Comparative Analysis Of Sequences And Physical Mapping By FISH Of 5S RDNA In Several Subspecies Maize

5S rDNA encodes 5S rRNA in cytoplasm,which is a kind of highly conserved tandem repeat in eukaryote.The length of repeat unit is 200～900 bp,and there are from 1000-50000 copies in haploid genome.Each repeat unit consists of a coding region and the nontranscribed intergenic spacer(NTS).Relative to the larger variation degree of copy number,5S rDNA sequence of coding region is quite conservative, which is about 120 bp.While the length of NTS varies from 100-700bp and the variations of 5S rDNA sequence are mainly from that.5S rDNA is an ideal molecular marker to study the species evolution.5S rRNA and the 5S NTS have been applied to gymnosperm,monocotyledon and dicotyledon.The experiment chose several subspecies maize materials which were in common use in our country.Analyzed and compared their 5S rDNA NTS as a molecular marker,and further located 5S rDNA on maize chromosomes to analyze their distribution by optimized fluorescence in situ hybridization(FISH)technique.The results were as follows:1 The primer was designed according to the highly conservative 5S rRNA sequence in maize.Long and short NTS fragments were obtained by PCR in seven cultivated subspecies of 14 materials.The length of long NTS was about 620 bp,while that of short NTS was about 250 bp.2 The NTS sequences of seven representative materials were determined by sequencing the clones of PCR products.Alignment of short NTS sequences revealed that the GC content(%)was 45.67%and there was from 1-15 variation sites of nucleotide acid.There were more transitions than conversions in the variation sites, and the ratios of transition/conversions were 0.83 to 2.0.The total size of short NTS conservative regions was from 212-213 bp,and the sequecnce length variation was only 1 bp,which were homologous as high as 98.6%-99.5%.There is no obvious repeat unit in the short NTS sequences.There were more absence in Zea mays indurate,Zea mays saccarata and Zea mays amylea saccharata.There was A at 37th site in Zea mays amylacea,while the other six materials were missing.There was A at 123rd site in Zea mays ceratina while T in the other materials.There was A at 168th in Zea mays indurate,but G in the rest.At 248th there was T in Zea mays indentata, while the others were missing.3 Alignment the long NTS revealed that it contained the 5S rRNA sequence.Their homology varied from 98.3%-99.2%.5S rDNA is the tandem repeat sequence of (5S rRNA-NTS)n,so long NTS is a false positive result by PCR.Alignment the 5S rRNA in the seven long NTS and the one published on the NCBI website revealed that:there were 2 variation sites in Zea mays indurate,Zea mays amylea saccharata and Zea mays indentata,only 1 variation site in other four cultivated subspecies materials,and the highest variation frequency was at the 264th site.4 Clustering analysis of short NTS with UPGMA showed that the 7 materials should be compartmentalized as two genus:the first genus contained Zea mays amylacea and Zea mays amylea saccharata,the rest maked up of the other genus. Clustering analysis of 5S rRNA with UPGMA showed that it was more closed among Zea mays saccarata,Zea mays ceratina,Zea mays everta and Zea mays amylea saccharata.The results were slightly different between the two clustering analysis,but there was a better consistency on the level of the genetic distances.5 Two sets of FISH system was established.The 5S rDNA probes were labled directly and chromosomes were colorated by PI in systemⅠ.In systemⅡthe probe was labled by bio-11-dUTP and chromosomes were colorated by DAPI.There were two different views of 5S rDNA on metaphase chromosomes of maize.Compared and analyzed the effects of the 5S rDNA signals between the two different systems.The method of Fluorescence-labeled system(systemⅠ)is fast and simple,suited to high repetitive DNA sequences,more multi-copy fragments and genome probe.The method of biotin-labeled system(SystemⅡ)is stable,highly sensitive,simple and convenient,which could be applicable in the detection and location of low/single copy. With improved computer scanning and image processing technology,it will be easiler to distinguish and more extensive in application.6 5S rDNA probe labled by bio-11-dUTP was located on metaphase chromosomes in 7 subspecies maize materials by FISH,magnified the signals to enhance the sensitivity,and observe the signals distribution of 5S rDNA in different materials.The results showed that each material contained only one pair of signals in accord with the results of NTS by PCR.There was a red signal on each chromatid,which was localized at the end of long arm of chromosome 2.There was a slight difference in brightness which may be caused by different copies.