基于RAPD标记的福建省稻曲病菌遗传多样性分析

为了解福建省稻曲病菌群体的遗传多样性和遗传组成,应用随机扩增多态性RAPD (random amplified polymorphic DNA)技术分析了来自福建省不同水稻种植区的102个稻曲病菌(Ustilaginoidea virens)的遗传多样性.从100条随机引物中筛选出10条扩增带清晰、重复性好的引物进行稻曲病菌多态性扩增,共扩增出157条带,多态性条带比率为82.17％,遗传距离变化范围为0.02～0.67.遗传多样性分析表明,福建省稻曲病菌具有丰富的遗传多样性,相对于其它地区而言,福建闽西地区分离的菌株遗传多样性水平最高PPB=76.43,H=0.2212,I=0.3383),晚稻分离的菌株群体遗传多样性(PPB=91.08,H=0.2402,I=0.3655)高于早稻群体(PPB=63.06,H=0.1892,I=0.2870).聚类分析显示,在遗传距离0.349水平上,供试的所有菌株可被划分成7个遗传聚类组(R1～R7),聚类组R1为优势聚类组,包含有80个菌株,其内又存有一些亚组.这些菌株的聚类与菌株的地理来源及水稻品种无明显相关性.但是在遗传距离0.330水平上,来自宁化的10个菌株可被明显划分成早稻群体和晚稻群体.初步分析认为菌株的地理来源、水稻品种及其生长季节是影响福建省稻曲病菌遗传多样性的主要因素,在稻曲病菌的遗传变异以及该病的发生和流行中可能起重要的作用.     

香稻品种RVA谱多样性研究

对来自不同种植区域的593份香稻材料(香糯稻152份,香籼稻342份,香粳稻99份)进行了RVA谱的多样性分析。结果表明,香糯稻以最终粘度的变异系数最大,为42.9,香籼稻和香粳稻以回生值的变异系数最大,分别为71.9和76.1。按遗传多样性指数(Shannon-wiener多样性指数)的分级标准计算,不同种质资源RVA谱特征值的分级情况复杂,除了峰值时间分布相对集中外,其他特征值的分级情况差别较大。RVA谱各特征值的遗传多样性指数均大于1.0,香糯稻峰值粘度和最终粘度的遗传多样性指数高达2.02和2.01,香籼稻和香粳稻均以回生值的遗传指数最高,分别为2.09和1.99。不同香稻种质间的淀粉糊化特性潜在分异明显。     

不同致病型水稻细条病菌的遗传多样性分析

采用引物BOX和ERIC对来自4省的35个细条病菌菌株进行Rep-PCR扩增,BOX引物扩增出14条指纹带,10种谱型,以致病型为单位计算遗传多样性值为0.63～1.00;ERIC引物扩增出19条指纹带,17种谱型,遗传多样性值为0.86～1;引物ERIC比BOX在遗传多样性方面有更好的分辨率;树状聚类图反映的遗传分簇差异,与菌株的致病型及其地理的来源之间没有相关性。     

我国南方部分稻区稻瘟病菌的群体结构

用重复序列探针ＭＧＲ－５８６，与限制性内切酶ＥｃｏＲＩ组合，分析了我国南方部分稻区８６个稻瘟病菌株的限制性片段长度多态性（ＲＦＬＰｓ）依其ＭＧＲ－ＤＮＡ指纹的相似性，结合病菌的致病型测定，将表现为２８个不同致病型的８６个稻瘟病菌株区分为１８个系谱，每个系谱的寄主范围有限，且显示出不同栽培稻区内稻瘟病菌的群体结构有明显差别。本研究通过分析一稻区内稻瘟病菌的群体遗传结构，用分子生态学的方法探索病流行的     

枯草芽孢杆菌SO113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化

枯草芽孢杆菌（Bacillus subtilis)SO113对水稻白叶枯病菌（Xanthomonas oryzaepv.oryzae）有强烈的抑菌作用（表现出一透明抑菌圈）。培养液上清中加入60％饱和度的硫酸铵沉淀所得的蛋白粗提液对热稳定，对链霉蛋白酶E不敏感，对蛋白酶K部分敏感，对中国各稻区白叶枯病菌的7种致病型都有强烈的抑杀作用，蛋白粗提液100℃加热15min后脱盐，经DEAE0－Sepharose Fast Flow柱层析得3个未分开的抗菌活性峰，示吸附部分经CM－Sepharose Fast Flow柱层析，得到1个主活性峰，由于B.subtilis SO113的分泌蛋白对水稻白叶枯病菌表现出良好的广谱抗性以及丰富的抗菌活性峰，因此对其进一步的研究和设法隆编码抗菌蛋白的基因都具有重要意义。     

水稻9311及其突变体响应白叶枯病菌的活性氧变化

为筛选水稻白叶枯病敏感型突变体材料,并确定其与白叶枯病菌互作早期活性氧代谢规律及其与抗病性的关系,用11个白叶枯病菌小种于分蘖期对野生型9311及其10个辐射突变体进行抗性评价,以接种后的病斑长度为依据,聚类分析发现突变体189抗病性与野生型差异最大,进而以突变体189为材料,比较研究接种P6小种后的活性氧变化。结果显示:接种后24 h,9311的O2·-和H2O2释放量达峰值,分别为突变体的120%和134%,差异达极显著水平;P6菌在189和9311叶中的生长趋势基本相同。推测9311可能是因高积累的活性氧分子抑制了白叶枯病菌在叶片中的扩展,从而限制了病斑长度。     

金福菇菌株的酯酶同工酶分析

本文采用酯酶同工酶分析和聚类分析技术,对15个金福菇菌株的遗传多样性和亲缘关系进行分析鉴定,以便为金福菇的遗传育种提供依据。试验结果表明：共检测到18条谱带,其中有2条谱带的分布频率为100%,是所有菌株的特征谱带;15个菌株共有14种酶带类型,不同菌株的酶带数在8条至12条之间。聚类分析结果表明,15个菌株间的遗传相似系数在0.33~1.00之间,在相似系数为0.62时,可将15个金福菇菌株划为3大类群。老挝金福菇TgLo菌株与国内金福菇的亲缘关系最远;同一菌株的不同分离株如Tg1.1与Tg1.2、Tg10.1、Tg10.2与Tg10.3间的遗传相似系数在0.94以上;菌株Tg3与Tg8,Tg12与Tg10.2或Tg10.3间的遗传相似系数也达到0.94,可认为是同物异名。上述结果表明,酯酶同工酶方法可有效应用于金福菇的菌株鉴定及亲缘关系分析。     

东南亚62个籼型水稻亲本SSR遗传多样性分析

为探讨东南亚水稻亲本间的遗传多样性,以东南亚62份籼型水稻亲本为材料,选择29对水稻功能基因标记以及平均分布于12条染色体、多态性较高和条带清晰的72对SSR引物进行遗传多样性和聚类分析。结果表明,32对多态性引物在62份水稻材料中检测到201个等位基因,每个位点等位基因数(Na)变异范围为2~12,平均为6.281;有效等位基因数(Ne)变异范围为1.067~5.399,平均为2.867;多态信息含量(PIC)变异范围为0.061~0.789,平均为0.515;平均Shannon信息指数(I)为1.176,变幅为0.143~1.908;观察杂合度(Ho)为0.977,范围为0.936~1.000;期望杂合度(He)为0.439,范围为0.179~0.937;固定指数(Fis)的范围为0.882~1.000;基因流(Nm)的范围为0~0.0157。聚类分析表明,在遗传相似系数为0.805处,东南亚水稻亲本被分为6大类,同一国家大多数品种聚为一类。相对于普通分子标记,功能基因标记的平均等位基因数不高,其遗传多样性参数也低于普通分子标记,东南亚各国水稻品种间的亲缘关系较近,遗传多样性程度较低。研究结果为水稻育种亲本选配、新种质资源的创制以及杂种优势利用等提供了科学依据。     

水稻品种资源抗稻褐飞虱不同生物型和稻瘿蚊核心样品的研究

以抗稻褐飞虱不同生物型和稻瘿蚊中国Ⅳ型的水稻品种资源454份为试验材料,评价10个主要农艺性状,按不同的抗性情况和水平进行分组,应用聚类分析方法对每一组进行聚类,从聚成一类的品种中随机选取1份品种构成核心样品。核心样品由53个品种组成。通过对10个性状的6个特征值、多样性指数进行比较,核心样品基本代表了总样品的遗传变异范围。     

湖南典型茶树地理种群遗传多样性研究

本文利用RAPD分子标记技术，对4个湖南典型茶树地理种群的240个单株的遗传多样性、种群内和种群间的遗传变异进行研究，结果表明：21个10碱基随机引物共检测到226条谱带，其中多态性谱带为201条，占88.9%。遗传多样性分析结果显示：Shannon's多样性指数为0.43，74.7%的变异分布于群体内，而种群间变异占了25.3%；Nei's指数群体总基因多样度(HT)为0.37，群体内平均基因多样度(HS)0.28，群体间的基因多样度(HST)0.09，群体Nei's基因分化系数（GST）为0.23，说明76.7%的变异存在于种群间，群体内的变异占了总变异的23.3%，与Shannon's多样性指数相比基本一致，均表明种群内有较丰富的遗传变异；种群间的基因流（Nm）为0.74，显示种群间的基因交流有限。     

新疆灰霉病菌多态性及其致病力分化分析

灰霉病菌(Botrytis cinerea)是引起植物病害的重要病原。本研究将来自新疆3个主要生态区域的12个灰霉病菌菌株, 经人工接种于7个寄主的离体叶片上测定病原菌的致病性, 并采用RAPD技术分析了12个菌株的遗传多态性。研究结果表明, 12个灰霉菌株的致病力存在明显差异, 可分为强、中、弱3种致病型, 其中来自和田的黄瓜灰霉菌和伊犁特克斯的番茄灰霉菌致病力最强; 阿克苏番茄灰霉菌的致病力最弱。根据在不同寄主上的致病反应, 12个菌株被划分为4个菌群。RAPD分析结果表明, 在0.65阈值下, 12个灰霉菌株可分为4个菌群, 说明供试灰霉菌株间有高度的遗传分化。灰霉病菌致病力强弱与菌株寄主、地理位置均无直接相关性, 但灰霉菌株的遗传距离与菌株来源存在明显相关性。     

绿色豌豆蚜不同地理种群的遗传多样性

豌豆蚜是我国苜蓿上危害最为严重的害虫之一。利用微卫星标记研究了我国绿色型豌豆蚜10个地理种群的遗传相似性、基因分化、遗传距离与地理距离和海拔之间的关系及其基因结构。选取15对引物扩增300个个体,共检测到41个多态性条带,多态性条带百分率(PPB)为100%。10个豌豆蚜地理种群观测等位基因数(Na)为1.592 7,有效等位基因数(Ne)为1.356 9,Nei’s基因多样性指数为0.206 6,Shannon-Wiener指数(I)为0.307 6。新疆维吾尔自治区、陕西省、河南省种群的遗传多样性较高,内蒙古自治区、山东省、青海省种群相对较低。然而,10个地理种群豌豆蚜(绿色型)的遗传分化较高(Gst=0.399 6)。种群聚类分析结果显示,全部豌豆蚜种群明显聚为两大类群,山东省、河南省种群为一大类群,其余为另一大类群。Mentel检测表明,遗传分化与地理距离、海拔无显著相关性。我国豌豆蚜种群具有非常丰富的遗传多样性,应加强豌豆蚜的监测和治理。     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Conservation genetics of evolutionary lineages of the endangered mountain yellow-legged frog, Rana muscosa (Amphibia: Ranidae), in southern California

Severe population declines led to the listing of southern California Rana muscosa (Ranidae) as endangered in 2002. Nine small populations inhabit watersheds in three isolated mountain ranges, the San Gabriel, San Bernardino and San Jacinto. One population from the Dark Canyon tributary in the San Jacinto Mountains has been used to establish a captive breeding population at the San Diego Zoo Institute for Conservation Research. Because these populations may still be declining, it is critical to gather information on how genetic variation is structured in these populations and what historical inter-population connectivity existed between populations. Additionally, it is not clear whether these populations are rapidly losing genetic diversity due to population bottlenecks. Using mitochondrial and microsatellite data, we examine patterns of genetic variation in southern California and one of the last remaining populations of R. muscosa in the southern Sierra Nevada. We find low levels of genetic variation within each population and evidence of genetic bottlenecks. Additionally, substantial population structure is evident, suggesting a high degree of historical isolation within and between mountain ranges. Based on estimates from a multi-population isolation with migration analysis, these populations diversified during glacial episodes of the Pleistocene, with little gene flow during population divergence. Our data demonstrate that unique evolutionary lineages of R. muscosa occupy each mountain range in southern California and should be managed separately. The captive breeding program at Dark Canyon is promising, although mitigating the loss of neutral genetic diversity relative to the natural population might require additional breeding frogs.     

Genetic and phenotypic diversity and random association of DNA markers of isolates of the fungal plant pathogen Sclerotinia sclerotiorum from soil on a fine geographic scale

Sclerotia of the soil-borne plant pathogen Sclerotinia sclerotiorum were collected from 1 m² area of the top 1.27 cm layer of soil in an alfalfa field in southeastern Washington state of the US. Out of 272 sclerotia collected, 40 were randomly selected and analyzed for genetic diversity in terms of microsatellite loci, mycelial compatibility groups (MCGs) and phenotypic diversity using five phenotypic traits (fungicide sensitivity, oxalic acid production, growth rate, colony color and virulence). Sixteen microsatellite haplotypes and 15 MCGs were found among the 40 isolates. The isolates showed three colony colors (beige, dark and white) on Difco PDA and exhibited significant differences in growth rate, oxalic acid production, and sensitivity to three fungicides, benomyl, fluazinam and iprodione. However, these isolates did not show differences in their ability to colonize detached pea leaves. No apparent relationship among the neutral genetic markers and the phenotypic traits was detected. Two of the haplotypes accounted 40% of the isolates, suggesting isolates of these haplotypes might be better adapted to the environmental conditions in this alfalfa field. Several lines of evidence indicated high levels of genetic diversity and potential outcrossing within the population of S. sclerotiorum: 1) high likelihood of five genetic populations based on Bayesian probability and the presence of admixed isolates; 2) random association of alleles in every pair-wise linkage disequilibrium test among eight independent microsatellite loci; 3) discordances between microsatellite haplotypes and MCGs and 4) lack of correspondence among the genetic markers and phenotypic traits. Multilocus Index of Association test suggested that outcrossing occurs only within interbreeding subpopulations of S. sclerotiorum.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

Diversity of Paenibacillus polymyxa populations in the rhizosphereof wheat (Triticum durum) in Algerian soils

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an API50CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson’s Chi-squared test. The strains isolated in D70, Z26, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.