Study On Karyotyping Of Brassica C Genome Based On Brassica A Genomic DNA As Block Reagent

Brassica oleracea is one of the three basic species in Brassica,which belongs to a great group of vegetable crops.Including various ecotypes and artificial crossing cultivars,no matter for evolution or breeding consideration,should be researched on the cytogenetics.However, chromosomes of B.oleracea are shorter and more homogeneous condensation in cell division phase than other species,so it is very difficult to accurately identify all chromosomes by traditional technique of banding.Recently fluorescence in situ hybridization technique has developed quickly. Mainly basing on the numbers and positions of 45S(or 25S)and 5S rDNA,some researchers have identified chromosomes of B.oleracea and analyzed karyotype by FISH technique.But there is not any report about identifying chromosomes of B.oleracea by systemically using the technique of genomic in situ hybridization.In this paper,we accurately identified nine pairs of B.oleracea chromosomes by GISH technique.We put forward a new method to discriminate species with small chromosomes comparing with traditional method.And this result of typing will be favorable for us to research localization of self-incompatible genes.The main results were as follows:1.B.oleracea K164 was used as experimental material,the best method on metaphase chromosome preparation of B.oleracea was found through the comparative studies of some important influencing factors.(1)When the seeds were germinated in dark with heterotherm,there were more metaphase cells in root than other conditions;(2)Comparing 8-hydroxyquinoline treatment with low temperature treatment,we found good shape of chromosomes could be obtained by pretreating with 0.002mol/L 8-hydroxyquinoline at 18℃for 1h;(3)Comparing acidolysis,enzymolysis,acidolysis and enzymolysis with enzymolysis and acidolysis,we found enzymolysis was the best treatment.2.The karyotype analysis of chromosomes at metaphase in B.oleracea K164 showed that:the karyotype formula was 2n=2x=18=4m+5sm,chromosomes 1,2,7 and 9 were metacentric chromosomes,and chromosomes 3,4,5,6 and 8 were sub-metacentric chromosomes.The length ratio of the longest and shortest chromosome was 2.3:1;Chromosome 1 was long chromosome, chromosomes 2,3 and 4 were sub-long chromosomes,chromosomes 5,6,7 and 8 were sub-short chromosomes,and chromosome 9 was short chromosome.3.Using genomic DNA of B.oleracea as probe,as well as genomic DNA of B.rapa as block reagent,we tried to identify metaphase chromosomes of B.oleracea by the technique of GISH.The best typing result was found through the comparative study of important parameters.(1)30μL hybridization solution:100%deionized formamide 12.7μL,probe 0.5μg and block reagent 2.25μg;(2)The denature of chromosome preparation was at 70℃for 2min,the probe was at 85℃for 10min,and then co-denature of them was at 85℃for 10min;(3)The elution after hybridization:Genomic DNA as probe,strong eluotropic strength was needed,2×SSC,4×SSCT and 1×PBS were at 37,37 and 25℃for 10,8 and 5min;Low copy gene as probe,weak eluotropic strength was needed,2×SSC,4×SSCT and 1×PBS were at 25℃for 4min.4.We applied the technique of GISH to successfully identify nine pairs of B.oleracea chromosomes,and carried out accurately typing on metaphase.Chromosomes 1,2 and 7 all had two hybridization signals.Signals of chromosome 1 were on long and short arm,respectively.One signal was strong and the other was weak.Signals of chromosome 2 were both very weak,near to the edge of long arm.Signals of chromosome 7 were on long and short arm,respectively.The one which on long arm was near to centromere,the other one was near to the edge of short arm.Chromosomes 3 and 9 both had three hybridization signals.Signals of chromosome 3 were strong and signals of chromosome 9 were weak.Chromosomes 4,5,6 and 8 all had one hybridization signal.Signals of chromosome 4 and 8 were both on the edge of short arm,the intensity of them was similar,but chromosome 4 was longer than chromosome 8.Signal of chromosome 5 was very weak,which was on short ann.Signal of chromosome 6 was near to the middle of chromosome.5.THL1 gene was cloned by PCR technique.We found that the size of gene fragment was 732bp,and then the gene fragment was labeled with DIG by PCR.The result of FISH on metaphase chromosomes of B.oleracea indicated that we initially located this gene on chromosome 1,but further researches should be carried out.