用于多基因表达检测的猕猴桃macroarray阵列

基因阵列是开展高通量功能基因组学研究的有效手段之一。对适用于多年生果树猕猴桃的macroarray阵列进行了探索,构建了含有42个基因的macroarray阵列,与α-32P标记的cDNA探针杂交产生清晰信号,有效分辨了不同基因或基因家族不同成员的表达差异。DNase处理可去除基因组DNA污染,提高结果准确性。     

Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.)

Here we characterized eight novel polymorphic SSR markers, developed from an enriched genomic library of garlic (Allium sativum L.). These SSRs produced a total of 64 alleles across 90 garlic accessions, with an average of 8 alleles per locus. Values for observed (H_O) and expected (H_E) heterozygosity ranged from 0.16 to 0.77 (mean = 0.44) and from 0.22 to 0.86 (mean = 0.65), respectively. Six loci deviated significantly (P < 0.05) from Hardy–Weinberg equilibrium (HWE). The averages of gene diversity and PIC values were 0.65 and 0.62, respectively. The mean genetic similarity coefficient was 0.4380, indicating that among garlic accessions existed wide genetic variation. Based on 64 alleles obtained by 8 SSRs, a phenogram was constructed to understand the relationships among the 90 accessions. These newly developed SSRs should prove very useful tools for genotypes identification, assessment of genetic diversity and population structure in garlic.     

A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars

Background  

Microsatellites are popular molecular markers in many plant species due to their stable and highly polymorphic nature. A number of analysis methods have been described but analyses of these markers are typically performed on cumbersome polyacrylamide gels or more conveniently by capillary electrophoresis on automated sequencers. However post-PCR handling steps are still required. High resolution melting can now combine detailed sequence analysis with the closed-tube benefits of real-time PCR and is described here as a novel way to verify the identity of plant varieties such as grapevine and olive.     

High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.     

RADIX: rhizoslide platform allowing high throughput digital image analysis of root system expansion

Background

Phenotyping of genotype-by-environment interactions in the root-zone is of major importance for crop improvement as the spatial distribution of a plant’s root system is crucial for a plant to access water and nutrient resources of the soil. However, so far it is unclear to what extent genetic variations in root system responses to spatially varying soil resources can be utilized for breeding applications. Among others, one limiting factor is the absence of phenotyping platforms allowing the analysis of such interactions.

Results

We developed a system that is able to (a) monitor root and shoot growth synchronously, (b) investigate their dynamic responses and (c) analyse the effect of heterogeneous N distribution to parts of the root system in a split-nutrient setup with a throughput (200 individual maize plants at once) sufficient for mapping of quantitative trait loci or for screens of multiple environmental factors. In a test trial, 24 maize genotypes were grown under split nitrogen conditions and the response of shoot and root growth was investigated. An almost double elongation rate of crown and lateral roots was observed under high N for all genotypes. The intensity of genotype-specific responses varied strongly. For example, elongation of crown roots differed almost two times between the fastest and slowest growing genotype. A stronger selective root placement in the high-N compartment was related to an increased shoot development indicating that early vigour might be related to a more intense foraging behaviour.

Conclusion

To our knowledge, RADIX is the only system currently existing which allows studying the differential response of crown roots to split-nutrient application to quantify foraging behaviour in genome mapping or selection experiments. In doing so, changes in root and shoot development and the connection to plant performance can be investigated.

     

Application of unmanned aerial systems for high throughput phenotyping of large wheat breeding nurseries

Background

Low cost unmanned aerial systems (UAS) have great potential for rapid proximal measurements of plants in agriculture. In the context of plant breeding and genetics, current approaches for phenotyping a large number of breeding lines under field conditions require substantial investments in time, cost, and labor. For field-based high-throughput phenotyping (HTP), UAS platforms can provide high-resolution measurements for small plot research, while enabling the rapid assessment of tens-of-thousands of field plots. The objective of this study was to complete a baseline assessment of the utility of UAS in assessment field trials as commonly implemented in wheat breeding programs. We developed a semi-automated image-processing pipeline to extract plot level data from UAS imagery. The image dataset was processed using a photogrammetric pipeline based on image orientation and radiometric calibration to produce orthomosaic images. We also examined the relationships between vegetation indices (VIs) extracted from high spatial resolution multispectral imagery collected with two different UAS systems (eBee Ag carrying MultiSpec 4C camera, and IRIS+ quadcopter carrying modified NIR Canon S100) and ground truth spectral data from hand-held spectroradiometer.

Results

We found good correlation between the VIs obtained from UAS platforms and ground-truth measurements and observed high broad-sense heritability for VIs. We determined radiometric calibration methods developed for satellite imagery significantly improved the precision of VIs from the UAS. We observed VIs extracted from calibrated images of Canon S100 had a significantly higher correlation to the spectroradiometer (r = 0.76) than VIs from the MultiSpec 4C camera (r = 0.64). Their correlation to spectroradiometer readings was as high as or higher than repeated measurements with the spectroradiometer per se.

Conclusion

The approaches described here for UAS imaging and extraction of proximal sensing data enable collection of HTP measurements on the scale and with the precision needed for powerful selection tools in plant breeding. Low-cost UAS platforms have great potential for use as a selection tool in plant breeding programs. In the scope of tools development, the pipeline developed in this study can be effectively employed for other UAS and also other crops planted in breeding nurseries.

     

Characterization of variability and genetic similarity of European pear using microsatellite loci developed in apple

Seven microsatellite loci (SSR) developed in apple were used for the identification of 63 European pear cultivars. A total of 46 fragments were amplified, with an average of 6.6 alleles per SSR. Only one microsatellite amplified more than one locus. The mean expected and observed heterozygosities over the six single-locus SSRs averaged 0.68 and 0.44, respectively, and the number of effective alleles per loci was 3.43. The amplified fragments produced 61 different fingerprinting patterns that allowed to unequivocally distinguish all the varieties analyzed. Only two varieties, which derive from mutations, could not be distinguished from the original variety. Cluster analysis of the estimated genetic similarity, grouped the varieties according to their pedigree and their geographic origin. The variability detected with the SSRs in European pear varieties was low when compared with the variability detected in other fruit crops in the Rosaceae.     

The influence of thematic resolution on metric selection for biodiversity monitoring in agricultural landscapes

The objective of this paper is to investigate the relationship between landscape pattern metrics and agricultural biodiversity at the Temperate European scale, exploring the role of thematic resolution and a suite of biological and functional groups. Factor analyses to select landscape-level metrics were undertaken on 25 landscapes classified at four levels of thematic resolution. The landscapes were located within seven countries. The different resolutions were considered appropriate to taxonomic and functional group diversity. As class-level metrics are often better correlated to ecological response, the landscape-level metric subsets gained through exploratory analysis were additionally used to guide the selection of class-level metric subsets. Linear mixed models were then used to detect correlations between landscape- and class-level metrics and species richness values. Taxonomic groups with differing requirements (plants, birds, different arthropod groups) and also functional arthropod groups were examined. At the coarse scale of thematic resolution grain metrics (patch density, largest patch index) emerged as rough indicators for the different biological groups whilst at the fine scale a diversity metric (e.g. Simpson’s diversity index) was appropriate. The intermediate thematic resolution offered most promise for biodiversity monitoring. Metrics included largest patch index, edge density, nearest neighbour, the proximity index, circle and Simpson’s diversity index. We suggest two possible applications of these metrics in the context of biodiversity monitoring and the identification of biodiversity hot spots in European agricultural landscapes.     

甜樱桃品种中新多态性SSR的鉴定、指纹图谱构建及聚类分析

【目的】应对迅速增加的甜樱桃品种及其分子鉴定需求,储备一批新多态性SSR,并获得其在甜樱桃品种中的基因型数据。【方法】从甜樱桃基因组测序开发的SSR库中,根据重复单元和重复次数在8条染色体上一共选择了96个SSR。用ABI3730,鉴定出其中9个SSR在4个代表甜樱桃品种中具有多态性。另外从305个李属SSR中又鉴定出9个多态性SSR。用S基因和多态性SSR排除了52个品种122个单株中S基因型不正确和混杂的品种或单株,然后用多态性信息比价高的11个SSR(其中包括8个新SSR)和S基因标记,构建了48个甜樱桃品种的指纹图谱库,并获得了可以区分48个品种的最少标记组合。【结果】聚类分析表明,48个甜樱桃品种组成了短低温组、中国组、北美组和东欧组。聚类分析结果可以和品种间亲缘关系及地域来源互相验证。【结论】鉴定了3个从未报道的甜樱桃品种的S基因型,验证了一种可以有效获得多态性SSR的途径,提供了9个新的多态性SSR及其等位基因大小,用12个分子标记构建了48个甜樱桃品种指纹图谱。本文提供的新SSR及其构建的甜樱桃品种指纹图谱真实可靠。     

Development of new microsatellite markers for molecular diversity analysis of Citrus species

Summary

Thirty microsatellite loci for further genetic analysis of Citrus species were developed by constructing a microsatellite-enriched library using capture with streptavidin-coated magnetic beads and used to assess genetic diversity in 40 Citrus accessions. In total, 150 alleles were detected, with an average of five alleles per locus. The average gene diversity and polymorphism information content values were 0.58 and 0.52, and ranged from 0.35 – 0.74 and from 0.32 – 0.70, respectively. Values for the observed and expected heterozygosities ranged from 0.13 – 1.00 and from 0.36 – 0.75, respectively. Fifteen loci deviated from the Hardy-Weinberg equilibrium (P < 0.05). The mean similarity coefficient among accessions was 0.5906. Based on the UPGMA algorithm, two main groups were successfully identified. These new microsatellite markers can be used to further investigate the genetics of, and phylogenic relationships in Citrus spp.     

Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR

Background

Abscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms.

Results

We report a new sensitive and high throughput quantitative real time immuno-PCR (qIPCR) method based on biotin–avidin linkage system for ABA determination in plants. ABA monoclonal antibody (McAb) coated on the inner surface of PCR well pretreated with glutaraldehyde. The pre-prepared probe complex, including biotinylated McAb, biotinylated DNA and streptavidin linker, was convenient for high throughput operations. Finally, probe DNA was quantified by real-time PCR. The detectable ranges were from 10 to 40 ng/L with a limit of detection (LOD) of 2.5 fg. ABA contents in plant sample were simultaneously analyzed using LC–MS/MS to validate the qIPCR method. The results showed that qIPCR method has good specificity and repeatability with a recovery rate of 96.9%.

Conclusion

The qIPCR method is highly sensitive for ABA quantification for actual plant samples with an advantage of using crude extracts instead of intensively purified samples.

     

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Background  

Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.     

Quantitative trait loci analysis of flowering-time-related traits in tomato

The regulation of flowering time has been studied mainly in photoperiod-sensitive model plants. Little is known about the environmental and genetic regulation of flowering time in photoperiod-insensitive plants. Here, we studied the genetic control of flowering time in day-neutral tomato using quantitative trait locus (QTL) analysis. We used a BC₁F₆ population developed from Solanum lycopersicum ‘M570018’ and its wild relative Solanum pimpinellifolium (PI124039) to measure days to flowering (DTF) and number of leaves preceding the first inflorescence (LN), as well as their underlying developmental processes, which include the number of leaves initiated (LI), percentage of flower-induced plants (reproductive index, RI), days to macroscopic flower bud appearance (DMB) and flower development duration (FDD: DTF − DMB). A composite interval mapping detected 12 QTLs for the six traits, which included two QTLs for DTF on chromosomes 1 and 6. The DTF QTLs explained 43% of the phenotypic variation of this trait. The presence of S. pimpinellifolium alleles in the detected QTLs increased the rate of leaf initiation, reduced LN and hastened flower induction, floral development and anthesis. Most QTLs for LN, LI, RI, DMB and FDD clustered with the DTF QTLs; dmb1, fdd1, li_14d1, li_19d1 and ri_19d1 clustered with dtf1 on chromosome 1, and ln6 and fdd6 clustered with dtf6 on chromosome 6. These results suggest that the QTLs on chromosomes 1 and 6 may form a functional “gene cluster” that drives tomato flowering in synergy. Alternatively, the two DTF QTLs may act as “master genes” that control flowering time through pleiotropic effects on multiple developmental processes.     

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test,comparison with pot grown plants and validation

Background

In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck.

Results

Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using “classic” growth conditions in pots.

Conclusions

This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

     

A new set of microsatellite markers for Fragaria species and their application in linkage analysis

Summary

This study reports the development of 68 new microsatellite markers. Of these, 45 were obtained, together with 20 others already published, from an AC-enriched genomic library of the wild strawberry Fragaria vesca. The 68 markers were tested for transportability to the cultivated strawberry F. ananassa ‘Miss’ and 83% gave positive amplifications. Twenty pairs of primers were selected and tested for their transportability to 16 Fragaria taxa and eight species of Rosaceae (peach, almond, apricot, European and Sino-Japanese plums, sweet and sour cherry, apple). The average proportion of primers amplifying loci in Fragaria was 69%, while the transportability to Rosaceae was very low and resulted in null amplification for 80% of the primer pairs. In addition, 23 microsatellite markers were developed from F. ananassa ‘expressed sequence tags’ databases. A total of 141 primer pairs from these and published primers, were tested for polymorphism in the two parents (91.333.2 and ‘Snovit’; both belonging to F. vesca) of a full sib population of 46 individuals. Fifty-eight percent of the primers were discarded because they were monomorphic, or were difficult to interpret, or their allelic conformation was not useful for mapping. The segregation of 73 primers was tested in the progeny and a partial map of the female parent was constructed, based on the segregation of 66 useful markers that were ordered into eight linkage groups of which four had from seven to 14 markers.     

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

Background

The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.

Results

In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.

Conclusions

The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.     

无籽西瓜三高栽培技术因子的研究与分析

无籽西瓜“三高”是指高品质、高商品果率、高产量。根据新农业、新市场的发展需要,通过试验比较分析,对无籽西瓜三高栽培技术因子种植密度、整枝方式、坐果节位、打顶处理等作了较全面的研究与分析。     

西瓜种质资源遗传多样性分析的SSR核心引物构建与验证

<正>目的与意义:西瓜作为重要的经济作物,在我国种植业结构调整和促进农民致富增收中发挥着重要作用。当前西瓜生产中种子质量问题比较突出和普遍,种子纯度不够和假种子坑农事件常有发生,严重干扰了种业的健康发展,也是对品种创新与知识产权保护的严重损害。常规的品种鉴定难以依靠品种形态特征进行准确、快速的品种鉴别。