Study On The Application Of Preparation Of DNA Fibers And Fiber-FISH In Loquat

DNA fiber fluorescence in situ hybridization (Fiber-FISH) is an important molecular cytogenetics technology, and is widely applied in construction of high-resolution physical maps, gene fine mapping, gene sequence analysis, and whole-genome sequencinget al. In this paper, using the diploid loquat’Xingning No. l’ as test material, DNA fiber preparation technology was used to deal with the material, SSR markersand45S rDNAsequences were usedto designprobes, the Fiber-FISH technology system was applied, and the main results are as follows:(1) Exploration and optimization ofthe loquat DNA fiber preparation technology, including the nuclei extraction, the test material selection, and the DNA fiber stretchingtechnology et al. The two different nuclei extraction methods, the Jackknife method and the liquid nitrogengrinding method, were compared in loquat nuclei extraction.The nuclei extracted fromloquat etiolated cotyledons of different developmental period were also compared. Jackknife nuclei extraction is generally better than the liquid nitrogen grinding method, since it is simple, non-toxic andwith high extraction rate, and the extracted nucleiare more integral; the latter method is more complex, using toxic agents, resulting in more impurities and having low yield, and the extracted nucleiare usually more or less damaged. Nuclei extracted from etiolated cotyledons of different developmental stages throughthe jackknife method were compared; nuclei extracted from4d stage etiolated cotyledonswere of low quality and mixed with impurities;nucleiwere failed to be extracted from10d stage etiolated cotyledons, probably due to the aging of cells and the decay of nuclei in the cotyledons; the nuclei extraction of best quality was drawn from7d stage etiolated cotyledons, with little impurity, the nuclei are generally complete, and the extraction amount is relatively high. The nuclei concentration was adjusted for DNA fiber preparation. For nuclei extracted from80mg etiolated cotyledons, when30μl nuclei storage solution was added, the concentration ofthe solution wastested to be5x106-7nuclei/ml, suitable for the preparation of DNA fibers, and when60μl of the nuclei storage solution was added to the same amount of extraction, the concentration would be too low to be used for DNA fiberpreparation. Different DNA fiber stretching methods were compared,the gravity stretching method and the slide smearing method were found to generate poor quality DNA fibers and should be unsuitable for loquat DNA fiber preparation.Cross, overlap and uneven distribution were observed for DNA fibers prepared by gravity stretching, and the slide smearing method did not only cause cross andoverlap of DNA fibers but alsocaused breakage and loss of content.The slide front drainage method was found to be the best in loquat DNA fiber preparation, generating straight stretches of DNA fibers with uniform distribution, seldom causing any overlap or fracture.(2) Probes designed from the’Dadu loquat’ SSR marker sequences SSR1and SSR2, as well as the45S rDNAwere used in FISH study of metaphase chromosomes and DNA Fibers, with optimization of the FISH system.Comparative analysis was conducted on the results.The optimization of FISH system mainly involved hybridization and denaturation conditions, the elution procedure, and hybridization signal detection et al. The procedures of SSR-FISH and Fiber-FISH were generally the same, except for therequirements of45S rDNA-FISH, several other requirements were also applied in SSR-FISH and Fiber-FISH. First, the denaturing temperature should be strictly controlled according to the time; second, after hybridization the elution temperature should be decreased from37℃to35℃, and the elution should be carried out twice rather than three times; third, anti-Dig-FITC antibody concentration was increased from2μg/ml to4μg/ml, and the reaction procedure was increased from1h to1.5h; in addition, in background dyeing of the DNA fiber, the DAPI concentration was increased from2μg/ml to5μg/ml, and the staining time was increased from1Omin to20min.Weak probing signals of SSR1and SSR2in the metaphase nucleiwere detected on two different pairs of homologous chromosomes, both of which wereadjacentto the centromere regions of the chromosomes; two pairs of45S rDNAprobe hybridization signals weredetected both in the metaphase nuclei and in the interphase nuclei, one pair of hybridization signals were relatively strong and covering a largestretch, and were located in chromosome terminus area;the other pair of signals were relatively weak and their distribution areasaremuch smaller and also located inchromosome terminus areas, suggesting that there should be more than one copy of45S rDNA in loquat genome.Atomic force microscopy was used to observe the hybridization signal and a much clearer picture was obtained, which clearly showed the locations of the hybridization signalson the metaphase chromosomes.Typical non continuous moniliform hybridization signals were observed forSSRl, SSR2 and45S rDNAprobes hybridizing with loquat DNA fibers, and the hybridization signals of the three probes extended15-30μm,20-35μm, and30-45μm, respectively.Combining theFISH results of SSR1, SSR2and45S rDNA probing on metaphase chromosomes and on DNA fibers,preliminary conclusions were that:①SSR1and SSR2probeswere successfully probed on metaphase chromosomes and DNA fibers, while Fiber-FISH has a higher resolution, and that results showed that the homozygous chromosomes of ’the Xingning No.l’had different copy numbers of the SSR marker fragments;②The45S rDNA probe were clearly and accurately positioned on metaphase chromosomes and DNA fibers.The resultswere highly consistent and should both be reliable, and the homologs of45S rDNAwere supposed to be distribution on at least two chromosomes, and the copy numbers of the45S rDNA were significantly different at the two locations.