| Fluorescence in situ hybridization(FISH)allows localization and analysis of gene positions by detecting fluorescence signals which are generated when fluorescence-labeled probes hybridize with targeted DNA sequences in accordance to base complementary paring rule.It has been widely applied to cytogenetic studies in plants because of the ability to specifically and accurately visualize the positions of target sequences with high sensitivity and effectiveness.Cucumber(Cucumis sativus L.,2n=14,which belongs to genus Cucumis(Cucur bitaceae),is one of the top ten vegetable crops in the world.Development of cucumber cytogeneics has lagged behind as compared to other crops,mainly due to two reasons:cucumber chromosomes are small and cytologically alike which makes them difficult to distinguish,and that information provided by traditional C-banding technique is very limit because there are relatively less repetitive sequences in cucumber genome.Recent achievements in the construction of cucumber draft genome assembly and saturated SSR-based genetic maps have provided an excellent platform for the development of cytogenetic studies.Integrated cytogenetic maps offer not only important biological information in genome composition,but a solid foundation for genetic and genomic studies.However,construction and analysis of cucumber cytogenetic maps is still not completed.In this study,we conducted FISH to firstly compare different resolution among three chromosomal stages(interphase,mitotic and pachytene chromosomes),and secondly constructed the physical maps of cucumber chromosomes 3 and 4.Moreover,integrated molecular cytogenetic maps were developed combining physical,genetic and sequence maps,which may provide abundant information for cucumber genomic research.1.Comparison of FISH resolution on different cucumber chromosome spreadsWe compared FISH resolution on interphase nuclei,mitotic metaphase chromosome and meiotic pachytene chromosome using two fosmid probes with a interval of 287 kb along cucumber chromosome 3.Results have shown that FISH signals of the two fosmid probes are seperated at a distance of 1.35±0.52 ?m in interphase nuclei,but overlapped on mitotic metaphase chromosomes,and adjacent to each other with a physical distance of 0.65 ?m.To further assess the highest resolution on metaphase and pachytene chromosomes,several pairs of fosmid clones from cucumber chromosomes 3 and 4 were selected and hybridized to metaphase and pachytene chromosomes.Results have shown that the highest resolutions of prometaphase and metaphase chromosomes are approximately 2.3 Mb and 3.0 Mb,and that of meiotic pachytene chromosomes is 150 kb.To better understand signal distribution of multiprobes in the above three chromosome stages,10 fosmids from chromosome 3 that covers the entire chromosome were selected for FISH analysis.FISH images indicated that probe signals distributed in certain regions instead of dispersing throughout the whole interphase nuclei.However,individual probe signal could not be identified although signals were not overlapped.On prometaphase and metaphase mitotic chromosomes,it was difficult to distinguish individual probe because of complete or partially overlapped signals.As on meiotic pachytene chromosomes,all 10 fosmid probes produced obvious and distinguishable signals.Therefore,it could be concluded that FISH resolution on pachytene chromosomes is significantly higher than that on interphase nuclei and mitotic metaphase chromosomes,which provides a basis for cucumber cytogenetics based on high resolution pachytene chromosome.2.Cytological characterization of cucumber pachytene chromosomes 3 and 4Cytological characters of cucumber '9930' chromosomes 3 and 4 were described using well-spread pachytene chromosome spreads.Total length of pachytene chromosome 3 was 55.3±3.4 ?m and that of chromosome 4 was 43.5±3.1 ?m.The lengths of long and short arm of chromosome 3 were 28.3±2.7 ?m and 27.1±2.1 ?m,respectively,with an arm ratio of 1:1,indicating a metacentric chromosome.The lengths of length of long and short arm of chromosome 4 were 27.3±2.3 ?m and 16.1±1.3 ?m,respectively,and the arm ratio was 1.6:1.Distribution patterns of euchromatin and heterochromatin along chromosomes 3 and 4 were quite different,despite of the similar heterochromatin domains at the ends of both chromosomes.Chromosome 3 was mainly euchromatic except for three main knobs adjacent to the centromere in the short arm,and one small knob in both the short arm and the long arm.For chromosome 4,bright pericentromeric heterochromatin region could be observed,and the transition between the heterochromatin and euchromatin regions of chromosome 4 was very sharp.In addition,two interstitial small knobs were consistently observed on short and long arm of chromosome 4.Heterochromatin accounted for 24.6%of the length of chromosome 4,which is much higher than that of chromosome 3(8.9%).These cytological characteristics could not only be used to distinguish individual cucumber pachytene chromosome,but also provide crucial information to explain the relationship between chromosomal structures and functions.3.Integrated cytogenetic maps of cucumber chromosomes 3 and 4We mapped twenty-three fosmids anchored by SSR markers from LG3(13 SSRs)and LG-4(10 SSRs),as well as satellite DNA sequences(including Type ?/?,Type ?,Type IV and 45S rDNA)on pachytene chromosomes 3 and 4 using FISH.Molecular cytogenetic maps of chromosomes 3 and 4 were constructed and integrated with published high density genetic map and sequence map.Comparison of genetic and physical maps of these chromosomes revealed significant difference in recombination frequency in different chromosomal regions,with a lower frequency in pericentric heterochromatin regions and a high one in euchromatin regions.Further analysis of the integrated cytogenetic map indicated a positive correlationship between recombination frequency and gene density.In addition,an obvious assembly gap was found in chromosome 4 when compared with genome sequence map.Thus,integrated cytogenetic maps can be used to assess the quality and integrity of genome sequence assembly,providing important information for polishing sequence assembly and further studies. |
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