【作者】 赵丽华；

【导师】 李名扬；

【作者基本信息】 西南大学 ， 园艺学， 2010， 博士

【摘要】 石榴(Punica granatum L.)为石榴科石榴属落叶灌木或小乔木植物,原产印度、伊朗、阿富汗等中亚地区,距今已有五千多年的栽培历史,引入中国至今已有2000余年的栽培历史。石榴在我国经长期天然杂交和基因突变,以及采用实生、分株、嫁接等多种繁殖方法,产生了复杂多样的品种和类型,据不完全统计,我国现有石榴品种资源约238个,分布遍及南北各地20多个省区。目前,国际上还没有建立统一的石榴品种分类方法和分类体系,大都根据果树学、农艺性状进行分类、鉴定,而且各地研究者对石榴品种资源进行调查研究时,都局限于某一地区,从不同的角度对石榴品种进行分类,导致品种混杂、同种异名、同名异种的现象出现,绝大多数品种的系统演化和品种间的亲缘关系也无文献资料可考证,给准确进行品种资源鉴定和利用带来了困难。本研究旨在应用ISSR标记技术对中国部分石榴品种进行遗传多样性、亲缘关系分析；应用AFLP标记技术对川滇石榴种质资源的遗传多样性和亲缘关系进行分析,揭示石榴品种的来源及其演变,为更合理地保护、开发石榴资源及进行石榴新品种选育提供遗传信息,为将来石榴种质资源研究和分子系统学研究提供依据。主要研究结果如下：(1)石榴基因组DNA提取：本研究针对石榴叶片富含多酚、多糖等次生物质的特点,对提取DNA的CTAB法进行改良：首先,选用1.5×10-2 mol. L-1 Na2B4O7.10H2O为多酚氧化酶抑制剂,完全抑制酚的氧化,消除多酚类物质氧化后对酶切、PCR扩增等操作产生不良影响；在细胞膜裂解前用缓冲液洗涤叶片粉末,将部分多糖、多酚、RNA等杂质除去；对NaCl浓度进行调节,增加氯仿／异戊醇抽提次数,增强除去蛋白质、多糖等杂质的能力；增加裂解液CTAB含量,控制悬浊液离心温度,提高DNA产率。对CTAB法改良Ⅱ所提DNA通过琼脂糖凝胶电泳后,点样孔干净、主带清晰、无拖带、无RNA条带；紫外分光光度计检测OD260m/OD280nm为1.7～1.9,OD260m／OD230nm比值大于2.0,DNA产率在96.4～127.2μg.g-1之间,结果表明CTAB法改良Ⅱ可从石榴叶片中提取高质量、高产率的基因组DNA。(2)石榴ISSR-PCR反应体系优化：本研究首次采用正交设计L16(45)探讨了模板DNA、Mg2+、dNTPs、引物、Tag DNA聚合酶五因素对石榴ISSR-PCR的影响,并利用加权平均法及百分制对ISSR-PCR反应体系扩增结果进行评分,采用DPS数据处理系统分析实验数据,探讨各因素及各因素不同水平对反应结果影响的内在规律性。结果表明：各因素对PCR反应影响的大小依次为：Mg2+>dNTPs>Tag DNA聚合酶>引物>模板DNA,五个因素不同水平间的差异都达到了极显著水平；建立石榴ISSR-PCR最佳反应体系为(25μ1):Mg2+1.75 mmol.L-1、dNTPs 0.15 mmol.L-1、Tag DNA聚合酶1 U、引物0.8 mmol.L-1、模板DNA 20 ng。对石榴ISSR-PCR最佳反应体系的退火温度进行梯度试验,确定各条引物最适退火温度。(3)石榴资源ISSR遗传分析：本研究从100条ISSR引物中筛选出重复性好、多态性高的6条引物：UBC 826、UBC 857、UBC 868、UBC 899、UBC 900,利用这6条引物对我国7个石榴主产区47个石榴品种进行了DNA多态性分析,共扩增出120条DNA条带,其中多态性带109条,多态性百分率为90.83%。利用PopGen32软件对试验数据进行分析,结果显示：7个石榴种群遗传多样性依次为：山东种群>陕西种群>云南种群>河南种群>安徽种群>四川种群>新疆种群；7个种群间的Nei’s基因分化系数(Gst)为0.191 1,表明分布在种群间的遗传变异占总遗传变异的19.11%,种群内遗传变异占81.89%,种群间表现出低水平的遗传分化；7个石榴种群间基因流的估测值(Nm)为2.116 9,种群间的遗传相似性(I)变化范围为0.921 8～0.975 9,表明种群间存在较强的基因流,遗传相似性很高。47个石榴品种中遗传相似系数(Sg)变异范围在0.600 0～0.9250之间,有效等位基因数(Ne)为1.2945±0.3094,Nei’s基因多样(H)为0.1897±0.1618、Shannon信息指数(I)为0.3091±0.2198,表明石榴在我国栽培过程中产生了较高的遗传变异,构成了较丰富的石榴品种资源基因库。用NTSYSpc-2.10 UPGMA法构建亲缘关系树状图,47个石榴品种被完全区分开,并分为5个类群。同时检测到15条特异性条带,可用于供试石榴品种中11个品种分子鉴定的参考性标记。(4)石榴AFLP体系优化：本研究选用MseⅠ为高频酶,EcoRⅠ为低频酶,确定石榴基因组DNA 20 u 1双酶切体系(MseⅠ为5U、EcoRⅠ为5 U、BSA为0.2μl、NEBuffer2为2 u1、基因组DNA为300 ng、ddH2O为14.05μl)最佳酶切时间为6 h；连接产物不稀释直接用于预扩增,预扩增程序为94℃2 min；94℃30 s,56℃30 s,72℃60 s, Go to 3 29 more times;72℃6 min,10℃hold;将预扩产物稀释20倍后进行选择性扩增,经检验优化的此AFLP体系能用于石榴遗传分析。(5)川滇石榴资源AFLP遗传分析：本研究从64对AFLP引物中筛选出重复性好、多态性高的5对AFLP引物为：E-AGG/M-CAA、E-AAG/M-CAA、E-ACA/M-CTC、E-AGG/M-CTG、E-AAG/M-CTG,应用这5对引物对四川、云南的42个石榴品种进行遗传多样性及亲缘关系分析,共扩增出362条DNA条带,其中多态性带235条,多态位点百分率(P)为65.66%。利用PopGen32软件对AFLP谱系进行分析,结果显示：每个位点有效等位基因数(Ne)为1.260 7、Nei’s基因多样性(H)为0.160 3、Shannon信息指数(I)为0.253 1；多态性百分率(P)为65.66%,表明四川、云南石榴品种之间的遗传变异较大,存在比较丰富的遗传多样性。用NTSYSpc-2.10软件对AFLP谱系进行分析,结果显示遗传相似系数(Sg)变异范围在0.712 7～0.925 4之间,UPGMA法构建亲缘关系树状图,5对引物可将42个石榴品种完全区分开,以遗传相似系数(Sg)0.82为阀值,42个石榴品种可分为4个类群,并在分子水平上显示供试品种间亲缘关系。同时检测到18条特异性条带,可用于供试石榴品种中的13个品种分子鉴定的参考性标记。确定蒙自的‘青皮石榴’与攀枝花的‘青皮石榴’为同名异物。更多还原

【Abstract】 Pomegranate(Punica granatum L.)is a small trees or deciduous shrub which belongs to Punicaceae,Punica L.,occurred originally Central Asia and native to Iran,Afghanistan etc.,in which it had already been cultivated more than five thousands of years and more than 2000 years in China.Pomegranate has formed a great deal of cultivars as long-term natural hybridization,gene mutation,seedling stand,grafting etc. in China.According to incomplete statistics, there are 238 cultivars of pomegranate throughout North and South,more than 20 provinces,in China.At present, there is no unified Classification and classification systems for pomegranate Cultivars internationally.The classification of the pomegranate is based mainly on the character of fruit trees and agronomic traits.Researchers who classify and identify it were limited to certain areas and classify it from different angles.These lead to the phenomenon that hybrid Cultivars,synonym and heterogeneous of the same name,which brought difficulties to identify and utilize accurately pomegranate resources.In order to protect and develop pomegranate and provide genetic information for breeding of new pomegranate cultivars and proof for future molecular systematic studies,ISSR markers is used to analyze the genetic diversity and genetic relationship for some pomegranate cultivars in China in this study;AFLP markers is used to analyze genetic diversity and genetic relationship for some pomegranate cultivars in Sichuan and Yunnan.The main results are as follows:(1)Extraction of genomic DNA from leaf of pomegranate:As pomegranate leaves contain lots of polyphenols, polysaccharides and other secondary material characteristics,CTAB method was improved in this study:First, 1.5×10-2 mol.L-1 Na2B4O7.10H2O was choosed as the polyphenol oxidase inhibitors to inhibit phenol oxidation completely and eliminate the negative impact of oxidation phenol on digestion, PCR amplification and other operations;In order to remove polysaccharides, RNA etc.,powder leaves was washed by buffer before the nuclear membrane lysis;In order to remove protein and polysaccharide,NaCl and KAc density are adjusted,extraction time of chloroform/isoamyl alcohol is increased;In order to increase the DNA yield,CTAB density of lysis is increased,centrifugal temperature of suspension is bellow 18℃.The results of agarose gel electrophoresis show that point hole of electrophoresis was clean,no towing,the results of spectrophotometer show that OD260nm/D280nm was 1.7～1.9, OD260nm/OD230nm was greater than 2,the yields ranged is 96.4～127.2μg.g-1.Conclusion:the improved CTAB II is the effective method of high-purity and high-yield extraction of genomic DNA from pomegranate leaves.(2) Optimization of ISSR-PCR amplification system of pomegranate:The orthogonal design was used to optimize the ISSR-PCR amplification system of Pomegranate in five factors (Taq DNA polymerase,Mg2+,DNA template,dNTPs and primer) at four factors respectively.The weighted average method and centesimal grade were used for the results of the orthogonal scoring, the DPS data processing systems is used to data analysis and study the inherent law of the results that different levels of each factor and factors influence on.The results showed that:the order of these five factors is:Mg 2+> dNTPs> Taq DNA polymerase> primer> template DNA,and these five factors among the different levels show a very significant level;The best ISSR-PCR reaction system(25μl)for Pomegranate is:Mg2+1.75 mmol.L-1、dNTPs 0.15 mmol.L-1,Taq DNA polymerase 1 U,DNA template 20 ng, and primer 0.8 mmol.L-1;the best annealing temperature of different primers is different..(3)Genetic analysis of pomegranate resources by ISSR:In this study, these 6 primers with good repetition and high polymorphism have been selected from 100 ISSR primers:UBC 826, UBC 857, UBC 868, UBC 899, UBC 900.The DNA polymorphism of 47 pomegranate cultivars from 7 main production areas in China is analyzed by these 6 primers and 120 bands were amplified,of which 109 were polymorphic loci.The average percentage of polymorphic bands was 90.83%. PopGen32 analysis indicated that the genetic diversity of 7 pomegranate populations as follows:Shandong>Shanxi>Yunnan>Henan>Anhui>Sichuan>Xinjiang.Nei’s gene differentiation coefficient of 7 populations (Gst) was 0.191 1,show that genetic variation of among populations is 19.11% and the genetic variation within populations is 81.89%,showing low levels of genetic differentiation among these populations;the gene flow of 7 pomegranate populations(Nm) was 2.116 9,the genetic similarity of these 7 populations (I) ranges from 0.921 8 to 0.975 9,it showed a strong gene flow and high genetic similarity among these populations and the diversity of genetic variation of pomegranates can be maintained.The genetic similarity coefficient (Sg) of 47 pomegranate cultivars ranges from 0.600 0 to 0.925 0,the effective number of alleles (Ne) is 1.2945±0.3094, the gene diversity of Nei (H) is 0.1897±0.1618,the information index of Shannon (I) was 0.3091±0.2198,which show pomegranate germplasm have greater genetic variation and richer genetic diversity among these cultivars.NTSYSpc-2.10 UPGMA method was used to contruct evolutionary trees,47 cultivars were divided into five taxa completely by six primers.As 15 specific bands could be used as referential identification tags for 11 pomegranate cultivars.(4) Optimization of pomegranate AFLP system:In this study, MseⅠwas selected as the high-frequency enzyme, EcoR I was selected as the low-frequency enzyme.The best time of double digestion system of 20μl (MseⅠwas 5 U、Eco RⅠwas 5 U、BSA was 0.2μl、NEBuffer 2 was 2μl、genomic DNA was 300 ng、ddH2O was 14.05μl) was 6～7 h;ligation product was not diluted but for pre-amplification straightly,pre-amplification process was:94℃2 min;94℃30 s,56℃30 s,72℃60 s, Go to 3 29 more times;72℃6 min,10℃hold.The product of pre-amplification is diluted 20-fold for the selective amplification.This AFLP system can be used for genetic analysis of pomegranates.(5) Genetic analysis of pomegranate in Sichuan-yunnan by AFLPIn this study,these 5 pairs of primers with good repetition and high polymorphism are selected from 64 pairs of primers:E-AGG/M-CAA、E-AAG/M-CAA E-ACA/M-CTC、E-AGG/M-CTG、E-AAG/M-CTG.The genetic diversity and the relationship of 42 pomegranate cultivars of Sichuan and Yunnan are analyzed by these 5 pairs of primers,PopGen32 analysis indicated that 362 bands are amplified by 5 pairs of polymorphic AFLP primers,of which 235 were polymorphic loci and the percentage of polymorphic bands is 65.66%.The effective number of alleles per locus (Ne),Nei’s gene diversity (H),Shannon information index (I) are 1.260 7,0.160 3,0.253 1 respectively,suggesting there have been greater genetic diversity among pomegranate germplasm in Sichuan and Yunnan cultivars;NTSYSpc-2.10 analysis indicate that the range of genetic similarity coefficient (Sg) is from 0.712 7 to 0.925 4 and with an average of 0.814 2;UPGMA method was used to construct phylogenetic tree,42 cultivars were seperated completely by 5 pairs of AFLP primers with the genetic similarity coefficient (Sg) 0.82 for the threshold value.42 pomegranate cultivars in Sichuan and Yunnan are divided into four taxa,which shows the genetic relationship among 42 pomegranate cultivars in Sichuan and Yunnan at molecular level.18 specific bands are detected,and which can be used as referential identification tags for the 13 pomegranate cultivars.Meng zi’Qing pi’pomegranate and Pan zhihua’Qing pi’ pomegranate are homonym.更多还原