Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.     

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1× coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40–70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP–dROX, we assembled a physical map for P.chrysogenum, with 2–3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

Secretome characteristics of pelletized Trichoderma reesei and cellulase production

Trichoderma reesei is an important cellulase producer and its secondary mycelial phase is responsible for cellulase expression and secretion in submerged fermentation. Little is known regarding the effects of fungal morphology on cellulase production by Trichoderma sp. In this study we aimed to extend the understanding of cellulase production by T. reesei, especially correlating cellulase productivity with pellet morphology and with its secretome characteristics. We found that T. reesei was more likely to form pellets in malt extract broth than in potato dextrose broth. CaCO(3) helped in formation of fine pellets in malt extract broth. 10(9) spores/ml resulted in formation of pellets with the size of 0.13 ± 0.047 mm. LC/MS spectrometry analysis indicated that the secretomes from pellet was different from that of mycelial mat under the same fermentation conditions. Optimization tests showed that lactose, xylose and Pluronic F68 are important for efficient production of cellulases with FPU activity in the pellets fermentation. This is the first report on the artificial formation of pellets by Trichoderma sp. as well as correlation between physiological characteristic of the pellets and cellulase production by T. reesei. The findings from this study can be used for improvement of cellulase productivity.     

中枢神经细胞的高尔基复合体ACP活性分布的电镜观察

本文采用电镜金属盐法—酸性磷酸酶(ACP)细胞化学技术,用30mmol/L pipes缓冲液配制低浓度戊二醛进行固定。对成年大鼠的大脑大锥体细胞,小脑浦肯野氏细胞,脊髓前角运动细胞的高尔基复合体的ACP活性进行了实验研究和探讨。结果发现ACP活性分布在高尔基复合体的部份转移泡、浓缩泡及GERL部位。高尔基复合体呈ACP阳性反应,并显示出多种形态。     

锌转运体ZNT7在Alzheimer病动物模型APP/PS1转基因鼠脑内的表达

目的研究阿尔茨海默病（Alzheimer disease,AD）模型小鼠APP/PS1转基因小鼠脑内锌转运体ZNT7的分布和表达,探讨ZNT7参与Aβ老年斑形成的机理。方法应用免疫组织化学染色观察ZNT7在脑内分布情况,应用Western Blot方法分析ZNT7在APP/PS1转基因小鼠大脑内的表达。结果ZNT7免疫阳性反应产物主要分布在APP/PS1转基因小鼠大脑皮层、纹状体和海马的老年斑内,强阳性的ZNT7免疫产物定位于老年斑的核心。Western Blot分析结果表明ZNT7在APP/PS1转基因小鼠大脑内的表达明显高于野生型小鼠。结论ZNT7在APP/PS1转基因小鼠大脑内的高表达以及在Aβ老年斑的定位,提示ZNT7可能参与了锌离子在老年斑内的聚集,进而参与了APP/PS1转基因小鼠大脑内老年斑的形成。     

Study of a two-stage growth of DHA-producing marine algae <Emphasis Type="Italic">Schizochytrium limacinum</Emphasis> SR21 with shifting dissolved oxygen level

The culture protocol of Schizochytrium limacinum SR 21, a known docosahexaenoic acid (DHA) producing marine algae was modified in this study to better fit fermentation parameters, particularly control of dissolved oxygen (DO) to the known reproductive and growth biology of the microorganism. The cultures controlled at 50% DO saturation produced a cell density of 181 million cells/ml, whereas cultures with 10% DO produced only 98.4 million cells/ml. A fixed-agitation rate of 150 rpm resulted in an even lower density of 22.5 million cells/ml. Fifty percent DO saturation level led to a decreased pH, as well as a negative correlation with lipid accumulation, while low oxygen concentration was obligatory for lipid accumulation. This study indicated that high DO was preferred for the cells’ reproduction via release of zoospores. Thus, the culture of S. limacinum SR21 should be best divided into two stages: (1) a cell-number-increasing stage in which cell reproduction and cell number increase with little increase in the size and weight of each cell; and (2) a cell-size-increasing stage in which cells stop reproduction but cell size enlarges due to lipids accumulation. With such a protocol, the production of algae biomass and DHA was improved to levels of 37.9 g/L and 6.56 g/L, respectively. The two-stage culture process could be potentially used not only for omega-3 PUFA production, but also in other single cell oil (SCO)-producing processes, including biodiesel production from algae.     

Cloning, characterization, and evolution of the NBS-LRR-encoding resistance gene analogue family in polyploid cotton (Gossypium hirsutum L.)

The nucleotide-binding site-leucine-rich repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. We cloned the NBS-LRR-encoding genes from polyploid cotton by a polymerase chain reaction-based approach. A sample of 150 clones was selected from the NBS-LRR gene sequence library and was sequenced, and 61 resistance gene analogs (RGA) were identified. Sequence analysis revealed that RGA are abundant and highly diverged in the cotton genome and could be categorized into 10 distinct subfamilies based on the similarities of their nucleotide sequences. The numbers of members vary many fold among different subfamilies, and gene index analysis showed that each of the subfamilies is at a different stage of RGA family evolution. Genetic mapping of a selection of RGA indicates that the RGA reside on a limited number of the cotton chromosomes, with those from a single subfamily tending to cluster and two of the RGA loci being colocalized with the cotton bacterial blight resistance genes. The distribution of RGA between the two subgenomes A and D of cotton is uneven, with RGA being more abundant in the A subgenome than in the D subgenome. The data provide new insights into the organization and evolution of the NBS-LRR-encoding RGA family in polyploid plants.     

The impact of population structure on genomic prediction in stratified populations

Key message

Impacts of population structure on the evaluation of genomic heritability and prediction were investigated and quantified using high-density markers in diverse panels in rice and maize.

Abstract

Population structure is an important factor affecting estimation of genomic heritability and assessment of genomic prediction in stratified populations. In this study, our first objective was to assess effects of population structure on estimations of genomic heritability using the diversity panels in rice and maize. Results indicate population structure explained 33 and 7.5 % of genomic heritability for rice and maize, respectively, depending on traits, with the remaining heritability explained by within-subpopulation variation. Estimates of within-subpopulation heritability were higher than that derived from quantitative trait loci identified in genome-wide association studies, suggesting 65 % improvement in genetic gains. The second objective was to evaluate effects of population structure on genomic prediction using cross-validation experiments. When population structure exists in both training and validation sets, correcting for population structure led to a significant decrease in accuracy with genomic prediction. In contrast, when prediction was limited to a specific subpopulation, population structure showed little effect on accuracy and within-subpopulation genetic variance dominated predictions. Finally, effects of genomic heritability on genomic prediction were investigated. Accuracies with genomic prediction increased with genomic heritability in both training and validation sets, with the former showing a slightly greater impact. In summary, our results suggest that the population structure contribution to genomic prediction varies based on prediction strategies, and is also affected by the genetic architectures of traits and populations. In practical breeding, these conclusions may be helpful to better understand and utilize the different genetic resources in genomic prediction.     

游离锌离子轴突运输的实验研究

目的：研究大鼠坐骨神经结扎后游离锌离子在轴突内的定位分布,探讨锌离子在含锌神经元内的轴突运输。方法：应用轴流阻滞／神经结扎术结合光、电镜锌金属自显影技术,检测含锌神经元轴突内的游离锌离子子。结果：锌阳性反应产物主要分布在靠近结扎点的坐骨神经近端和远端轴突内,并且随着结扎时间的延长锌离子在轴突近端和远端的积累逐渐增加。此外,电镜结果表明锌离子主要定位于无髓神经纤维以及薄髓鞘的有髓神经纤维轴突内。结论：游离锌离子在含锌神经元轴突内进行双向轴突运输,即顺行运输和逆行运输。