【作者】 李媛媛；

【导师】 郭修武；

【作者基本信息】 沈阳农业大学 ， 果树学， 2009， 博士

【摘要】 本研究以树莓属植物资源为试材,探索建立并优化树莓的RAPD分析体系和SSR分析体系,在此基础上,利用这两种分子标记技术分析了沈阳农业大学树莓资源圃保存的101份树莓属植物资源的遗传多样性。同时进行了树莓叶片离体再生体系建立和多倍体诱导的研究。主要结果如下:1.改进的CTAB法（提取缓冲液中含3%的CTAB）和TIANGEN试剂盒法是适宜的树莓总DNA提取方法。改进CTAB法提取的树莓总DNA的产率较低,但是纯度较高,OD₂₆₀/OD₂₈₀比值在1.729～1.911之间;TIANGEN试剂盒法提取的树莓总DNA的产率和纯度均较高,OD₂₆₀/OD₂₈₀比值在1.727～1.901之间。以上述两种方法提取的树莓总DNA为模板进行RAPD和SSR扩增,能扩增出多条清晰的谱带,多态性、稳定性、重复性良好。2.对反应体系中dNTP的种类和浓度、DNA模板浓度、Taq DNA聚合酶的种类和浓度、Mg²⁺浓度、不同公司的PCR Buffer和Mg²⁺,以及循环次数等多个影响RAPD反应结果的因子进行了研究,建立了优化的树莓属植物RAPD分析体系,即:PCR反应体积为20μl,内含1×PCR反应缓冲液,2.0 mmol/L MgCl₂,0.2 mmol/L dNTP,0.3μmol/L引物,0.5 U Taq DNA聚合酶,50 ng树莓总DNA;扩增程序为93℃2 min;93℃1 min,35℃1 min,72℃2 min,42个循环;72℃5 min。根据谱带数目、多态性和清晰度,从100个A系列RAPD引物中筛选出16个适宜于树莓属植物遗传分析的引物。3.对反应体系中Mg²⁺浓度、dNTP浓度、引物浓度、Taq DNA聚合酶浓度、DNA模板浓度及退火温度和循环次数等影响SSR反应结果的因子进行了研究,建立了优化的树莓属植物SSR分析体系,即:PCR反应体积为15μl,内含1×PCR反应缓冲液,0.9 mmol/L MgCl₂,0.25 mmol/L dNTP,引物各0.2μmol/L,0.5 U Taq DNA聚合酶,60ng总DNA;扩增程序为扩增程序为:95℃5 min;95℃45 s,50～66.5℃45 s,72℃45 s,30个循环;72℃延伸5 min。根据谱带数目、多态性和清晰度,从60对SSR引物中筛选出38对适宜于树莓属植物遗传分析的引物。4.回收了50个树莓RAPD多态性谱带,然后对其进行克隆和测序,共获得了27个不同的树莓基因组序列。基于解析的树莓序列设计特异引物,成功地将16个RAPD标记转化成SCAR标记。5.利用分子标记技术对树莓属植物资源进行遗传多样性研究,用16个RAPD引物在101份树莓属植物资源上共检测到207个多态性位点,不同树莓种质资源之间的相异系数在0～0.73之间,UPGMA法聚类分析结果显示不同树莓种质资源之间距离系数在0～0.63之间;用38对SSR引物在101份树莓属植物资源上共检测到241个等位变异,Shannon遗传多样性指数范围为0.0953～0.8591,平均值是0.6324。不同树莓种质资源之间的相异系数在0～0.86之间,UPGMA法聚类分析结果显示不同树莓种质资源之间距离系数在0～0.74之间,表明树莓属植物存在较高的遗传多样性。6.分别绘制了基于RAPD标记和基于SSR标记的树莓属植物分子系统进化树,聚类结果基本一致。表明两种标记技术都适合于树莓属植物的种内、种间的遗传变异分析。7.系统地进行了树莓叶片离体再生的研究,以红树莓品种‘秋福’（Rubus L.AutumnBliss）离体叶片为外植体,接种于MS+TDZ 2.00 mg/L+IAA 0.10 mg/L的培养基,暗培养2～3周后转移至正常光照下培养,愈伤组织形成率、不定芽再生率和外植体不定芽数分别为100.00%、95.83%和5.57±0.27个。将再生芽接种于1/2 MS+IBA 0.10mg/L的培养基中,35 d后生根率达100.00%。8.用含秋水仙碱45 mg/L的固体培养基处理‘秋福’树莓叶片8 d后,转移至正常叶片再生培养基上处理3周,将再生的不定芽在成苗培养基（MS+0.5 mg/L BA+0.2mg/L IAA+1.0 mg/L GA）上培养40 d左右分化成苗。通过观察染色体数目对再生植株进行倍性鉴定,20个检测植株中有5株是四倍体,四倍体诱导率高达25%。更多还原

【Abstract】 Rubus germplasm resources were taken as materials and RAPD system and SSR system of raspberry were established and optimized.Genetic diversity of the genus Rubus was analyzed by RAPD markers and SSR markers with the 101 accessions of the genus Rubus growing in Shenyang Agricultural University raspberry germplasm nursery.The in vitro leaf regeneration system of raspberry was established and the polyploid plants were inducted.The main results were as follows:1.The modified CTAB method（3%CTAB in the extraction buffer） and the TIANGEN DNAsecure Plant Kit were the suitable protocols for the extraction of total DNA of raspberry. The production of total DNA was lower with the modified CTAB method,but the purity was relatively high and OD₂₆₀/OD₂₈₀ value was from 1.729 to 1.911.The production and the purity of total DNA was both high with the TIANGEN DNAsecure Plant Kit,and OD₂₆₀/OD₂₈₀ value was from 1.727 and 1.901.RAPD and SSR amplifications were carried on using total DNA of raspberry with the modified CTAB method and the TIANGEN DNAsecure Plant Kit, and several clear bands with better polymorphism,stability and repeatability could be obtained.2.A number of factors affected the amplification results of RAPD were investigated such as kinds and concentration of dNTP,DNA concentration,kinds and concentration of Taq DNA polymerase,Mg²⁺ concentration,kinds of PCR buffer and Mg²⁺ and cycle numbers,etc.The optimized RAPD analysis system of the genus Rubus was established:reaction mixture contained 1×PCR buffer,2.0 mmol/L MgCl₂,0.2 mmol/L dNTPs,0.3μmol/L primer,0.5 U Taq enzyme and 50 ng total DNA of raspberry in a total volume of 20μl.The cycling conditions consisted of an initial denaturation step at 93℃for 2 min,followed by 42 cycles at 93℃for 1 min,35℃for 1 min and 72℃for 2 min,and then a final elongation step at 72℃for 5 min.Sixteen primers screened out from 100 RAPD primers were suitable for the genetic analysis of the genus Rubus based on the number,polymorphism and clarity of the bands.3.A number of factors affected the results of SSR were investigated such as concentration of Mg²⁺,dNTP,primer,Taq DNA polymerase and DNA,annealing temperature and cycle numbers,etc.The optimized SSR analysis system of the genus Rubus was established:reaction mixture contained 1×PCR buffer,0.9 mmol/L MgCl₂,0.25 mmol/L dNTP,0.2μmol/L of each primer,0.5 U Taq enzyme and 60 ng total DNA of raspberry in a total volume of 15μl.The cycling conditions consisted of an initial denaturation step at 95℃for 5 min,followed by 30 cycles at 95℃for 45 s,suitable annealing temperature 50～66.5℃for 1 min and 72℃for 45 s,and then a final elongation step at 72℃for 5 min.Thirty eight primers were screened out from 60 SSR primers were suitable for the genetic analysis of the genus Rubus based on the number,polymorphism and clarity of the bands.4.After 50 polymorphism bands of RAPD were recovered,cloned and sequenced,27 different genome sequences of raspberry were obtained.Based on the analysis of genome sequences of raspberry,specific primers were designed and SCAR conversions of 16 RAPD markers were done successfully.5.The genetic diversity of Rubus germplasm resources were analyzed with molecular marker techniques.Two hundreds and seven polymorphism loci were detected from the 101 accessions of the genus Rubus with 16 RAPD primers.The dissimilarity coefficients of different raspberry germplasm resources were from 0 to 0.73,and dendrogram with UPGMA method displayed that distance coefficients of different raspberry germplasm resources were from 0 to 0.63;241 loci were detected from the 101 accessions of the genus Rubus with 38 SSR primers.Shannon genetic diversity ranged from 0.0953 to 0.8591,with the average of 0.6324.The dissimilarity coefficients of different raspberry germplasm resources were from 0 to 0.86,and dendrogram with UPGMA method displayed that distance coefficients of different raspberry germplasm resources were from 0 to 0.74,which indicated that the genus Rubus had abundance genetic diversity.6.Dendrograms of the genus Rubus were drawn separately based on the RAPD marker and the SSR marker.Dendrogram based on the RAPD marker was nearly the same as that based on the SSR marker.The two markers were both suitable to study on the genetic diversity of intraspecies and interspecies of the genus Rubus.7.In vitro regeneration from leaves of raspberry was investigated in this study.The whole leaf of red raspberry cutivar‘Autumn bliss’ inoculated on MS medium containing 2.00 mg/L TDZ and 0.10 mg/L LAA,dark incubated for the first 2 to 3 weeks and then transferred to light was the most optimum for shoot regeneration,and the callus percentage,the shoot percentage and average shoots number per explants were 100.00%,95.83%and 5.57±0.27, respectively.Regenerated shoots were cut and transferred to 1/2 MS medium containing 0.10 mg/L IBA for rooting.After cultured for 35 days,the rooting percentage reached up to 100.00%.8.The leaves of‘Autumn bliss’ was disposed by the solid medium containing 45 mg/L colchicine for 8 d and transferred to common medium for 3 weeks.The regenerating buds were inoculated on MS medium containing 0.5 mg/L BA,0.2 mg/L IAA and 1.0 mg/L GA for 40 d to induce plant differentiation.The result of chromosome numbers indicated that 5 out of 20 tested plants were tetraploid and the variation rate reached 25%.更多还原