Detection of the biocontrol agent Colletotrichum coccodes (183088) from the target weed velvetleaf and from soil by strain-specific PCR markers

Diagnostic molecular markers, generated from random amplified polymorphic DNA (RAPD) and used in polymerase chain reaction (PCR), were developed to selectively recognize and detect the presence of a single strain of the biocontrol fungus Colletotrichum coccodes (183088) on the target weed species Abutilon theophrasti and from soil samples. Several isolates of C. coccodes, 15 species of Colletotrichum, a variety of heterogeneous organisms and various plant species were first screened by RAPD-PCR, and a strain specific marker was identified for C. coccodes (183088). No significant sequence similarity was found between this marker and any other sequences in the databases. The marker was converted into a sequence-characterised amplified region (SCAR), and specific primer sets (N5F/N5R, N5Fi/N5Ri) were designed for use in PCR detection assays. The primer sets N5F/N5R and N5Fi/N5Ri each amplified a single product of 617 and 380 bp, respectively, with DNA isolated from strain 183088. The specificity of the primers was confirmed by the absence of amplified products with DNA from other C. coccodes isolates, other species representing 15 phylogenetic groups of the genus Colletotrichum and 11 other organisms. The SCAR primers (N5F/N5R) were successfully used to detect strain 183088 from infected velvetleaf plants but not from seeded greenhouse soil substrate or from soil samples originating from deliberate-released field experiments. The sensitivity of the assay was substantially increased 1000-fold when nested primers (N5Fi/N5Ri) were used in a second PCR run. N5Fi/N5Ri selectively detected strain 183088 from seeded greenhouse soils as well as from deliberate-released field soil samples without any cross-amplification with other soil microorganisms. This rapid PCR assay allows an accurate detection of C. coccodes strain 183088 among a background of soil microorganisms and will be useful for monitoring the biocontrol when released into natural field soils.     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.     

Fine mapping of the rice Bph1 gene, which confers resistance to the brown planthopper (Nilaparvata lugens stal), and development of STS markers for marker-assisted selection

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.     

RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different.

Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period.     

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

陆地棉HB红花性状特异SCAR分子标记的开发

利用Operon系列引物筛选到1个与HB红花性状基因连锁的RAPD标记OPA15^1160,对差异条带进行克隆与核苷酸测序,根据测序结果设计SCAR引物,在HB红花近等基因系及其白花轮回亲本中进行PCR扩增程序优化和鉴定,筛选出一对引物可稳定扩增出与HB红花性状基因连锁的特异片段,获得了与HB红花性状基因紧密连锁的SCAR标记HB^-330。利用具黄色花瓣紫红色基斑的海岛棉与粉红花瓣的红叶棉等种质材料以7LHB红花近等基因系与白花轮回亲本杂交的F1、BC1F1、F2群体,对该SCAR标记的特异性与准确性进行了鉴定与验证,在红花植株中扩增出了330bp大小的片段而在白花植株中未扩增出,证明该标记准确性高、重复性好。HB红花是通过远缘杂交转自野生二倍体比克氏棉的性状,已成功地应用于性状标记杂交棉育种。该SCAR标记不仅为HB红花标记杂交种的纯度鉴定提供了有效技术手段,也为新品种保护提供了技术支持,促进了红花性状杂交种的分子标记辅助育种进程。     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.     

Design of grapevine (Vitis vinifera L.) cultivar-specific SCAR primers for PCR fingerprinting

Among 34 grapevine cultivars (Vitis vinifera L.), eight putative genotype-specific RAPD markers, from ’Albariño’, ’Caíño blanco’, ’Chardonnay’, ’Folle blanche’, ’Grenache blanc’, ’Malvasía Sitges’, ’Torrontés’ and ’Treixadura’ respectively, were selected to transform into SCAR markers. Of these, seven markers were cloned and then five which showed a positive specific hybridization signal were sequenced. For these five markers, 30 sequence-specific primers ranging from 14 to 29 bases were designed to amplify genomic DNA from 64 grapevine cultivars under more-stringent PCR conditions. Only, two primer pairs, OpA11₁₁₇₅p17R/ p17F and OpD10₈₀₀p14R/p14F, still produced a specific SCAR marker, the ’Folle blanche’ ScA11₁₁₇₅ and the ’Malvasía Sitges’ ScD10₈₀₀ respectively. Moreover, the ScA11₁₁₇₅ marker was amplified only in ’Folle blanche’ among the 64 cultivars tested with a large annealing temperature range using either two different Taq DNA polymerases or two separate thermocyclers. In addition, we discuss the initial polymorphism originated by the RAPD technique and suggest a new design of SCAR primers to obtain reliable cultivar-specific SCAR markers from single PCR-based bands for identification purposes.     

葡萄感霜霉病基因的分子标记(英文)

 在葡萄抗病育种中 ,幼苗期排除感霜霉病的后代具有特别重要的意义 .用 BSA,RAPD和SCAR方法研究了葡萄感霜霉病基因的分子标记 .分析了两个种间杂交组合 [毛葡萄 (抗病 )×欧洲葡萄 (感病 ) ]88- 1 1 0和 88- 84与 88- 1 1 0的 F1代自交或互交所得的 3个 F2 代 ,以及欧洲葡萄品种和中国野生葡萄种 .共筛选了 2 80个随机引物 .引物 OPO1 0产生了一个 RAPD标记 OPO1 0 - 80 0与葡萄感霜霉病主效基因紧密联锁 .将该 DNA片段克隆并测序 .OPO1 0 - 80 0的实际长度为 835bp,所以 OPO1 0 - 80 0应为 OPO1 0 - 835.据其两端序列 ,设计了一对长度为 2 6bp和 2 8bp的特异引物分别扩增上述试材 ,获得了与该 RAPD标记相同大小的一条带 ,将 RAPD标记转化为 SCAR标记SCO1 0 - 835.并证实了此 SCAR标记的通用性 ,该 SCAR标记可用于葡萄抗病育种中杂种后代对霜霉病的抗病与感病性鉴定 .     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strain's virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.     

黄瓜霜霉病抗病基因的RAPD及SCAR标记

以感霜霉病黄瓜L18-10-2和抗霜霉病黄瓜129为亲本构建F2代分离群体,以F3代植株霜霉病抗性鉴定表示F2代各单株抗病性并得以区分各单株杂合或纯合感病性,采用RAPD技术和转SCAR的方法筛选黄瓜抗霜霉病基因分子标记.结果显示,在318条RAPD引物中有18条引物表现出两亲本间多态性,其中引物P18的SB-SP18561扩增片段与霜霉病抗病基因之间紧密连锁,根据交换率和Kosambi函数公式计算其遗传距离为7.85 cM.回收SBSP18561片段并克隆和测序,其准确长度为561 bp.将该RAPD标记转换为SCAR标记,长度为494 bp,命名为SSBSP18494.     

与小麦白粉病抗性基因Pm2紧密连锁RAPD标记的筛选研究

以２５６个随机引物对含小麦抗白粉病基因Ｐｍ２近等基因系进行ＲＡＰＤ分析,发现１７个随机引物的扩增产物在抗、感ＮＩＬｓ材料间表现多态性,且其中５个引物经４次以上重复,均获相同结果,其多态性标记分别为ＯＰＭ０８(１６００)、ＯＰＩ０４(１７００)、ＯＰＨ１９(１１００、ＯＰＥ０９(９００)及ＯＰＭ１６(８５０)。当以这５个随机引物对１４个已知含Ｐｍ２基因的抗病材料及９个不含Ｐｍ２基因的感病材料进行检测时,只有标记ＯＰＩ０４(１７００)在１２个抗病材料中出现（另两个抗病材料中未检测到）,而在９个感病材料中均未出现。进一步用 ＯＰI(０４)对１０２株（Ｃｈａｎｃｅｌｌｏｒｘ Ｕｋａ／８＊Ｃｃ）Ｆ２分离群体进行分析,估算出标记ＯＰＩ０４(１７００)与Ｐｍ２基因间的遗传距离为１２．２±３．３ｃＭ。     

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.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.     

SCAR markers for discriminating species of two genera of medicinal plants, Liriope and Ophiopogon

The development of DNA markers that can closely discriminate between Liriope and Ophiopogon species is vital for efficient and accurate identification of these species, and to ensure the quality, safety, and efficacy of medicines made from these plants. We developed species-specific molecular markers for these two genera. Forty RAPD primers were tested to detect polymorphism; species-specific RAPD bands were gel-purified, cloned, and sequenced. Primers for sequence-characterized amplified regions (SCARs) were then designed, based on nucleotide sequences of specific RAPD primers. SCAR markers SA06 and SB05, specific to Ophiopogon japonicus, amplified 460- and 553-bp DNA fragments, respectively. The marker SA12 amplified a 485-bp fragment specific to Liriope platyphylla. This is the first report of a species-specific SCAR marker for this group. These markers will be useful for rapid identification of closely related Liriope and Ophiopogon species.