Specific Sequence Characterized Amplified Region Research Articles

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance

Development of DNA markers associated with sunburn resistance in pomegranate (Punica granatum L.) using bulk segregant analysis

Production and consumption of pomegranate have been increasing in the world in recent years due to its health benefits to humans. Sunburn is an important physiological disorder caused by high temperature, light, and radiation. With the changes in climatic conditions that have occurred in recent years, the effect of sunburn is increasing and limiting the production and quality of pomegranate. Therefore, development of pomegranate cultivars resistance to sunburn is one of the main objectives of pomegranate breeding program. However, the development of a new variety takes a very long time. In order to shorten this time, molecular markers have been used in plant breeding in recent years. In this study, molecular markers associated with sunburn resistance in pomegranate fruit were developed using bulked segregant analysis technique (BSA) in a pomegranate progeny population whose morphological, pomological, and phenological evaluations were completed. Two RAPD fragments (OPAI-08-650 and OPPP-14-900) associated with sunburn resistance or sensitivity were determined and used as markers. One of these RAPD fragments was purified, cloned and sequenced to produce a more specific sequence characterized amplified regions (SCAR) marker, designated as PgSCARGYH1. The markers developed in this study will shorten the breeding process of sunburn-resistant pomegranate cultivars. In addition, it will also contribute to the increase in pomegranate production in terms of quality and quantity.

Identification of SCAR markers for genetic authentication of Dendrobium nobile Lindl.

Dendrobium nobile Lindl. is an orcid plant with important medicinal values. This is a colourful houseplant, and also a popular herb in traditional Chinese medicine (TCM). The variants of this plant from different geographic regions might be high, and in this study, we aimed to develop specific sequence characterized amplified region (SCAR) markers for the identification of specific variant of this plant. Different cultivars of D. nobile were collected from nine different places of China, and one cultivar from Myanmar. DNA materials were extracted from the plant samples, random amplified polymorphic DNA (RAPD) were developed, cloned and sequenced for the development of SCAR markers. We have developed four SCAR markers, which are specific to the cultivar from Luzhou China, and clearly distinguishable (genetically) from other cultivars. These SCAR markers are deposited in GenBank (accession number MZ417502, MZ484089, MZ417504 and MZ417505). Four SCAR markers for D. nobile are effective molecular technique to genetically identify the different cultivars or species, and this method is applicable for genetic characterization and identification of other plant species too.

Registration of ‘White Pearl’ great northern common bean cultivar with upright plant architecture and high yield

AbstractGreat northern common bean (Phaseolus vulgaris L.) cultivar ‘White Pearl’ (Reg. no. CV‐343, PI 698189) was developed by the dry bean breeding program at the University of Nebraska and released in 2018. It was bred specifically for adaptation to western Nebraska growing conditions and for upright plant architecture, high yield, and enhanced resistance to bean rust, common bacterial blight (CBB), and Bean common mosaic virus (BCMV). Seed yield of White Pearl was comparable and slightly higher than other great northern cultivars in on‐station trials conducted in Nebraska and did not differ significantly in yield from ‘Aries’ and ‘Matterhorn’ in regional trials. Seed size (100‐seed weight) of White Pearl was larger than ‘Marquis’, ‘Orion’, ‘Gemini’, ‘Beryl‐R’, and Aries in trials grown in Nebraska. White Pearl carries the sequence characterized amplified region (SCAR) marker SAP6 linked to a major quantitative trait locus (QTL) for resistance to CBB. White Pearl had a CBB disease rating of 6.0 (intermediate) under field conditions at the Scottsbluff and Mitchell Ag Labs in 2020. White Pearl also has the Ur‐3 gene for resistance to bean rust and carries the SS68 Kompetitive Allele Specific polymerase chain reaction (KASP) and SK14 SCAR markers linked to the Ur‐3 locus. It also contains the single dominant hypersensitive I gene that confers resistance to all non‐necrotic strains of BCMV and the SW13 SCAR marker linked to the I gene. White Pearl has bright white seed, blooms 46 d after planting, and is a midseason bean, maturing 89 d after planting.

Morpho-physiological characterization of bread wheat genotypes and their molecular validation for rust resistance genes Sr2, Sr31 and Lr24

Leaf rust caused by Puccinia recondita and stem rust by Puccinia graminis triticii are among the most destructive diseases of wheat worldwide. The selection of genotypes carrying two or more genes using traditional host-parasite interaction is very time consuming and often not possible due to lack of isolates with specific virulence and difficulty of identifying one seedling resistance gene in the presence of another gene. Identification of molecular markers for resistance genes can facilitate selection of gene positive plants at early stage to complete the study in short time and cost effective manner. In current investigation assessment of 51 diverse genotypes of wheat (Triticum aestivum L.) was carried out using 16 morpho-physiological yield attributing traits. Biological yield was found highly correlated and positively significant with grain yield (r = 0.410) and spike length (r = 0.601) at 1% significant level. It was significantly correlated with number of grains per spike (r = 0.333) and canopy temperature (r = 0.325) at 5% significant level. Validation of genes Lr24, Sr31 and Sr2 in these genotypes was done using gene specific Sequence Characterized Amplified Region (SCAR) and Simple Sequence Repeats (SSR) markers. The SSR marker Xgwm533 showed highest genetic diversity (0.6290) and PIC value (0.5542), whereas the SCAR primer SCS30.2 and SCSS26.1 revealed lowest gene diversity (0.1769) and PIC value (0.1612). The major allele frequency for Xgwm533 was observed (0.4706) which is lowest; while primer SCS30.2 and SCSS26.1 showed highest major gene frequency (0.9020).

Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD

BackgroundLeishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD.MethodsRAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics.ResultsThe cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein.ConclusionsThe RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.

Validation of Molecular Markers Genetically Linked to S-Cytoplasm and Restoration-of-fertility (Rf) Loci in Hot Pepper (Capsicum annuum L.)

Existence of CGMS system in hot pepper is due to the rearrangements in the mitochondrial genome and is largely used in economized and pure F 1 hybrid seed production around the world. The orf456, a new ORF present at flanking region of the coxII gene at the 3’ end, was distinguished male sterile cytoplasm in hot peppers along with atp6-2gene. In the current study, eighteen pepper genotypes (nine each of A and corresponding B lines) of varied origin were used to validate with two male sterile cytoplasm (S-cytoplasm) specific sequence characterised amplified region (SCAR) markers viz., atp6-2 (875 bp) and orf456 (456 bp) and one restoration-of-fertility (Rf) locus specific marker, CRF (550 bp). The results clearly showed that the presence of CMS-S-cytoplasm and absence of restoration-of-fertility (Rf) gene in the pepper genotypes studied and is comparable with the phenotypic data. In view of the outcomes it has been reasoned that the accessible S and Rf markers available in the public domain are reproducible and can be promptly utilized for marker assisted selection (MAS) in hot pepper crop improvement program.

Validation of Molecular Markers Genetically Linked to S-Cytoplasm and Restoration-of-fertility (Rf) Loci in Hot Pepper (Capsicum annuum L.)

Existence of CGMS system in hot pepper is due to the rearrangements in the mitochondrial genome and is largely used in economized and pure F 1 hybrid seed production around the world. The orf456, a new ORF present at flanking region of the coxII gene at the 3’ end, was distinguished male sterile cytoplasm in hot peppers along with atp6-2gene. In the current study, eighteen pepper genotypes (nine each of A and corresponding B lines) of varied origin were used to validate with two male sterile cytoplasm (S-cytoplasm) specific sequence characterised amplified region (SCAR) markers viz., atp6-2 (875 bp) and orf456 (456 bp) and one restoration-of-fertility (Rf) locus specific marker, CRF (550 bp). The results clearly showed that the presence of CMS-S-cytoplasm and absence of restoration-of-fertility (Rf) gene in the pepper genotypes studied and is comparable with the phenotypic data. In view of the outcomes it has been reasoned that the accessible S and Rf markers available in the public domain are reproducible and can be promptly utilized for marker assisted selection (MAS) in hot pepper crop improvement program.

South American species <i>Solanum alandiae</i> Card. and <i>S. okadae</i> Hawkes et Hjerting as potential sources of genes for potato late blight resistance

For several decades, wild species of Solanum L. section Petota Dumort. have been involved in potato cultivar breeding for robust resistance to pests and diseases. Potato late blight (LB) is caused by oomycete Phytophthora infestans (Mont.) de Bary, and the genes for race-specific resistance to P. infestans (Rpi genes) have been introgressed into cultivated potatoes by remote crosses and trans- or cisgenesis, first from S. demissum Buk. and, more recently, from other wild species, such as S. bulbocastanum Dun., S. stoloniferum Schlechtd. et Bché, and S. venturii Hawkes et Hjerting (according to the nomenclature by Hawkes, 1990). Most wild species already involved in breeding for LB resistance came from North and Central Americas: series Bulbocastana (Rydb.) Hawkes, Demissa Buk. and Longipedicellata Buk., and some Rpi genes of these species have been already characterized in much detail. Rpi genes of South American species, including the series Tuberosa (Rydb.) Hawkes, have not been sufficiently investigated. Among the latter, this study focuses on the Rpi genes of S. alandiae Card. and S. okadae Hawkes et Hjerting. Four accessions of S. alandiae, one accession of S. okadae and 11 clones of interspecific potato hybrids comprising S. alandiae germplasm from the VIR collection were PCR-screened using specific SCAR (Sequence Characterized Amplified Region) markers for eight Rpi genes. SCAR amplicons of five Rpi genes registered in this study were validated by comparing their sequences with those of prototype genes deposited in the NCBI Genbank. Among the structural homologues of Rpi genes found in S. alandiae and S. okadae, of special interest are homologues of CC-NB-LRR resistance genes with broad specificity towards P. infestans races, in particular R2=Rpi-blb3, R8, R9a, Rpi-vnt1 and Rpi-blb2 (94–99, 94–99, 86–89, 92–98 and 91% identity with the prototype genes, respectively). Our data may help to better understand the process of Rpi gene divergence along with the evolution of tuberbearing Solanum species, particularly in the series Tuberosa.

Development of the sequence-characterized amplified region (SCAR) marker for distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries.

Distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries is important for the development of new intergeneric hybrids. The aim of this study was to develop a simple and cost-effective DNA marker to distinguish between a large number of putative hybrids of A. frutescens × R. gayanum during primary selection. We designed sequence-characterized amplified region (SCAR) markers based on the genetic sequences of the internal transcribed spacer (ITS) region for A. frutescens and R. gayanum. Specific SCAR markers for A. frutescens amplified bands in A. frutescens and hybrids but not in R. gayanum. In contrast, specific SCAR markers for R. gayanum amplified bands in R. gayanum and hybrids but not in A. frutescens. Additionally, we confirmed the reproducibility of the SCAR marker using 31 cultivars. The findings demonstrated that these SCAR markers could be used to distinguish putative hybrids between A. frutescens and R. gayanum. This is the first report of the development of a SCAR marker to facilitate distinction of these hybrids.

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random amplified polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh (P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

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.

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.

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.

Analysis of Genetic Diversity and Development of a SCAR Marker for Green Tea (Camellia sinensis) Cultivars in Zhejiang Province: The Most Famous Green Tea-Producing Area in China

Camellia sinensis (L.) O. Kuntze is one of the most important non-alcoholic beverage crops in Asian and African countries. In recent years, many green tea cultivars have been released and played an important role in improving the production and quality of tea trees. The objectives of this study were to assess the genetic diversity of the eighteen main green tea cultivars in Zhejiang Province-the most famous green tea-producing area of China-using start codon-targeted (SCoT) markers and to develop a specific sequence-characterized amplified region (SCAR) marker for application in cultivar diagnosis. Thirty-one SCoT primers produced 264 loci, 226 of which were polymorphic. The genetic similarity coefficients among these green tea cultivars ranged from 0.587 to 0.814, indicating that a high level of genetic diversity was present. Both a UPGMA dendrogram and a PCoA plot grouped the tea cultivars into three groups. The partitioning of groups in the UPGMA and PCoA was similar, and much of the clustering was highly consistent with the classification of tea cultivars according to their genetic backgrounds. A unique SCoT band, SCoT4-1649, specific to the tea cultivar 'Yingshuang,' was transformed into a SCAR marker. This SCAR marker is highly useful for the identification and germplasm conservation of green tea cultivars.

Development of a PCR test for detection of Xanthomonas campestris pv. raphani

Leaf spot of Brassica is an important bacterial seed borne disease caused by X. campestris pv. raphani (Xcr), one of the three pathovars of Xanthomonas campestris species which makes significant economic loss throughout the world. A rapid and reliable detection method will be helpful for early diagnosis of the disease. To do that, we designed Xcr specific sequence-characterized amplified region (SCAR) markers from the Xcr specific genomic fragments, identified by aligning the whole genome sequences of several closely related bacterial pathovars and other bacterial species. Our primer set Xcr_59 produced Xcr specific amplicon of 679 bp in PCR assays. This primer pair was also capable of detecting Xcr strains directly from the artificially infected leaf samples prepared by suspending the diseased leaf area in sterile water without extracting bacterial DNA. This rapid, sensitive and reliable detection technique will be useful for adopting any firsthand plant protection measures against the disease.

Development of species specific SCAR based molecular marker for the detection of Pseudocercospora eumusae, the causal agent of eumusae leaf spot disease in banana

Eumusae leaf spot disease caused by Pseudocercopra eumusae is one of the destructive leaf spot diseases in banana. A diagnostic method based on conventional and quantitative real-time PCR (qPCR) assay was developed in the present study for the specific detection of P. eumusae from pure cultures and leaf spot tissues. The sequence characterized amplified region (SCAR) based molecular marker and TaqMan probe was designed and validated using the genomic DNA of different Pseudocercopra spp. and 11 other leaf spot pathogens of banana. The primers and TaqMan probe proved to be highly specific to the target pathogen P. eumusae and no cross reactivity was encountered with any of the other leaf spot pathogens tested. Both conventional and qPCR assay developed detected the target pathogen P. eumusae directly from single leaf spot tissue and thus simplifies the identification by eliminating the need for culturing of pathogen. However, the sensitivity of qPCR (1 pg/μL) is higher compared to conventional PCR (100 pg/μL). To the best of our knowledge, this is the first report on the use of SCAR based molecular marker for the specific and early detection of P. eumusae in banana.

Development of race-specific molecular marker forXanthomonas campestrispv.campestrisrace 3, the causal agent of black rot of crucifers

Race-specific molecular markers were established to distinguish Xanthomonas campestris pv. campestris (Xcc) race 3, the causal agent of black rot disease of crucifers. The available genome sequences of Xcc races were aligned and identified three DNA fragments specific to Xcc race 3. The identified race-specific DNA fragments namely XccR3-49, XccR3-52, and XccR3-55 were used for designing the race-specific primers to detect and identify Xcc race 3. The specificity of race-specific primers was tested against the genomic DNA extracted from Xcc (races 1–7), Xcc strains, Xc pathovars, and other bacterial species. XccR3-49, a specific sequence characterized amplified region (SCAR) primer set, gave a single band with 867 bp length for Xcc race 3 only. The remaining two markers XccR3-52 and XccR3-55 showed polymorphic amplification with amplicon sizes of 1889 and 2109 bp for Xcc race 3, respectively. Additionally, the SCAR primer set detected Xcc race 3 rapidly and efficiently in artificially infected cabbage leaves with bio-PCR. This result showed that the newly developed race-specific markers can successfully and efficiently detect and identify Xcc race 3 from Xanthomonas campestris pv. campestris races, Xanthomonas species/pathovars, as well as other plant pathogenic bacteria (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). Up to now, this is the first report describing the race-specific marker for the detection of Xcc race 3.

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.