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Application of Target Region Amplification Polymorphism and EST in Auricularia Auricula (L.ex Hook.) Underw Cultivars

Author: YangXiaoBing
Tutor: SongHui
School: Jilin Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Esterase Isozyme Auricularia auricula (L.ex Hook.)Underw TRAP cluster analysis polymorphism
CLC: S646.6
Type: Master's thesis
Year: 2011
Downloads: 28
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Abstract


To study the phylogenetic relationships among 52 Auricularia auricula (L.ex Hook.)Underw cultivars, target region amplified polymorphism (TRAP) was employed. This technique adopted two primers of 18 nucleotides, a fixed primer and a random primer, which can detect the polymorphisms in the genomes. Auricularia auricula (L.ex Hook.)Underw is one of the main variety types in China. The main results of this study were as follows:1. Analysis of Esterase Isozyme in fifty two cultivars of Auricularia auricula suggested high polymorphism on the whole and high similarity anmong some strains, which indicated possible synonyms.2. Target region amplified polymorphism (TRAP) was developed for in Auricularia auricula (L.ex Hook.) Underw. The system was established, basing on reported study, and optimized two through orthogonal tests.. The 15μ1 TRAP reaction system includes 50 ng DNA templates,2.5 mmol/L of Mg2+,0.2 mmol/L of dNTP,1.5 U of Taq DNA polymerase,600 nmol/L of each primer. The conditions for PCR were as follows:initial denaturing step was held at 94℃for 4 min, followed by 5 cycles at 94℃for 45 sec,35℃for 45 sec, and 72℃for 1 min, followed by 35 cycles at 94℃for 45 sec,50℃for 45 sec, and 72℃for 1 min and with a final extension step at 72℃for 7 min. Silver staining was employed to detect the polymorphic bands.The amplicons were run on 6.5%(w/v) polyacrylamide denaturing gel for 1.5h at 75w.3. The results of the study suggested that TRAP is polymophic at detecting unique bands. The 5 primer combinations produced 103 (Table 3) markers with 91 (87.8%) of them polymorphic. The bands ranged in size from 150 to 2000bp. The number of bands produced by individual primer combinations range from 15 (ras2+em6) to 27 (ras3+em3) with an average of 20.6. Polymorphism was generally high, ranging from 80.0% to 94.1%. The high level polymorphism could be attributed to breeding region and environment of A. auricular. All the strains are divided into three groups, TRAP showed power ability for polymorphism detection and high efficiency..4. Both the results of Esterase Isozyme and TRAP markers suggsted high polymorphism on the whole and high similarity anmong some strains, there are almost no difference between somestrains, NT1 and NT3 for example, which indicated possible synonyms, further study would make our choice.

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