Genetic variation and structure of Solanum elaeagnifolium in Australia analysed by amplified fragment length polymorphism markers

Solanum elaeagnifolium is a weed of national significance in Australia. However, the genetic diversity of S. elaeagnifolium is poorly understood. Four amplified fragment length polymorphism primer combinations were utilised to investigate the genetic variation and structure of 187 S. elaeagnifolium individuals collected from 94 locations in Australia. High genetic diversity was found, with an average Jaccard's genetic similarity at 0.26. Individuals were assigned to two genetic clusters or considered as admixed according to their membership coefficient value (q) calculated by Bayesian model‐based genetic structure analysis. This suggested that Australian S. elaeagnifolium may have originated from two distinct gene pools. These results were further supported by principal co‐ordinates analysis. Large spatial groups of individuals assigning to these two gene pools were found in western Victoria and south‐western New South Wales (NSW) and northern NSW, which correlated well with the early records of S. elaeagnifolium in both regions. The high genetic diversity found here could add difficulties to effective control of S. elaeagnifolium across regions.     

Genetic diversity of the weed species,Stellera chamaejasme,in China inferred from amplified fragment length polymorphism analysis

Stellera chamaejasme is a perennial weed with a wide geographic range that is found from the Altai of eastern Russia, northern China and Mongolia southwards as far as the western Himalayas of the Qinghai–Tibet and Yungui Plateaus. The genetic diversity and population structure of 17 populations of S. chamaejasme, represented by 349 individuals, were assessed by using amplified fragment length polymorphism markers. The results showed a relatively high level of genetic variation at the species level. The proportion of total diversity among populations was 0.4370, suggesting significant genetic differentiation and a low gene flow among the populations of this species. The Mantel test indicated that genetic differentiation among populations was significantly correlated with geographic distance. Genetic drift through range expansion and a low gene flow among populations might result in a lower diversity in peripheral populations, compared to central populations. A Bayesian analysis revealed two potential gene pools in S. chamaejasme, which was confirmed by neighbor‐joining clustering and principal coordinate analysis. These results demonstrate that it is necessary to develop suitable biocontrol agents for populations with different gene pools.     

松材线虫种群遗传多样性AFLP标记的建立及其应用

 松材线虫是极具危险性的外来入侵生物,其引起的松材线虫病,目前正在我国部分地区迅速扩展和蔓延,对我国林业生产造成了严重的经济损失。开展松材线虫的种群遗传学研究,是了解其成功入侵和爆发成灾内在机理的重要途径。但迄今为止,尚未找到很有效的分子标记方法来检测松材线虫入侵种群的遗传变异。本文采用AFLP分子标记技术,通过对各反应体系和反应条件的优化及对多态性引物组合的筛选,成功地建立了松材线虫的AFLP分子标记实验体系,并筛选出52对高效多态性的引物组合。应用4对引物对27个松材线虫种群样品进行遗传多样性检测,结果表明,AFLP是进行松材线虫种群遗传学研究的一种很灵敏和可靠的分子标记。此外,本文还对AFLP技术在松材线虫研究中的应用前景进行了讨论。     

Extent and pattern of genetic differentiation within and between European populations of Phelipanche ramosa revealed by amplified fragment length polymorphism analysis

The extent and pattern of genetic differentiation between Phelipanche ramosa populations colonising tobacco in different European regions were investigated by amplified fragment length polymorphism (AFLP) analysis, in order to determine levels of variation for tobacco resistance breeding and management programmes. Four different AFLP primer pairs amplified a total of 1050 clear and reproducible bands, of which 962 (91.62%) were polymorphic among the 35 individuals taken from four P. ramosa populations collected in Spain, Italy, Bulgaria and Germany. Cluster analysis based on the AFLP data categorised the plants into distinct groups, in line with their geographical origin, denoting clear genetic differentiation among the four populations. This differentiation was supported by both high bootstrap values and significant results of the analysis of molecular variance. The most divergent population was the one from Bulgaria. The majority of the genetic diversity was attributable to differences among populations (77.80%), as expected from the predominant autogamous behaviour of this species. Populations differed significantly in within-population diversity, as measured by Shannon's information index. The German population presented the lowest genetic diversity and the Italian population harboured the highest level of within-population genetic diversity. There are significant differences in genetic diversity level among the studied P. ramosa populations and clear population-specific genetic diversity structures. These need to be taken into account, together with the potential differences in parasite aggressiveness, when planning breeding and management strategies for P. ramosa control in tobacco cultivation.     

Incidence of thiophanate-methyl resistance in Cercospora kikuchii within a single lineage based on amplified fragment length polymorphisms in Japan

We collected 247 isolates of Cercospora kikuchii from soybean seeds with typical purple stain symptoms from 15 prefectures in Japan. Of the 247 isolates, 93 were sensitive to thiophanate-methyl, a benzimidazole used to control this soybean disease; the remaining 154 were highly resistant to the fungicide. To examine genetic variability among the population of 247 isolates, we developed amplified fragment length polymorphism (AFLP) markers. An AFLP primer pair generated DNA fingerprint polymorphisms among the sample isolates, and with the unweighted pair-grouping method to cluster arithmetic means of the similarity coefficients among all pairs of the fingerprint patterns, the isolates were divided into four lineages (I to IV). Of the 247 isolates, 225 belonged to lineage I, including all isolates that were resistant to thiophanate-methyl. To determine whether the resistance of these isolates was related to mutations in the β-tubulin gene, we amplified partial nucleotide sequences of the gene from 29 representative isolates, including 12 that were resistant to thiophanate-methyl, by means of the polymerase chain reaction. The resistant isolates had identical nucleotide sequence with a one-step change at codon 198, in which the amino acid glutamic acid had been replaced by alanine. The evidence thus suggests that thiophanate-methyl resistance might have arisen in lineage I, the largest of the four lineages. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB214511 to AB214515     

Dairyland populations of bur cucumber (Sicyos angulatus) as a possible seed source for riverbank populations along the Abukuma River,Japan

In 2006, the distribution of bur cucumber (Sicyos angulatus L.) along the river bank of the Abukuma River, Japan, was examined. Twenty‐six populations were found in the area surrounding the upper reach of the river and they were limited to dairy land. On the basis of polymerase chain reaction–restriction fragment length polymorphism analysis for four of the six polymorphic loci that were detected in 16 non‐coding regions of the chloroplast DNA from 300 samples, five multilocus haplotypes (A–E) were identified. Despite its smaller size, the surrounding area's population showed a higher genetic diversity at the haplotype level than did the riverbank population. The haplotype composition of the surrounding area's population was similar to that of the riverbank population in the upper‐stream region. The distribution of haplotype D was biased towards the upper reach of the river, possibly because of migration from the surrounding area's population. The distribution of haplotype E was biased towards the middle‐to‐lower reaches of the river and was associated with the existence of source populations other than the surrounding area's population. The results also showed that the estimated seed flow from another dairy land could transport seeds into the middle reach of the river at a high frequency, suggesting that dairy land is largely responsible for the development of the riverbank populations. The eradication of dairyland populations should be given priority in order to prevent future migration along the river bank and to protect the existing endangered wetland species from invasive S. angulatus.     

江苏茶园秋季杂草群落及外来入侵杂草的数量生态学分析

为明确除草剂使用、翻耕、树龄、坡度和郁闭度等杂草管理措施及环境因子对江苏茶园秋季杂草群落和外来入侵杂草物种组成、群落结构的影响,于2013年10月分别对江苏茶叶主产区仪征、金坛和宜兴3地共90个样地进行了杂草调查。通过主成分分析和典范对应分析,90块样地可以依据优势杂草种类和杂草丰富度被分成3个类群。类群Ⅰ为荩草、野老鹳草、酢浆草、狗尾草、蓬蘽、麦冬杂草群落,该类群翻耕和化学除草频次少,树龄较大,郁闭度高,田间多年生杂草多,杂草种类多,田间杂草密度低;类群Ⅱ为马唐、小旱稗、小飞蓬、野老鹳草、狗尾草杂草群落,该类群以化学除草为主,田间一年生禾本科恶性杂草和抗除草剂杂草多,杂草优势种突出;类群Ⅲ为马唐、小旱稗、牛筋草、野老鹳草、酢浆草、虮子草杂草群落,该类群茶园管理较为精细,田间优势杂草种类少,杂草密度低。对外来入侵杂草与环境因子关系的数量分析发现,化学除草剂的使用和翻耕频率显著影响外来入侵杂草在江苏茶园的发生和分布。研究表明,不同管理措施,如化学除草剂和翻耕频率,对杂草群落的形成起决定性作用。     

Genetic diversity of Sicyos angulatus in central and north-eastern Japan by inter-simple sequence repeat analysis

For the development of effective procedures to control invasive plants, it is necessary to learn about their mechanism of spread, for which an understanding of the plant's genetic variation may be important. Sicyos angulatus is a widespread and invasive weed that grows among forage crops and natural vegetation in Japan. Inter-simple sequence repeat (ISSR) genotyping was used to detect the regional patterns of genetic variation of S. angulatus in its introduced range in Japan. The goal of this research was to assess the introduction dynamics and mechanism of spread of S. angulatus in Japan. Four screened ISSR primers produced 15 reliable bands, and 12 of these were polymorphic among six areas in central and north-eastern Japan. The analysis of molecular variance ( amova ) revealed that 88.4% of genetic variation occurred within areas rather than between the two regions (1.1%), or among areas within regions (10.5%). While the total gene diversity among areas ( Ht ) was 0.1684, the coefficient of gene differentiation was low among two geographically distinct regions ( Gst = 0.053). The genetic diversity indices ( h  = 0.116; I  = 0.173) of the Fuji river area, assumed to be near the first introduction site, were not higher than other areas (total h  = 0.165; total I  = 0.263). In contrast, those of the Chikuma river and Tenryu river areas were relatively high ( h  = 0.173 and 0.187; I  = 0.276 and 0.277 respectively). The Mantel test showed no significant correlation between geographical and genetic distances. These results suggest multiple introductions from the same gene pools into both central and north-eastern regions of Japan.     

Population structure of Phytophthora infestans in China – geographic clusters and presence of the EU genotype Blue_13

The population structure of Phytophthora infestans in China was studied and three mitochondrial haplotypes (Ia, IIa, IIb) were observed. Genetic analysis with 10 highly informative SSR markers identified 68 different genotypes, including three dominant clonal lineages. In the Chinese P. infestans population, the genotypes were strongly clustered according to their geographic origin. One of dominant clonal lineages was genetically similar to Blue_13, a dominant clonal lineage found in Europe since 2004. This is the first report of Blue_13 outside Europe. Only one mating type (A1) was found in the northern and southeastern provinces, but in southern and northwestern China both mating types were observed. The mating type ratio and SSR allele frequencies indicate that in China the sexual cycle of P. infestans is rare. These results emphasize that the migration of asexual propagules and the generation of subclonal variation are the dominant driving factors of the population structure of P. infestans in China. They may also have implications for the role of monitoring P. infestans populations in potato late blight management strategies in China.     

Genetic structure and pathogenicity of populations of Phytophthora infestans from organic potato crops in France, Norway, Switzerland and the United Kingdom

Genetic variation and pathogenicity of Phytophthora infestans isolates collected from organic potato crops of the susceptible cv. Bintje and the moderately resistant cv. Santé were assessed in France, Norway, and the United Kingdom in 2001 and in Switzerland in 2001 and 2002. Population structures differed considerably between the four P. infestans populations. Those from France, Switzerland and the UK were mainly clonal populations showing restricted levels of genetic diversity, whilst those from Norway were mixed A1 and A2 mating type populations with high levels of genetic diversity, suggesting periodical sexual reproduction. Isolates collected from cv. Bintje were on average more aggressive than or comparable to isolates from cv. Santé. Race complexity varied considerably between the regional P. infestans populations, with isolates from France and Switzerland showing the highest number of virulence factors. In all pathogen samples but the French, isolates collected from cv. Santé were more complex than isolates collected from cv. Bintje. No directional selection towards increased aggressiveness towards the more resistant cultivar Santé was observed. This suggests that there is no shift towards increased levels of pathogenicity in P. infestans populations following the large-scale introduction of more resistant potato varieties in organic production systems in Europe.     

Population structure of the ash dieback pathogen,Hymenoscyphus fraxineus,in relation to its mode of arrival in the UK

The ash dieback fungus, Hymenoscyphus fraxineus, a destructive, alien pathogen of common ash (Fraxinus excelsior), has spread across Europe over the past 25 years and was first observed in the UK in 2012. To investigate the relationship of the pathogen's population structure to its mode of arrival, isolates were obtained from locations in England and Wales, either where established natural populations of ash had been infected by wind‐dispersed ascospores or where the fungus had been introduced on imported planting stock. Population structure was determined by tests for vegetative compatibility (VC), mating type and single‐nucleotide polymorphisms (SNPs). VC heterogeneity was high at all locations, with 96% of isolate pairings being incompatible. Frequencies of the MAT1‐1‐1 and MAT1‐2‐1 idiomorphs were approximately equal, consistent with H. fraxineus being an obligate outbreeder. Most SNP variation occurred within study location and there was little genetic differentiation between the two types of location in the UK, or between pathogen populations in the UK and continental Europe. There was modest differentiation between UK subpopulations, consistent with genetic variation between source populations in continental Europe. However, there was no evidence of strong founder effects, indicating that numerous individuals of H. fraxineus initiated infection at each location, regardless of the route of pathogen transmission. The ssRNA virus HfMV1 was present at moderate to high frequencies in all UK subpopulations. The results imply that management of an introduced plant pathogen requires action against its spread at the continental level involving coordinated efforts by European countries.     

Population structure,races, and host range of <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> from alfalfa production fields in the central USA

Aphanomyces euteiches (races 1 and 2) causes root rot of alfalfa; however, its population biology and distribution are poorly understood where alfalfa is a major crop. The objectives of this study were to (1) characterise the distribution and frequency of races of A. euteiches in Illinois alfalfa fields, (2) determine host range of A. euteiches on cultivated and native legumes, and (iii) to describe genetic diversity and population genetic structure of A. euteiches in alfalfa fields. To accomplish this, soil samples (n = 103) were collected from 30 alfalfa fields in 18 Illinois counties. Using the susceptible cv. ‘Saranac’, 148 isolates of A. euteiches were baited from the soil. The virulence phenotype of isolates representing all 18 counties was tested, and 54% were R1 and 46% were R2. Both races were detected in 61% of the counties, whereas only R1 was detected in 22% and R2 in 17%. Thirteen legume hosts for isolates from alfalfa fields were identified based on symptoms and/or production of oospores in roots. In addition to six previously known hosts, seven species were susceptible to infection: kura clover, purple prairie clover, white prairie clover, ladino clover, hairy vetch, Canadian milk vetch, and Illinois tick trefoil. AFLP analysis revealed high levels of genetic diversity among the isolates from different fields and counties and a lack of genetic structuring of populations based on race or geographical origin. The results suggest that populations of A. euteiches in alfalfa fields are diverse, often composed of races 1 and 2, and create risk for alfalfa and to multiple cultivated and native legume species.     

我国大麦条纹病菌(Pyrenophora graminea)群体遗传多样性AFLP分析

条纹病是重要的种传真菌病害之一,能对大麦生产造成严重产量损失。本研究利用AFLP方法对来自青海、河南等10省份大麦种植区的条纹病菌菌株进行遗传多样性分析,从分子水平上揭示我国不同大麦产区的条纹病菌群体遗传差异。结果显示,利用8对AFLP选择性引物组合对19份大麦条纹病菌进行了全基因组多态性扩增,共获得144条可统计的条带,其中112条具有多态性,多态性条带占77.8%。在相似系数为0.83时,可将19份大麦条纹病菌菌株划分为4个类群,多数菌株聚类在类群Ⅰ和类群Ⅱ中,类群Ⅲ和类群Ⅳ中仅各包含1个菌株,且与其他2个类群中的成员亲缘关系较远。因此,我国大麦条纹病菌群体中存在一定程度的遗传变异。另外,发现菌株间亲缘关系远近与其分布地域无明显规律。     

湖北省随州地区稻瘟病菌群体结构分析及生态模式菌群的建立

采用人工接种方法,用7个中国鉴别品种和95个自选资源材料对2014年至2015年间采自湖北省随州地区的76个稻瘟病菌单孢菌株进行致病型鉴定。分别利用传统生理小种种群划分方法和近邻传播聚类法(affinity propagation clustering,AP聚类法)进行菌群结构分析,比较两种方法的差异。结果表明,传统生理小种种群划分方法将76个菌株分为6个菌群,14个中国小种, ZB菌群为优势种群,占比65.79%,ZB15为优势生理小种,占比达到60.53%;近邻传播聚类法将76个菌株分为14个菌群,Cluster 2、Cluster 6、Cluster 7、Cluster 8为随州地区优势菌群,占比分别为18.42%、19.74%、22.37%、17.11%,Cluster 6、Cluster 7和Cluster 8具有相对较高的致病力;两种方法划分结果虽不完全一一对应,但划分趋势相同,近邻传播聚类法在稻瘟病菌种群结构研究中具有一定应用前景;根据试验结果筛选出1个代表随州地区稻瘟病总致病谱的菌群,菌群由12个菌株组成,将其命名为“随州生态模式菌群”。     

枯草芽孢杆菌SN-02抑菌物质的分离、纯化及结构测定

为明确生防菌株Bacillus subtilis SN-02产生的抑茵物质的种类和组成,对其进行分离、纯化及结构测定.发酵滤液经盐酸沉淀,有机溶剂提取,薄层层析显色分析,初步确定该活性物质是一种具有闭合肽键的脂肽类物质.经Pharmadex LH-20精制后得到纯度为95.7%的淡黄色结晶状物质,液质联用检测到活性成分是分子量为1080、1035和1021 D的生物表面活性素.紫外吸收光谱表明该物质在210nm处有吸收峰,红外吸收光谱证实该分子的亲水基是一条肽链,疏水基部分是一脂肪酸分子,且为一种环状脂肽类物质.氨基酸分析表明该活性物质由4种氨基酸组成,且4种氨基酸的摩尔比为Glu∶Leu∶Val∶Asp=1∶4∶1∶1.     

Genetic analysis of the population structure of the rice false smut fungus,Villosiclava virens,in China using microsatellite markers mined from a genome assembly

Studies on population genetics of Villosiclava virens are limited because of the lack of polymorphic markers. Based on a draft genome sequence of isolate HWD‐2 produced in this study, 20 of 403 potential simple sequence repeats (SSR) loci showed polymorphisms in preliminary screening using eight diverse V. virens isolates. Among polymorphic loci, most of them with tetra‐ to hexanucleotide unit motifs showed higher levels of polymorphism than loci with smaller motifs. After testing with 20 polymorphic SSR markers, the 87 isolates of V. virens from eight populations in China showed a high level of genetic diversity, with each as a unique haplotype. This differs from some previous findings showing little to no genetic variation based on random amplified polymorphic DNA and amplified fragment length polymorphism analyses. Among eight populations from major rice production areas of China, the population from Guangxi province in south China showed the highest levels of polymorphism, which led to the speculation that it might be closer to the centre of origin of this pathogen. The northern, central and eastern populations (Jilin, Liaoning, Hubei, Hunan, Jiangxi and Zhejiang), when considered together as a group, showed significant molecular variation compared to the southern populations (Fujian and Guangxi) (Φ_PT = 0·043, P = 0·037). A significant relationship (Mantel test, P = 0·027) but with low correlation (R² = 0·23) was also found between geographic distance and genetic distance. The 20 polymorphic SSR primer pairs designed in this study provide a tool for further research on the population diversity of this emerging fungal pathogen of rice.     

Genetic differentiation and gene flow in Colletotrichum sublineolum in Ethiopia,the centre of origin and diversity of sorghum,as revealed by AFLP analysis

Isolates of Colletotrichum sublineolum were collected from different sorghum‐producing regions of Ethiopia and divided into five groups based on their geographic origin. The growth rate of 50 isolates showed considerable variation: 1·7–5·8 mm day^?1, mean 3·3 mm day^?1. However, the isolates displayed little variation in colony colour and colony margin, except for isolates from the north, which were different from the others. Amplified fragment length polymorphism analysis of 102 isolates revealed much greater variations among the different groups. Dice similarity coefficients ranged from 0·32 to 0·96 (mean 0·78). Cluster analysis and principal coordinate analysis revealed a differentiation of the isolates according to their geographic origin, and both methods clearly indicated a genetic separation between the southern, the eastern and the other isolates. Analysis of molecular variance (amova ) indicated a high level of genetic variation both among (42%) and within (58%) the C. sublineolum sampling sites in Ethiopia. The amova also indicated a high level of genetic differentiation (F_ST = 0·42) and limited gene flow (Nm = 0·343). The results of this study confirmed the presence of a highly diverse pathogen, which is in agreement with the existence of diverse host genotypes and widely ranging environmental conditions in sorghum‐producing regions of the country. Such diversity should be taken into account in future breeding programmes to achieve an effective and sustainable disease management strategy.