Oligonucleotide Probe-Based Chromosome Painting Technique And Its Application In Chromosome Research Of Cucumis

Visualizing specific DNA sequences on chromosomes by fluorescence in situ hybridization(FISH)is the basis for genetics,genomics,and cell biology.The technique of using FISH to visualize a certain chromosome segment,chromosome arm or whole chromosome under a fluorescence microscope is called chromosome painting.The emergence of chromosome painting technique has greatly enhanced our understanding of chromosome structure and changes.For example,using this technique can analyze chromosome recombination,aberrations,and homologous genes.In addition,chromosome painting technique has provided a new method for comparative study of closely related interspecies genomes without genome sequencing,through chromosome comparison between species.Cross-species chromosome painting can visually show the difference in chromosome structure between species,so that we can analyze the rearrangement of chromosomes among closely related species.However,due to the presence of cell walls and a large number of tandem repeats in plant chromosomes,plant chromosome painting technique has been difficult to develop for a long time.In recent years,the development of oligonucleotide synthesis technology has opened up a new way to solve this problem.By screening specific oligonucleotide probes in the genomes of sequenced plant species,it is easy to complete the painting of specific chromosome segments or the full length of chromosomes.In addition,the probes can also be used to track homologous chromosome pairings at early meiotic stages.Therefore,this method has great potential for the study of sequenced plant chromosomes.At the same time,the synthesis technology of oligonucleotide probes is still somewhat inadequate.The synthesis step is time-consuming and labor-intensive,and the in vitro transcription and reverse transcription involved are strict in the experimental environment.What’s more,the generated RNA is easily degraded.In addition,the one-time input cost in the synthesis process is relatively high.All of the above have affected the widespread use of oligonucleotide probe-based chromosome painting techniques.In this study,based on cucumber genome sequence information,we screened and built cucumber chromosome oligonucleotide library,and proposed a method of synthesizing oligonucleotide probes based on multiple PCR.This method can synthesize a large number of oligonucleotide probes in a short time,and can independently select the synthesized chromosome segments according to the needs of the experiment.It can quickly accomplish the painting and recognition of specific chromosomes and play an important role in the analysis of inter-species chromosome rearrangement.This study taking cucumber chromosome 4 as an example,the chromosome rearrangement of cucumber chromosome 4 with C.hystrix and C.melo was analyzed by using segmented oligonucleotide probes synthesized by multiplex PCR.The main findings are as follows:1 Design of oligonucleotide probesThe oligonucleotide probes for the library of cucumber chromosome 4 were screened by Chorus software.Firstly,RepeatMasker was used to remove all the repetitive sequences in cucumber genome,and then the genomic sequences were divided into 48nt oligonucleotides,and the oligomers containing more than 6 nucleotides were discarded.Next,each oligonucleotide was compared with‘9930’reference genome using BLAT,and oligonucleotides with a similarity of>75%were selected.In addition,a probe database was constructed by using primer3 to retain dTm>10℃ oligonucleotides,and probes related to specific chromosome or genome regions were selected from the probe database.Finally,88670 oligonucleotide probes covering cucumber chromosome 4(about 23.3 Mb)were screened,averaging about 3.8 probes per Kb.In the process of designing oligonucleotide probes,the oligonucleotide libraries were segmented by double primers,and the oligonucleotide probes could be synthesized freely according to the needs of experiments.2 Synthesis of Oligonucleotide Probes Based on Multiplex PCRIn the synthesis process,oligonucleotide probes are synthesized by multiplex PCR,which greatly simplifies the experimental steps,saves experimental materials and costs,and improves the synthesis efficiency of oligonucleotide probes.The whole and segmented chromosomes of cucumber at metaphase and pachytene were dyed and good results were obtained.The oligonucleotide probe optimized by the synthesis method is more conducive to the promotion and application of the probe.3 Segmented oligonucleotide probe painting based on multiplex PCR revealed chromosome rearrangement in CucumisCross-species FISH was performed on the mitotic metaphase and meiotic pachytene chromosomes of C.hystrix and C.melo by segmented cucumber chromosome 4 oligonucleotide probes.According to the signal distribution,it was found that chromosome 4 of cucumber corresponded to the chromosomes 7,8 and the terminal segments of 5 of C.hystrix and C.melo.An insertion event on chromosome 7 of C.hystrix and C.melo was found.Besides,three major rearrangement events occurred between C.hystrix and C.melo chromosome 8.