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.     

Development of IRAP-SCAR marker for strain identification in Lentinula edodes

Rapid and correct authentication of commercial strains is still important in today’s mushroom industry. Here for the first time we reported the using of sequence characterized amplified region (SCAR) marker developed from inter-retrotransposon amplified polymorphism (IRAP) marker to identify Lentinula edodes strain. Genomic polymorphism among 44 shiitake cultivars in China were surveyed by 24 IRAP primer combinations, from which primer combination LTR1L/MarY1R could generate a unique and reproducible 1712 bp fragment to distinguish strain No. 4 from other 43 strains. Based on this strain-specific fragment, a SCAR primer pair was designed and its validity was verified by correctly amplifying a single strain-specific fragment from DNA samples of 100 L. edodes strains. Our study lays the foundation for developing strain-specific SCAR marker by retrotransposon-based marker technique in fungi.     

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.     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.     

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.     

Development of SCAR marker for simultaneous identification of Miscanthus sacchariflorus, M. sinensis and M. x giganteus

The sequence-characterized amplified region (SCAR) marker for simultaneous identification of Miscanthus sacchariflorus, Miscanthus sinensis, and Miscanthus x giganteus was developed. In this study, it was attempted for the first time to develop the SCAR marker for detecting the molecular phenotypes among Miscanthus species. Randomly amplified polymorphic DNA technique was applied for this study and one fragment which is unique to M. sacchariflorus was identified and then sequenced. Based on the specific fragment, one SCAR primer pair designated as MS62-5F and MS62-5R was designed to amplify an approximately 1,000 bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. Using this SCAR marker, approximately 1,000 bp and 1,200 bp DNA fragments were obtained in M. sacchariflorus and M. sinensis, respectively. Moreover, M. x giganteus was obtained both bands at the same time. The result showed that this SCAR marker can clearly distinguish the M. sacchariflorus, M. sinensis, and M. x giganteus, respectively.     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.     

Isolation and characterization of an AHL lactonase gene from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

An AHL lactonase gene (aiiA) was PCR amplified from the genomic DNA of Bacillus amyloliquefaciens, with the intact open reading frame of 753 base pair. The gene shares high identity to its homologues present in different Bacillus species. The expression plasmid carrying a tact aiiA-PEBA gene was constructed and the gene was overproduced in Escherichia coli BL21 (DE3). The product expressed resulted in attenuation and suspension of the infection of Pectobacterium carotovorum subsp. carotovorum on carrot. This study verified the existence of the aiiA gene in B. amyloliquefaciens and provided a prospect of the strain as biocontrol agents with quorum quenching property on bacterial disease control.     

菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

Identification of a male-specific AFLP marker in a functionally dioecious fig,<Emphasis Type="Italic"> Ficus fulva</Emphasis> Reinw. ex Bl. (Moraceae)

A male-specific amplified fragment length polymorphism (AFLP) marker was identified in the functionally dioecious fig species, Ficus fulva. A total of 89 polymorphic fragments from three primer combinations were produced, of which one (246 bp) was present in all males (n=23) and absent in all females (n=24) of two populations. This strong association suggests a tight chromosomal linkage between the AFLP marker and the sex-controlling locus. Further analysis indicated that the marker segregated in open-pollinated progenies from natural populations in a 1:1 ratio (n=156), implying that males are the heterogametic sex. Chromosome preparations showed no evidence for morphologically distinct sex chromosomes. The low frequencies of associated markers argue against a morphologically cryptic non-recombining sex chromosome. The sex-locus is therefore likely to be autosomal. The male-specific AFLP marker was sequenced and converted into a sequence characterised amplified region (SCAR) marker. This SCAR marker produced a fragment of equal size in males and females, suggesting that sequence divergence between male- and female-specific chromosomal regions is low.Publication 3311 NIOO-KNAW Netherlands Institute of Ecology     

Characterization of a RAPD fragment unique to species with hairy fruit skin in the genus<Emphasis Type="Italic">Actinidia</Emphasis>

To develop a SCAR primer related to the hairy-fruit trait in the genusActinidia, we took a PCR-RAPD approach using arbitrary 10-mer primers. PCR with the UBC 376 primer generated specific fragments from three species with hairy fruit skin. Those fragments were then cloned to determine their nucleotide sequences. Two SCAR primers were designed from the UBC 376 primer and nucleotide sequences were obtained from the PCR fragments. A SCAR primer, OKC385, specifically amplified a 385-bp fragment from one clone ofActinidia eriantha, four ofActinidia chinensis, and four ofActinidia deliciosa. Deduced amino acid sequences of this fragment showed high sequence homology with plant cellulose synthases, which are involved in the biosynthesis of cellulose, a major cell wall component. The 385-bp fragment was specifically detected only in the seriesPerfectae C.F. Liang of sectionStellatae Li. This type has many hairs on the leaves, fruits, and stems, suggesting that the gene containing the PCR fragment is involved in hair formation in this phylogenetic group. Taken together, our results suggest that the SCAR primer, OKC385, can be used as a specific primer for early selection of the non-hair trait in breeding of the genusActinidia.     

Molecular detection of Colletotrichum falcatum causing red rot disease of sugarcane (Saccharum officinarum) using a SCAR marker

Red rot disease of sugarcane caused by Colletotrichum falcatum is one of the most destructive diseases of sugarcane (Saccharum officinarum) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease‐free planting materials is essential for preventing disease development in the field. In the present study a polymerase chain reaction (PCR) assay was developed for accurate and sensitive detection of C. falcatum in planting materials. Randomly amplified polymorphic DNA (RAPD) analysis identified a 566 bp PCR fragment that was specific to C. falcatum. The DNA sequence of this fragment was determined and used to design oligonucleotides amplifying a 442 bp sequence characterised amplified region (SCAR). The specificity of the SCAR primers was evaluated using purified DNA from C. falcatum and other Colletotrichum spp. as templates in PCR. The results indicated that the SCAR primers were highly specific to C. falcatum since the 442 bp fragment was amplified only from DNA of isolates and races of C. falcatum but not from any other Colletotrichum spp. tested. The detection sensitivity of C. falcatum was 0.1 ng for genomic DNA of C. falcatum and 5 ng for DNA extracted from infected sugarcane tissue. This new PCR‐based assay is a convenient tool for detection of this important pathogen in seed canes to ensure production of sugarcane.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

Development of a species-specific AFLP-based SCAR marker for authentication of black muntjac (Muntiacus crinifrons)

Black muntjac is a rare and endangered deer endemic to eastern China. Due to the economic and pharmaceutical value of the meat, antlers and skin, the species has chronically suffered from poaching though it is regarded as the state key protected animal. To provide an effective molecular method for authentication of tissue specimen (such as meat, skin etc.) of the species, we developed a Sequence Characterized Amplified Region (SCAR) derived from a species specific Amplification Fragment Length Polymorphism (AFLP) marker. Initially, a 707-bp species specific DNA fragment of the animal was detected by a pair of AFLP primers (Ep7/Mp8). Subsequently, a species-specific primer pair (P-F/P1-R) was designed based on the specific AFLP fragment sequence, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified by two separate PCRs using the designed SCAR primers and a cyt b universal primer pair. As expected, all black muntjac samples presented two bands but the others failed to produce the SCAR by merely showing one band. Our results indicated that the SCAR primers developed in this study may provide a useful tool for forensic authentication of black muntjac samples though further testing with larger sample sizes is warranted.     

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.     

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan® PCR

Aims: To develop a strain‐specific TaqMan^® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan^® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan^® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.     

耐盐绒毛白蜡特异性SCAR分子标记的鉴定

该研究以耐盐型和盐敏感型绒毛白蜡及其F1代为材料,采用混合品系分析法进行RAPD分析。结果显示:在随机选取的150个10碱基随机引物中,仅有引物S20在耐盐基因池和盐敏感基因池间扩增出特异而可重复的592bp的多态性片段,命名为S20-592。获得的RAPD标记S20-592经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记。通过F1代个体验证,耐盐型个体均能扩增出此差异条带而盐敏感型个体中不能扩增出此差异条带,证明该SCAR标记的特异引物可用于耐盐绒毛白蜡物种的快速分子鉴定。