Establishment And Optimization Of FISH Technology System In Actinidia L.

Kiwifruit(Actinidia Lindl.)belongs to the Actinidiaceae.It is a dioecious,perennial climbing deciduous vine fruit tree.And it is one of the four most successful fruit tree species for the domestication and cultivation of wild fruit trees in the 20 th century.There are many kinds of kiwis,which are widely distributed,and have different ecological habits.They contain a large number of wild resources and related species,and have relatively complex genetic diversity.Due to factors such as natural crossing,seed selection and cross breeding,interspecific and intraspecific gene infiltration is frequent,resulting in extremely rich inter-and intra-species variants.Therefore,it is obviously important and necessary to carry out further research on the genetic diversity of kiwifruit and the inter-and intra-species genetic relationships.At present,domestic and foreign researchers have conducted a lot of studies on genetic diversity and genetic relationship of kiwifruit and its related wild species from different levels,aspects and perspectives by using morphology,palynology,cytology,biochemistry,and molecular biology techniques,and made important progress.FISH(Fluorescence in situ hybridization),as an important research method in molecular cytogenetics,has a wide range of applications in the construction of genetic maps of animals and plants,genetic diversity and genetic relationship analysis,gene mapping,and hybrid identification.However,due to the small(0.6 ~ 1.5 ?m)and many(2n=58,116,174,etc.)chromosomes of kiwifruit,FISH is almost blank in kiwifruit research.In this research,the current main variety 'Hongyang'(2n=2x=58)was selected as the test material.And the progeny of sexual hybrids between 'Hongyang' and wild kiwifruit(A.eriantha Benth.,2n=2x=58)were also selected as the test material.Using genomic DNA and 45 S rDNA as probes,the GISH(Genome in situ hybridization),45 S rDNA-FISH and DNA fiber-FISH technology systems were studied,and the system was established for kiwi chromosome in situ hybridization technology system of chromosome and DNA fiber.It provides an important research approach for the analysis of genetic diversity and genetic relationship,genetic map construction,gene mapping and hybrid identification of kiwifruit.The main research results are as follows: 1.Establishment and optimization of GISH technology systemThe two key influencing factors in the GISH technology system,chromosomal degeneration and blocking DNA concentration,were optimized.In this study,we adopted a method of denaturation,in which the probe and chromosomal DNA were denatured separately.The probe DNA was denatured by conventional denaturation,that is,the PCR instrument was reacted at 97? for 10 min.Chromosome degeneration was critical.Insufficient denaturation temperature and time,the DNA double helix cannot be completely dissociated,and excessive temperature or time may cause severe loss of DNA on the chromosome or excessive swelling,which may cause loss or reduction of hybridization targets.By screening the denaturation temperature and time,the suitable temperature range for kiwifruit chromosome denaturation was about 70-75?,and the denaturation time was about 2 min.Using 'Hongyang' kiwifruit genomic DNA as a probe and Actinidia eriantha genomic DNA as a block,screening different blocking ratios.The blocking DNA concentration is 50 times the probe concentration,which is most suitable for kiwifruit GISH analysis.It can effectively distinguish different genomic sources,with clear hybridization signal and clean background.2.Establishment and optimization of 45 S rDNA-FISH technology systemBased on the main technical steps such as screening and optimizing chromosomal degeneration,the young roots of 'Hongyang' kiwifruit was used as the research material and 45 S rDNA was used as the probe to optimize the 45 S rDNA-FISH technology system of kiwifruit.The main purpose was to compare and screen the elution strength after hybridization.Compared with the 42? elution in GISH analysis,the elution temperature at 37? was better for small fragments of DNA such as 45 S rDNA after hybridization.It is suitable for kiwi fruit FISH analysis,and hybridization results with clean background and obvious signal can be obtained.3.Establishment and optimization of Fiber-FISH technology systemUsing the ?Hongyang? Kiwifruit young root system as a test material,the nucleus extraction,DNA fiber preparation and the key influencing factors of the Fiber-FISH technology system(including DNA fiber denaturation,elution after hybridization and dyeing conditions,etc.)were studied.The main results are as follows:(1)The Jackknife method was more effective than the liquid nitrogen grinding method for cell nucleus extraction.The former was simple and non-toxic,the nucleus was intact and not damaged,and the extraction rate was 100%.The latter was complicated and toxic,with many impurities,the nucleus was easily damaged and incomplete,and the extraction rate was lower relatively.(2)During the preparation of DNA fibers,the incubation time of the lysate was screened.Five reaction gradients were set,that is,10,15,20,25,and 30 min respectively.The results showed that the best effect was obtained at 20 min,and the quality of the obtained DNA fiber was the best,showing flat,straight,uniform and high density.(3)In the Fiber-FISH technology system,the denaturation temperature and time of DNA fibers were strictly controlled at 68°C for 2 min.After hybridization,the best elution temperature was 37? for 2 times.In addition,when DNA fiber was dyed,the DAPI concentration was selected to be 5 ?g/mL,and the dyeing time was 20 min,the effect was the best.