彩色马铃薯优良新品系培育及花青素组分的HPLC-MS分析

发布时间：2017-12-27 05:10

本文关键词：彩色马铃薯优良新品系培育及花青素组分的HPLC-MS分析　出处：《内蒙古农业大学》2017年博士论文　论文类型：学位论文

【摘要】：彩色马铃薯富含类黄酮化合物花青素等营养成分,不仅是一种特色作物,还是提取天然色素的重要原料。花青素具有很强的自由基清除能力和抗氧化功能,在人体保健、药用、食品和化妆品添加等方面具有极高的利用价值。目前,国内外严重缺乏高产、高花青素含量、综合性状突出且具有大面积推广种植潜力的彩色马铃薯优良品种。为培育花青素含量高、综合农艺性状优良的彩色马铃薯新品种,近年来我们根据优缺点互补、地理间隔远和生态型差异大的亲本选配原则,将美国引进的四倍体马铃薯品种Red-P1、MIN-021、Russet-01分别与国内育成品种黑美人相组配,通过人工去雄授粉套袋杂交,得到了 "MIN-021×黑美人"、"Red-P1×黑美人"、"Russet-01×黑美人"3个杂交组合的杂种F1代实生种子。经温室栽培及田间试验选择,从3个组合的F1代单株分离群体中选育出5个彩色马铃薯杂种新品系蒙彩-01、蒙彩-02、蒙彩-03、蒙彩-04和蒙彩-05。本试验以彩色马铃薯品种红美和CY-1作对照,重点分析评价这5个新品系的产量、营养品质特性、抗旱能力、花粉育性、染色体配对构型、SSR指纹特征及遗传差异,并利用高效液相色谱与质谱联用技术(HPLC-MS),对5个新品系中花青素含量最高的蒙彩-01和花青素含量相对较低的蒙彩-02的块茎花青素组分进行了鉴定。得到以下主要结果。1.从3个杂交组合F1代分离单株群体中培育出5个彩色马铃薯优良新品系,即蒙彩-01、蒙彩-02、蒙彩-03、蒙彩-04和蒙彩-05。其株型均为直立型,结薯集中整齐、芽眼浅,薯形椭圆或长圆,块茎薯肉均呈不同深浅程度的紫色。2.新品系蒙彩-04为中早熟型,蒙彩-01为中晚熟型,蒙彩-02、蒙彩-03和蒙彩-05均为中熟型。5个新品系的平均单株产量和商品薯率均较高,在1.11 kg和80.66%以上。3.新品系蒙彩-01的块茎花青素含量高达626.71 mg/kg·FW,蒙彩-02、蒙彩-03和蒙彩-05在200mg/kg·FW以上,蒙彩-04为118.15mg/kg·FW。新品系蒙彩-01和蒙彩-02的块茎干物质含量和淀粉含量均较高,分别为21.32%、22.38%和17.11%、18.81%,其余3个新品系在19%和15%以上。5个新品系粗蛋白含量变幅为0.98%～1.67%,还原糖含量为0.015%～0.028%,Vc含量为18.67%～21.20%,酚类物质含量为 0.12%～0.19%。4.蒙彩-01、蒙彩-02和蒙彩-05的花粉育性均在76.07%以上,蒙彩-03和蒙彩-04的育性分别为33.39%和58.74%,均可作杂交育种的中间材料加以利用。新品系蒙彩-01 的染色体配对构型为 1.18 Ⅰ +14.65Ⅱ+1.28Ⅲ+3.42Ⅳ,蒙彩-02为 1.33 Ⅰ+13.97Ⅱ+1.22Ⅲ+3.77Ⅳ,蒙彩-03为9.12 Ⅰ+7.71Ⅱ+6.88Ⅲ+0.70Ⅳ,蒙彩-04为3.96Ⅰ+11.25Ⅱ+4.91Ⅲ+1.70Ⅳ,蒙彩-05为3.82 Ⅰ +13.05 Ⅱ+2.31Ⅲ+2.79Ⅳ。5.利用筛选出31对SSR适宜引物,PCR扩增共得到多态性位点328个,其占总位点数89.86%。用C54、C57、STI027和S153这4个SSR引物构建了 5个新品系及其亲本和对照品种的DNA指纹图。以GD值0.40为基准将10个材料聚为3类:蒙彩-05、Red-P1、MIN-021和CY-1为第一类,蒙彩-01、蒙彩-02、蒙彩-03、蒙彩-04和黑美人为第二类,品种红美为单独的一类。6.试验建立了各新品系茎尖脱毒培养的再生体系,筛选出最适激素浓度配比的培养基为 MS+6-BA0.1 mg/L+NAA0.2 mg/L+GA30.1 mg/L。经 DAS-ELISA 检测,得到了 5个新品系的脱毒组培苗,为其无毒苗的扩繁和原原种繁殖奠定了基础。7.利用不同浓度的PEG溶液处理新品系蒙彩-01、蒙彩-02和蒙彩-05及其亲本的脱毒组培幼苗,测定不同干旱胁迫强度下幼苗叶片的细胞膜相对透性、丙二醛含量和脯氨酸含量等指标,综合评定6个材料幼苗的抗旱能力强弱顺次为:蒙彩-01蒙彩-05MIN-021Red-P1蒙彩-02黑美人。8.HPLC-MS分析结果表明:块茎薯肉呈紫色的马铃薯新品系蒙彩-01和蒙彩-02均含有矮牵牛素-3-香豆酰-芸香糖苷、矮牵牛素-3-香豆酰-芸香糖基-5-葡萄糖苷和矮牵牛素-3-阿魏酰-芸香糖基-5-葡萄糖苷,其中矮牵牛素-3-香豆酰-芸香糖基-5-葡萄糖苷为主要组分;此外,蒙彩-01特有锦葵素-3-香豆酰-芸香糖基-5-葡萄糖苷组分,蒙彩-02中特有芍药素-3-咖啡酰-芸香糖基-5-葡萄糖苷和芍药素-3-香豆酰-芸香糖基-5-葡萄糖苷2种组分。块茎薯肉呈红色的品种'红美'含有天竺葵素-3-芸香糖苷、天竺葵素-3-阿魏酰-芸香糖基-5-葡萄糖、天竺葵素-3-香豆酰-芸香糖基-5-葡萄糖苷和矢车菊素-3-香豆酰-葡萄糖苷3种组分,其中组分天竺葵素-3-香豆酰-芸香糖基-5-葡萄糖苷和矢车菊素-3-香豆酰-葡萄糖苷为主要组分。
[Abstract]:Color potatoes are rich in anthocyanins and other nutrients, which are not only a special crop but also an important raw material for the extraction of natural pigment. Anthocyanin has strong free radical scavenging and antioxidant function. It has great value in health care, medicine, food and cosmetics. At present, there is a serious lack of colorful potato varieties with high yield, high anthocyanin content, high comprehensive characters and large area to popularize planting potential at home and abroad. For the new color, high anthocyanin content of potato varieties cultivation agronomic traits, in recent years we have complementary advantages and disadvantages, according to the geographic distance and the ecotype differences in the principle of parental selection, will be the introduction of Tetraploid Potato Varieties Red-P1, MIN-021 and Russet-01 respectively with the domestic varieties of black beauty is matched by artificial pollination. Bagging hybridization, got the "MIN-021 * Black Beauty" and "black beauty" Red-P1 * Russet-01 *, "black beauty" of 3 combinations of F1 hybrid seeds. Through greenhouse cultivation and field experiments, 5 color potato hybrids, Mongolian color -01, Mongolian color -02, Mongolian color -03, Mongolia color -04 and Mongolia color -05, were selected from 3 combinations of F1 generation segregating populations. In this experiment, color potatoes and red beauty CY-1 as control, focus on the analysis and evaluation of the 5 varieties of yield, nutritional quality characteristics, drought resistance, pollen fertility, chromosome pairing of SSR fingerprint and genetic diversity, configuration, and use of high performance liquid chromatography and mass spectrometry (HPLC-MS), on the anthocyanin content 5 new lines in the highest Mongolia color -01 and anthocyanin content is relatively low by color -02 tuber anthocyanins were identified. The following main results are obtained. 1., from the 3 cross combinations of F1 generation, 5 new elite strains of color potato were bred, namely Mongolia -01, Mongolia -02, Mongolian color -03, Mongolia color -04 and Mongolia color -05. The plant is erect, Microtuberizing concentrated neat, Yayan shallow, elliptic or oblong shape, tuber flesh showed different shades of purple. 2. new product line -04 is medium and early maturing type, and Mongolian color -01 is medium and late maturing type, Mongolian color -02, Mongolian color -03 and Mongolian color -05 are medium mature. The average yield of single plant and the rate of commercial sweet potato were higher in the 5 new lines, at 1.11 kg and more than 80.66%. 3., the content of tuber anthocyanin in the new line of Mongolia -01 is as high as 626.71 mg/kg FW. The Mongolia color -02, Mongolian color -03 and Mongolia color -05 are above 200mg/kg. FW, and Mongolia color -04 is 118.15mg/kg. The dry matter content and starch content of the new line Mongolia -01 and Mongolia -02 were higher than those of the new line, which were 21.32%, 22.38% and 17.11%, 18.81% respectively, and the other 3 new strains were 19% and 15% above. The crude protein content of 5 new strains ranged from 0.98% to 1.67%, the content of reducing sugar was 0.015% to 0.028%, the content of Vc was 18.67% to 21.20%, and the content of phenols was 0.12% to 0.19%. 4., the pollen fertility of Mongolia lottery -01, Mongolian color -02 and Mongolia -05 is over 76.07%, and the fertility of Mongolian color -03 and Mongolian color -04 is 33.39% and 58.74%, respectively, which can be used as intermediate materials for cross breeding. The new Department of Mongolia color -01 chromosome configuration was 1.18 +1.28 +3.42 I +14.65 II III IV, Mongolia color -02 1.33 +1.22 +3.77 I +13.97 II III IV, Mongolia color -03 9.12 +6.88 +0.70 I +7.71 II III IV, Mongolia color -04 3.96 +4.91 +1.70 I +11.25 II III IV, Mongolia color -05 3.82 1 +13.05 +2.31 +2.79 II III IV. 5. using 31 pairs of appropriate primers for SSR, 328 polymorphic loci were obtained by PCR amplification, which accounted for 89.86% of the total point number. 4 SSR primers of C54, C57, STI027 and S153 were used to construct the DNA fingerprints of 5 new strains, their parents and the control varieties. Based on GD value 0.40, 10 materials were clustered into 3 categories: Mongolian color -05, Red-P1, MIN-021 and CY-1 as the first category, Mongolian color -01, Mongolia color -02, Mongolia color -03, Mongolian lottery -04 and black beauty second categories, and Hongmei as a separate category. 6., we established the regeneration system of virus-free culture of every new line, and screened the optimum medium concentration of MS+6-BA0.1 mg/L+NAA0.2 mg/L+GA30.1 mg/L. By DAS-ELISA detection, we obtained 5 strains of virus-free plantlets, which laid the foundation for the propagation of virus-free seedling and seed breeding. 7. using different concentrations of PEG solution by -01, color varieties by tissue culture seedling de -02 and Mongolia Mongolia color color -05 and its parents, the determination of different drought stress intensity of seedling leaf cell membrane relative permeability and MDA content and proline content index, drought resistant ability in comprehensive evaluation of 6 seedlings as the material -01 -05MIN-021Red-P1: Mongolia Mongolia Mongolia color color -02 color black beauty. The results of 8.HPLC-MS analysis showed that potato tuber meat is potato purple color -01 and -02 color Mongolia Mongolia contain Petunia -3- p-coumaric acyl rutinoside, Petunia -3- p-coumaric acyl rutinose group -5- glucoside and Petunia -3- feruloyl rutinose group -5- glucoside, the in Petunia -3- p-coumaric acyl rutinose group -5- glucoside as the main component; in addition, Mongolia color -01 unique malvidin -3- p-coumaric acyl rutinose group -5- glucoside group, Mongolia color -02 unique paeonidin -3- caffeoyl - rutinose -5- glucoside and peonidin -3- sweet bean acyl rutinose based -5- glucoside 2 components. The meat is red tuber potato cultivar 'red beauty' containing pelargonidin rutinoside, pelargonidin -3- -3- feruloyl rutinose base -5- glucose, pelargonidin -3- p-coumaric acyl rutinose -5- glucoside and cyanidin -3- p-coumaric acyl glucoside 3 components, including components of pelargonidin -3- p-coumaric acyl rutinose based -5- glucoside and cyanidin -3- p-coumaric acyl glucoside was the main component.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S532

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