SSR标记用于玉米自交系遗传变异与优势类群划分的研究

采用SSR和杂种优势聚类方法分析我国15个玉米骨干自交系的遗传变异,并初步进行了杂种优势类群划分,从62个SSR引物中筛选的40对有效引物对15个玉米自交系进行了分析,共检测到188个等位基因变异,每个SSR座位的等位基因数2－9个,平均为4．7个,SSR位点的多态信息含量PIC值平均为0．675,分布范围在0．360－0．851之间,根据SSR数据对供试材料进行遗传相似性分析,Nei氏相似性系数分布在0．574－0．777之间,10对多态性高的SSR引物可有效区分15个自交系,应用SSR聚类分析的结果与系谱关系相一致,与杂种优势类法相比较,SSR方法具有效率高,结果可靠,可标准化的特点,对SSR方法在玉米育种实践上的应用进行了初步探讨。     

铁皮石斛微卫星SSR设计与应用

通过Websat对来源于NCBI公共数据库的2 447条石斛属(Dendrobium)核苷酸序列进行简单重复序列SSR的搜索,剔除冗余序列后,找到124个SSR位点。利用primer3.0软件设计引物75对,并通过改良的方法提取铁皮石斛DNA作为模板,对铁皮石斛(Dendrobium officinaleKimuraetMigo)的SSR引物进行筛选,选出21对有较清晰且稳定的目标扩增产物的引物,对8个种源的铁皮石斛进行多态性分析和聚类分析,得到8个种源的铁皮石斛进行遗传多样性和亲缘关系。     

Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining

Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60–96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced costs.     

塔玛亚历山大藻遗传多样性的微卫星分析

应用12个微卫星标记对9株来自欧洲和中国等不同海域的塔玛亚历山大藻(Alexandrium tamarense)进行了遗传多样性分析,探讨了不同地理藻株之间的遗传分化程度和基因流水平,分析了我国沿海塔玛亚历山大藻的遗传多样性。结果表明：9株塔玛亚历山大藻共检测出26个等位基因,其中9个位点具有多态性,多态比率75%。有效等位基因数1.3243～3.2667,平均为1.8774。塔玛亚历山大藻种内基因多样性为0.3630。9株塔玛亚历山大藻大致可以分为3个进化支,进化支与藻株的地理位置相关联。其中,中国海域的塔玛亚历山大藻至少可分为2个进化支。不同地理分布的塔玛亚历山大藻的遗传分化水平较高,达0.7522。种群间的基因流估算水平较低,提示3个种群间可能不存在基因交流。     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends

Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala ‘Maxxa’, 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Dynamic metabolic flux analysis (DMFA): a framework for determining fluxes at metabolic non-steady state

Metabolic flux analysis (MFA) is a key tool for measuring in vivo metabolic fluxes in systems at metabolic steady state. Here, we present a new method for dynamic metabolic flux analysis (DMFA) of systems that are not at metabolic steady state. The advantages of our DMFA method are: (1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; (2) flux estimation is achieved without integration of ODEs, or iterations; (3) characteristic metabolic phases in the fermentation data are identified automatically by the algorithm, rather than selected manually/arbitrarily. We demonstrate the application of the new DMFA framework in three example systems. First, we evaluated the performance of DMFA in a simple three-reaction model in terms of accuracy, precision and flux observability. Next, we analyzed a commercial glucose-limited fed-batch process for 1,3-propanediol production. The DMFA method accurately captured the dynamic behavior of the fed-batch fermentation and identified characteristic metabolic phases. Lastly, we demonstrate that DMFA can be used without any assumed metabolic network model for data reconciliation and detection of gross measurement errors using carbon and electron balances as constraints.     

葡萄品种DNA指纹数据库的构建及遗传多样性分析

利用SSR分子标记技术对314份葡萄品种进行了DNA指纹数据库构建和遗传多样性分析,为葡萄品种鉴定、亲缘关系分析和植物品种权保护提供科学依据。结果表明:9对引物共扩增出199个等位基因,多态性位点为199个,多态性比率达100%,每个标记检测到的位点数在17~31之间,平均为22.1个;多态性信息含量(PIC)值变幅在0.793~0.886之间,平均值为0.839。本研究发现3组同名异物品种和9组疑似同物异名品种,除此之外的290份品种中,70份品种仅需1对引物即可区分开,其余品种需要引物组合来实现品种之间的区分。最少选用8对引物即可完全区分开290份葡萄品种。最终利用8对多态性SSR引物构建了314份供试材料的DNA指纹数据库,聚类分析结果表明:263份二倍体供试材料可被分为真葡萄亚属和圆叶葡萄亚属两大类,而真葡萄亚属又被分为15个亚类。51份多倍体供试材料被分为3组,聚类结果与供试材料已知的系谱来源基本吻合。     

小麦-黑麦小片段易位系的分子细胞遗传学分析

为选育具有经济价值的带有黑麦R染色体组小片段的小麦-黑麦育种基础材料,对小麦-黑麦5R/5A×6R/6A代换系杂交后代的8份高代材料6-30、6-31、7-1、7-9、7-13、7-21、7-22和7-28进行形态学、细胞学观察,及SSR分析和GISH检测。结果表明,8个品系田间生长整齐、育性正常,具有大穗、多小穗,抗白粉病、叶锈病等优良性状;对其中2个品系7-1和7-9进行花粉母细胞减数分裂观察,发现大多数细胞染色体构型为2n=21Ⅱ,具有良好的遗传稳定性;选择黑麦R染色体通用引物及5R、6R染色体上的微卫星引物共8对,对8个品系进行SSR分析,结果表明8个品系都有黑麦5R或6R染色体片段的导入,进一步进行GISH检测,发现5个品系6-31、7-1、7-13、7-21、7-22都存在黑麦杂交信号,为小麦-黑麦小片段易位系。本研究综合多种手段鉴定的8份材料皆为小麦-黑麦小片段易位系,在育种上具有利用价值。