海南普通野生稻不同居群rDNA ITS区序列的比较分析

以转bar基因粳稻(O.astiva ssp.japonica)YOO3为外类群,采用PAUP4.0等软件,对海南7个主要普通野生稻(O.rufipogon)核糖体基因(nrDNA)的内转录间隔区(ITS)全序列信息进行比较分析.结果表明,ITS全长为586～589 bp,其中ITS1为193～195 bp,共有20个变异位点数,G/C含量为72.02%～74.74%;ITS2为229～231 bp,共有12个变异位点数,G/C含量为75.55%～76.08%:离栽培稻越近的类群G/C含量越高.所有材料5.8S rDNA序列完全一致,全长164 bp,G/C占97 bp,含量为59.15%.各类群间ITS序列信息较丰富,且显示ITS1比ITS2进化速度要快.利用ITS差异信息序列进行聚类分析为鉴别不同居群的普通野生稻提供了分子依据.     

基于nSSR和cpDNA序列的城步峒茶群体遗传多样性和结构研究

采用简单重复序列标记(nSSR)与叶绿体DNA序列(cpDNA)分析技术,对城步峒茶群体进行了遗传多样性、遗传结构和遗传分化等研究。结果表明,15对nSSR引物在参试81份资源中共获得142个等位位点,平均每对引物9.47个,城步峒茶群体的观测杂合度(Ho)、期望杂合度(He)和Nei期望杂合度(Nei)分别为0.49、0.62和0.62,具有较高的遗传多样性。Structure分析将79份峒茶资源分成3个类群,但各类群的遗传背景较为复杂,没有明显的群体结构。F检验表明,城步峒茶群体的近交系数F_IS为正值(F_IS=0.177 5),群体间的遗传分化系数F_ST较小(F_ST=0.034 5),分化程度较低,基因流Nm较高(Nm=7.01)。3对cpDNA引物分别获得了473 bp(rbcL)、704 bp(matK)和320 bp(psbH-trnA)的片段序列,变异位点占总位点的比例分别为0.42%、0.71%和1.25%。将3个序列依次拼接,共产生了9个单倍型,单倍型数由多到少的居群依次为TXZ(6)、DZC(4)、DPS(4)、TYS(3)、HJZ(2),群体的单倍型多样性(Hd)和核苷酸多样性(π)分别为0.732和0.001 39。9个单倍型中,单倍型H1和H5处于进化网络图的中心节点上,并且包含资源数量最多,属于比较原始的单倍型。同时,nSSR和cpDNA的AMOVA分析结果基本一致,居群内的变异百分比分别达到96.69%和80.54%,城步峒茶的遗传变异主要存在于居群内。     

苏皖鲁豫麦田小麦孢囊线虫调查以及rDNA ITS序列特征分析

为给小麦孢囊线虫综合治理提供一定的理论依据,通过对苏皖鲁豫接壤地区30块田的调查和分析,探讨了这些地区麦田小麦孢囊线虫的发生情况及线虫群体之间亲缘关系。结果表明,采集的30份样品中都能检测到孢囊线虫,孢囊密度为每100g土壤1～80个。进一步对30个小麦孢囊线虫群体的rDNA-ITS区PCR扩增、测序,对其序列进行比较分析发现,30个分离物的核苷酸序列同源性为97.1%～100%,与已报道的中国菌株（AY148382,EU106175）、澳大利亚菌株（AY148395）和俄罗斯菌株（AY148351）群体的进化关系最为接近,且位于进化树的同一个分枝,都属于禾谷孢囊线虫Avenae组。     

Development of a PCR Assay for the Rapid Detection and Differentiation of ‘Candidatus Liberibacter solanacearum’ Haplotypes and Their Spatiotemporal Distribution in the United States

Zebra chip, or zebra complex (ZC) has become an important invasive disease of potato in the United States and New Zealand and is caused by a phloem-limited bacterium, ‘Candidatus Liberibacter solanacearum’ (Lso). A PCR assay using a single pair of simple sequence repeat (SSR) primers was developed for simultaneous detection and genotype differentiation of Lso haplotypes associated with zebra chip disease of potato. The sensitivity of the SSR PCR was similar to a 16S PCR assay, with detection limit of 100 copies of the Lso genome in haplotype A infected potato and psyllid samples and 10 copies of Lso genome in haplotype B potato and psyllid samples. The Lso detection frequency of the SSR PCR assay was 79.1 % in potato and 26.4 % in psyllid samples, respectively; whereas the detection frequency of the 16S PCR assay 59.0 % in potato and 25.9 % in psyllid samples, respectively. Samples of Lso positive potato plants and psyllids from multiple states in the US were demonstrated to have either haplotype A or haplotype B Lso and occasionally both haplotypes were found in individual samples. This is the first report that co-infection of the two haplotypes of Lso exists in potato and potato-psyllid samples. Only haplotype A Lso was detected in North Dakota psyllid samples collected in 2010, in Idaho and Washington ZC potato samples sampled from storage in 2011, and in Idaho ZC potato samples in 2012. Haplotype A Lso was also detected in New Zealand ZC affected potato samples and psyllid samples collected in 2010 and 2011. The PCR assay developed is as sensitive as previously developed assays and has the advantage of simultaneously detecting and differentiating Lso haplotypes of the ZC bacterium, thus making it extremely useful for epidemiological studies.     

9种热区植物白粉病菌的rDNA-ITS序列及系统发育分析

为弄清橡胶树、野艾蒿、大尾摇、胡萝卜、车前草、马占相思、红木、苦瓜和九里香等热区植物上白粉病菌的种类和彼此之间的系统发育关系,对这些白粉病菌的rDNA-ITS序列进行扩增和测序,与GenBank上公布的序列进行同源性比对,并进行分类.结果表明:橡胶树、马占相思、红木和九里香上的白粉病菌相似性非常高,均在99％以上;野艾蒿、大尾摇和苦瓜白粉病菌与叉丝单囊壳白粉菌的亲缘关系较近,分别达到100％和99％;胡萝卜白粉病菌与白粉菌属的独活白粉菌相似性在99％以上;而车前草白粉病菌与污色白粉菌的相似性达100％.利用供试菌和GenBank上公布的rDNA-ITS序列构建了系统发育树,将供试白粉菌分为4个类群,确定供试白粉菌之间的亲缘关系.     

橡胶灵芝菌（Ganoderma pseudoferreum）DNA 条形码检测 筛选

选用 10 对 DNA 条形码标准序列 ITS、COI、nrDNA-LSU、nrDNA-SSU 和 trnL DNA 对海南和云南红根病区 分离的 21 个橡胶灵芝菌进行检测。除 COI 和 trnL DNA 外，其他 8 对引物均扩增得到 PCR 产物，产物大小范围为 268~1 355bp，种内相似度为 98.05%~99.54%。分别将扩增得到的序列与 GenBank 公布上的 Ganoderma 属和常见真 菌属序列，共 77 个 ITS、74 个 nrDNA-LSU 和 74 个 nrDNA-SSU 序列进行系统进化树构建与分析。结果表明：nrDNA-LSU 序列和 nrDNA-SSU 序列种间差异较小，不能准确区分 Ganoderma 属及其他属；5 对 ITS 序列均能区分 Ganoderma 属，但仅 ITS1/ITS4 序列能将 Ganoderma 属下 16 个种归入不同分支。ITS1/ITS4 序列表现出适宜的种内与种间差异， 适宜作为 G. pseudoferreum 的 DNA 条形码。     

咖啡驼孢锈菌 ITS 序列特征及系统发育关系

为了了解咖啡驼孢锈菌 ITS 序列特征及系统发育关系,本研究对来自中国咖啡栽培区的咖啡驼孢锈菌 ITS 序 列进行了克隆。将克隆序列与来自不同咖啡种植区的咖啡驼孢锈菌菌株序列多样性进行了比较,并于 NCBI 数据库中 下载其他锈菌菌株 ITS 序列进行系统发育关系研究。结果表明,来自中国咖啡栽培区的咖啡驼孢锈菌 ITS 序列全长 950 bp。 其中 ITS1 长为 224 bp,GC 含量介于 30.36%~31.25%之间;5.8S 长为 153 bp, GC 含量为 37.25%;ITS2 长 483~485 bp, GC 含量介于 24.12%~25.05%。来自不同咖啡种植区咖啡驼孢锈菌 ITS 核苷酸序列多态性虽然存在一定差异,但是其多 样性十分低且不存在明显的区域分化。在系统发育关系方面,咖啡驼孢锈菌与其他锈菌 ITS 序列差异明显,能独立聚 类成一个分支。     

橡胶树多主棒孢菌rDNA–ITS区的分子鉴定及检测

以来自国内外的18个多主棒孢病菌[Corynespora cassiicola(Ber.&Curt.)]菌株为研究材料,提取其基因组DNA进行rDNA-ITS区序列扩增,并进行了5.8S rDNA及其两侧ITS序列分析.序列分析结果表明,种内各菌株间ITS序列同源性高达99.9%以上,部分菌株间出现个别碱基的差异.根据ITS区序列设计的一对特异引物(CorP1/CorP2)可以在供试的多主棒孢病菌中扩增出1条约277 bp的特异谱带,其余18个参试菌株和橡胶树叶片组织未能扩增出该特异谱带,检测灵敏度可达浓度为0.1 pg的目标DNA.     

利用ITS1和ITS4通用引物扩增香蕉枯萎病菌核酸片段鉴定其生理小种

利用真菌核糖体rDNA区通用引物ITS1和ITS4,扩增采集自香蕉的13株镰刀菌包括18S rDNA部分序列、ITS1-5.8S-ITS2全部序列和28S rDNA部分序列的片段,通过BLAST序列比对分析,确定13株菌为尖孢镰刀菌[Fusarium oxysporum]。采用最大简约法,以层出镰刀菌[F.proliferatum(EU151490)]和茄病镰刀菌[F.solani(GQ376116)]为外群,将13株菌的序列与BLAST检索获得的尖镰孢古巴专化型(F.oxysporum f.sp.Cubense)相应序列构建了系统发育树。系统发育分析结果显示,13株菌与NCBI登录的4个尖镰孢古巴专化型菌株一起被分成3个亚群,亚群聚类结果与回接鉴定结果及文献报道的生理小种结果完全一致。     

腐烂茎线虫rDNA-ITS序列分析

利用一对通用引物rDNA1/rDNA2扩增6个腐烂茎线虫供试群体的rDNA-ITS区域,获得序列长度不等的片段.Des-1、Des-2和Des-3群体扩增片断长度均为1 130 bp.而Des-5、Des-6和Des-7群体的扩增片段长度均为942 bp,与以上群体相比,在ITS区具有一个188 bp的缺失片段.序列比对后确定缺失片段位于ITS1区,而且该片段含有多个可重复小片段,这可能是造成碱基缺失的原因.根据遗传进化分析可将供试线虫分为两个大的群体.     

Haplotype Diversity at Sub1 Locus and Allelic Distribution Among Rice Varieties of Tide and Flood Prone Areas of South-East Asia

Single nucleotide polymorphisms and restriction digestion-based haplotype variations among 160 flood prone rice varieties were analyzed with enzymes Alu I and Cac8 I to generate polymorphisms at Sub1A and Sub1C loci(conferring submergence tolerance), respectively. Haplotype associated with phenotype was used to study the haplotype variations at Sub1A and Sub1C loci and to determine their functional influence on submergence tolerance and stem elongation. Three patterns at Sub1A locus, Sub1A0(null allele), Sub1A1(does not cut) and Sub1A2(one SNP), and four patterns at Sub1C locus, Sub1C1, Sub1C2,Sub1C3 and Sub1C4, were generated. Both tolerant Sub1A1 and intolerant Sub1A2 had the same length, but the difference was presence of a restriction site in the Sub1A2, but absent at the Sub1A1. Further, two types of polymorphism were detected at the Sub1C, one included major length polymorphisms(165, 170 and 175 bp) and the other was a single restriction site at different position. Eight haplotypes(different combinations of the two loci), A1C1, A1C2, A1C4, A2C2, A2C4, A0C2, A0C3 and A0C4, were detected among 160 varieties. Haplotype A1C1 was comparatively more related to haplotypes A1C2 and A1C4, having the same Sub1A allele, and these haplotypes were found only in Bangladeshi, Sri Lankan and Indian varieties. Most tolerant varieties in A1C1 haplotype showed slow elongation, having tolerant specific Sub1A1 and Sub1C1 alleles. Further, the varieties Madabaru and Kottamali(A2C2) also showed moderate level of tolerance without Sub1A1 allele. These varieties were different with FR13 A and also suspected to carry different novel tolerant genes at other loci. These materials could be used for hybridization with Sub1varieties for pyramiding additional tolerant specific alleles into a single genotype for improving submergence tolerance in rice.     

Assessing Potato Psyllid Haplotypes in Potato Crops in the Pacific Northwestern United States

The potato psyllid, Bactericera cockerelli (?ulc), is a vector of the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) that has been linked to the economically devastating zebra chip disease of potato. To date, four haplotypes of the potato psyllid have been identified and include Central, Western, Northwestern, and Southwestern haplotypes. Zebra chip was reported in potato crops in the Pacific Northwestern United States for the first time in 2011, and the Lso-infected psyllids collected from zebra chip-affected fields were identified as the Western haplotype. Additional studies have reported a mix of the Western and Northwestern psyllid haplotypes in the Pacific Northwest. The present study further examined psyllid population dynamics over the duration of the 2012 potato season in the Pacific Northwest by haplotype analysis of 864 potato psyllids collected from potato fields in Washington, Oregon, and Idaho. In the Yakima Valley of Washington and the lower Columbia Basin of Washington and Oregon, the Northwestern haplotype was predominant (78 %), and was detected earlier in the season than the Western haplotype. Interestingly, in south-central Idaho, all four psyllid haplotypes were identified, but the predominant haplotype was the Western haplotype (77 %). Here, Northwestern psyllids were detected early in the season from June to mid-August, whereas Central psyllids were detected in late July and thereafter. These results suggest that haplotype composition of psyllid populations in potato fields throughout the 2012 growing season in south-central Idaho differed greatly from those in Washington and Oregon. Additionally, all psyllids were analyzed for the presence of Lso, and no Lso-positive psyllids were found in Washington and Oregon, whereas Lso-positive psyllids were found in south-central Idaho. These Lso-positive psyllids consisted of the Western, Northwestern, and Central haplotypes.     

河南省玉米穗粒腐病病原串珠镰刀菌鉴定

河南省玉米穗粒腐病一般发病率为10%～30%,串珠镰刀菌是优势病原菌。从河南郑州感病的玉米穗轴和穗粒上分别分离出两个镰刀菌菌株。运用培养性状和形态特征综合分析的方法进行种类鉴定,并提取基因组DNA,对其内转录间隔区(ITS)序列进行PCR扩增和序列测定。结果鉴定出1个种为串珠镰刀菌。两个菌株rDNA间区ITS2可变区属I型,均能产伏马菌素。     

Virulence and diversity of Peronospora viciae f. sp. pisi in Alberta,Canada

Downy mildew, caused by Peronospora viciae f. sp. pisi, has emerged as a destructive disease of field pea in central Alberta, Canada. The objectives of the current study were to identify the predominant P. viciae pathotypes in Alberta and to assess genetic diversity in regional populations of the pathogen. Three pathotypes (UKP1, UKP2 and UKP11) were identified when nine populations of P. viciae were tested on a host differential set, with pathotype UKP1 being predominant. A collection of 30 P. viciae isolates clustered into three main groups when subjected to random amplified polymorphic DNA (RAPD) marker analysis. Comparison of the internal transcribed spacer (ITS) and 5.8S ribosomal RNA (rRNA) gene sequences revealed a high degree of homology among the isolates, suggesting a common origin. This is the first report on the pathotype composition and genetic diversity of P. viciae in western Canada.     

橡胶树炭疽病菌rDNA-ITS区序列分析

应用核糖体rDNA-ITS区的序列分析研究18个不同地区来源的橡胶树炭疽病病原菌。结果表明:测试菌株的ITS扩增区(ITS3-ITS4)约为300bp,无太大的长度变异。ITS核酸序列聚类分析的结果表明:18个测试菌株被聚为2个类群,群与群之间差异不明显,2组之间同源率大约为99.3%,通过在NCBI网站的blast比对分析,结果显示2组均为胶孢炭疽菌(Colletrichum gloeosporioides)。     

Molecular characterization,vector identification and partial host range determination of phytoplasmas associated with faba bean phyllody in Iran

During surveys of phytoplasma diseases, faba bean phyllody (FBP) was observed in several locations in Fars and Bushehr provinces (southern Iran). Samples of affected plants from Borazjan (Bushehr province) and Fasa (Fars province) were used to transmit the phyllody agent to faba bean, mung bean, pea, alfalfa and periwinkle by grafting, dodder and/or vector insect. Orosius albicinctus (Distant) was identified as the vector of Borazjan (BFBP) and Fasa (FFBP) faba bean phyllody. Naturally affected faba bean plants and all inoculated plants were positive for phytoplasma by direct PCR using the P1/P7 primer pair and nested PCR using P1/P7 and R16F2n/R16R2 primer pairs. Sequencing of PCR products identified the associated phytoplasmas as members of 16SrII phytoplasma group. Phylogenetic analysis using full length 16S rRNA gene sequences also confirmed similarity of the BFBP and FFBP phytoplasmas to the 16SrII group phytoplasmas. In this analysis the FFBP phytoplasma was grouped with ’Candidatus Phytoplasma australasia’, representative of 16SrII-D subgroup, while the BFBP phytoplasma formed a discrete group close to the 16SrII-C subgroup. Restriction fragment length polymorphism (RFLP) confirmed that BFBP and FFBP phytoplasmas belong to 16SrII group. Virtual RFLP confirmed that as members of peanut witches’ broom (16SrII) phytoplasma group, BFBP and FFBP phytoplasmas belonged to 16SrII-C and16SrII-D subgroups, respectively. Phytoplasmas associated with BFBP and FFBP were shown to be serologically related to the Fars alfalfa witches’broom phytoplasma, a member of 16SrII-C subgroup. It seems that witches’broom affected alfalfa fields are natural reservoirs of the FBP phytoplasma in southern Iran.     

Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

利用SSR标记研究甘蓝型油菜叶绿体基因组DNA多态性

叶绿体基因组具有母性遗传、单倍性、高度的保守性和明显的种内差异等特点。研究不同品种叶绿体基因多样化程度，可为甘蓝型油菜的收集与引种及遗传改良提供指导。利用15对特异性叶绿体SSR标记对287份国内外甘蓝型油菜叶绿体基因组多样性进行了分析，结果显示，在15对叶绿体SSR特异引物中，5对引物扩增产生多态性条带合计19条，平均每对引物检测出3.8条多态性条带；287份材料共划分成14个单倍型，优势单倍型H01占比74.91%、H02占比13.59%、H03占比4.88%，其余11种单倍型（H04-H14）占比合计6.62%；H02为波里马、陕2A胞质类型，为中国特有单倍型；国外甘蓝型油菜中，俄罗斯与瑞典的单倍型较丰富，引进俄罗斯与瑞典的资源能更高效拓宽中国甘蓝型油菜的遗传多样性本底。本研究还获得2对引物（MF-4和ccmp2）得到特异条带，并为此建立了快速鉴定波里马、陕2A胞质类型的方法，为波里马与陕2A细胞质类型材料的鉴定与保护提供了方法。     

Carrot Pathogen ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ Haplotype C Detected in Symptomless Potato Plants in Finland

‘Candidatus Liberibacter solanacearum’ (CLso) haplotype C, a bacterial pathogen transmitted by the carrot psyllid Trioza apicalis, causes yield losses in carrot production. Due to concerns that this pathogen might also threaten potato (Solanum tuberosum) production, the occurrence of CLso in cultivated and volunteer potatoes in Tavastia Proper and Satakunta regions of Finland was studied. Volunteer potato plants were found in 13 of the 27 inspected carrot fields. Of the 148 potato samples tested by PCR, eight volunteer potato plants and one cultivated potato grown at the edge of a carrot field were found to be CLso positive. The PCR products obtained from these potatoes with primers OA2/OI2c, LpFrag4-1611F/LpFrag4-480R and CL514F/CL514R all showed 100% sequence identity to CLso haplotype C. This is the first observation of CLso haplotype C in field-grown potatoes. In addition, transmission experiments were performed. Attempts to transmit CLso into potato with carrot psyllids were not successful; however, CLso haplotype C was transmitted from infected carrots to potato plants by leaf grafting and by phloem connection formed by dodder, a parasitic plant, and found to survive in the potato plants for several weeks after transmission. However, the bacterial colonisation progressed slowly in the potato phloem and the amount of bacteria detected was low. The plants produced from the daughter tubers of the CLso-positive potato plants were all CLso negative, suggesting that CLso haplotype C was not able to pass to the daughter plants. None of the CLso-positive potatoes inoculated in greenhouse or collected from fields showed symptoms characteristic of zebra chip disease, associated with CLso haplotypes A and B.     

海南长春花变叶病病原物的分子鉴定

本研究分别利用植原体16S rDNA和核糖体蛋白(ribosomal protein,rp)基因的通用引物对自然表现变叶症状的海南长春花样品总DNA进行PCR扩增,获得16S rDNA基因片段长为1 432 bp,rp基因片段长为1 211 bp。BLAST程序比较、系统进化树构建及iPhyClassifier分析表明:海南长春花变叶病是由植原体引起的,该植原体株系属于16S rⅠ-B亚组,与候选种Ca.Phytoplasma asteris相关,将其暂命名为长春花变叶病植原体海南株系(Periwinkle phyllody strain Hainan,PP-Hn)。