Cross-amplification of EST-derived markers among 16 grass species

The availability of a large number of expressed sequence tags (ESTs) has facilitated the development of molecular markers in members of the grass family. As these markers are derived from coding sequences, cross-species amplification and transferability is higher than for markers designed from genomic DNA sequences. In this study, 919 EST-based primers developed from seven grass species were assessed for their amplification across a diverse panel of 16 grass species including cereal, turf and forage crops. Out of the 919 primers tested, 89 successfully amplified DNA from one or more species and 340 primers generated PCR amplicons from at least half of the species in the panel. Only 5.2% of the primers tested produced clear amplicons in all 16 species. The majority of the primers (66.9%) were developed from tall fescue and rice and these two species showed amplification rate of 41.6% and 19.0% across the panel, respectively. The highest amplification rate was found for conserved-intron scanning primers (CISP) developed from pearl millet (91%) and sorghum (75%) EST sequences that aligned to rice sequences. The primers with successful amplification identified in this study showed promise in other grass species as demonstrated in differentiating a set of 13 clones of reed canary grass, a species for which very little genomic research has been done. Sequences from the amplified PCR fragments indicated the potential for the transferable CISP markers for comparative mapping purposes. These primer sets can be immediately used for within and across species mapping and will be especially useful for minor grass species with few or no available molecular markers.     

Prevalence of Endosymbionts in Polish Populations of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) is one of the most serious insect pest feeding on wild and cultivated Solanaceae plants. This pest poses a significant threat to potato crops. CPB originated from North America but has become widespread and has adapted in new localizations. Currently, it is reported in many countries worldwide. Endosymbiotic bacteria might have an influence on insect adaptation to new conditions. They are known to play a role in invasiveness of insect hosts and to facilitate colonization of new niches; however, information on endosymbionts of the CPB is very limited. In this study, we screened CPB populations collected from 20 evenly distributed locations in Poland for the presence of Arsenophonus, Cardinium, Wolbachia, and Flavobacterium. We found the presence of Flavobacterium in the studied insects. Little is known about CPB–endosymbionts interactions, thus this study may provide a reference for future studies in this subject.     

花生微卫星DNA分子标记研究进展

微卫星DNA广泛存在于真核生物基因组中,由于其多态性高,结果稳定,重复性好,操作简单等优点,而成为目前植物DNA标记研究中应用最多和发展最快的标记技术之一。本文就花生中微卫星分子标记的开发,及其在花生遗传多态性、亲缘关系鉴定、花生指纹图谱构建等方面的应用研究进展进行了综述。     

Identification of the Population Structure of Myzus persicae (Hemiptera: Aphididae) on Peach Trees in China Using Microsatellites

In this study, we characterized the genetic structure of Myzus persicae (Sulzer) (Hemiptera: Aphididae) populations in China using microsatellites. We expected that these data will reveal the genetic relationships among various populations of M. persicae and will be of value in the development of better methods for pest control. Four hundred sixty individuals from 23 areas over 13 provinces were collected in the early spring of 2010, all from their primary host, Prunus persicae. The markers analyzed were highly polymorphic, as demonstrated by the expected heterozygosity value (H_e = 0.861) and the Polymorphism Information Content (PIC = 0.847), which indicated that M. persicae maintains a high level of genetic diversity. Analysis of molecular variance revealed an intermediate level of population differentiation among M. persicae populations (F_ST = 0.1215). Geographic isolation existed among these populations, and, consequently, the genetic structure of the populations was split into a southern group and a northern group divided by the Yangtse River.     

Sequential sampling plans for estimating density of glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae) on citrus

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is a serious pest of grapes and other crop and ornamental plants mainly through its role as a vector of the bacterium Xylella fastidiosa Wells. Citrus harbors large populations of this insect throughout much of the year in areas where the pest is problematic and improved understanding of the population dynamics and management of H. vitripennis on citrus may be key to its management in the broader agricultural landscape. In turn, the study of population dynamics and the development of management strategies require effective and efficient sampling methods. Within-tree sampling distribution studies revealed that adults and nymphs were more abundant and less variable in the upper strata of citrus trees (>1.5 m). They occurred in greater numbers on the southern quadrants of trees but relative variability did not differ due to cardinal direction. We developed and validated several fixed-precision sequential sampling plans for estimating the density of nymphs and adults of H. vitripennis using a pole bucket sampling method. Based on validation from resampling of independent data sets, Green’s sequential sampling model, based on the Taylor’s power law, provided the best overall performance in terms of providing mean density estimates with levels of precision equal to or better than the desired precision over a range of possible insect densities. Average sampling costs varied from about 21 to 189 min for a desired precision of 0.25 depending on insect density and whether the goal is to sample nymphs, adults or both stages combined. Further, the sampling plans developed on orange trees were robust, being equally effective on orange and lemon trees and on trees treated or not with insecticides.     

Genetic structure of the oak wilt vector beetle Platypus quercivorus: inferences toward the process of damaged area expansion

Background  

The ambrosia beetle, Platypus quercivorus, is the vector of oak wilt, one of the most serious forest diseases in Japan. Population genetics approaches have made great progress toward studying the population dynamics of pests, especially for estimating dispersal. Knowledge of the genetic structuring of the beetle populations should reveal their population history. Using five highly polymorphic microsatellite loci, 605 individuals from 14 sampling sites were assessed to infer the ongoing gene flow among populations as well as the processes of expansion of damaged areas.     

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. Totally 5 screening elements (P–35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P–35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.     

玉米自交系RAPD分析中的样品选择

近年来分子标记技术在我国农业科研中的应用已受到了广泛的重视。分子标记样品的代表性如何将对研究结果产生影响，因此，研究分子标记的取样问题具有一定的现实意义。本文对以玉米自交系为样品，进行分子标记分析时的取样技术进行了探讨。在对玉米自交系中黄204基因组DNA进行RAPD分析时，同一引物8个单株及其混合样品的扩增结果中，单株6在564bp处比其它单株多一条较强的谱带，而8个单株的混合样品的扩增结果中也有一条同样的谱带。而丹340、旅9、5003等其它自交系的10个单株及其混合样品的RAPD谱带是一致的。说明中黄204单株6的遗传背景与其它单株不同，不能代表中黄204，如用它们的混合样品进行RAPD分析，就会得出错误的结果。可以看出，分子标记分析中的取样技术是检测结果可靠性不可忽视的重要因素之一。     

基于茶树SNP的dCAPS标记体系研究

从茶树EST序列中搜寻出候选SNPs位点,在候选SNPs两侧设计测序引物进行PCR扩增,并对扩增产物双向测序验证;然后从每个扩增子中选择一个确证的SNPs,设计dCAPS引物,并进行扩增和酶切验证,将SNPs转化为dCAPS标记;最后,在长叶白毫×福鼎大白的F1群体中检测了标记的分离情况。结果发现,20对测序引物中有11对扩增成功,共确证了17个SNPs,占检测总数的54.8%;在设计的11对dCAPS引物中,有8对在长叶白毫、福鼎大白和龙井43等8个品种中表现出多态性,成功转化为dCAPS标记,转化成功率72.7%;8个dCAPS标记中的DCC229和DCC371在长叶白毫和福鼎大白之间表现有多态性,经检测,它们在这2个品种的F1群体中均符合孟德尔1︰1分离。     

Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.     

利用SSR标记分析栽培种花生多态性及亲缘关系

利用11对SSR引物对24个栽培种花生品种(包括四大类型)进行PCR扩增分析,其中4对检测到明显的多态性,共检测到33个等位基因变异,每一个位点上检测到的等位变异数为5～13个,平均为8.25个.根据扩增结果可以将24个品种中的21个相互区分.供试品种间的遗传相似系数值在0.2～1.0之间,平均为0.4788.根据UPGMA聚类分析结果显示供试品种大多数按亚种聚为两大类群(Ⅰ、Ⅱ);在两大类群下,大多数品种也基本上按类型分类.本研究结果表明,SSR在分析栽培种花生DNA多态性和遗传关系方面非常有用.     

Seasonal dynamics of Microcystis spp. and their toxigenicity as assessed by qPCR in a temperate reservoir

Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC)-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes) to non-toxic (without mcy genes) genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4-6 weeks before and 8-10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems.     

Genetic variation in a population of tetraploid potatoes: Response to the potato leafhopper and the potato flea beetle

Progenies from a group of tetraploid parental clones from the USDA potato breeding program were used to investigate variation in resistance to the potato leafhopper,Empoasca fabae (Harris), and the potato flea beetle,Epitrix cucumeris (Harris). The study utilized two mating designs: i) selfing and testing both the parents and the S₁ progeny; and ii) nine clones, used as males, were each crossed to three different clones, and progenies from the resulting families were tested. Statistically significant differences between families were measured in each test for both insects. Non-additive genetic variance was larger than additive genetic variance in progeny reaction to leafhopper infestation and hopperburin, but was smaller in progeny reaction to flea beetles. Environmental variation contributed heavily to the total variation of plant reaction to both insect species. Selecting individuals was indicated to be slightly more effective than selecting males on half-sib progeny performance but not as effective as selecting clones on S₁ progeny performance. Because of the large environmental variance and small additive variance for both the leafhopper and flea beetle, slow progress in increasing the level of resistance to these two species in this sample population was predicted. Resistance to leafhopper infestation was genetically quite highly correlated (positive) with resistance to hopperburn, but phenotypically the correlation was considerably smaller. Negative genotypic, phenotypic, and environmental correlations between leafhopper infestation and flea beetle infestation suggest that selecting for resistance to one of these species, in the population sampled for these tests, would tend to increase susceptibility to the other.     

Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg²⁺ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.     

Evaluation of augmentative release of Zygogramma bicolorata Pallister (Coleoptera: Chrysomelidae) for biological control of Parthenium hysterophorus L.

Augmentative release of biocontrol agents has been largely successful for the management of insect pests but it has not been a common approach for weed management. Augmentation methods need to be developed for weed management, especially for pernicious weeds like Parthenium hysterophorus L., commonly known as pathenium or carrot weed. The leaf beetle Zygogramma bicolorata is a potential biocontrol agent of P. hysterophorus. Initial release of biocontrol agents is subject to uncertainties as to whether timely population built-up will take place in sufficient numbers. Several augmentative releases may be required to ensure early establishment of the biocontrol agents, for successful biological control of noxious weeds including pathenium. We made augmentative releases of larvae or adults of Z. bicolorata each to three sites, severely infested with pathenium at Jabalpur, India consecutively for a period of three years. Initially 10 larvae or adults per sq m were released in each plot, followed by a second, third and fourth release of 3, 1.5 and 1.5 larvae or adults per sq m at an intervals of 3, 7 and 14 days after the first augmentation. The pathenium at augmented sites were completely defoliated in 45 and 60 days by larvae and adults respectively. There was also a reduction in the pathenium density and plant height in the augmented sites as compared to the non-augmented sites. Over a period of 3 years augmentation resulted in a noteworthy negative effect on the weed.     

Structural Analysis and Anti-Complement Activity of Polysaccharides from Kjellmaniella crsaaifolia

Two polysaccharides, named KCA and KCW, were extracted from Kjellmaniella crassifolia using dilute hydrochloric acid and water, respectively. Composition analysis showed that these polysaccharides predominantly consisted of fucose, with galactose, mannose and glucuronic acid as minor components. After degradation and partial desulfation, electrospray ionization mass spectrometry (ESI-MS) was performed, which showed that the polysaccharides consisted of sulfated fucooligosaccharides, sulfated galactofucooligosaccharides and methyl glycosides of mono-sulfated/multi-sulfated fucooligosaccharides. The structures of the oligomeric fragments were further characterized by electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS² and ESI-CID-MS³). Moreover, the activity of KCA and KCW against the hemolytic activity of both the classical and alternative complement pathways was determined. The activity of KCA was found to be similar to KCW, suggesting that the method of extraction did not influence the activity. In addition, the degraded polysaccharides (DKCA and DKCW) displayed lower activity levels than the crude polysaccharides (KCA and KCW), indicating that molecular weight had an effect on activity. Moreover, the desulfated fractions (ds-DKCA and ds-DKCW) showed less or no activity, which confirmed that sulfate was important for activity. In conclusion, polysaccharides from K. crassifolia may be good candidates for the treatment of diseases involving the complement pathway.     

基于SSR标记的橡胶树无性系鉴定方法的建立

从88对橡胶树SSR引物中筛选出5对产物清晰、扩增稳定的引物.采用6%变性聚丙烯酰胺凝胶电泳结合快速银染检测的方法,构建了87份橡胶树的DNA指纹图谱.5对引物共扩增出16条带,平均每对引物可扩增出3.2条带,多态性条带为15条,扩增条带分布在148～342 bp,无性系之间只存在1～2个片段差异,说明遗传差异小;根据建立的无性系数字指纹图谱,能逐一区分65个无性系,表明应用SSR分子标记技术进行橡胶树无性系的鉴定是可行的.     

海南黎药胆木PsbA-trnH-PCR体系的建立与优化

为了建立适合黎药胆木的PsbA-trnH-PCR体系来研究不同地理居群胆木遗传多样性,本研究以植物基因组试剂盒法提取胆木基因组DNA为模板,采用单因素实验和正交试验对PsbA-trnH-PCR过程中的关键影响因素进行优化,并对PsbA-trnH-PCR产物进行测序鉴定。结果表明,最佳PsbA-trnH-PCR反应体系(25μL)为:Taq酶1.0 U,dNTPs 0.4 mmol/L,Mg~(2+)0.75 mmol/L,引物0.15μmol/L,模板20 ng,10×PCR Buffer(不含Mg~(2+))2.5μL;采用该最佳体系对胆木基因组DNA进行PCR扩增,获得扩增产物,经单向测序获得了胆木PsbA-trnH部分序列;并建立了稳定的PsbA-trnH-PCR体系,为胆木的药材鉴别及其遗传多样性研究奠定了基础。     

玉米对几种主要害虫的抗性QTL分析

综述了玉米对4种主要害虫抗性基因定位的最新研究结果。受环境、样本及分析方法等因素的影响,不同的试验检测到的QTL结果不尽相同。通过引进染色体箱的概念,对各检测结果进行比较分析发现,玉米对同一种害虫的食叶抗性和茎秆抗性之间没有相关性,但对不同种害虫的食叶抗性和茎秆抗性却有相关性。这些抗性QTL不是随机分布的,而是以基因簇的形式存在于染色体上。玉米的抗虫性与植株组织硬度、蛋白含量、丁布和细胞壁成分相关,从分子水平上揭示了玉米的抗虫机制,为进一步的遗传分析研究和抗虫育种提供理论依据。