Exploration Of High-resolution Low-copy And Single-copy FISH Technology System For Wheat And Rye

With the development of cytogenetics and modern molecular biology,chromosome identification technology has developed from extensive morphological feature identification to DNA-based molecular cytology methods,such as in situ hybridization and molecular markers.In situ hybridization has become the most commonly used method to identify chromosomes of wheat and its close relatives,but in recent years the commonly used fluorescence in situ hybridization(FISH)technology often encounters the problem of weak signal.At present,a SABER-FISH(Signal amplification by exchange reaction,SABER)technology has been reported in the literature.This technology combines ordinary FISH oligonucleotide probes with long single-stranded DNA concatemers and gathers multiple complementary Fluorescently modified strands to amplify FISH signal.However,this technique has not yet been applied on plant chromosomes.This study is mainly based on SABER-FISH technology,using wheat and rye low-copy oligonucleotide sequences and single-copy oligonucleotide sequences as probes to explore the application of this method on wheat and rye chromosomes,aiming at Establishment of high-resolution FISH methods applicable to low-copy,single-copy sequences of plant chromosomes.The specific results are as follows:1.According to the literature,explore the system of oligonucleotide sequence tailing reaction,and determine the final concentration of each component in the tailing reaction system: 1× PBS,10 m M/L Mg SO4,800U/m L of Bst LF polymerase,600μM/L each of d ATP、d TTP、d CTP,100 n M/L of Clean.G,50 n M of Hairpin,10 μM/L of Primer(pool).2.Perform SABER-FISH on the probes Oligo-5BL.46,Oligo-5A8080.1,Oligo-4BS,Oligo-5BS,Oligo-5AS,Oligo-5AL that have been reported to produce weak signals on chromosomes of rye and wheat for optimization.In rye,after chromosome denaturation treatment,the probe was diluted in a mixed solution containing 10% dextran sulfate,50% deionized formamide,2× SSC,0.1% Tween-20.The signal of the identical probe by SYBER-FISH was significantly enhanced compaired with that of ND-FISH when hybridized overnight.In wheat under denatured conditions,the probe is diluted in buffer containing 10% dextran sulfate,30% deionized formamide,2× SSC,0.1% Tween-20.The signal of the identical probe by SYBER-FISH was significantly enhanced compaired with that of ND-FISH when hybridized overnight.3.Preliminary exploration of single-copy sequence SABER-FISH detection method:(1)After screening the single-specific oligonucleotide sequence with a length of 30-50 bp and tailing by PER as a probe,no clear signal can be observed after SABER-FISH detection.(2)The pool of specific oligonucleotide sequence was tailed and used as a probe for SABER-FISH.In rye under denatured condition,the pool of 11 probes was diluted in10% dextran sulfate,50% deionized formamide,2× SSC,0.1% Tween-20 mixed solution.Weak fluorescent signal could be observed hybridizing at 53℃,but still showed a high noise signal.Low-copy repetitive sequences can detect signals by using SABER-FISH technology,and have a certain enhancement effect,indicating that SABER-FISH technology can be successfully applied to the detection of plant chromosomes.However,for single-copy sequences,if single specific oligonucleotide sequence is tailed as a probe,no clear signal can be detected by SABER-FISH.While the specific oligonucleotide sequences(Currently,up to 11 probes)are tailed as a probe pool,weak fluorescent signals could be observed by SABER-FISH detection,but there are many noise signals.This method still needs to be further optimized.The breakthrough point is to find a single-copy oligonucleotide probe with strong specificity.