Establishment And Application Of DNA Fiber-FISH System In Asparagus Officinalis

DNA fiber in situ hybridization?Fiber-FISH?has important application value in high-resolution physical map construction,fine positioning,sequence analysis and chromosome evolution.In particular,the combination of Fiber-FISH and immunofluorescence hybridization can be used to perform epigenetics studies such as genomic methylation and histone modification from chromosomal levels.The plant sex chromosomes originated from a pair of autosomes.With the accumulation of repeats such as the retrotransposon sex chromosome were gradually heterochromatinized and heteromorphize.It is important for analyzing the origin and evolution of sex chromosomes to analyze the pattern of insertion of repetitive sequences on the sex chromosomes and the structure changes of chromosomes.However,metaphase chromosome fluorescence in situ hybridization analysis is difficult to achieve because of the low resolution.Therefore,in this paper,the preparation technology of the DNA fiber was established by the optimization of the key technical details using Asparagus officinalis?2n=20?.The main findings are as follows:1.To obtain high stability of DNA fiber,DNA fiber stretching method and other key technical details are optimized including nuclear extraction methods,yellowing seedling culture time,etc.The results showed that the dendritic cotyledons of 2 g were developed with"knife-cut method"for 7 days,and the nuclei obtained after the disruption were intact.The nuclei density was about 5×10^6-7/ml by diluting with20?l.Compared with the DNA fiber stretching method,the DNA fibers prepared by the front-end drainage method were straight,distributed,no overlapping,no fracture and complete.2.To further detect the degree of extension of the preparation of Asparagus officinalis fiber,We constructed the fosmid library of the Asparagus officinalis male genome,35 fosmid clones were randomly selected for induction and amplification.Plasmids were extracted and labeled by the notch translation method.The labeled fosmid clone probe was hybridized with the mid-chromosome.The results showed that the 3 fosmid clones were mainly located in the centromere of the chromosomes,indicating that the inserted DNA fragments of the three fosmid clones were derived from the centromere region of the stone pilgrimage.Four clones were distributed in other regions of the chromosomes except telomere,which is a typical distribution of the repeats of the transposon type on plant chromosomes.While the other 1 fosmid clone located in short arm of chromosomes 8,the length of these fragments on the fiber were 15.1?m.which provided the basis for the karyotype analysis of Asparagus officinalis.At the same time these 4fosmid clones labeled probe,conducted Fiber-FISH detection.The results show that these fragments can hybridize clearly on the fiber,the length of these fragments on the fiber were 12.8?m,12.6?m and11.9?m,the average length of about 12.4?m.Since the insert size of the fosmid clone is about 40 kb,it is calculated that the resolution of our stretched asparagus fiber is about 3.1 kb/?m.3.Based on the preparation of high quality Asparagus genome fiber,the FISH system was optimized,FISH studies on DNA fiber were performed using the optimized system with genomic,45S rDNA and 5S rDNA as probes.The results showed that the genome,45S rDNA and 5s rDNA showed typical non-continuous rosary hybridization signal on the DNA fiber.The genome had hybridization signal on the genomic fiber of Asparagus,and the signal of 45S rDNA was clear,bright and beaded.Besides,the average length of rosary is about 465?m.5S rDNA average length of rosary is about 79.2?m,and according to the resolution of DNA fiber in this article is about 3.1 kb/?m,The results of that this copy number is 762.4.To establish DNA Fiber methylation analysis system,we optimized the technology system including5-methylcytocine antibody concentration,hybridization time,washing temperature and time etc.The results of hybridization showed that the signal was unevenly distributed on the fiber,and its brightness was not uniform,indicating that the methylation level of DNA in different regions of the genome was different.