ISSR标记在小麦指纹图谱分析中的应用研究初探

对ISSR标记技术在构建多态水平很低的小麦种内指纹图谱方面做了初步研究。发现（AG）n,(AC)n,(TG)n等二核苷酸重复引物类型在小麦中多态水平最高。能将亲缘关系很近的品种或品系区分区。结果有明：与其它常规分子标记技术如RFLP、RAPD等相比，ISSR标记具有简便、快捷、成本低、多态水平高等特点，是一种植得进一步研究的分子标记技术。     

基于ISSR指纹的甘薯食用品种的遗传多样性分析

运用ISSR分子指纹技术检测甘薯基因组DNA的多态性,是鉴定甘薯遗传多样性和亲缘关系有效的方法之一。本研究选用6个ISSR标记对17份甘薯品种进行了DNA指纹扩增,共扩增出55条谱带,其中多态性谱带50条,多态性水平为90.91%;UBC825引物扩增出带型最多,有16种;根据Nei’s遗传距离计算获得的聚类树状图表明,在GS为0.625时,参试的17份甘薯品种分为4个组群;遗传相似性分析表明,顶芽菜用品种间遗传相似系数最高0.877,叶柄食用品种和烤薯型品种之内的遗传相似系数分别为0.778和0.740。结果表明甘薯主要食用间具有较好的遗传多样性,但同类型品种间却又有较高的遗传相似性,这类品种需要引进或创制新资源。     

SSR标记的彩色马铃薯遗传多样性分析及指纹图谱构建

彩色马铃薯(指块茎的皮或肉为红、蓝、紫、橙色等)近年来日益为育种工作者所关注,很多彩色马铃薯品种(系)从形态学上难以鉴定是否为同一基因型,给育种工作带来诸多不便。本研究利用SSR标记对50份彩色马铃薯(Solanum tuberosumL.)材料进行了遗传多样性分析及指纹图谱构建。研究筛选出56对马铃薯SSR引物,对50份材料的基因组DNA进行PCR扩增,共检测出236个等位位点,其中多态性位点230个,多态性比率达97.46%。分析显示,基因型间遗传相似性系数在0.50~1.00之间。UPGMA聚类分析表明,在相似系数0.63处可将全部材料分为3大类。利用5对核心引物构建了50份供试材料的指纹图谱,并证明其属于44个基因型的,为彩色马铃薯资源鉴定和利用提供了依据。     

河南粳稻品种SSR指纹图谱的构建及遗传差异分析

为了建立河南粳稻品种的DNA指纹图谱,了解河南粳稻种植品种的遗传多样性,本研究利用均匀分布于水稻(Oryza sativaL.)12条染色体的37对SSR引物,以河南1963～2010年间主要推广种植的37个粳稻(O.sativaL.ssp.japonica)品种为材料,进行简单序列重复(SSR)指纹图谱构建和遗传多样性分析。结果表明,33对引物具有特异多态性片段,共检测到114个等位基因,平均每位点3.53个等位基因;引物多态性频率为0.054～0.739。37个品种的遗传相似系数为0.627～0.992,平均为0.812。17个品种都至少具有1对SSR特征引物,其余20个品种的鉴定需要结合不同的SSR引物。利用19对核心SSR引物构建的指纹图谱能逐一区分出36个粳稻品种。采用非加权类平均法(UPGMA)进行聚类分析,在遗传相似系数0.710处可将37个品种分为2大类,河南选育品种基本都聚到同一大类群,表明河南推广的粳稻品种遗传基础狭窄。     

滇青、青饼和普洱茶（熟饼）近红外指纹图谱分析

为深入了解滇青（晒青毛茶）、青饼和普洱茶（熟饼）在化学组成方面的异同,为茶类的区分与界别提供进一步的科学依据,对滇青、青饼和普洱茶（熟饼）的近红外化学指纹图谱采用欧氏距离、主成分分析和系统聚类等方法进行了定性判别分析。结果发现,普洱茶（熟饼）近红外指纹图谱与滇青、青饼图谱间的欧氏距离均大于各自组内最大偏差的绝对阈值,而滇青和青饼的组间差异小于各自的组内阈值;在第一主成分和第二主成分得分值散点图上,熟饼样品与青饼和滇青样品间界限分明,而滇青和青饼样品间无明显界限;所有的熟饼样品在系统聚类分析中聚为一类,而青饼与滇青样品聚为一类。因此,青饼的近红外光谱在各方面都与普洱茶（熟饼）样品差异显著,而与其原料滇青（晒青毛茶）十分接近。从化学本质上来说,青饼更接近于绿茶类产品。     

节水抗旱稻品种DNA指纹图谱的构建

为了解节水抗旱稻品种的多样性,利用SSR分子标记技术对24份节水抗旱稻和2份普通水稻品种进行DNA指纹图谱构建和遗传多样性分析。结果表明,24对引物共扩增出96个多态性片段,平均每对引物可检测到4个等位基因,每个SSR位点可以检测到2~6个等位基因。引物多态信息含量(PIC)的变化范围为0.36~0.75,平均值为0.58。指纹图谱显示至少可以利用RM71、RM72、RM336、RM337、RM1195和RM5414这6个核心标记的不同组合鉴别区分26份供试材料。聚类分析结果表明,26份材料间遗传相似系数为0.54~0.98,在遗传相似系数0.65处可以将供试材料分为籼、粳两类,较好地反映了供试材料的亲缘关系。本研究结果为节水抗旱稻新品种保护、真伪鉴定及亲本选配提供了参考。     

山东省46个花生品种SSR指纹图谱构建与遗传多样性分析

为从分子水平上快速鉴别花生品种和选配优良杂交组合,以山东省审定的46个花生品种为材料,利用微卫星(SSR)标记进行DNA指纹图谱的构建和遗传多样性分析。从788对SSR引物中筛选出50对多态性高、稳定性好、谱带清晰的引物,共检测到175个等位位点,其中122个为多态性位点,多态性比率达70.52%;每对SSR引物扩增出的等位位点数为2~7个,多态性信息量变化范围为0.6753~0.8412,平均为0.823。此外,利用14对引物可将46份材料完全区分开。聚类分析表明,在相似系数0.77处,所有供试材料聚为一类,在相似系数0.80处,仍有76%的材料聚在一起。利用SSR标记构建的指纹图谱可为花生种质资源管理及育种实践提供依据。     

茶叶氨基酸特征指纹图谱的初步研究

通过酸水解法处理茶叶样品,以阳离子交换—茚三酮柱后衍生法分析水解液中的氨基酸含量,以归一法计算各氨基酸的峰面积,绘制了茶叶水解氨基酸的指纹图谱,进行了主成分分析.结果表明,茶叶中水解氨基酸总含量在18％～30％之间,水解氨基酸指纹图谱能够较好区分龙井群体和龙井43,同时也可区分龙井茶与其他茶叶,与主成分分析结果基本一致.该法的建立为研究茶叶品质提供了一个新的思路,同时也为我国名优茶叶的真伪鉴别和追踪溯源提供了参考.     

基于SSR标记的我国主栽日本栗品种(系)遗传结构分析和指纹图谱构建

为解析我国主栽日本栗品种资源的遗传多样性并构建其指纹图谱,本研究通过毛细管电泳和高质量的17个SSR分子标记对日本栗品种资源进行位点检测。结果显示,日本栗与茅栗具有较近的种间亲缘关系。在59份日本栗品种(系)中共检测到131个等位位点,每个标记平均有7.706个等位位点;多态信息含量(PIC)变幅为0.375~0.815,平均值为0.605;供试日本栗品种表现出高的遗传多样性。系统发育树、群体结构和主坐标分析结果一致,支持中国境内的部分日本栗品种(系)独立形成一个分支,且大多品种为杂交资源。此外,本研究根据多位点匹配分析确定了CmSI0922、CmSI00702和CmSI0658为核心引物,并利用这3个核心引物构建了59份日本栗品种(系)的指纹图谱。综上所述,相比其他栗属植物,日本栗分布范围较狭窄,但其具有丰富的遗传多样性,且品种资源间存在广泛的基因流。本研究成功构建了59份日本栗品种(系)资源的具有唯一对应关系的指纹图谱,为栗属植物的资源鉴定和保护利用提供了有力支撑。     

利用SSR技术构建多花黑麦草品种指纹图谱

通过构建我国多花黑麦草主栽品种的SSR标记指纹图谱数据库,以实现对多花黑麦草品种的快速、准确鉴定.本研究利用SSR标记,基于多态性高、稳定性强和连锁群分布均匀的原则,从100对多花黑麦草(Lolium multiflorum Lam.)基因组来源的SSR引物中,筛选出12对引物用来构建了21个多花黑麦草品种(系)的指纹图谱.12对SSR引物共扩增出108种多态性基因型,多态性比率达94.15％,每对引物的基因型从5～22种不等,平均每对引物扩增出9种基因型,多态性信息量(PIC)变幅为0.744～0.934,平均为0.843,此12对引物可以在构建多花黑麦草品种DNA指纹数据库作为核心引物推荐使用.21个多花黑麦草品种(系)基因型间遗传相似性系数在0.4956～0.8571之间,UPGMA聚类分析表明,在相似系数0.69处可将全部材料分为3大类.7对引物在9个品种上具有唯一特征带,采用15-08C单对引物即可将21个多花黑麦草品种完全区分开,该对引物不仅多态性高(PIC=0.934),且具备多个品种的特征谱带.基于该引物建立了供试品种指纹图谱标准模式图,每个品种具有唯一的指纹图谱(带型),为牧草品种的审定和保护以及选配优良杂交组合培育新品种提供了重要的理论依据.     

Constructing DNA fingerprinting of Hemarthria cultivars using EST-SSR and SCoT markers

DNA fingerprinting of known cultivars may provide much-needed data to assist with the identification of such cultivars. From 86 pairs of expressed sequence tag SSRs (EST-SSR) and 45 start-codon targeted polymorphism (SCoT) primers, eight pairs of EST-SSR primers and seven SCoT primers were chosen to construct the DNA fingerprinting of six Hemarthria cultivars in this study. Using genomic DNA from six cultivars, a total of eight pairs of EST-SSR primers were able to amplify 193 bands, producing an average of 24.1 bands per primer pair. The percentage of polymorphic bands (PPB) was 83.4 %, and the polymorphism information content (PIC) ranged from 0.480 to 0.695, with an average of 0.602. A total of seven SCoT primers amplified 105 bands with an average of 15 bands per sample. The PPB was 84.8 %, and the PIC ranged from 0.471 to 0.758, with an average of 0.612. The unweighted pair-group method with arithmetic mean dendrogram from EST-SSR and SCoT markers grouped the six Hemarthria cultivars into two major groups of the same. These clusters are in accordance with their known species and origin. Our results indicate a rich genetic diversity in these six Hemarthria cultivars. The six cultivars we assayed could be easily identified using these eight pairs of EST-SSR and seven pairs of SCoT primers.     

Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis

Metabolite fingerprints, obtained with direct injection mass spectrometry (MS) with both positive and negative ionization, were used with analysis of variance-principal components analysis (ANOVA-PCA) to discriminate between cultivars and growing treatments of broccoli. The sample set consisted of two cultivars of broccoli, Majestic and Legacy, the first grown with four different levels of Se and the second grown organically and conventionally with two rates of irrigation. Chemical composition differences in the two cultivars and seven treatments produced patterns that were visually and statistically distinguishable using ANOVA-PCA. PCA loadings allowed identification of the molecular and fragment ions that provided the most significant chemical differences. A standardized profiling method for phenolic compounds showed that important discriminating ions were not phenolic compounds. The elution times of the discriminating ions and previous results suggest that they were common sugars and organic acids. ANOVA calculations of the positive and negative ionization MS fingerprints showed that 33% of the variance came from the cultivar, 59% from the growing treatment, and 8% from analytical uncertainty. Although the positive and negative ionization fingerprints differed significantly, there was no difference in the distribution of variance. High variance of individual masses with cultivars or growing treatment was correlated with high PCA loadings. The ANOVA data suggest that only variables with high variance for analytical uncertainty should be deleted. All other variables represent discriminating masses that allow separation of the samples with respect to cultivar and treatment.     

Genetic diversity analysis of yams (Dioscorea spp.) cultivated in China using ISSR and SRAP markers

Yam (Dioscorea spp.) is widely cultivated in China and many landraces are maintained by local farmers. However, there is little information available about their diversity and species identity. In this study, inter simple sequence repeat (ISSR) and sequence related amplified polymorphism (SRAP) techniques were used to assess genetic diversity within 21 yam landraces from seven cultivated populations. We observed high level of polymorphism among these landraces, specifically, 95.3 % for ISSR and 93.5 % for SRAP. Analysis of molecular variance revealed a significantly greater variation among the four yam species (40.39 %) and their populations (35.78 %) than within the populations (23.83 %). The unweighted pair group method arithmetic averages clusters and principal component analysis for 21 landraces formed four well-separated groups containing landraces of each of the four species, namely, Dioscorea opposita Thunb., Dioscorea alata L., Dioscorea persimilis Prain et Burkill, and Dioscorea fordii Prain et Burkill. The ISSR and SRAP primers were highly discriminatory among the 21 landraces; all 21 landraces could be easily differentiated using these primers. The average mean of gene flow (Nm = 0.1081) estimated from high genetic differentiation (Gst = 0.8222) suggested that gene flow among the populations was relatively restricted. The lack of genetic diversity within individual yam species suggests that it is critical to develop long-term strategies for enhancing genetic diversity within various yam species.     

Nitrogen requirement of fodder and sugar beet (Beta vulgaris L.) cultivars under high-yielding conditions of northwestern Europe

This study provides current data on plant nitrogen (N) uptake required for maximum sugar yield (PNUp_max) and the corresponding fertilizer N dose (ND) (optimum N dose [ND_opt]) for high-yielding beet crops (sugar yield up to 20 Mg ha^?1). In 2010 and 2011, field experiments were conducted with four cultivars from Beta genus differing in dry matter composition, and six mineral NDs (0–200 kg N ha^?1) at three sites (The Netherlands, Germany, Denmark). Differences between cultivars in PNUp_max and ND_opt were small; however, environments (defined as combination of site and year) substantially differed from each other: highest PNUp_max and lowest ND_opt occurred at environments supplying high amounts of N from soil resources, and vice versa. The level of maximum sugar yield (SY_max) was related neither to PNUp_max (200–270 kg N ha^?1) nor to ND_opt. However, N dose and plant N uptake required for 95% of maximum sugar yield was 50–80 kg N ha^?1 lower than for maximum sugar yield. To conclude, accepting a slight reduction in sugar yield might allow for a substantial decrease in the ND. Cultivar choice and yield level need not to be taken into account at present.     

Biodegradable composites from sugar beet pulp and poly(lactic acid)

Sugar beet pulp and poly(lactic acid) (PLA) composites were prepared by compression-heating. The resultant thermoplastics had a lower density, but they had tensile strength similar to that of pure PLA specimens as well as the same geometric properties. Tensile properties depended on the initial water content of sugar beet pulp and the process by which composites were manufactured. In comparison with sugar beet pulp, the composite showed improved water resistance. This can be attributed to the hydrophobic character of PLA and pulp-matrix interactions. The composite thermoplastics showed suitable properties for potential use as lightweight construction materials.     

Quality evaluation of sugar beet (Beta vulgaris) by near-infrared spectroscopy

The legal method (polarimetric measurement) for the determination of sucrose content and the wet chemical analysis for the quality control of sugar beet uses lead acetate. Because heavy metals are pollutants, the law could forbid their use in the future. Therefore, near-infrared spectroscopy (NIRS) was evaluated as a procedure to replace these methods. However, there are alternatives to lead clarification, such as the use of aluminum salts, which have been applied at many sugar companies. The real advantage of NIRS is in speed and ease of analysis. The aim of this study was to determine simultaneously the concentration of several components which define the industrial quality of beets. The first objective was the determination of sucrose content, which determines the sugar beet price. The standard error of prediction (SEP) was low: 0.11 g of sucrose/100 g of fresh beet. NIRS was also able to determine other beet quality parameters: brix, marc, glucose, nitrogen, sodium, potassium, sugar in molasses (i.e. sucrose in molasses), and juice purity. The results concerning brix, marc, sugar in molasses, and juice purity were satisfactory. NIRS accuracy was lower for the other parameters. Nevertheless, RPD (ratio standard deviation of concentration/SEP) and RER (ratio concentration range/SEP ratio) show that NIRS might be used for the sample screening on nitrogen, potassium, sodium, and glucose content.     

甜菜谷氨酸合成酶活性与块根产量、含糖率的相关性分析

采用双丰16(偏丰产型)和甜单1号(偏高糖型)两个甜菜(Beta vulgaris L.)品种作为试材,在5个氮素水平(0、60、120、180和240 kg.hm-2)下,研究甜菜功能叶片与块根中谷氨酸合成酶(glutamate syn-thase,GOGAT)在生育进程中的活性变化动态及其与块根产量、含糖率和产糖量的关系。结果表明:在不同氮素水平下,两个品种功能叶片和块根的GOGAT活性均呈双峰曲线变化,两峰值分别出现在6月下旬和8月下旬,其中,功能叶GOGAT前一峰值高于后一峰值;块根GOGAT则与之相反。在不同氮素水平下,功能叶片GOGAT活性表现为N180>N120>N240>N60>N0,块根GOGAT表现为N120>N180>N240>N60>N0。GOGAT的活性与块根产量及含糖率关系紧密,块根产量随GOGAT活性的增加而提高,但是GOGAT活性过高反而降低了块根含糖率,进而导致甜菜产糖量的降低。     

基于无人机影像的冠层光谱和结构特征监测甜菜长势

甜菜是中国北方地区重要的经济作物。快速、准确、高通量的获取甜菜的地上部和块根鲜质量、块根含糖率、叶绿素含量对甜菜生产具有重要意义。该研究采用无人机搭载数码和多光谱相机,获取甜菜叶丛快速生长期、块根及糖分增长期和糖分积累期的数码影像和多光谱影像,提取了冠层的结构特征和光谱特征。选择随机森林回归（Random Forest Regression,RFR）和偏最小二乘回归（Partial Least Squares Regression,PLSR）2种建模方法基于获取的冠层特征,构建甜菜全生育期的地上部和块根鲜质量、块根含糖率和SPAD（Soil and Plant Analyzer Development）值估算模型。研究结果表明,随机森林回归模型和偏最小二乘回归模型对地上部和块根鲜质量、含糖率都做出较好的预测,R2范围分别为0.9～0.94、0.88～0.9,rRMSE范围分别为7.6%～17%、8.8%～20%。对SPAD值的预测均较弱,R2分别为0.66和0.67。为了减小输入变量集的大小以及去掉对预测不敏感的变量,该研究采用置换重要性（Permutation Importance,PIMP）来筛选冠层光谱特征和结构特征中对预测有重要影响的变量。结果表明基于筛选出的重要性特征构建的随机森林回归模型和偏最小二乘回归模型对地上部和块根鲜质量、含糖率都做出较好的预测,R2范围分别为0.89～0.94、0.74～0.91,rRMSE范围分别为7.3%～19%、7.6%～19%。对SPAD值的预测均较弱,R2分别为0.65和0.68。进一步表明随机森林回归模型在精度上略好于偏最小二乘回归模型。同时基于PIMP筛选变量的方法在保持原有精度的同时能实现降低数据收集复杂性的目的。研究结果为基于无人机遥感技术快速、准确监测甜菜长势和估测块根类作物的根部活性物质提供了参考。