| 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.
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