【作者】 夏岩石；

【导师】 宁正祥；

【作者基本信息】 华南理工大学 ， 食品科学， 2012， 博士

【摘要】 大麦（Hordeum vulgare L.）是重要的粮食作物和啤酒酿造原料，同时富含β-葡聚糖、黄酮类化合物、多酚、甜菜碱等多种生物活性成分，具有抗氧化、降胆固醇和增强免疫等多种生理活性。随着经济的发展和人们生活水平的提高，以大麦为原料的营养保健品得到了迅速的发展，综合有效地开发利用大麦丰富的种质资源，培育高产、优质、富含生物活性成分的新品种已成为大麦育种的重要课题。大麦的生物活性成分多表现为数量性状，是由多个基因和环境共同作用的结果，对其遗传基础的研究比较困难。近年来，随着基因组学的发展和生物统计软件的完善，特别是分子标记技术在遗传育种领域的广泛应用，以连锁不平衡(linkage disequilibrium，LD)为基础的关联分析(association analysis)方法的出现为数量性状遗传的研究提供了新途径。本研究利用关联分析探讨了大麦部分生物活性成分的分子遗传基础，并选择具有优异等位基因的大麦材料加工麦苗营养保健品——麦绿素（barley green），对其最佳浸提条件和贮藏稳定性进行分析。其主要结果如下：1.利用分布于全基因组上的21个微卫星标记（simple sequence repeat, SSR），对来自35个国家和地区的292份大麦材料（包括地方品种、野生品种和改良栽培品种）进行全基因组扫描，分析群体遗传多样性，以构建大麦生物活性成分关联分析的定位群体。结果表明，这个大麦群体具有广泛的遗传变异，除2对SSR引物无扩增结果外，19对SSR引物共扩增出135个等位变异，平均每对引物检测到7.1个，平均遗传多样性指数（Hi）为0.73。比较不同地理生态大麦群体的多样性显示，亚洲中东、亚洲东北及阿拉伯半岛地区的大麦材料遗传多样性最丰富，这些地区具有选取高生物活性成分含量遗传稳定的大麦材料的潜力。292个大麦材料基于SSR数据的聚类分析发现，三个大类的划分与野生型、地方型和改良栽培型大麦等三个基因型群体存在显著的相关性，11个亚类的划分与7个地理生态群体也极显著相关。因此，这个群体适合于全基因组范围上的SSR标记及候选基因与大麦生物活性成分进行关联分析。2.为建立适合于大麦的EcoTILLING技术体系，用于鉴定大麦目的基因或特定区域的自然突变。利用M13通用接头作荧光标记，采用巢式PCR对大麦目的基因进行扩增，从PCR扩增的退火温度及模板量、CELⅠ的用量和酶切处理时间等方面对大麦EcoTILLING技术体系进行了优化。结果显示，用于第一次PCR扩增（10μL反应体系）的大麦基因组DNA模板最佳用量为20ng，第一次PCR扩增产物稀释3倍后作为第二次PCR扩增的模板，2次PCR扩增的适宜退火温度均为58℃；在20μL CELⅠ酶切体系中，最适酶量为0.4μL（3U·μL~-1），45℃条件下最佳酶切处理时间为17.5min。优化的EcoTILLING技术体系能有效地发现大麦自然群体中特定区域的DNA多态性，降低了试验成本，为大规模检测与大麦生物活性成分相关候选基因内的核苷酸多态性提供了技术平台。3.利用高通量的EcoTILLING技术检测与生物活性成分合成相关的四个候选基因，即△1-吡咯啉-5-羧酸合成酶基因（P5CS）、甲基固醇羟化酶单加氧酶基因（CSMO）、叶绿素a/b结合蛋白基因（CABP）和热激蛋白17.8基因（HSP17.8）在292个大麦材料中的核苷酸变异。结果显示，四个基因中共67个核苷酸变异被鉴定，其中包括28个插入缺失（Indel）和39个单核苷酸多态性位点（SNP）。25个SNP和28个Indel分布在基因的内含子或非编码区，14个SNP分布在基因的外显子区域，其中7个非同义突变对大麦生物活性成分的合成可能有严重的影响。统计分析显示四个基因进化过程承受了不同的选择压力，P5CS基因内高水平的核苷酸多样性可能是平衡选择的结果，而HSP17.8，CSMO和CABP等三个基因内低水平的核苷酸多样性可能是中性选择的结果。因此，这些被鉴定的自然等位变异需进一步通过关联分析来评价候选基因在大麦生物活性成分积累中的潜在作用。4.基于群体结构的基础上，利用分布于全基因组的19个SSR标记和4个候选基因内的67个SNP标记，对292个大麦材料的4个农艺性状和4个生物活性成分的表型值进行标记与表型的关联分析。结果显示，大麦群体中累计有12个SSR位点和16个SNP位点与7个表型性状的相关性在P<0.01水平上显著，其中4个SSR位点和4个SNP位点与大麦的四个生物活性成分相关联，关联标记对表型性状的解释率在1.99%~15.77%之间，平均值为5.80%。对与性状关联位点的等位变异作进一步解析显示，37个SSR等位变异和8个SNP稀有变异对表型性状具有增效效应，其中5个SSR等位变异和3个SNP稀有变异对大麦四个生物活性成分的含量具有增效效应，这些优异等位基因可为选育高营养品质大麦提供有效的参考，促进大麦保健食品加工专用的基因型品种的发展。5）选用叶面积宽、生长快、生物活性成分含量高且具有优异等位基因的4个大麦品种（138256、138277、26229和BMZ05-234）进行大田种植，对麦苗不同生长期的株高、营养及生物活性成分比较分析发现，大麦品种26229适合作为麦绿素加工专用品种，六叶期麦苗最适作为麦绿素加工原料。与常规加热浸提相比，超声波和微波辅助浸提能显著提高麦绿素产品营养成分的含量；麦苗汁在40℃条件下超声波处理30分钟后，300W功率的微波再萃取8分钟是超声波-微波协同浸提麦绿素的最佳条件，所得麦绿素总黄酮含量高，抗氧化活性强。在室温和冷藏条件下，麦绿素营养成分和抗氧化性随着保存时间的延长都有明显的下降，其中总黄酮和甜菜碱两种物质的稳定较好，而可溶性蛋白质、可溶性总糖及SOD酶的稳定性较差。与室温条件相比，冷藏能有效保证麦绿素的营养成分和抗氧化活性，因此尽可能低温保藏是麦绿素产品保持营养及生理活性的有效方法。更多还原

【Abstract】 Barley is an important cereal crop and brewing raw material. There are many kinds ofbioactive components in barley, such as β-glucan, flavonoids, polyphenol, betain and others.Many claims have been made regarding the health benefits of barley supplements. Some ofthe suggested benefits include antioxidation, cholesterol lowering and boosting energy andimmunity. With the development of economy and the people’s living standards rising quickly,the production with barley bioactive components have been developed rapidly. It has becomean important topic to develop and utilize the rich germplasm resources of barley effectively,and cultivate barley varieties with high yield, high quality, and being rich in functionalcomponents. Most bioactive components of barley are quantitative traits. Dissection of thegenetic basis for these traits is difficult, because they are controlled by multi-genes and areaffected by environmental factors. With the development of genomics and biostatisticssoftware and applications of molecular markers in crop breeding, association analysis basedon the linkage disequilibrium (LD) offers a new method for identifying of loci controllingthose traits. In the present study, association analysis was used to study genetic basis forbioactive components of barley, and the extraction condition of barley green andstorage-stability of barley green were investigated. The main results are summarized asfollowings:1. Twenty-one genome-wide simple sequence repeat (SSR) markers were used to assessthe genetic diversity and population structure of a set of292barley accessions includinglandraces, cultivars and spontaneum. The results showed that the population included adiverse genetic variation. A total of135alleles were detected by19SSR markers with anaverage of7.1per marker. The average value of genetic diversity index (Hi) for19SSRs was0.73. While comparing the genetic diversity, the accessions coming from Middle East Asia,North East Asia and Arabian Peninsula showed more diversity as compared to that of othergeographic regions. A dendrogram was derived from UPGMA cluster analysis based on thegenetic similarity coefficient matix for292barley accessions by using allelic of19SSR markers. Three major clusters were found to be associated with three genepools of barley andseveral subclusters containing genotypes from the same geographic regions were observed.The results suggested that the population could be used for detection of genome-wide SSRmarker-phenotype association mapping with bioactive component in barley.2. The goal of this to establish an optimized EcoTILLING protocol for identification ofnatural variations within target genes and specified regions of genomes in barley. Theuniversal adapter M13was labeled with fluorescent dyes, the nested PCR with specific primercombination and M13adapter was performed for the targeted region. The EcoTILLINGprotocol for barley was optimized, including the template amount and annealing temperaturein PCR amplification, CEL I amount and reaction time in cleavage of mispaired heteroduplexDNA. The results showed that the optimal amount of genomic DNA in a10μL of reactionsystem was20ng as template for the first round of PCR, and the first PCR product was diluted3-fold and used as a template for the second round. The proper annealing temperature for bothfirst-and second-round PCR was58℃. Optimum amount of CEL I enzyme was0.4μL (3U·μL-1) in a20μL of digestion reaction system, and17.5min at45℃was the optimal time forthe cleavage of heteroduplex DNAs. The experiment indicates that the optimizedEcoTILLING protocol can effectively detect DNA polymorphisms for natural population ofbarley with lower cost, and it could provide a foundation for larger scale efforts in reversegenetics and characterization of natural nucleotide variation in barley.3. The high-throughput reverse genetic method called EcoTILLING was used for SNPdiscovery of four bioactive component-related candidate genes, which encoded HSP17.8(heat-shock protein17.8), CABP (a chlorophyll a/b-binding protein), CSMO (C-4sterolmethyl oxidase) and P5CS (Δ′-pyrroline-5-carboxylate synthetase), across292barleyaccessions collected from35different countries. A total of67variations including28insert/deletions (Indels) and39single nucleotide polymorphisms (SNPs) in which14SNPs inthe coding region were identified. Seven of missense changes are predicted to be deleteriousto synthesis of bioactive component in barley. Statistical results of nucleotide diversityshowed that different genes were seemingly under different selective pressures. Balancingselection resulted in high nucleotide diversity in P5CS region, and nucleotide polymorphismsin HSP17.8, CSMO and CABP region were not significant deviated from neutral variation. These natural allelic variants could be used to make association analysis with phenotypictraits for studying these candidate gene’s potential contribution and possible role in bioactivecomponents.4. To determine the population structure and its impact on marker-trait association, the292barley accessions was assessed by19simple sequence repeat (SSR) markers and67polymorphism sites in four candidate gene related with bioactive components. The associationanalysis was tested with the TASSEL v3.0software using the general linear model (GLM). Atotal of28distinct markers significantly associated (p<0.01) with one or more phenotypictraits were detected, which included16single nucleotide polymorphisms (SNPs) and12SSRmarkers. Of those,4SNPs and4SSRs were significantly associated with phenotypic traits offour bioactive components. The percentage of variation of a given trait explained by eachassociated marker spanned from1.99%to15.77%with the average level of5.80%. Thephenotypic allele effect was estimated through comparison between the average phenotypicvalue over accessions with the specific allele and that of accessions with “common allele”.The results showed that37alleles of9SSRs and8rare SNPs had positive effect onphenotypic traits. Of these,4alleles of4SSRs and4rare SNPs had positive effect onbioactive components. These excellent alleles identified in our study will provide informationfor further breeding and genetic research and for improvement of barley bioactivecomponents.5. According to special quality requirements of barley green,4barley genotypes(ICARDA No.138256、138277、26229and BMZ05-234) were primary selected from292accessions based on the results of association analysis. Under field growing conditions,according to plant height and nutrient components of seedling，6-leaves seedling of barleyvariety26229fitted for producing barley green were screened out in this study. Compared toheating solvent extraction, microwave-assisted extraction and ultrasonic-assisted extractionon barley leaf exhibited more amount of barley green. Under ultrasonic treatment30min at40℃, followed by microwave-assisted extract8min at300W power, the maximum amount offlavonoid and the highest antioxidant activity were attained. The nutrient components andantioxidant activity of barley green descended obviously at cold storage and roomtemperature. Betain and flavonoid in barley green were easy to keep, while soluble protein, soluble total sugar and super-oxide dismutase (SOD) were hard to keep at2differenttemperature conditions. Compared with the room temperature, the cold storage could betterkeep the nutrient components and antioxidant activity of barley green.更多还原