RAPD and SCAR Markers Linked to a Gene Conferring Resistance to Angular Leaf Spot in Common Bean

Angular leaf spot, caused by the fungus Phaeoisariopsis griseola , is one of the most important bean diseases in Brazil. The objectives of this study were to determine the inheritance of angular leaf spot resistance and to identify random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) markers linked to the resistance gene present in cv. Cornell 49-242, in a cross between this cultivar and susceptible cv. Rudá. The parents, F₁, F₂ and backcross-derived plants were inoculated with P. griseola pathotype 31-17 under environmentally controlled greenhouse conditions. The results indicate that one single dominant gene controls the resistance in Cornell 49–242. Two RAPD markers, OPN 02_890c and OPE 04_650c, were found to be linked in the coupling phase, at 3.2 and 12.5 c m of the resistance gene, respectively. To increase the reproducibility of the detection of marker OPN02_890c it was converted into a SCAR marker. It is proposed that the designation of Phg-2 be used for the angular leaf spot resistant gene present in Cornell 49-242.     

Identification of a specific scar marker for detection of Tilletia foetida (Wall) Liro pathogen of wheat

Common bunt is one of the most important destructive diseases of wheat worldwide and is a domestic quarantined disease in China. However, a rapid and efficient method to identify the corresponding pathogens is currently limited. The objective of the present study was to develop a diagnostic molecular marker specific towards Tilletia foetida (Wall) Liro, a causal agent of the bunt disease. One specific DNA fragment for T. foetida (286 bp in length) was amplified using an Amplified Fragment Length Polymorphism (AFLP) assay and, this fragment was cloned and sequenced. One pair of specific primers (SC(286-1)/SC(286-2)), which was designed according to the sequence, could specifically amplify the corresponding fragment in all of the T. foetida isolates employed from both the People's Republic of China and United States, whereas this fragment could not be amplified by the other fungal species tested. Therefore, a specific Sequence Characterized Amplified Region (SCAR) marker was developed. This SCAR marker could distinguish T. foetida from related pathogenic fungi efficiently and could be used for the early diagnosis of the common bunt of wheat in the field, and provide an efficient way for disease surveillance and disease forecasting in cereal crop.     

Development of a SCAR marker by inter-simple sequence repeat for diagnosis of dwarf bunt of wheat and detection of <Emphasis Type="Italic">Tilletia controversa</Emphasis><Emphasis Type="SmallCaps">Kühn</Emphasis>

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important internationally quarantined disease in many countries. The primer ISSR818 generated a polymorphic pattern displaying a 867-bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF3/TCKSR3) designed for use in PCR detection assays; they amplified a unique DNA fragment in all isolates of T. controversa but not in the related pathogens. The detection limit with the primer set (TCKSF3/TCKSR3) was 5 ng of DNA which could be obtained from 5.5 μg of teliospores in a 25-μL PCR reaction mixture.     

Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

A genetic linkage map of the sea cucumber, Apostichopus japonicus (Selenka), based on AFLP and microsatellite markers

We present the first genetic maps of the sea cucumber ( Apostichopus japonicus ), constructed with an F₁ pseudo-testcross strategy. The 37 amplified fragment length polymorphism (AFLP) primer combinations chosen identified 484 polymorphic markers. Of the 21 microsatellite primer pairs tested, 16 identified heterozygous loci in one or other parent, and six were fully informative, as they segregated in both parents. The female map comprised 163 loci, spread over 20 linkage groups (which equals the haploid chromosome number), and spanned 1522.0 cM, with a mean marker density of 9.3 cM. The equivalent figures for the male map were 162 loci, 21 linkage groups, 1276.9 and 7.9 cM. About 2.5% of the AFLP markers displayed segregation distortion and were not used for map construction. The estimated coverage of the genome was 84.8% for the female map and 83.4% for the male map. The maps generated will serve as a basis for the construction of a high-resolution genetic map and mapping of the functional genes and quantitative trait loci, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.     

An ISSR‐based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat,Caused by Tilletia controversa Kühn

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is an important international quarantine disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from intersimple sequence repeat (ISSR) for rapid identification of T. controversa. A total of 60 primers were tested by ISSR to detect DNA polymorphisms between T. controversa and related species. The primer ISSR818 generated a polymorphic pattern displaying a 952‐ bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF2/TCKSR2) were designed for use in a PCR detection assay. Its detection limit was 1 ng of DNA, which could be yielded by 1.1 μg of teliospores in a 25‐ μl PCR. Conclusively, a method to distinguish T. controversa from similar pathogenic fungi has been successfully developed based on the use of a SCAR marker.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

Development of a PCR marker for rapid identification of the Bt-10 gene for common bunt resistance in wheat.

In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based on the segregation analysis. Segregation analyses of Bt-10 and the 275-bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3:1 for the molecular marker in two of the populations. These results demonstrated that the PCR marker system using the FSD and RSA primer pair permitted a rapid and reliable identification of individual lines carrying the Bt-10 gene for resistance to common bunt.     

Response of plankton communities to whole-lake Ca(OH)2 and CaCO3 additions in eutrophic hardwater lakes

1. The impact of whole-lake lime (slaked lime, Ca(OH)₂, and/or calcite, CaCO₃) addition on plankton communities was evaluated in eutrophic hardwater lakes on the North American Boreal Plain.
2. Two lakes received a single treatment of lime (Ca(OH)₂ at 74 or 107 mg L^–1), two lakes received multiple treatments with Ca(OH)₂ and/or CaCO₃ (5–78 mg L^–1), and four lakes were untreated and served as reference systems.
3. Over the long-term (> 1 year), phytoplankton biomass was reduced in multiple-dose lakes, but not in single-dose lakes. Cyanobacteria typically dominated the algal community in the years before, during and after lime treatment in both single- and multiple-dose lakes.
4. In the single-dose lakes, randomized intervention analysis showed no significant change in the biomass of zooplankton after lime addition.     

Evaluation of genetic diversity and germ plasm identification of 44 species, clones, and cultivars from 5 sections of the genusPopulus based on amplified fragment length polymorphism analysis

The genusPopulus L. (Salicaceae) can be divided into 5 sections with distribution throughout the world. Accurate identification ofPopulus clones and species is essential for effective selection, breeding, and management of genetic resources. In this study, amplified fragment length polymorphism (AFLP) analysis, which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct analyses of genetic diversity and variety identification of 44 species, clones, and cultivars ofPopulus that represent a wide range of breeding and commercially available germplasms. Cluster analysis of the 44 samples was carried out, and a dendrogram of genetic relatedness was developed on the basis of the AFLP data. DNA fingerprints of the 44 samples were developed from 12 selected bands amplified with 2 primer combinations (M-CAG/E-TA and M-CAG/E-TC). Each sample has its unique fingerprint pattern and can be distinguished from the others. Furthermore, 1 specific AFLP band of the cultivarPopulus canadensis cl. Guariento coming from fragments amplified by primer combination M-CTC/E-AG was successfully converted into a sequence-characterized amplified region (SCAR) marker. The results indicate that AFLP analysis should be considered as the preferred technique for the study of polymorphism inPopulus. This research is the first report concerning the use of AFLP analysis in genetic diversity and germplasm identification among all sections ofPopulus.     

A qRT-PCR-based method for the measurement of rrn operon copy number

Aim:  To develop a convenient and accurate method for estimating the rrn operon copy number ( Y _rrn ) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR).
Methods & Results:  Using Escherichia coli, the Y _rrn of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers ( C _t ), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y _rrn of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli . Using this method, the Y _rrn values of four species, i.e. Xanthomonas campestris , Staphylococcus aureus , Aeromonas hydrophila and Pseudomonas fluorescens , were estimated as 1·80, 4·73, 8·58 and 5·13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%.
Conclusions:  The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells.
Significance and Impact of the Study:  qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.     

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

番茄抗青枯病基因的AFLP分子标记

用番茄高抗青枯病品种“T51A”与高感青枯病品种“T9230”配制杂交组合,接种鉴定其正反交Ｆ₁代及Ｆ₂代分离群体的青枯病发生情况。结果表明,T51A对青枯病的抗性属于细胞质遗传,受1对杂合基因加性控制。用64个EcoRＩ/ＭseＩ引物组合对“T51A”、“T9230”两个亲本及其Ｆ₂代抗病和感病基因池进行AFLP分析,共扩增出约4200条可分辨的带,其中2条为稳定的差异。用“T51A”和“T9230”杂交产生的Ｆ₂代分离群体对2个特异条带与目的基因的遗传连锁性进行分析,发现特异条带AAG/CAT与暂定名为RRS-342的抗青枯病基因紧密连锁,二者之间的遗传距离为6.7 cM。将AAG/CAT片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对番茄青枯病基因的标记辅助选择。      

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Last male sperm precedence in a damselfly demonstrated by RAPD profiling

We used the random amplified polymorphic DNA technique to determine last male sperm precedence ( P ₂) in the damselfly Calopteryx splendens xanthostoma (Charpentier). We amplified DNA from mothers, putative fathers and from the embryos of individual offspring, and subsequently calculated band-matching coefficients between known first-order relatives (offspring within a clutch) and non-relatives (mothers and fathers) to estimate last-male paternity. The data indicate that, as in other Calopterygidae, P ₂ is high (0.98) in the bout of oviposition immediately following copulation, despite the fact that the males of this species do not completely remove the sperm of previous males (Siva-Jothy & Hooper 1995).     

利用小麦微卫星引物建立簇毛麦染色体组特异性标记

选位于普通小麦1A-7A、1B-7B、1D-7D染色体上的102对微卫星引物对多年生簇毛麦、二倍体簇毛麦、小麦-簇毛麦双二倍体与后代和普通小麦中国春、R25、R111、MY11进行了PCR扩增, 发现引物对Xgwm301可以在含簇毛麦染色体的材料中扩出一条长415 bp的特异片段(命名为Xgwm301/415), 而所有供试小麦均未扩出此片段。进而用一套中国春-二倍体簇毛麦附加系来进行扩增, 发现1V-7V染色体均可以扩出该片段, 说明该片段为簇毛麦1V-7V染色体所共有。因此, Xgwm301/415是簇毛麦染色体组上的一个特异片段, 可以用来快速跟踪检测导入到普通小麦背景中的簇毛麦染色体。     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism (AFLP)

The utility of the new polymerase chain reaction (PCR)-based multilocus DNA fingerprinting technique amplified fragment length polymorphism (AFLP) for paternity analysis in natural plant populations was assessed. In a natural population of 25 plants of Persoonia mollis (Proteaceae), three AFLP primer pairs generated 147 dominant loci. Of these, 125 (85%) were polymorphic, with a mean recessive allele frequency of 0.735. The theoretical expected percentage of offspring for which all males except the true father can be excluded ( P _ET) was 99.9% for this population. The estimates of P _ET drop marginally to 99.6% and 97.6% for larger populations of 100 and 1000 individuals, respectively. A preliminary investigation confirmed the power of AFLP for paternity analysis by assigning paternity, or excluding all known potential sires, for 242 of 252 (96.0%) naturally pollinated seeds. Ambiguous paternity for the remaining 10 seeds was quickly resolved by utilizing two further AFLP primer pairs, ultimately generating over 200 polymorphic loci and resulting in the exclusion of all nonsires for all 252 (100%) seeds. This study highlights the utility of AFLP for paternity analysis because: (i) it generates sufficiently large numbers of highly reproducible polymorphic loci, that are (ii) quickly and accurately scored using an automated DNA sequencer and dedicated software, and (iii) unlike microsatellites, requires no sequence knowledge so it is more easily applied to new study species.