Sequence Characterized Amplified Region Markers Research Articles

Polymorphisms and Evolution of Solanum bulbocastanum Genes for Broad-Spectrum Resistance to Phytophthora infestans

For two decades, Solanum bulbocastanum Dun. has been challenging potato geneticists with its durable resistance to Phytophthora infestans Mont. de Bary, the causal agent of late blight, the most devastating potato disease. In this study, highly specific SCAR (sequence characterized amplified region) markers for three S. bulbocastanum genes of broad-spectrum resistance to P. infestans,RB/Rpi-blb1,Rpi-blb2 and Rpi-blb3, were employed to screen a vast collection of wild tuber-bearing species from several series of Solanum L. section Petota Dumort. and also potato hybrids and varieties. In S. bulbocastanum and S. stoloniferum Schlechtd. et Bche, the sequences of the markers Rpi-blb1-820 and Rpi-sto1-890, which recognize the LRR and CC regions respectively differed inconsiderably in the orthologous genes RB/Rpi-blb1 and Rpi-sto1. These two markers were tightly linked in diverse wild Solanum genotypes. These markers as well as RB-226, another marker recognizing the LRR region of the Rpi-blb1 gene, were obviously related to high resistance to P. infestans. One more marker recognizing the CC region of the gene, RB-629, was found in much wider range of Solanum genotypes; it was not related to resistance to P. infestans and is therefore considered non-specific. In addition to Rpi-blb1 = Rpi-sto1, the Rpi-blb2 and Rpi-blb3 genes were registered in both S. bulbocastanum and S. stoloniferum accessions. In most cases, three bulbocastanum genes were not linked together. The markers specific for Rpi-blb1 = Rpi-sto1 genes were found in the Solanum series Bulbocastana (Rydb.) Hawkes (genome designation BB) and Longipedicellata Buk. (genome designation AABB) and were absent from five Pinnatisecta Rydb. species also comprising genome B. The marker for Rpi-blb2 was found in the series Bulbocastana and Longipedicellata and also in three accessions of Pinnatisecta, while the Rpi-blb3 gene, the orthologue of widely spread R2 gene for resistance to P. infestans, was present in all three Solanum series. In contrast with Rpi-blb1 = Rpi-sto1, two other functional RB homologues, Rpi-bt1 in S. bulbocastanum and RBver in S. verrucosum Schlechtd. (genome AA), apparently diverged earlier in the evolution of tuber-bearing Solanum species. Our evidence on functional Rpi-blb1 = Rpi-sto1 genes in three Petota series substantiates the assumption that the B genome of tetraploid Longipedicellata evolved from diploid Bulbocastana rather than from diploid Pinnatisecta progenitors. Similar to wild Solanum species, the markers Rpi-blb1-820 and Rpi-sto1-890 were tightly linked in advanced potato hybrids and established varieties bred from S. stoloniferum as the conventional source of bulbocastanum genes for late blight resistance, but did not always match the markers for the Rpi-blb2 and Rpi-blb3 genes and the marker for the Rysto gene as an independent indicator of stoloniferum germplasm.

Authentication of traditional Chinese medicinal herb “Gusuibu” by DNA-based molecular methods

Ensuring the authenticity of raw materials used as herbs is a key step prior to producing medicines. “Gusuibu” is a traditional Chinese medicine for the treatment of bone diseases. Drynaria roosii Nakaike is the botanical origin of “Gusuibu”. However, many “Gusuibu” adulterants which are morphologically similar, have been widely used in China. It is important to develop DNA-based markers to efficiently distinguish authentic “Gusuibu” from adulterants. In this study, 21 chloroplast genomes from seven species including D. roosii and six “Gusuibu” adulterant species were sequenced. The chloroplast genomes of D. roosii, D. sinica Diels, D. bonii Christ, D. delavayi Christ, D. quercifolia (L.) J. Sm., D. propinqua (Wall.) J. Sm., and Pseudodrynaria coronans (Wall.) Ching were 154,181 bp, 151,711 bp, 151,542 bp, 151,709 bp, 151,570 bp, 152,442 bp, and 151,466 bp in length, respectively. Phylogenetic analysis indicated that whole chloroplast genomes could be used to distinguish D. roosii from adulterants and between each adulterant. Comparing chloroplast genome sequences, 12 protein-coding genes and eight intergenic sequences with high divergence in chloroplast genomes were identified to exploit specific DNA barcodes and sequence characterized amplified region (SCAR) markers. One specific DNA barcode and three SCAR markers, which were available to distinguish D. roosii from adulterants, were developed and four primer pairs were designed. The primer pairs for amplification of DNA barcode and SCAR markers designed in this study will be useful for economically and effectively distinguishing D. roosii from adulterants, and for guaranteeing the quality, safety, and effectiveness of “Gusuibu” herbs.

Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples

At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and sensitivity in fungal DNA-spiked blood and sputum samples, and compare it with previously described molecular markers. Specific amplified fragment length polymorphism (AFLP) amplicons for Coccidioides immitis and Coccidioides posadasii were cloned into the vector pGEM® -T Easy vector and sequenced to develop a SCAR marker. Oligonucleotides were designed to identify Coccidioides spp. by polymerase chain reaction (PCR), and the specificity and sensitivity of these oligonucleotides were tested with the DNA from related pathogens. The specificity and sensitivity of the SCAR marker was evaluated with blood and sputum samples spiked with Coccidioides DNA and compared with other previously described markers (621, GAC2, and Ag2/PRA). In addition, the conditions for its use were established using biological samples. A specific marker named SCAR300 was obtained to identify Coccidioides spp. that exhibited good sensitivity and specificity. The results showed that all of the markers tested in this study can identify Coccidioides spp. However, the SCAR300 and 621 markers were the most sensitive, whereas the SCAR300 marker was the most specific. Thus, the SCAR300 marker is a useful tool to identify Coccidioides spp.

Registration of Great Northern Common Bean Cultivar ‘Panhandle Pride’ with Enhanced Disease Resistance to Bean Rust and Common Bacterial Blight

‘Panhandle Pride’ (Reg. no. CV‐325, PI 691461) great northern common bean (Phaseolus vulgaris L.) cultivar was developed by the dry bean breeding program at the University of Nebraska and released in 2016. It was bred specifically for adaptation to western Nebraska growing conditions and for enhanced resistance to bean rust and common bacterial blight (CBB). Seed yield of Panhandle Pride was comparable to the other cultivars in the on‐station and on‐farm trials conducted in Nebraska and was greater than ‘Orion’ and lower than ‘Matterhorn’ in regional trials. Seed size (100‐seed weight) for Panhandle Pride was larger than Marquis and Beryl‐R in trials grown in Nebraska, and larger than Orion and Matterhorn in regional trials. Panhandle Pride carries the sequence characterized amplified region (SCAR) markers SAP6 and SU91 linked to a major CBB resistance quantitative trait locus, and its reaction to the CBB pathogen under field conditions was consistent across 3 yr at the West Central Research and Extension Center, North Platte, NE, with disease ratings of 6.0, 4.3, and 5.2. Panhandle Pride has the Ur‐3 and Ur‐6 genes for resistance to bean rust and carries the SS68 Kompetitive Allele Specific polymerase chain reaction (KASP) and SK14 SCAR markers linked the Ur‐3 bean rust resistance locus. It also carries the single dominant hypersensitive I gene that provides resistance to all non‐necrotic strains of the Bean common mosaic virus (BCMV) and the SW13 SCAR marker linked to the I gene for resistance to BCMV. Panhandle Pride has bright white seed, blooms 44 d after planting, and is a midseason bean, maturing 87 d after planting.

Genetic relationship of orchids in the Calanthe group based on sequence-related amplified polymorphism markers and development of sequence-characterized amplified regions markers for some genus/species identification

The Calanthe comprises a group of terrestrial orchid species known for their attractive flowers consisting of three genera (Calanthe, Phaius and Cephalantheropsis) which share many similar characteristics causing difficulty in identification. Here, the genetic relationship of the Calanthe group was studied using sequence-related amplified polymorphism (SRAP) markers. Using 18 primer pairs, 565 fragments were generated in which 562 were polymorphic (99.45%) indicatinga high level of polymorphism in the Calanthe. A dendrogram was constructed using the unweighted pair group method with arithmetic mean method. Calanthe orchids were separated into five clades consisting of three groups of the genus Calanthe, one Phaius group and one consisting of Calanthe and Cephalantheropsis species. Overall clusters were in agreement with their morphologies and other reports using DNA sequences of the chloroplast genome, indicating the effectiveness ofthe SRAP markers; however, C. rubens var. alba was not closely related to C. rubens, suggesting a possible identification error. Sequence characterized amplified regions (SCAR) primers for P. flavus and C. herbacea species identification were also developed. The results showed that PFLF/PFLR primers could be used to amplify the specific fragment of 240 bp only in P. flavus. CHEF/CHER primers were used to amplify the fragment of 430 bp in three species (C. masuca, C. triplicata and C. herbacea) which were in the same clade. SCAR markers were successfully developed for P. flavus identification and detection of the C. masuca-C. triplicata-C. herbacea clade.online

SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.

This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set. The RAPD primer, OPA4, amplified a specific fragment of c. 400bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5pgµl-1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants. A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots. A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.

Development of a sequence-characterized amplified region marker for detection of Ascochyta rabiei causing Ascochyta blight in chickpea.

Ascochyta blight of chickpea is caused by Ascochyta rabiei (Pass.) Labr. which is primarily seedborne. For rapid detection and precise identification of A. rabiei, a sequence-characterized amplified region (SCAR) marker was developed for detection of genomic DNA and infected plant DNA. An SSR primer amplified monomorphic band was cloned in pGEM®-T easy vector and sequenced. The best primer pair was selected and validated on A. rabiei. The specificity and sensitivity of the SCAR-based marker designated as MBAR was evaluated using conventional PCR and real-time PCR. The marker produced consistently an amplicon size of 196bp in all A. rabiei isolates tested. The sensitivity of the marker was 0.1ng of genomic fungal DNA and 0.5ng of plant DNA by conventional PCR and 0.5pg of A. rabiei DNA and 1.0pg of plant DNA by real-time PCR. This is the first SCAR marker having high specificity and sensitivity towards A. rabiei. The marker may be useful in detecting the pathogen before the disease appearance and in plant quarantine program to detect the pathogen in seed lots.

Comparison of Anthracnose Resistance with the Presence of Two SCAR Markers Associated with the Rca2 Gene in Strawberry

Strawberry anthracnose diseases are caused primarily by three Colletotrichum species: C. acutatum J.H. Simmonds, C. fragariae A.N. Brooks, and C. gloeosporioides (Penz.) Penz. & Sacc. Molecular markers are being used in breeding programs to identify alleles linked to disease resistance and other positive agronomic traits. In our study, strawberry cultivars and breeding germplasm with known anthracnose susceptibility or resistance to the three anthracnose-causing Colletotrichum species were screened for two sequence characterized amplified region (SCAR) markers linked to the Rca2 gene. The Rca2 resistant allele SCAR markers were associated with varying degrees of significance for a strawberry plant’s anthracnose resistance to C. fragariae but not to C. acutatum or C. gloeosporioides. Although the presence or absence of the markers associated with the Rca2 resistance gene is an imperfect indicator of anthracnose resistance, it may serve as a useful starting point in selecting germplasm for breeding programs.

The Rx gene derived USDA 41956 and Rx1 gene derived CPC 1673 confer equal resistance to the migration of Potato virus X from potato leaves to tubers

Genes conferring resistance to Potato virus X (PVX) are known, but it remains unknown whether these genes also confer resistance to the migration of PVX from potato leaves to tubers. Potato leaves of 43 cultivars and breeding lines were inoculated with PVXo and PVXb strains and their subsequent appearance in tubers was assessed. Then, a sequence-characterized amplified region (SCAR) marker was developed, and the nucleotide sequence of the SCAR marker was compared with the sequences of known PVX resistance genes. Eleven cultivars and breeding lines exhibited resistance to viral migration from leaves to tubers. The SCAR marker was detected in all 11 potato cultivars and breeding lines that exhibited resistance to both PVXo and PVXb migration, as well as an additional six lines that had not been screened. Comparisons of the nucleotide sequences of the SCAR markers from Atlantic, Multa, Saco, and USDA 41956 revealed a sequence similarity of 100% with Rx1 resistance gene derived from Solanum tuberosum ssp. andigena CPC 1673 for all cultivars and breeding lines and a sequence similarity of 99.5% with the Rx2 resistance gene derived from Solanum acaule. The Rx gene was inherited as immune resistance by a single dominant gene. Within the SCAR marker, Rx had a higher sequence similarity with Rx1 than with Rx2 and it was suggested that Rx and Rx1 are the same gene derived from the S. tuberosum ssp. andigena. Introducing Rx or Rx1 into cultivars to obtain PVX free potato tubers is effective in potato breeding.

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

Genetic variation and development of a SCAR marker of anemone-type flower in chrysanthemum

An anemone shape featuring elongated and pigmented disk florets is an important target in breeding for flower shape in chrysanthemum. A good understanding of the inheritance of anemone-related traits and specific molecular markers are vital for accelerating the breeding progress. In this study, two segregating intercultivar F1 populations of the anemone-type ‘Nannong Xuefeng’ × the nonanemone-type ‘QX096’ (80 lines) and the same anemone-type ‘Nannong Xuefeng’ × ‘Monalisa’ (144 lines) were employed to investigate the genetic variation of anemone traits, and the anemone-specific markers were developed via the bulked segregant analysis (BSA) method by constructing two extreme bulks from each 10 anemone-type and nonanemone-type F1 lines, respectively. A moderate high level of variation coefficient (~ 24 to 57%) was obtained for most tubular floret traits relevant to the anemone type in both segregating populations. The anemone morphology of the hybrids was intermediate between that of their parents, and some transgressive individuals were observed, indicating great potential for the selection of lines with the desirable anemone type. In the BSA, four sequence-related amplified polymorphism (SRAP) primer combinations were identified as informative between the bulks of the anemone and nonanemone F1 individuals. After cloning and sequencing, only one SRAP marker, i.e., M11E1_272, was successfully converted to the sequence-characterized amplified region (SCAR) marker SCAR168. The SCAR marker was validation in the two F1 populations demonstrated that the marker SCAR168 could truly discriminate anemone and nonanemone types with a high coincidence ratio 87.86~92.5%. In addition, a general linear model (GLM)-based association analysis (P < 0.01) revealed that the SCAR marker exhibited a positive effect on the anemone traits and explained 20.83~33.66% phenotypic variation. Results of the current study show the inheritance pattern of anemone traits, and the developed anemone-specific SCAR marker paves the way for the marker-assisted selection of flower shape in chrysanthemum.

Developing tools for evaluating inoculation methods of biocontrol Streptomyces sp. strains into grapevine plants.

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.

Development of molecular markers for the early detection of scion roots in Shiranuhi Mandarin hybrid and other citrus cultivars

Recently, scion rooting at the grafted region of Poncirus trifoliata rootstocks has been found to decrease fruit setting and quality. Under cultivation conditions, the identification of scion-rooted Shiranuhi mandarin hybrid [(Citrus unshiu Marcov. × C. sinensis Osbeck) × C. reticulata Blanco] trees is difficult. Therefore, we aimed to develop a novel method for rapid and accurate identification of scion roots based on sequence-related amplified polymorphism (SRAP) in combination with a sequence-characterized amplified region (SCAR) marker. The genomic DNA was extracted from the normal rootstocks of P. trifoliata and scion roots of Shiranuhi. The sequence of amplified DNA fragments from scion root samples was analyzed using SRAP F4 and R15 primer combination, and was used to construct nine combinations of SCAR primers. Five markers (P1, P2, P3, P4, and P6) were obtained, of which SCAR-P3 presented the best results. We also efficiently screened the scion roots in Kiyomi and Settokka. These SCAR markers accurately identified scion rooting in Shiranuhi mandarin hybrid and other citrus cultivars, thus allowing the implementation of improved cultivation techniques to ensure normal fruit setting. The proposed method can also be applied to control the damage caused by scion rooting in other Citrus cultivars.

SCAR Marker for Gender Identification in Date Palm (Phoenix dactylifera L.) at the Seedling Stage

Date palm (Phoenix dactylifera L.) is cultivated in arid and semiarid regions worldwide. Given the dioecious nature of this plant, gender identification is very important at the seedling stage. Molecular markers are very effective tools that help in gender identification at this stage. A sequence characterized amplified region (SCAR) marker linked to sex-specific regions in the genome of date palm was developed. Of the 300 tested randomly amplified polymorphic DNA (RAPD) primers, only one primer (OPC-06) produced reproducible band (294 bp) in male plants. The PCR product of this primer was cloned and sequenced. The specific primers were synthesized for amplification of a 186 bp fragment in male date palm plants. These primers were validated in male and female date palm plants, wherein the designed SCAR marker was reported only in male plants and no amplification was observed in female plants. The developed SCAR marker was used with seedlings of date palm and proved very effective in identification of gender.

Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers.

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 104 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 103 cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.

Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method.

A rapid and sensitive loop-mediated isothermal amplification (LAMP) method was developed for the specific detection of Pseudocercospora eumusae. The LAMP primers designed based on the specific SCAR (Sequence Characterized Amplified Region) marker sequence of P. eumusae proved highly specific to P. eumusae and there was no cross reactivity with closely related Pseudocercospora spp. (P. fijiensis and P. musicola) and 17 other leaf spot causing fungal pathogens of banana. The developed LAMP method exhibited greater sensitivity as the minimum detectable concentration of P. eumusae genomic DNA was 10 ƿg/µl which was 100 times lower than that of conventional PCR (1ng/µl).This method also detected the target pathogen from crude DNA of the mycelium and single leaf spot tissues which eliminates laborious purification steps in DNA isolation and requires less operational time. To the best of our knowledge, this is the first report on the development of this LAMP method to specifically detect and diagnose P. eumusae pathogen from pure cultures and leaf spot tissues.

Development of seven novel specific SCAR markers for rapid identification of Phytophthora sojae: the cause of root- and stem-rot disease of soybean

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root and stem rot. In this study, we developed seven pairs of polymerase chain reaction primers derived from sequence-characterized amplified regions (SCAR). These seven SCAR markers allowed discrimination of P. sojae from 17 different Phytophthora species and three other soilborne pathogens (Pythium ultimum, Fusarium solani and Rhizoctonia sp.) which also induce root rot in soybean. Among those 17 Phytophthora species, P. melonis has approximately 98% similarity in ITS sequences; P. drechsleri requires an annealing temperature up to 66 °C with an ITS-targeting diagnostic marker (PS primers) developed by Wang et al. (2006) for P. sojae; and P. sansomeana is a newly described soybean-infecting Phytophthora species. These three Phytophthora species could be specifically distinguished against P. sojae by these seven SCAR markers. After screening 100 random amplified polymorphic DNA (RAPD) primers, eight primers clearly produced specific bands only for P. sojae rather than other Phytophthora species tested. Subsequently, seven of eight P. sojae-specific RAPD markers were successfully converted into SCAR markers, namely, Scar276, Scar304, Scar333, Scar37, Scar519, Scar57 and Scar78. These SCAR markers were used to detect 75 isolates of P. sojae specifically, while no products were obtained for 29 additional isolates representing 17 other Phytophthora species and three other soilborne pathogens. Furthermore, Scar333 successfully allowed the detection with a sensitivity of 100 pg from genomic DNA of P. sojae, Scar276 had a higher sensitivity of 10 pg, and four specific SCAR primers (Scar304, Scar37, Scar519 and Scar78) had a sensitivity of 100 fg, which is the highest for detecting P. sojae until now. Six of the seven SCAR markers, with the exception of Scar57, were also used to detected P. sojae in artificial or naturally infected soybean seedlings and infested soil. Our findings demonstrate that SCAR markers provide a rapid and sensitive molecular diagnostic tool for the detection of P. sojae in plants, and will play a key role in effective management of the disease.

Molecular markers and cytogenetics to characterize a wheat-Dasypyrum villosum 3V (3D) substitution line conferring resistance to stripe rust.

Dasypyrum villosum has been used as a valuable gene resource for disease resistances, yield increase and quality improvement in wheat. A novel wheat-D. villosum alien introgression line CD-3 was generated through hybridization between the common wheat Chinese Spring (CS) and a CS- D. villosum 3V addition line having considerably high stripe rust resistance, which enable the characterization of a potential new stripe rust resistance gene (s) derived from D. villosum. The results of non-denaturing fluorescent in situ hybridization (ND-FISH) showed that CD-3 contained 42 chromosomes, including a 3V chromosome pair, and the absence of both of the 3D chromosomes. PCR-based Landmark Unique Gene (PLUG) molecular marker analysis supported results from the FISH analysis, revealing CD-3 was a wheat-D. villosum 3V (3D) disomic substitution line. Resistant test of stripe rust on 52 plants of F2 generation (CD-3/CS), CD-3, CS and D.villosum have been conducted at seedling stage. 7 plants of F2 generation possessing two 3V chromosomes exhibited high resistance to stripe rust as CD-3 and D.villosum, 10 plants carrying one 3V chromosome and 35 plants without 3V chromosome were susceptive to stripe rust as CS. The result implied the high stripe rust resistance of CD-3 should be controlled by recessive gene(s) originating from D.villosum. To rapidly detect chromosome 3V in the genetic background of wheat, we developed a novel Sequence Characterized Amplified Region (SCAR) marker specific for 3V chromosome based on the sequence of a grain size-related gene DvGS5 in D. villosum, an orthologue of TaGS5 from wheat. The SCAR marker was designated DvGS5-1443, which could successfully amplify a unique 3V-specific fragment in CD-3 and D. villosum, suggesting that this SCAR marker could facilitate targeting the chromosome 3V in the genetic background of wheat for wheat improvement.

Authentication of Herbal Medicines Dipsacus asper and Phlomoides umbrosa Using DNA Barcodes, Chloroplast Genome, and Sequence Characterized Amplified Region (SCAR) Marker.

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

Development of a SCAR Marker-Based Diagnostic Method for the Detection of the Citrus Target Spot Pathogen Pseudofabraea citricarpa.

Target spot, a recently observed citrus disease that is caused by Pseudofabraea citricarpa, can cause substantial economic losses in citrus production. In this study, a 797 bp marker specific to Ps. citricarpa was identified via random amplified polymorphic DNA (RAPD) technique. The primer pair Pc-SFP/Pc-SRP, which was designed from RAPD amplicons, was utilized as a sequence-characterized amplified region (SCAR) marker. This marker identified Ps. citricarpa with a single and distinct band of 389 bp but did not amplify DNA from other tested fungal species. The PCR assay was highly sensitive to the target DNA at picogram levels and could reliably amplify Ps. citricarpa sequences with the Pc-SFP/Pc-SRP primer pair. The SCAR marker that was identified in the present study can facilitate rapid decision-making and precise disease forecasting and management.