Investigation of the phenotypic and genetic properties of Brenneria nigrifluens strains,the causal agent of walnut bark canker in Kurdistan province,Iran

Phenotypic and genetic properties of Brenneria nigrifluens, the causal agent of walnut bark canker disease were studied using a combination of key biochemical tests, analysis of whole‐cell protein extracts and genetic fingerprints based on DNA primers corresponding to conserved motifs in bacterial repetitive (repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus and BOX) elements and to the 16s rRNA gene. Phenotypic properties indicated that 11 strains showed a high degree of similarity and <5% of isolates showed different properties such as utilization of dulcitol and inositol. A majority of isolates produced rep‐PCR profiles similar (>80%) to a B. nigrifluens reference strain. This study showed that the majority of strains had 0–3 plasmids with estimated sizes ranging 9–32 Kb. Strains were clustered into two and three groups by whole‐cell protein analysis and rep‐PCR, respectively.     

Detection and characterization of Ralstonia pseudosolanacearum infecting Eucalyptus sp. in Brazil

Ralstonia solanacearum sensu lato causes bacterial wilt in many agronomic crops and tree species economically important worldwide. It is a species complex that has been divided into phylotypes and sequevars, commonly related to geographic distribution. Knowledge of the phylotype composition and genetic variability in populations of this phytopathogenic bacterium is useful for implementing effective control measures. In a survey conducted in 2019, six bacterial strains were obtained from wilted Eucalyptus urophylla trees in plantations located in the municipality of Dom Eliseu, Pará state, Brazil. Multiplex PCR based on the internal transcribed spacer (ITS) indicated that the bacterial strains belonged to two different species, namely R. pseudosolanacearum (phylotype I) and R. solanacearum (phylotype II). In a phylogenetic analysis, the nucleotide sequence of the endoglucanase (egl) gene from eucalypt strains of phylotype I clustered together with sequevar 18 sequences from GenBank. Separation of the strains into two different species was confirmed by repetitive element palindromic PCR (rep‐PCR). Pathogenicity tests demonstrated that the R. solanacearum and R. pseudosolanacearum strains recovered from E. urophylla cause disease in both tomato and eucalypt plants. Until now, only R. solanacearum (Phylotype II) has been reported causing wilt symptoms on Eucalyptus spp. in Brazil. Therefore, the presence of R. pseudosolanacearum and a need for better understanding of its genetic and aggressiveness variability as well as possible differences between the two species should be considered in breeding programmes aimed at the deployment of host resistance.     

Molecular characterization of Ralstonia solanacearum infecting Eucalyptus spp. in Brazil

Among the bacterial pathogens of Eucalyptus in Brazil, Ralstonia solanacearum is considered one of the most important because of the characteristics of the pathogen, like the high diversity among the strains related to host range, high virulence, broad geographical distribution and its damage to the crop in recent years. Given its importance and the lack of research on this pathosystem, the present study aimed to perform a molecular characterization of different strains of infected Eucalyptus plants in Brazil. A total of 19 bacterial cultures isolated from Eucalyptus in different regions of Brazil were analysed. A 372‐bp product generated by multiplex‐PCR amplification using Nmult primers identified all the strains analysed as belonging to phylotype II. Eighteen strains were grouped into subclade IIA and one into subclade IIB. The phylogenetic tree generated from the gene sequences of endoglucanase (egl) confirmed the classification of the strains into phylotype II and separated the strains into sequevars. Strains AMC22, IBSBF2568 and IBSBF2576 were grouped into a single clade, as were strains UFV18 and UFV20, with 89% and 78% a posteriori probability, respectively, forming two new potential sequevars not yet defined. We also identified strains belonging to sequevars 41 (100% probability) and 37 (88% probability). However, most of the strains did not fit into any previously described sequevar and did not form distinct clades. The results of the analysis of fragments amplified using the ERIC‐PCR technique indicated the existence of genetic diversity among the strains studied, with a generally high correlation between similarity and the geographical origin of the strains.     

Genetic diversity of sea buckthorn (Hippophae rhamnoides) populations in northeastern and northwestern China as revealed by ISSR markers

As a N₂-fixing tree species, sea buckthorn (Hippophae rhamnoides) is well adapted to arid regions and is utilized for multiple purposes in China. Current knowledge of genetic variability of H. rhamnoides is limited in terms of rangewide distributions. Eleven natural populations of sea buckthorn in northeastern and northwestern China were analyzed to detect genetic variation among and within populations, by use of ISSR (inter-simple sequence repeats) markers. Using eight primers, 207 polymorphic loci were observed, ranging in size from 250 to 2500 bp. The coefficient of gene differentiation (Gst = 0.0679) showed that the total molecular variance of 11 populations was mainly existed within populations. The genetic variation within and among the 11 populations was 93.21 and 6.79%, respectively. No significant correlation between genetic and geographic distances of the populations was found using ISSR markers. Our study provides a population-level genetic profile for further investigation and conservation of genetic diversity of sea buckthorn.     

湛江高桥红海榄群落样方遗传相似性特征研究

遗传相似性特征及分布的研究对保护生物多样性、维护森林生态服务功能有积极的意义。选择广东湛江高桥红海榄Rhizophora stylosa纯林的400 m²固定样地，采用简单重复序列间区扩增ISSR和相关序列扩增多态性SRAP两种分子标记技术开展研究。用筛选出的10个ISSR引物和14个SRAP引物组合进行PCR扩增，分别共扩增出73个和111个清晰可读的位点，多态性位点数分别是60和88，多态性位点百分率分别是82.2%和79.3%。样方红海榄遗传相似系数变异范围为0.327～0.741，平均值为0.563，差值为0.414。在相似系数0.55的水平下，分为3个类群，遗传相似度为第一类群>第二类群>第三类群，遗传相似变动度第三类群>第二类群>第一种类群。在相似系数0.60的水平上，可分为8个类群。相似系数0.55的水平下红海榄聚集度越强，遗传相似度越高，遗传相似变动度越低。高桥红海榄群落样方分属于不同的类群，在遗传上有差异性，存在一定的基因交流；亲缘关系为远和较近的分布区间，遗传相似度和变动度中等，第三类群为优势种。初步证明红海榄聚集度与遗传相似度成正比，与遗传相似变动度成反比。     

A comparison of UP‐PCR and RAPD markers to study genetic diversity of Fusicladium effusum (G. Winter), cause of pecan scab

Fusicladium effusum infects pecan causing yield loss, but no information is available on the genetic diversity of F. effusum. Randomly amplified polymorphic DNAs (RAPDs) and universally primed polymerase chain reaction (UP‐PCR) were compared to detect polymorphisms on a group of 20 isolates of F. effusum from 11 geographical locations in the southeastern USA. Two tests (run 1 and 2) of both the RAPD and UP‐PCRs were conducted to assess the repeatability of the methods, and the markers scored on agarose gels. In addition, the UP‐PCR markers from run 1 were scored using an automated capillary system. Both RAPDs and UP‐PCR markers detected a high level of polymorphism among the scored markers (92 and 91% of RAPD markers, and 86 and 87% of manually scored UP‐PCR markers in run 1 and 2 were polymorphic, respectively; 93% of UP‐PCR markers were polymorphic when scored using the automated system). Unweighted paired group method of arithmetic averages (UPGMA) analysis showed both RAPDs and UP‐PCR markers individually identified each isolate, producing three groupings, but only the groupings based on run 1 and 2 of the UP‐PCR contained the same isolates. Bootstrap analysis based on the Dice coefficient produced phenograms from the UP‐PCR data with weak to moderate node support (≥54) for the primary branch, but no support for the RAPDs data (≤34). A Mantel test of runs 1 and 2 using RAPDs or UP‐PCR showed good agreement (r = 0.8761 and 0.8289, p < 0.0001), but poor agreement between RAPDs and UP‐PCR. UP‐PCR results based on the interisolate Dice coefficients showed a weak to strong association with distance. Based on these results, both RAPDs and UP‐PCR markers were capable of demonstrating polymorphisms and identifying relationships among isolates of F. effusum; however, UP‐PCR markers appear to be more reliable.     

Characterization of Phytophthora cinnamomi from common walnut in Southern Europe environment

Forty‐eight isolates of Phytophthora cinnamomi obtained from common walnut were analysed according to their variability in growth at different temperatures, virulence, sensitivity to metalaxyl and in genomic DNA. Isolates were obtained from commercial common walnut orchards located in northern Italy and in southern France. Inter‐simple sequence repeat (ISSR) were analysed for the 49 isolates, 43 of which were Italian, 6 French; an isolate of the same species obtained from Viburnum spp. was used as an outgroup. ANOVA on phenotypic characters showed a significant impact of the geographic location of the orchard on isolate variability in terms of reaction to temperatures and aggressiveness. In turn, clustering obtained with UPGMA analysis on genetic data was almost exclusively dependant on isolate variability, nevertheless the 48 isolates seem to share a common variability that differentiates the group from the isolate from Viburnum spp. Correlation between phenotypic and genetic traits was not statistically significant. In conclusion, phenotypic variability like virulence seemed to be conditioned from geographic origin while the genetic variability of P. cinnamomi isolates from walnut was associated to the single genotype.     

51个木麻黄无性系遗传多样性的ISSR分析

采用ISSR分子标记对51个木麻黄无性系进行遗传多样性和亲缘关系分析。结果表明:ISSR适用于木麻黄无性系遗传分析,22个ISSR引物在供试无性系中共扩增出199个位点,多态性位点154个,多态位点百分率为77.4%;平均有效等位基因数为1.5,Nei’s基因多样性指数为0.174 1 0.389 1,Shannon信息指数为0.273 20.556 0,相似系数为0.467 3 0.995 0,平均为0.743 0,表明供试无性系的遗传基础已相对比较狭窄。ISSR聚类分析表明:参试无性系并不能按来源地各自单独聚为1类,无性系亲缘关系的远近与来源相关不大;在相似系数为0.678时,可将所有供试材料分为2大类群;亲缘关系树状图在分子水平上显示了供试无性系间的亲缘关系,为今后木麻黄无性系的推广应用及育种亲本的选配提供了理论依据。     

Genetic diversity of the hyperparasite Sphaerellopsis filum on Melampsora willow rusts

The non‐specific rust hyperparasite Sphaerellopsis filum occurs naturally on Melampsora rusts of many species of the genus Salix as well as on a large range of other rust genera worldwide. To study the genetic diversity of the hyperparasitic fungus 77 S. filum isolates collected from rusts on willow clones from plantations, clone collections and natural habitats of different sites were investigated using polymerase chain reaction ‐ restriction fragment length polymorphism (PCR‐RFLP) analysis of the rDNA internal transcribed spacer regions including 5.8S rDNA and sequence analysis. Additionally, strains from Melampsora poplar rusts (4) and strains of Puccinia abrupta from Parthenium hysterophorus (5) and of P. obscura from Bellis perennis (1) were used for comparisons. Results of genetic analysis demonstrated distinct variation within the S. filum isolates. Two main groups with more than 32% difference between their nucleotide sequences were distinguished, indicating two taxa within S. filum. Within the first main group three profiles (I, II, III) were detected. The differences between these profiles were about 12%. The variation within each profile was very low (less than 2%). The second main group comprised two profiles (IV, V), which differed in 12 to 16% of their nucleotide positions. The isolates of group IV possessed a higher variation (up to 5%) within the group than those of the first main group (I, II, III). Group V was only represented by a single isolate. Neither interrelations between the S. filum profiles and the Melampsora genotypes nor a spatial distribution could be detected. It is remarkable that the six strains of S. filum from Puccinia rusts belong to one subgroup.     

桂花优良品种‘珍珠彩桂’遗传多样性的ISSR分析及指纹图谱构建

利用ISSR分子标记技术对44个桂花品种进行遗传多样性研究,从100条引物中筛选出10条引物对供试材料基因组总DNA进行PCR扩增,共扩增出条带122条,其中多态性条带112条,多态性比例为91.8%。经Pop Gen32软件包分析,44个桂花品种的平均有效等位基因数为1.592 8,平均Nei’s基因多样性指数为0.341 9,平均Shannon信息指数为0.506 3,遗传一致度介于0.475 4~0.926 2之间,说明桂花品种间遗传多样性较高。UPGMA聚类将44个品种分成7类。建立了新品种‘珍珠彩桂’和其它43个桂花品种的DNA指纹图谱,可从分子水平将‘珍珠彩桂’与现有其它桂花栽培品种进行鉴别。研究结果为桂花分类、品种鉴定、分子育种和子代鉴定提供了重要依据。     

Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.     

Genetic diversity analysis in a seed orchard of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

India is the largest grower of Eucalyptus with an area of 3.943 million hectares under plantations and E. tereticornis is the predominant species in the plains of southern India with an average productivity of 12–25 m³ ha⁻¹ year⁻¹. With the aim to establish seedling seed orchards of the species, seed lots of fifteen provenances were imported from Australia and a trial was laid. In the present study the genetic diversity existing in the seed orchard was estimated using ISSR–PCR. Seven ISSR primers amplified 663 amplicons in the size ranging from 255 to 2,711 bp. The total number of polymorphic bands varied from 59 to 123 with 100% polymorphic banding profiles. The average gene diversity (Hj) of all the provenances ranged from 0.0589 to 0.1109 and the total gene diversity estimated was low (H _T = 0.130) when compared to the earlier reports from other eucalypts species. Analysis of Molecular Variance partitioned the ISSR variation into inter and intra population components. The inter population component accounted for 55.2% of the variation and the intra population component accounted for 46.3% (P < 0.001). A neighbour-joining analysis was done using the dissimilarity matrix to determine the aggregation of the individuals into clusters. Existence of population structure among the populations was revealed in STRUCTURE analysis but geographical region based clustering was not observed. The assessment of intra and inter genetic variation documented in the present study suggests that, along with the phenotypic traits, knowledge about genetic diversity measured at the DNA level in individuals of seed orchards provide an objective guide for selective culling of trees for maintaining optimal diversity for enhanced genetic gains.     

AFLP fingerprinting of <Emphasis Type="Italic">Populus deltoides</Emphasis> and <Emphasis Type="Italic">Populus </Emphasis>×<Emphasis Type="Italic"> canadensis</Emphasis> elite accessions

Amplified fragment length polymorphism (AFLP) was used in genetic fingerprinting of 22 elite Chinese Populus deltoides and Populus × canadensis accessions. The results indicated that each of the nine AFLP primer pairs selected generated fingerprint profiles that were unique to each of the accessions. Therefore, each accession was definitively identified by any of the nine primer pairs. Furthermore, the inter-accession genetic relationships inferred based on 461 polymorphic fragments from the nine AFLP primer pairs were largely consistent with phylogenetic relationships based on morphologic traits. Bootstrap analysis showed that three AFLP primer pairs were required to obtain genetic similarity values with a maximum CV of 10% while 10 AFLP primer pairs could give a maximum CV of 5%. Thus, AFLP can readily be applied for a rapid and accurate evaluation of the degree of similarity between poplar cultivars. In this study, the number of AFLP fragments used was sufficient to establish a reliable estimate of genetic similarity among accessions, with a maximum CV of 5.12%. Therefore, the information on the genetic relationships among the poplar accessions generated in this study in connection with knowledge on agronomic traits may have an impact on poplar breeding and planting in China.     

Genetic variation in Polish strains of Gremmeniella abietina

Thirty-nine strains of Gremmeniella abietina were isolated between 1980 and 2005 from diseased or symptomless Pinus sylvestris and Pinus nigra, or sporadically Pinus jeffreyi and Pinus armandii from four regions of Poland with varying climatic conditions. Thirty-five of the strains were genetically similar to the G. abietina A type found in Scandinavia, whereas four were similar to the B type. On the basis of random amplified microsatellite markers, very high intrapopulation and interpopulation genetic variation was detected among the strains. The average value of the Jaccard genetic similarity coefficients among strains was 0.56 for A type and 0.47 for B type, and the monomorphic loci partition was small, at 14% of all analysed loci for the A type. The degree of genetic variation of the fungal strains within each of the four regions was similar, showing no significant differences. The Jaccard coefficient for the strains isolated from P. sylvestris and P. nigra differed significantly, at 0.57 and 0.54 respectively. The genetic distance among A type strains from the investigated regions, expressed using Nei’s coefficient, was not correlated with geographic distance; however, it was highly dependent on elevation (correlation coefficient r = 0.92).     

Molecular Discrimination of Eucalypt Clones Using the ISSR Marker System

Clonal forestry captures genetic gain generated by skillful selection and is now well recognized as a method for mass production of desired trees to obtain increased economic benefits. In Eucalyptus, clonal plantations have enhanced the productivity twofold, compared to plantations of unimproved seed origin. The present investigation was carried out to assess the genetic relationship of 41 eucalypt clones using the intersimple sequence repeat (ISSR) marker system. The ISSR-derived dendrogram and principal coordinate analysis (PCA) clustered 39 clones into two groups of E. camaldulensis and E. tereticornis in agreement with their taxonomic classification. Further, a total of three clone specific diagnostic markers and five unique profiles were identified using five ISSR primers. The eucalypt clones analyzed in the present investigations are derived from the breeding populations of both the species and are presently being used for large-scale plantation and hybridization studies. The molecular marker based clonal discrimination can be used to avoid duplication of clones and quality control of planting stock.

[Supplementary materials are available for this article. Go to the publisher's online edition of Journal of Sustainable Forestry for the following free supplemental resource(s): ISSR Profiles of Eucalyptus Clones]     

乐昌含笑不同类型鉴定的ISSR-PCR分析

利用inter -简单重复序列 (ISSR)标记对乐昌含笑 6种类型的遗传变异进行了研究 ,从 30个引物中筛选出 12个多态性引物用于正式扩增 ,共扩增出 134条DNA带 ,其中多态性DNA带 6 7条 ,占 5 0 % ,平均每个引物扩增的DNA带的数目为 11 16 7条。其中引物ISSR16、ISSR19能区分全部类型。引物ISSR16、ISSR19和ISSR2在不同的类型中扩增出了一些特有条带 ,对乐昌含笑类型和品种鉴定以及检验品种的真实性方面非常有价值。本研究对ISSR分析技术的关键步骤进行了讨论 ,并利用DNA扩增结果对供试类型进行了聚类分析     

花吊丝竹居群遗传多样性的ISSR分析

[目的]研究花吊丝竹居群遗传多样性和遗传结构,为种质资源有效利用和良种选育提供理论指导。[方法]利用12条ISSR引物对48份种质(共3个居群)花吊丝竹居群进行遗传多样性和遗传距离分析。[结果]共检测到124个位点,其中,多态性位点为102个,种质和居群水平上的多态位点百分比(PPB)分别为82. 26%和50. 27%,Ne’基因多样性指数(He)分别为0. 220 4和0. 206 6,Shannon’s信息指数(I)分别为0. 349 4和0. 300 5,表明花吊丝竹居群间存在中等水平的遗传变异。根据Nei’s遗传多样性计算出不同居群间分化水平(Gst)=0. 163 3,表明16. 33%的遗传变异存在于居群间,居群内的遗传变异为83. 67%。居群间的基因流Nm为2. 562 1,表明花吊丝竹居群间存在较大基因流,很大程度减少居群间遗传差异。基于遗传距离的UPGMA聚类结果表明,48份种质可分为3组,3个居群可分为2组,居群间地理距离与亲缘关系无显著相关性。[结论]虽然花吊丝竹主要靠营养生殖来繁衍后代,其居群遗传多样性较丰富,且居群内遗传多样性大于居群间。此外,福建居群遗传多样性明显高于广西和广东地区居群。     

Detection and genetic variability of European mountain ash ringspot‐associated virus (EMARaV) in Sweden

European mountain ash ringspot‐associated virus (EMARaV) is a plant virus inducing characteristic ringspots and mottling in Sorbus aucuparia L. For the first time, EMARaV was detected in mountain ash in Sweden. All four genomic segments of the virus were detectable by RT‐PCR after total RNA extraction from leaves showing chlorotic ringspots, mottling or necrotic lesions. The samples originated from southern and northern Sweden. Sequence analyses of amplified fragments revealed low genetic variability of the virus at nucleotide as well as protein level. All investigated coding regions of EMARaV were under strong purifying selection pressure.     

金花茶组物种遗传关系的ISSR分析

利用ISSR分子标记技术对广西南宁金花茶公园29份金花茶组物种进行遗传关系分析。筛选出的14条引物扩增得到133条清晰条带,其中126条为多态性条带,多态性位点百分比为94.74%。29份金花茶种质材料的Nei’s基因多样性指数为0.360 6,Shannon’s 信息指数为0.531 4,遗传相似系数介于0.481 0.835,金花茶组物种的遗传基础较宽。用NTSYS软件对样品进行UPGMA聚类分析,29份金花茶样品聚为3大类群,其中扶绥中东金花茶单独为一类,顶生金花茶和龙州金花茶聚为一类,其它金花茶聚为一类。分析结果表明：夏石金花茶和小花金花茶的遗传相似系数最高,支持将两者归并到柠檬黄金花茶;弄岗金花茶和毛籽金花茶亲缘关系很近,支持合并到同一个种;龙州金花茶和薄叶金花茶分别归为单独的种。     

枫香优良观赏种质的遗传多样性分析

利用ISSR标记技术对12份枫香观赏种质进行遗传多样性分析,通过正交实验优化枫香的ISSR-PCR扩增体系,即在20μL体系中加入PCR反应缓冲液2μL、镁离子1.5 mmol.L-1、Taq DNA聚合酶0.75 U、dNTP 0.15 mmol.L-1、引物0.4mmol.L-1、DNA模板80 ng。筛选出的19条ISSR引物共得到131条扩增带,其中多态性条带为94条,多态性条带比率为71.75%。12份种质的有效等位基因数是1.482,基因多样性0.3012,Shannon信息指数0.4674,表明遗传多样性水平相对较高。进一步的聚类将这些种质分成3类,其中4号样本和11号样本的遗传相似度最高。