| Vaccinium Linn.also called blueberry,is a genus of perennial deciduous or evergreen shrubs belonging to the Ericaceae originating from North America.In the world,more than 400 species have been found and most of them distribute in North America.Today,blueberry are recognized worldwide as one of the foremost healthy food because of its multiple vitamins,microelements and pharmacologically active compounds,and it is of great value in nutrition and medicine.Highbush blueberry cultivars are divided into three types: northern,intermediate and southern.The southern highbush blueberry cultivar V3 and Legacy have been introduced to south China for many years,and they grow well.Nevertheless,the fruits of V3 are not big enough and production are limited.When Legacy go through spring and summer rainy season in chongqing,their blade is easy to appear a large number of brown disease spot.Compared with conventional breeding methods,colchicine inducing polyploidy plant can quickly get new cultivar.The lab has researched polyploidy blueberry induced by colchicine for many years and has gotten several mutant plants.Currently,the research about the mutant of blueberry is reported less,and the traditional methods for classification of highbush blueberry cultivars using morphological and flavour traits are largely unsuccessful due to environmental influence.Understanding the genetic relationship within blueberry germplasm is important to establish a broad genetic basis for safeguarding and future use of the existing genetic resources.The objective of this study is to assess the genetic variability within different cultivars and their mutants,discuss the botanical,cytological characters and genetic differences among mutants from V3,Legacy and their control,and further improve the screening accuracy of mutants.The main research results are as follows:1.From morphological identification: V3 Mutant was induced by colchicine,then transplanted to field and grew for three years,of which height,leaf length and width had a significant increase contrast with the control.The growth condition of mutants was better than the control,but the fruits of mutants were smaller than the control.There were no significant differences among three mutants.Mutant L1 was induced by colchicine,then transplanted to field and grew for one year,of which height,leaf length and width had a significant increase contrast with the control.The height of mutant L2 declined 32.5%;however the number of branch increased 194.4% and the leaf length,width increased 34%,38%,respectively.2.The stoma density of mutant V3 declined 20.5%,and of L1 declined 11.6%.The number of chloroplast in guard cell of V3,L2 and L1,among which showed highly significant difference,increased 14.6%,24.6%,and 35.6%,respectively.The guard cell length and width of L1 increased 16.7%,18.4%,respectively.The morphological characteristic of guard cell had no difference between L2 and the control.The plant anatomy results showed the leaf vein thickness of epidermis,palisade tissue and spongy parenchyma of V3 and Legacy significantly increased.That the CTR of Legacy mutants significantly increased,it indicated that the drought-resistant ability of Legacy mutants had been improved.3.The chromosome number count results showed that the chromosome number of diploid cell was 2n=2x=24,the tetraploid cell was 2n=4x=48,and V3 mutants and L1 mutants were chimaeras whose tetraploid cell ratios were 45.52%,38.62%,respectively.The ratio of tetraploid cell showed a reduction compared with before(70%,49.06%).This indicated that the cell proliferation speed of diploid cell was faster than tetraploid cell and mutant plants had the degeneration tendency in cytochimera of Vaccinium Linn.4.The plant physiological indexs between V3 mutants and control showed that the content of chlorophyll,soluble suger and proline increased significantly,while the MDA declined.The plant physiological index between Legacy mutants and control showed highly significant difference on the content of chlorophyll,soluble suger,proline and MDA.5.Based on modified CTAB method,through the intuitive analysis and the variance analysis,a suitable ISSR-PCR reaction system was established,namely 10×buffer 2ul;0.75 UTaq DNA polymerase;1.875 mmol.L-1 Mg2+;80ng template DNA;0.1mmol.L-1 d NTPs;0.2μmol.L-1 primer.The annealing temperature,the number of cycles and the extension time of primer UBC835 were also investigated and the optimal parameter of PCR was: 54.2℃,35 cycles,60 s.The results indicated that the optimized reaction system was very stable.6.10 primers were screened from 70 ISSR primers to analyze the genetic diversity.A total of 95 bands were obtained,of which 90(94.7%)were polymorphic.From the UPGMA,the coefficiet of genetic similarity of the 21 Vaccinium Linn.ranged from 0.57 to 0.83.The 21 Vaccinium Linn.resources were divided into two groups: V.macrocarpon L.(Bluecrop,Blueray,Brigitta Blue,Darrow)and V.Australe.The coefficiet of genetic similarity between A1,A2,A3 mutants and V3 was 0.800,0.711,0.722,respectively;the coefficiet of genetic similarity between L1,L2 mutants and Legacy was 0.800、0.767,respectively.The obvious differences on morphological characters,leaf anatomical structure,physiological indexs and the results of ISSR indicated that colchicine mutagenesis could change the DNA molecular genetic structure of plant. |
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