybridization technology could be employed for the cytogenetic and further chromosomes structure evolution researches in the species that lack of genomic information.In this study,firstly based on the C.sativus genome information which belong to the same genus with C.anguria,the chromosome-specific single copy genes of C.anguria were selected to construct the karyotype for C.anguria metaphase chromosomes.Further the selected single copy gene probes were used to identify individual pachytene chromosome of C.anguria,and chromosome morphology and heterochromatin distribution were analyzed.Then based on the position of single copy genes in C.anguria and C.sativus,the syntenic relationship between these two species was analyzed.Finally,the repetitive sequences of C.anguria were analyzed and identified by FISH.The main results are as follows:1.Construction of the Metaphase Chromosomes Karyotype of C.anguria Based on the Selected Chromosome-Specific Single Copy GenesAbout 4-5 single copy gene probes were selected from each chromosome of C.sativus,and then were used to identify the individual metaphase chromosome of C.anguria.Each chromosome of C.anguria was identified by 14 selected single copy genes combining with 45S rDNA and 5S rDNA loci,and karyotype was analyzed.The results showed that one or two cucumber single copy gene probes gave clear signals on C.anguria indi-vidual chromosome,and each chromosome was clearly identified by different single copy gene signals.The relative length of each chromosome and the arm ratio were measured.And the karyotype pattern of C.anguria was established,and the 12 chromosomes of C.anguria were identified as A01-A 12.2.FISH Identification and Morphological Analysis of C.anguria pachytene ChromosomesA total of 14 single-copy genes screened on the metaphase chromosomes of C.anguria,were used as probes to identify the corresponding 12 chromosomes on pachytene stage of C.anguria,and then the distribution of heterochromatin along chromosomes was analyzed.The results showed that the heterochromatin distributions were varied in 12 chromosomes.Heterochromatin regions in chromosomes A01,A02,and A11 were closed to the long arm end of chromosomes.For chromosomes A03,A05,A06,and A08,very bright DAPI staining pericentromeric heterochromatin regions were observed.About two thirds of chromosome A04 was occupied by heterochromatin region except two ends.Heterochromatin regions in chromosomes A07 and A12 were mainly located on the short arm and pericentromeric heterochromatin regions.There were obvious heterochromatin distributions in the middle regions of long arm and short arm of chromosome A09.Chromosome A10 was mainly euchromatin,and had less scattered heterochromatin.3.The Chromosomes Syntenic Relationship Analysis of C.anguria and C.sativusThe chromosomes syntenic relationship between C.sativus and C.anguria can be obtained based on the position of the single copy gene sequences on C.sativus and C.anguria.The results showed that each chromosome of C.sativus corresponded to one or two chromosomes of C.anguria.Among C.anguria chromosomes,A06 + A09,A03 + A12,A02 + A04,and A01 +A11,corresponded to C.sativus chromosomes(represented as C1-C7)Cl,C2,C3,and C6,respectively.Chromosomes A08,A10,and A05 corresponded to C.sativus chromosomes C4,C5,and C7,respectively.4.Repeat Sequence Analysis of C.anguriaBased on Hig2000 platform and graph-based clustering analysis,major families of repetitive DNAs in C.anguria genome were obtained.The results showed that about 84%of the genome of C.anguria consisted of repetitive sequences,and long terminal repeat(LTR)retrotransposable elements were major repetitive DNAs.Other repeats included DNA transposons,rDNA,low complexity,simple repeat etc.Based on the reads graphs and annotation,some different types of repetitive sequences were selected,like LTRs,low complexity,and simple repeats,and their chromosomal distributions and characteristic were analyzed through FISH mapping.The results showed that a LTR type repeat,CL 159 was observed to locate on the terminal region of most chromosomes.And a low complexity type repeat,CL133 was mapped on pericentromeric heterochromatin regions of all chromosomes.The tandem repeats,CL255,CL194,and CL 192,were found to position on some specific chromosomes. |
