基于高通量测序及分离培养初步研究美国大豆中真菌多样性

中国是全球最大的大豆进口国,进境大豆所携带的病原真菌传入我国的风险极高。基于高通量测序技术对6批美国大豆真菌多样性进行分析,同时采用分离培养获得单一菌株,依据菌落形态、显微结构及分子技术进行鉴定。高通量测序结果显示,大豆中所有真菌共计5门15纲35目63科112属155种,主要为链格孢属(Alternaria)、球腔菌科(Mycosphaerellaceae)、小戴卫霉科(Davidiellaceae)、小囊菌科(Plectosphaerellaceae)、赤霉属(Gibberella)、附球霉属(Epicoccum)、间座壳属(Diaporthe)、隐球菌属(Cryptococcus)。分离培养结果显示,得到真菌共计40株10种,分别是大豆北方茎溃疡病菌(Diaporthe phaseolorum var.caulivora)、大豆南方茎溃疡病菌(Diaporthe phaseolorum var.meridionalis)、大豆拟茎点种腐病菌(Diaporthe longicolla/Phomopsis longicolla)、大豆炭腐病菌(Macrophomina phaseolina)、尖孢镰刀菌(Fusarium oxysporum)、菌核菌(Sclerotinia sclerotiorum)、镰刀菌(Fusarium sp.)、附球菌(Epicoccum sp.)、交链孢菌(Alternaria sp.)、小双胞腔菌(Didymella sp.)。     

马尾松叶表微生物多样性

为发掘对林木生长发育有利的优良微生物资源,并筛选适合叶表微生物的收集方法,以马尾松针叶为试验材料,分别用悬摇法和超声波法收集马尾松叶表微生物,用扩增子高通量测序技术、MUSCLE和Qiime软件研究马尾松叶表微生物的多样性。结果表明:扫描电镜观测结果显示,马尾松针叶表面定殖有大量微生物,包括真菌(菌丝及孢子)和细菌。扩增子高通量测序结果表明,马尾松叶表微生物物种丰富,包含细菌运算分类单位(OTUs)490个,真菌OTUs 1273个。马尾松叶表细菌以未分类的蓝细菌属(unidentified_Cyanobacteria)(36.53%)、未分类的拜叶林克氏菌属(unidentified_Beijerinckia)(28.60%)、鞘氨醇单胞菌属(Sphingomonas)(2.35%)为优势属;叶表真菌以枝孢属(Cladosporium)(2.45%)、拟盘多毛孢属(Pestalotiopsis)(0.92%)、无头孢菌属(Capnobotryella)(0.91%)为优势属。针对叶表细菌多样性的研究表明,悬摇法和超声波法均有较高的物种检出度;在叶表真菌多样性的研究中超声波法优于悬摇法,但超声波法样品间数据变异性较大,测定结果不稳定。     

Improvement,identification, and target prediction for miRNAs in the porcine genome by using massive,public high-throughput sequencing data

Despite the broad variety of available microRNA (miRNA) research tools and methods, their application to the identification, annotation, and target prediction of miRNAs in nonmodel organisms is still limited. In this study, we collected nearly all public sRNA-seq data to improve the annotation for known miRNAs and identify novel miRNAs that have not been annotated in pigs (Sus scrofa). We newly annotated 210 mature sequences in known miRNAs and found that 43 of the known miRNA precursors were problematic due to redundant/missing annotations or incorrect sequences. We also predicted 811 novel miRNAs with high confidence, which was twice the current number of known miRNAs for pigs in miRBase. In addition, we proposed a correlation-based strategy to predict target genes for miRNAs by using a large amount of sRNA-seq and RNA-seq data. We found that the correlation-based strategy provided additional evidence of expression compared with traditional target prediction methods. The correlation-based strategy also identified the regulatory pairs that were controlled by nonbinding sites with a particular pattern, which provided abundant complementarity for studying the mechanism of miRNAs that regulate gene expression. In summary, our study improved the annotation of known miRNAs, identified a large number of novel miRNAs, and predicted target genes for all pig miRNAs by using massive public data. This large data-based strategy is also applicable for other nonmodel organisms with incomplete annotation information.     

四倍体马铃薯块茎蛋白含量分子标记的开发与验证

块茎蛋白含量是马铃薯的重要品质性状之一,相关分子标记的开发研究较少。本研究以马铃薯高蛋白品种大西洋、低蛋白品种定薯1号及包含173份子代的蛋白含量分离群体为材料,通过高通量简化基因组测序技术与集群分离分析(BSA)相结合,开发了SCAR8-107标记,并以此标记对分离群体的74份高蛋白、42份低蛋白子代及54个四倍体马铃薯品种验证表明,SCAR8-107标记在后代分离群体和四倍体马铃薯品种中的检测结果与表型蛋白含量的对应度分别达到了90.51%和72.97%,经过Pearson’s双侧相关性分析,相关性均达到了极显著水平。SCAR8-107的开发对于四倍体马铃薯块茎蛋白含量标记辅助选择与加速马铃薯品质育种具有重要意义。     

Detection and characterization of a rhabdovirus causing mortality in black bullhead catfish,Ameiurus melas

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993–2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities.     

基于高通量测序的露地菊(Chysanthemum×grandiflora)盐胁迫转录组分析

为揭示露地菊生长发育及耐盐胁迫的应答机制和分子基础,本研究以盐胁迫处理的露地菊及其对照为材料,使用Illumina Hiseq2500高通量测序平台对转录组进行测序,分别获得了60370448和71415448条Clean reads,通过序列拼接组装得到45591条Unigene,平均长度724 bp。有37675条Unigene在七大数据库(COG,GO,KEGG,KOG,Pfam,Swiss-Prot,NR)中得到注释。通过比对露地菊盐胁迫处理组和对照组样品间Unigene的表达量及在各数据库中的注释情况,统计得到:有4143条差异表达基因获得注释;有2441条差异表达基因在GO数据库中获得功能注释;注释到COG数据库中的2281条差异表达基因依据功能可分为25类;有1062条差异基因映射到KEGGPathway数据库中,涉及了199个代谢通路,包括核糖体途径、植物激素信号传导途径、淀粉蔗糖代谢、碳代谢、氨基酸的生物合成等。本研究获得的转录组数据将有助于揭示露地菊生长发育及耐盐胁迫的应答机制和分子基础,及相关抗性基因的挖掘和分子辅助育种等方面的研究。     

High-throughput screening reveals small molecule modulators inhibitory to Acidovorax citrulli

Acidovorax citrulli is a seed-transmitted gram-negative bacterium that can cause substantial economic yield loss in watermelon and melon production worldwide. Four small-molecule libraries containing 4,952 compounds were selected for high-throughput screening against A. citrulli wild-type strain Xu3-14 by evaluation of growth inhibition. One hundred and twenty-seven molecules (2.5% hit rate) were identified as bactericidal or bacteriostatic against A. citrulli at 100 μM. Secondary screens indicated that 27 candidate compounds were more effective against A. citrulli Group II strains than Group I strains (classified using repetitive element PCR). Several compounds were inhibitory to other pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Xanthomonas campestris pv. campestris, but did not affect the growth of plant beneficial bacteria Pseudomonas fluorescens and Bacillus subtilis. More than half of the compounds did not inhibit germination of Arabidopsis or watermelon seeds. The effect of small molecules on A. citrulli seed-to-seedling transmission was evaluated by applying each compound to inoculated watermelon seeds and assessing seedling infection. Nine compounds were chosen for further investigation based on their reduction of percentage seedling infection and compiling scores on their specificity, sensitivity, and phytotoxicity obtained in the secondary screens. The five best compounds were selected (thiamphenicol, nadifloxacin, pipemidic acid, ciclopirox, and zinc pyrithione) for greenhouse tests and were found to effectively reduce the seed-to-seedling transmission of A. citrulli in both artificially and naturally infested seeds. These top five compounds provide a basis for future development of an A. citrulli-specific bactericide.     

Soil Characteristics Overwhelm Cultivar Effects on the Structure and Assembly of Root-Associated Microbiomes of Modern Maize

Modern breeding primarily targets crop yield traits and is likely to influence root-associated microbiomes, which play significant roles in plant growth and health. The relative importance of soil and cultivar factors in shaping root-associated microbiomes of modern maize (Zea mays L.) remains uncertain. We conducted a pot experiment in a controlled environment using three soils (Mollisol, Inceptisol, and Ultisol) and four contrasting cultivars, Denghai 605, Nonghua 816, Qiaoyu 8, and Zhengdan 958, which are widely planted in China. We used 16S rRNA gene amplicon sequencing to characterize the bacterial communities in the bulk soil, rhizosphere, and endosphere. Our results showed that the four cultivars had different shoot biomass and root exudate total organic carbon and organic acid contents. The microbiomes in the bulk soil, rhizosphere, and endosphere were different. We observed apparent community divergence between soils rather than cultivars, within which edaphic factors substantially contributed to microbiome variation. Moreover, permutational multivariate analysis of variance corroborated significant contributions of soil type but not cultivar on the root-associated microbiome structure. Differential abundance analysis confirmed that each soil presented a distinct root microbiome, while network analysis indicated different co-occurrence patterns of the root microbiome among the three soils. The core root microbiome members are implicated in plant growth promotion and nutrient acquisition in the roots. In conclusion, root-associated microbiomes of modern maize are much more controlled by soil characteristics than by cultivar root exudation. Our study is anticipated to help improve breeding strategies through integrative interactions of soils, cultivars, and their associated microbiomes.     

基于DNA宏条形码的莼菜大田土壤真菌多样性研究

高通量测序与DNA条形码结合产生的DNA宏条形码技术(DNA-Metabarcoding),能快速鉴定混合样本中的物种,现已成为检测群类物种多样性和丰富度的常用方法.本试验采用这一方法分析了莼菜大田不同种植年限土壤真菌多样性,结果表明:10年生土壤中含有最多的真菌种类,多样性及丰富度最高;在属水平上表现为蛙粪霉属、裂梗霉属、酵母属等占优势;群类组成分析显示,种植年限的变化对土壤真菌群落组成有一定的影响;聚类分析中10年与15年生莼菜土壤物种多样性相似度较高.