Ontology-oriented retrieval of putative microRNAs in Vitis vinifera via GrapeMiRNA: a web database of de novo predicted grape microRNAs

Background

Peanut (Arachis hypogaea L.) is widely used as a food and cash crop around the world. It is considered to be an allotetraploid (2n = 4x = 40) originated from a single hybridization event between two wild diploids. The most probable hypothesis gave A. duranensis as the wild donor of the A genome and A. ipaënsis as the wild donor of the B genome. A low level of molecular polymorphism is found in cultivated germplasm and up to date few genetic linkage maps have been published. The utilization of wild germplasm in breeding programs has received little attention due to the reproductive barriers between wild and cultivated species and to the technical difficulties encountered in making large number of crosses. We report here the development of a SSR based genetic map and the analysis of genome-wide segment introgressions into the background of a cultivated variety through the utilization of a synthetic amphidiploid between A. duranensis and A. ipaënsis.

Results

Two hundred ninety eight (298) loci were mapped in 21 linkage groups (LGs), spanning a total map distance of 1843.7 cM with an average distance of 6.1 cM between adjacent markers. The level of polymorphism observed between the parent of the amphidiploid and the cultivated variety is consistent with A. duranensis and A. ipaënsis being the most probable donor of the A and B genomes respectively. The synteny analysis between the A and B genomes revealed an overall good collinearity of the homeologous LGs. The comparison with the diploid and tetraploid maps shed new light on the evolutionary forces that contributed to the divergence of the A and B genome species and raised the question of the classification of the B genome species. Structural modifications such as chromosomal segment inversions and a major translocation event prior to the tetraploidisation of the cultivated species were revealed. Marker assisted selection of BC₁F₁ and then BC₂F₁ lines carrying the desirable donor segment with the best possible return to the background of the cultivated variety provided a set of lines offering an optimal distribution of the wild introgressions.

Conclusion

The genetic map developed, allowed the synteny analysis of the A and B genomes, the comparison with diploid and tetraploid maps and the analysis of the introgression segments from the wild synthetic into the background of a cultivated variety. The material we have produced in this study should facilitate the development of advanced backcross and CSSL breeding populations for the improvement of cultivated peanut.     

Embryogenic cell lines from somatic embryos of grape (Vitis vinifera L.)

Somatic embryo formation occurred on leaf callus of grape (Vitis vinifera cv. Koshusanjaku). An embryogenic callus was induced from somatic embryo clusters cultured on vitamin-, inositol- and glycine-free Nitsch and Nitsch (1969) medium supplemented with 1.0M 2,4-D. This callus has retained a high embryogenic activity after repeated subculture on the same medium for over two years, and has produced numerous embryos after transfer to a hormone-free medium. The effect of cytokinin treatment on somatic embryogenesis from leaf callus was also examined. N-(2-chloro-4-pyridyl)-N-phenylurea (KT-30) and N-(1,2,3-thiadiazol-5-yl)-N-phenylurea (TAG), both synthetic cytokinins, were found to be effective for the induction of somatic embryogenesis. When leaf callus was induced by these cytokinins combined with 2,4-D at either 5.0 or 10.0M, somatic embryos were produced.Abbreviations B₅ Basal medium, Gamborg et al. (1968) - BA 6-benzylaminopurine - 2,4-D 2,4- dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - KIN kinetin - KT-30 N-(2-chloro-4-pyridyl)-N'-phenylurea, also called 4PU-30, Kyowa Hakko Kogyo Co., Japan - NAA 1-naphthaleneacetic acid - NN Basal medium, Nitsch and Nitsch (1969) - MS Basal medium, Murashige and Skoog (1962) - TAG N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea, also called thidiazuron or TDZ, Tomono Noyaku Co., Japan - ZEA zeatin     

Current challenges and solutions of de novo assembly

Background: Next-generation sequencing (NGS) technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads. However, numerous technical or computational challenges in de novo assembly still remain, although many new ideas and solutions have been suggested to tackle the challenges in both experimental and computational settings.Results: In this review, we first briefly introduce some of the major challenges faced by NGS sequence assembly. Then, we analyze the characteristics of various sequencing platforms and their impact on assembly results. After that, we classify de novo assemblers according to their frameworks (overlap graph-based, de Bruijn graph-based and string graph-based), and introduce the characteristics of each assembly tool and their adaptation scene. Next, we introduce in detail the solutions to the main challenges of de novo assembly of next generation sequencing data, single-cell sequencing data and single molecule sequencing data. At last, we discuss the application of SMS long reads in solving problems encountered in NGS assembly.Conclusions: This review not only gives an overview of the latest methods and developments in assembly algorithms, but also provides guidelines to determine the optimal assembly algorithm for a given input sequencing data type.     

Using ESTs to improve the accuracy of de novo gene prediction

Background  

ESTs are a tremendous resource for determining the exon-intron structures of genes, but even extensive EST sequencing tends to leave many exons and genes untouched. Gene prediction systems based exclusively on EST alignments miss these exons and genes, leading to poor sensitivity. De novo gene prediction systems, which ignore ESTs in favor of genomic sequence, can predict such "untouched" exons, but they are less accurate when predicting exons to which ESTs align. TWINSCAN is the most accurate de novo gene finder available for nematodes and N-SCAN is the most accurate for mammals, as measured by exact CDS gene prediction and exact exon prediction.     

SSR-based assessment of genetic diversity in South American Vitis vinifera varieties

Six SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability and cultivar relatedness in a collection of 25 autochthonous Vitis vinifera varieties from Perú and Argentina.

The number of alleles per locus ranged from 6 to 13, while the number of microsatellites genotypes varied between 9 and 16. The expected heterozygosity varied between 0.71 and 0.89 and the polymorphism information content ranged from 0.70 to 0.88 indicating that the SSRs were highly informative. It was possible to identify 76 different genotypes, with all accessions showing-at least one-specific combination of alleles. Triallelic loci were observed with some SSR. Sequence analysis revealed that variation in the number of repeats and insertion/deletions (InDels) accounted for the polymorphisms observed. Clustering analysis resulted in four separate groups of varieties sharing at least 75% of the markers. A few cases of synonymies were found within the Peruvian accessions. Varieties were clustered following a general pattern of shared morphological and enological traits, rather than geographical origin.     

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3′-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′-O-β-diglucoside by spectroscopical methods.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

A neonatal polyvisceral failure linked to a de novo homoplasmic mutation in the mitochondrially encoded cytochrome b gene

Mutations within the mitochondrially encoded cytochrome b (MTCYB) gene are heteroplasmic and lead to severe exercise intolerance. We describe an unusual clinical presentation secondary to a novel homoplasmic mutation within MTCYB. The m.15635T > C transition (S297P) was carried by a newborn who presented with a polyvisceral failure. This mutation was responsible for a complex III deficiency. It was homoplasmic in all tissues tested and was undetectable in patient’s mother. Functional analyses, including studies on patient’s cybrid cell lines, demonstrate the pathogenicity of this variant. Our data show that mutations within MTCYB can be responsible for severe phenotype at birth.     

Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression

Background  

Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana.     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

基于转录组测序的葛根SSR标记研究与利用

该研究以‘桂粉葛1号’为材料，通过转录组测序的方法测得8.9 Gb clean reads，组装成137 629个转录本，最终得到83 811个Unigene序列。进化树分析表明，葛根和苜蓿、花生聚为一支。用MISA软件在83 811个序列中检测到25 452个简单重复序列(SSR)位点，三核苷酸重复的SSR数量最多，其次是二核苷酸重复。三核苷酸重复中，(AAG)_n是最普遍的重复单元(27.87%)。共设计了229对SSR引物，其中28对引物可以产生清晰的条带和丰富的多态性，被用于检测44份葛根资源的遗传多样性。在44个葛根资源的基因组DNA中共扩增出90个片段，其中89个条带有多态性，平均等位基因数为3.178 6。多态性信息量范围为0.083 0~0.774 2(平均数为0.455 7)。聚类分析显示遗传相似性系数范围为0.266 7~1.000 0。这些结果提示所检测的葛根资源在DNA分子水平存在着丰富的遗传多样性。当阈值为0.58时，44个资源可以划分为2个类群，且44份资源的类群划分与地理来源之间没有直接关系，但这些标记将是葛根遗传多样性研究可用的基因组资源。     

The dominant mutation Suppressor of black indicates that de novo pyrimidine biosynthesis is involved in the Drosophila tan pigmentation pathway

A deficiency in the production of -alanine causes the black (b) phenotype of Drosophila melanogaster. This phenotype is normalized by a semi-dominant mutant gene Su(b) shown previously to be located adjacent to or within the rudimentary (r) locus. The r gene codes for three enzyme activities involved in de novo pyrimidine biosynthesis. Pyrimidines are known to give rise to -alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to -alanine synthesis during the tanning process. In this report we show that flies carrying Su(b) can exhibit an additional phenotype, resistance to toxic pyrimidine analogs (5-fluorouracil, 6-azathymine and 6-azauracil). Our interpretation of this observation is that the pyrimidine pool is elevated in the mutant flies. However, enzyme assays indicate that r enzyme activities are not increased in Su(b) flies. Genetic mapping of the Su(b) gene now places the mutation within the r gene, possibly in the carbamyl phosphate synthetase (CPSase) domain. The kinetics of CPSase activity in crude extracts has been studied in the presence of uridine triphosphate (UTP). While CPSase from wild-type flies was strongly inhibited by the end-product, UTP, CPSase from Su(b) was inhibited to a lesser extent. We propose that diminished end-product inhibition of de novo pyrimidine biosynthesis in Su(b) flies increases available pyrimidine and consequently the -alanine pool. Normalization of the black phenotype results.     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。     

Cryptobiosis in the Eutardigrade Adorybiotus (Richtersius) coronifer: Tolerance to Alcohols, Temperature and de novo Protein Synthesis

The eutardigrade Adorybiotus (Richtersius) coronifer survives cryptobiosis for years. During entrance into anhydrobiosis this species accumulates the disaccharide trehalose reaching a maximum content of 2.3% d.w.

In the present study we examined the survival of anhydrobiotic A. (R)coronifer during exposure to alcohols of various polarity, and to high temperatures, as well as qualitative changes in protein synthesis during entrance into anhydrobiosis. Results showed that A. (R) coronifer in anhydrobiosis survived exposure to ethanol for less than 10 minutes whereas exposure to 1-butanol only decreased survival to 40% after the first 7 days and 1-hexanol did not change survival from the controls after the first 7 days. A. (R) coronifer survived temperatures up to approximately 70 °C for 60 minutes without any decrease in survival. However, survival decreased rapidly when the exposure temperature was increased to above 70 °C and no animals survived exposure to 100 °C. During the entrance into anhydrobiosis a protein with a molecular weight of approximately 71 kDa appeared on acryl amide gels showing protein bands after the animals had been incubated with ³H-Leucine. This protein may belong to the Heat-shock protein (Hsp) 70 family. The results on the survival of A. (R) coronifer during exposure to alcohols and high temperature are discussed in light of the trehalose content earlier described in this animal during anhydrobiosis.     

葡萄原生质体分离及瞬时转化体系的建立

为了建立葡萄原生质体进行遗传转化的技术,该研究以葡萄品种‘黑香蕉’的叶片和愈伤组织为材料,分析纤维素酶和离析酶的浓度与配比、渗透压和酶解时间等主要因素对葡萄原生质体分离的影响,探讨建立稳定、高效的葡萄原生质体分离与瞬时转化体系,为鉴定目标基因的功能奠定基础。结果表明:(1)葡萄叶片原生质体的分离以3.0%纤维素酶和0.75%离析酶的酶组合,在0.6mol/L甘露醇溶液中,酶解14h为宜,每克游离产量为4.09×106个原生质体,活力为83.12%。(2)葡萄愈伤组织原生质体的分离以2.0%纤维素酶和0.5%离析酶的酶组合,在0.5mol/L甘露醇溶液中,酶解14h为宜,每克游离产量为6.05×106个原生质体,活力为84.13%。(3)利用该方法得到的葡萄原生质体为受体,采用40%PEG-4000介导转化质粒载体pEZS-NL,目标基因瞬时表达产物检测表明,GFP蛋白表达稳定、清晰。该研究建立的葡萄原生质体制备和转化体系,可以用较少量的质粒DNA获得外源基因在原生质体内的表达,为葡萄功能基因的研究提供技术支持。     

根域限制对葡萄根中IAA含量及其代谢相关基因表达的影响

以1年生"玫瑰香"葡萄为试验材料,进行根域限制栽培处理,以传统地栽为对照,研究根域限制对葡萄根系构型、细胞结构、IAA含量及其代谢和根系生长发育相关基因表达丰度的影响,以探讨根域限制栽培影响葡萄根系构型变化的内在机制.结果表明:(1)根域限制处理后,葡萄根系构型发生显著变化,主要表现在根尖处出现大量集群根、不定根持续发...     

施氮时期对酿酒葡萄叶片氮代谢酶及相关基因表达的影响

以10年生‘蛇龙珠’葡萄为材料,在萌芽期(S1)、新梢旺长期(S2)、开花期(S3)、果实第一次膨大期(S4)、副梢生长旺期(S5)和果实第二次膨大期(S6)分别一次性施入尿素300kg·hm-2,以不施氮肥为对照(CK),分析了花前5d(DBF5)、花后25d(DAF25)、花后55d(DAF55)和花后85d(DAF85)叶片的各项指标,以明确氮素施用时期对葡萄叶片氮代谢的调控与影响。结果表明:(1)S1和S2处理葡萄叶片中总氮及可溶性蛋白含量在DAF25时显著增加。(2)S3和S4处理的NR及GS活性在DAF85时显著高于其他处理;在DAF25时,S1和S2处理的GOGAT活性,以及S3和S4处理的GDH活性均显著高于同期对照和其他施肥处理。(3)各施肥处理叶片VvNR表达水平在不同时期均高于同期对照,S3处理VvNR表达水平在DAF25和DAF85时分别为对照的3.4倍和2.7倍;S3和S4处理的VvGS表达水平分别在DAF55和DAF85时达到最高值,S3处理的VvGOGAT和S4处理的VvGDH表达水平在DAF55和DAF85均显著高于其他处理,S3处理的VvGDH表达水平在DAF55和DAF85仅次于S4处理。研究表明,氮素通过诱导叶片氮素代谢基因的响应,从而调控叶片中氮素代谢酶活性增加,促进了氮素的积累,S3和S4处理在不同时期氮代谢酶活性和对应的基因表达水平均较高,更有利于叶片中氮素的转化和代谢。     

无核白葡萄愈伤组织瞬时转化体系的优化与应用

为了优化根癌农杆菌介导的葡萄愈伤组织瞬时转化体系,该研究以欧洲葡萄品种无核白(Vitis vinifera L.cv.Thompson Seedless)单芽茎段诱导的愈伤组织为材料,探讨重悬液pH、菌液浓度、真空渗透时间等主要因素对葡萄愈伤组织瞬时转化效率的影响。结果表明:(1)以激素组合分别为1.0mg/L BAP、1.0mg/L BAP+0.02mg/L NAA、2.0mg/L BAP+0.02mg/L NAA和4.0mg/L BAP+0.02mg/L NAA的系列培养基更适合无核白葡萄单芽茎段逐步诱导胚性愈伤组织。(2)葡萄愈伤组织瞬时转化体系中,重悬液pH 5.1,菌液浓度OD6001.0,真空渗透20min为转化效率最佳条件。(3)利用优化的瞬时转化体系瞬时转化无核白葡萄的不同组织,发现在不同器官中转化效率存在显著差异。其中以愈伤组织为受体的转化效率显著高于其他器官(65 231.99±3 339.29mU/g),而且愈伤组织的GUS组织化学染色最深,以叶片为受体的转化效率则最低。利用该体系转化质粒载体pCAMBIA0390∷GUS,瞬时表达产物经过GUS蛋白活性检测,结果表明该研究优化的葡萄愈伤组织瞬时转化体系有助于外源基因在葡萄愈伤组织内的表达,为后期通过转基因技术研究目标基因功能奠定了技术基础。     

Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction

A method is described, which consistently yields high quality total RNA from grapevine. Dissolving of crude RNA pellets in borate-containing buffer, instead of normally used water before a selective lithium chloride precipitation, was found to be a critical step, leading to a 2.5-fold increase of yield. The resulting RNA preparations were suitable for standard downstream applications and also for cDNA library construction. The method worked efficiently and reproducibly and could easily be scaled from milligram to gram quantities of plant material grown in hydroponic culture, sandy soil or Perlite. It was applied to different kinds of grapevine tissues (leaves, stem) and, after additional adaptation of the protocol, to roots.     

Teleomorph of Erysiphe necator var. necator on Vitis vinifera and Ampelopsis brevipedunculata var. heterophylla (Vitaceae) newly found in Japan

 The teleomorph of Erysiphe necator var. necator [≡Uncinula necator var. necator], hitherto unknown in Japan, was found on Vitis vinifera and Ampelopsis brevipedunculata var. heterophylla (Vitaceae) in Okayama, Japan. Morphological characteristics of the fungus are described and illustrated. Received: December 13, 2002 / Accepted: January 22, 2003 Correspondence to:Y. Nomura