本文選題：靈芝 + 表型性狀��； 參考：《東北農(nóng)業(yè)大學(xué)》2013年碩士論文

【摘要】：本研究以8個野生靈芝菌株和9個栽培菌株作為研究對象,通過形態(tài)學(xué)方法和分子標(biāo)記方法,對它們的遺傳多樣性及親緣關(guān)系展開分析,并構(gòu)建各菌株的分子身份證,對保護靈芝菌種的知識產(chǎn)權(quán)、加強靈芝菌種的分類和鑒定具有理論意義。 本文主要展開了以下工作： 1)采用覆土栽培法進行栽培試驗,對各菌株的菌絲體長勢,子實體形態(tài),產(chǎn)量,孢子形態(tài)等表型性狀進行了觀察分析,初步確定供試菌株為三個親緣較近的種類。并且菌株GL-1、GL-2、GL-3、GL-5、GT、HZ和菌株SYLZ表現(xiàn)出較優(yōu)良農(nóng)藝性狀,可以進行工廠化規(guī)模生產(chǎn)。菌株GL-4、GL-A、Han G、Tai-1、XLLZ和菌株JHHZ等子實體規(guī)整,芝型好,多叢生等特點可以用作觀賞靈芝的栽培親本。 2)采用改良的CTAB法提取供試菌株的基因組DNA,進行ITS序列克隆、測序。通過序列比對,17個靈芝菌株的ITS序列長度均為546bp,菌株間ITS序列的長度無變異。靈芝菌株ITS區(qū)G+C含量在48.1%-50.6%。采用Neighbor-Joining法對17個靈芝菌株和GenBanK數(shù)據(jù)庫中登錄的7條相關(guān)序列進行聚類分析,所有菌株均與登錄號為HQ235632的菌株處在一個分支,表明它們之間的遺傳差異性較小,可以判定它們均為Ganoderma lucidum種。 3)采用SSR分子標(biāo)記對17個靈芝菌株進行遺傳多樣性分析。篩選出了20對能夠有效鑒別菌種且鑒別能力較強的SSR引物。通過平均遺傳相似系數(shù)和平均遺傳距離的分析,得知野生靈芝菌株間的親緣關(guān)系比較遠(yuǎn),栽培靈芝菌株間的親緣關(guān)系比較近。結(jié)合聚類分析和菌株特異性指數(shù)研究,野生組的遺傳多樣性要高于栽培組,遺傳多樣性更豐富,更具有資源開發(fā)的價值。 4)應(yīng)用軟件ID Analysis1.0,在相似系數(shù)設(shè)為0.8,缺失引物百分比為0.05的情況下,篩選出9對引物的13個位點用于構(gòu)建17個靈芝菌株的分子身份證。其中菌株GL-3、GL-4、GL-5、GL和HZ各檢測出1個特異性條帶,能夠直接對它們用相應(yīng)的引物進行鑒定。用這9對引物共同擴增同一品種,經(jīng)電泳檢測、照相得出各菌株特有的分子身份證圖片,使菌株的特征數(shù)字化,可以直觀有效的鑒定菌株。
[Abstract]:In this study, 8 wild Ganoderma lucidum strains and 9 cultivated strains were used as research objects. Their genetic diversity and genetic relationship were analyzed by morphological method and molecular marker method, and molecular identity cards of each strain were constructed.It is of theoretical significance to protect the intellectual property of Ganoderma lucidum strains and strengthen the classification and identification of Ganoderma lucidum strains.The main work of this paper is as follows:1) the phenotypic characters of mycelium growth, fruiting body morphology, yield and spore morphology of each strain were observed and analyzed by using soil covering cultivation method. The strains were preliminarily identified as three closely related species.The strain GL-1 GL-2GL-3 GL-5 GL-5 and strain SYLZ showed better agronomic characters and could be used for industrial scale production.The characters of GL-4 strain GL-AHG Tai-1XL LZ and strain JHHZ, such as regular fruiting body, good type of lucidum and multiple tufts, can be used as cultivated parents of Ganoderma lucidum.2) the genomic DNA of the tested strain was extracted by modified CTAB method, the ITS sequence was cloned and sequenced.By sequence alignment, the ITS sequence length of 17 Ganoderma lucidum strains was 546 BP, and there was no variation in ITS sequence length among the 17 Ganoderma lucidum strains.The content of Ganoderma lucidum in the ITS region was between 48.1 and 50.6.The cluster analysis of 17 Ganoderma lucidum strains and 7 related sequences in GenBanK database was carried out by using Neighbor-Joining method. All strains were in one branch with HQ235632, indicating that the genetic difference between them was small.They are all Ganoderma lucidum species.3) genetic diversity of 17 Ganoderma lucidum strains was analyzed by SSR molecular marker.Twenty pairs of SSR primers were screened to identify the bacteria effectively.Through the analysis of the average genetic similarity coefficient and the average genetic distance, it was found that the genetic relationship between wild Ganoderma lucidum strains and cultivated Ganoderma lucidum strains was far away and that between cultivated Ganoderma lucidum strains was close.Combined with cluster analysis and strain specificity index, the genetic diversity of wild group was higher than that of cultivated group, and the genetic diversity was more abundant and valuable for resource development.4) with software ID analysis 1.0, when the similarity coefficient was 0.8and the percentage of missing primers was 0.05, 13 loci of 9 pairs of primers were selected to construct molecular identity cards of 17 Ganoderma lucidum strains.One specific band was detected from GL-3 GL-4GL-5 and one specific band from HZ, which could be directly identified by corresponding primers.Using these 9 pairs of primers to amplify the same variety together, the unique molecular ID image of each strain was obtained by electrophoretic detection, and the characteristic of the strain was digitized, which could be used to identify the strain intuitively and effectively.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：S567.31

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