Sequence Characterized Amplified Region Primers Research Articles

Discrimination of lethal Russula subnigricans from wild edible and morphologically similar mushrooms in the genus Russula using SCAR markers

Russula is a diverse genus of mushrooms that is consumed worldwide. Russula subnigricans is a lethal species that causes myotoxicity upon ingestion. It is morphologically similar to wild foraged species, including R. nigricans and R. densifolia. Misidentification of these mushrooms can lead to severe food poisoning. Several PCR-based approaches have been used to overcome this problem. In this study, 22 samples of wild edible Russula species were collected, and five clinical samples of R. subnigricans leftovers were obtained. Based on the sequence data of the internal transcribed spacer, the phylogenetic trees revealed that these 27 samples could be classified into well-established clades of six subgenera. Fifty arbitrary primers were used in high annealing temperature rapid amplified polymorphic DNA (HAT-RAPD) technique to screen polymorphic bands among R. subnigricans, R. nigricans, R. densifolia, and other edible species. Distinctive RAPD fragments were obtained from 11 primers and used in sequence characterized amplified region (SCAR) technique. Three pairs of species-specific SCAR primers were selected to distinguish lethal R. subnigricans, R. nigricans, and R. densifolia with SCAR markers of 213, 420, and 609 bp, respectively. Ten-fold sensitivity test revealed that the SCAR marker of R. subnigricans could be detected at DNA template concentrations as low as 30 pg/μL. This marker could detect the presence of lethal R. subnigricans even at 1% in a cooked sample of mixed mushroom remnants. The developed SCAR markers ensured rapid and accurate diagnosis of suspected myotoxic mushrooms, thereby reducing the risk of severe foodborne illness and death.

First Report of Root-Knot Nematode (Meloidogyne incognita) on Dioscorea opposita in Henan Province, China.

Dioscorea opposita is an annual twining plant in China that is used for consumption and medicinal purposes. The planting area of D. opposita is near 500,000 hectares in China, mainly in Shangdong, Hebei, Henan, Jiangxi and Yunnan provinces. In August 2021, we observed that some D. opposita plants grew poorly with smaller and chlorotic leaves in Changyuan (35°8'12"N; 114°43'52"E), Henan Province, China. Galls with hook-shaped roots and tuber damage were also observed, typical of root-knot nematode. Thirty tubers were randomly collected and 60% were infested with root-knot nematodes. During a disease survey in Changyuan, the incidences of root-knot nematode damage were 31.5%, 21%, and 18% in three fields (0.33, 0.67, and 4 ha, respectively) at harvest. The average tuber length of infected plants was decreased by 65.8%, and the average weight was decreased by 70.1% compared to the healthy plants. Males, females, second-stage juveniles (J2s), and eggs were extracted from individual diseased tubers from the three fields for morphological identification. Females were white, pear-shaped with a projecting neck. Males showed a trapezoidal labial region with prominent stylet knobs, including a high head cap which had a stepped outline and was centrally concave in lateral view. Morphological measurements are described in the supplementary material. All data and descriptions conformed to the morphological characteristics of Meloidogyne incognita. Genomic DNA was extracted from J2s (n=9) using PCR lysis buffer, and used for PCR amplification of the sequence characterized amplified region (SCAR) markers specific for M. incognita. Two pairs of the SCAR primers, Mi-F/Mi-R, and Inc-K14-F/Inc-K14-R, were used to diagnose whether these nematodes from D. opposita were M. incognita (Meng et al. 2004; Randig et al. 2002). The PCR produced expected amplification products of 955 and 399 bp, confirming the nematode to be M. incognita. Primers specific for M. arenaria (Far/Rar) and M. javanica (Fjav/Rjav) were used but failed to amplify fragments (Randig et al. 2002; Zijlstra et al. 2000). The obtained PCR fragments were sequenced and deposited in GenBank (accession no. OQ420602.1, OQ427638.1). They showed 99.9 and 100% identity to the available GenBank M. incognita sequence (accession no. MK410954.1, ON861825.1), respectively. A pathogenicity test was conducted in greenhouse conditions. Bulbils of D. opposita were sown in the pots filled with 2,000 ml of autoclaved soil mixture (loamy soil/sand, 1:1). One month later, 15 seedlings (five to six leaf stage) were inoculated with 1,000 M. incognita J2s individually. Five plants without nematode inoculation were used as the control. Two months after inoculation, all of the inoculated roots had galling symptoms similar to those observed in the field, and 100% of root system tissues had galls. The root gall index was ~6 according to a 0 to 10 RKN damage rating scale (Poudyal et al. 2005). No symptoms were found on the control plants. The nematodes were reisolated from root tissue and identified. M. incognita has a broad host range in many species of economic importance including Salvia miltiorrhiza (Wen et al. 2023), Ipomoea batatas (Maleita et al. 2022), and Zea mays (López-Robles et al. 2013). So far, M. incognita has been reported in D. alata and D. rotundata in Africa (Onkendi et al. 2014). To our best knowledge, this is the first record of M. incognita on D. opposita in Henan Province, China. With the increased planting area of D. opposita in China, root-knot nematodes are becoming more serious and reducing tuber production, with yield losses more than 60%. This identification is a preliminary step in developing effective disease management schemes. Declaration of interest The authors declare no conflict of interest. Funding This work was financially supported by the Key Scientific Research Projects of Higher Education Institutions of Henan Province (21A180013), China Agriculture Research System (CARS-21), The Zhongyuan high level talents special support plan-Science and Technology Innovation Leading Talents (224200510011) and Science and Technology Research Project of Henan Province (222102310211). References López-Robles, J., et al. 2013. Plant Dis. 97:694. https://doi.org/10.1094/PDIS-07-12-0674-PDN. Maleita, C., et al. 2022. Plant Dis. 106:2536. https://doi.org/10.1094/PDIS-12-21-2680-PDN. Meng, Q. P., et al. 2004. Acta Phytopathol. Sinica 34:204. https://doi.org/10.13926/j.cnki.apps.2004.03.003. Onkendi, E. M., et al. 2014. Plant Pathol. 63:727. https://doi.org/10.1111/ppa.12202. Poudyal, D. S., et al. 2005. Australas. Plant Pathol. 34:181. https://doi.org/10.1071/AP05011. Randig, O., et al. 2002. Genome 45:862. https://doi.org/10.1139/g02-054. Wen, Y., et al. 2023. Plant Dis. Accepted. https://doi.org/10.1094/PDIS-05-22-0997-PDN. Zijlstra, C., et al. 2000. Nematology 2:847. https://doi.org/10.1163/156854100750112798.

Enzymatic and Molecular Identification of Meloidogyne Species in Tomato Orchards in Paraguay

Tomato is a major crop in Paraguay, where it provides a source of employment and income for households. Tomato production can be affected by root-knot nematodes, especially Meloidogyne spp. The unequivocal identification of Meloidogyne spp. in Paraguay has not been conducted yet. This study aims to identify Meloidogyne species in eight tomato production districts of this country by biochemical and molecular techniques. Females of Meloidogyne spp. were extracted from tomato roots and characterized using esterase isozyme phenotypes. In addition, DNA was extracted from nematode eggs, and species-specific SCARs (sequence-characterized amplified regions) were used to confirm the diagnosis. Nematodes were detected in 100% of studied samples (prevalence), of which M. incognita (Est: I2, Rm: 1.1;1.2) and M. javanica (Est: J3, Rm: 1.0, 1.20, 1.35) were present in 39.13% and 26.08% of samples, respectively. One population (8.69%) of Meloidogyne sp. presenting an atypical esterase profile (Rm: 1.0 and 1.3) was only detected in Julián Augusto Saldívar District. Mixed populations, mostly M. incognita and M. javanica, were observed in 26.08% of samples. The SCAR primers incK14F/incK14R amplified specific fragments for M. incognita (399 bp) and M. javanica (670 bp), confirming the enzymatic results. Here, we present the first study of root-knot nematode identification at the species level in Paraguay.

Development and Application of a Cultivar-Specific Sequence-Characterized Amplified Region (SCAR) Marker for the Detection of Chrysanthemum morifolium Ramat. 'Daboju'.

Chrysanthemummorifolium Ramat. ‘Daboju’ is a C. morifolium cultivar with important ornamental and medicinal values, and is often used in the treatment of colds, blurred vision, dizziness, and itchy skin. As the morphological characteristics of C. morifolium ‘Daboju’ are very similar to those of other C. morifolium cultivars, they are often confused in practice. However, the medicinal value and practical use of C. morifolium depends on using the correct rapid and accurate identification of C. morifolium ‘Daboju’ and its differentiation from other, morphologically similar C. × morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific DNA marker was developed with the aim of the rapid and accurate identification of C. morifolium ‘Daboju’ and its differentiation from other, similar C. morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific 385-bp amplicon (named SCoT36-385), amplified only in C. morifolium ‘Daboju’ (and in all samples of this cultivar), was identified, cloned, and sequenced. Subsequently, a sequence-characterized amplified region (SCAR) marker (named DBJF/DBJR), generating a 360-bp amplicon, was developed from SCoT36-385 and tested for amplification in all 21 C. morifolium cultivars, ten C. morifolium ‘Daboju’ populations, and different simulated adulterations of ‘Daboju’ with other cultivars. The primers amplified the specific 360-bp-long DNA fragment in all the tested C. morifolium ‘Daboju’ samples but failed in the absence of ‘Daboju’. The detection limit of the SCAR primer pair (DBJF/DBJR) was 100 pg of DNA extracted from C. morifolium ‘Daboju’. Hence, this SCAR marker has a very high detection sensitivity, and can be used for accurate and rapid identification of C. morifolium ‘Daboju’. It can play an important role in ensuring the quality of medicinal preparations and protecting C. morifolium ‘Daboju’ germplasm resources in breeding programs and in identifying lines generated from this cultivar.

PCR-based rapid diagnostic tools for the authentication of medicinal mistletoe species

BackgroundTaxilli Herba (TH) and Visci Herba (VH), defined as the leaves and branches of the mistletoe species Taxillus chinensis and Viscum coloratum, respectively, are popular herbal medicines in East Asia. However, commercial TH and VH products are frequently adulterated with related inauthentic mistletoe species, posing efficacy and safety concerns. Accurate species identification of herbal medicinal products is a prerequisite for quality control, but traditional morphological identification methods are hampered by difficulties in discriminating among closely related species and in identifying the source materials in processed products. PurposeThis study aimed to develop sequence-characterized amplified region (SCAR) markers and a multiplex-SCAR assay for rapid and accurate identification of authentic TH and VH. MethodsThe matK region was sequenced in a total of 20 samples from five mistletoe species, namely T. chinensis and V. coloratum, and three species often found in adulterated herbal medicines, T. sutchuenensis, V. articulatum, and Macrosolen tricolor. Species-specific nucleotide polymorphisms were identified and short regions (21–22 bp) containing at least two species-specific nucleotides close to the 3ʹ end were incorporated into SCAR primers that produced uniquely sized PCR amplicons for each species. The five SCAR primer sets were also combined into a multiplex-SCAR assay. ResultsThe SCAR primers successfully generated amplicons of the expected size for each target species even with low-DNA templates or with templates containing DNA from multiple samples. No amplification was observed in non-target species. The SCAR markers and the multiplex-SCAR assay successfully identified commercial TH and VH products that were counterfeit or adulterated in both dried and processed products. ConclusionThis is the first report to illustrate discrimination of genuine medicinal mistletoe species with DNA-based marker assays, enabling rapid and accurate species identification. The SCAR assays developed in this study will facilitate the standardization of commercial mistletoe products.

English

Sugarcane smut caused by Sporisorium scitanmineumis the most severe sugarcane disease that causes major economic losses in sugarcane production in China, and disease resistance breeding is an important way of preventing and controlling this disease. In this study, BC3F1lines derived from the cross between YC 73-226 and YCE 06-111 were used to generate sugarcane smut-resistant and -susceptible gene pools using bulked segregant analysis (BSA). Eighty-nine random primers of start codon targeted (SCoT) polymorphisms were screened, whereas only primer SCoT44 could stably amplify the specific fragment (HE-Ss44) in the resistant pool. Then, several primer pairs of sequence characterized amplified regions (SCARs) were designed based on the sequence alignment of HE-Ss44 (920 bp), which was recovered after purification, and only one pair of SCAR primers (Ss44-F2/R2, forward: 5'-GGCGGGCACCGTCGAGTCCACAT-3'; reverse: 5'-CCGTCCGTCGG TCTCGTCCTTACG-3') could stably amplify a 400-bp specific band in resistant gene pool and its individuals. A validation test of SCAR marker Ss44-F2/R2 was performed using 34 sugarcane cultivars with known smut resistance, which revealed a selection accuracy of 82.35% between marker detection and known smut resistance. Moreover, Pearson’s correlation analysis also showed that the SCAR marker Ss44-F2/R2 was significantly correlated (r= 0.583, P= 0.0003 < 0.01) with the smut resistance trait in sugarcane. In addition, the nucleotide sequence of HE-Ss44 linked with smut-resistancewas not aligned to the homologous sequence in GenBank (NCBI), and the accession number was MG740763. The SCAR marker Ss44-F2/R2 developed in this study can be used for the rapid detection of smut resistance in sugarcane and may be utilized as reference for the improvement of sugarcane smut resistance based on molecular marker-assisted selection.© 2021 Friends Science Publishers

Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas.

Xanthomonas campestris pv. campestris (Xcc), the pathogen of black rot which is the most destructive disease of Brassica vegetables throughout the world. Here, we reported two novel sequence-characterized amplified region (SCAR) markers (i.e., XccR6-60 and XccR6-67) for the detection of Xcc race 6 via re-alignment of the complete genome sequences of Xcc races/strains/pathovars. The specificity of SCAR primer sets was verified by mean of PCR amplification using the genomic DNA template of Xcc races/strains/pathovars and two other plant infecting bacterial strains. The PCR result revealed that the XccR6-60 and XccR6-67 primer sets amplified 692-bp and 917-bp DNA fragments, respectively, specifically from race 6, while no visible amplification was detected in other samples. In addition, the SCAR primers were highly sensitive and can detect from a very low concentration of genomic DNA of Xcc race 6. However, the complete genome sequence of Xcc race 6 is not yet publicly available. Therefore, the cloning and sequencing of XccR6-60 and XccR6-67 fragments from race 6 provide more evidence of the specificity of these markers. These results indicated that the newly developed SCAR markers can successfully, effectively and rapidly detect Xcc race 6 from other Xcc races/strains/pathovars as well as other plant pathogenic bacteria. This is the first report for race-specific molecular markers for Xcc race 6.

Sequence-characterized amplified region markers and multiplex-polymerase chain reaction assays for kiwifruit cultivar identification

A simple and reliable multiplex polymerase chain reaction (PCR) method was developed using sequence-characterized amplified region (SCAR) markers to identify kiwifruit (Actinidia spp.) cultivars. We observed 183 different polymorphic random amplified polymorphic DNA (RAPD) markers using 23 arbitrary primers in 30 kiwifruit cultivars. The number of polymorphic bands per primer varied from five (OPM-15, OPN-05, and OPN-19) to 12 (OPG-01), with an average of eight. Fifty-eight cultivar-specific polymorphic RAPD fragments were converted into 16 SCAR markers after validating their specificity. Among them, 15 SCAR markers retained the dominant behavior of the original RAPD fragment, whereas the KA11_774 SCAR marker showed a co-dominant polymorphism. Based on RAPD and SCAR data sets, cluster analyses were performed with the unweighted pair-group method of arithmetic averages. The mean genetic similarities were estimated as 0.703 and 0.677 using the RAPD and SCAR markers, respectively. Thirty kiwifruit cultivars were divided into three clusters for RAPD analysis and into two clusters for SCAR analysis, based on a similarity value of 0.67. Overall, the kiwifruit cultivars with a similar breeding background tended to cluster together. Therefore, RAPD and SCAR markers could be successfully applied to assay genetic relationships for kiwifruit cultivar identification. Cluster analysis of combined RAPD and SCAR markers showed very similar results when compared to the RAPD analysis in dendrogram form. The kiwifruit cultivars tested in this study could be identified using only 10 different SCAR markers (KB08_182, KK08_287, KG19_295, KN15_316, KG01_411, KF07_433, KG09_468, KG09_477, KL01_764, and KA11_774). Furthermore, we optimized SCAR markers for simultaneous identification of the 30 kiwifruit cultivars by multiplex PCR, using two sets of five SCAR primer pairs. The developed SCAR markers and multiplex PCR approach facilitate efficient identification of kiwifruit cultivars.

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 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.

Quick and accurate detection of Fusarium oxysporum f. sp. carthami in host tissue and soil using conventional and real-time PCR assay.

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40-80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213bp in all the 75 Foc isolates. The primer set was able to detect as low as 10pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.

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.

Meloidogyne izalcoensis parasitizing coffee in Minas Gerais state: the first record in Brazil

Root-knot nematodes (RKNs) are among the most economically important plant-parasitic nematodes parasitizing coffee (Coffea arabica) in the Americas, including Brazil. Meloidogyne izalcoensis is a parasite of coffee, which was first reported in El Salvador. Biochemically, the esterase phenotype is effective to differentiate M. izalcoensis from other species described from coffee. Two years ago, this nematode was detected in a sample containing coffee roots from the region of Indianopolis (Minas Gerais State, MG, Brazil). The esterase phenotype (Est I4, Rm: 0.86, 0.96, 1.24, 1.30) of this population supported the species identification of M. izalcoensis. To further confirm this identification, PCR (Polymerase Chain Reaction) using SCAR (Sequence Characterized Amplified Regions) primers amplified a specific fragment of expected size (e.g. 670 bp) typical of M. izalcoensis. Morphologically, this population was also identified as M. izalcoensis. In Minas Gerais, coffee plants were erradicated and soybean planted in the infested area. The root symptoms are completely different from other RKNs of coffee in Brazil. This is the first record of M. izalcoensis occurring on coffee in Brazil. Although the overall distribution of this species in Brazil is unknown, its occurrence on coffee would indicate a potentially important threat to the commercial cultivation of coffee, particularly in the state of Minas Gerais. This find resulted in the removal of the coffee plantation where this nematode was found.

Development of DNA and GC-MS Fingerprints for Authentication and Quality Control of Piper nigrum L. and Its Adulterant Carica papaya L.

Piper nigrum L. (family: Piperaceae) is cultivated for its mature fruit/berries. Generally dried, they are one of the most important commercial spice/food additives. The dried fruit of P. nigrum is almost similar in morphology to seeds of Carica papaya L. and is used as an adulterant of P. nigrum. Molecular technology and plant metabolomics are effective tools for unequivocal authentication and detection of food ingredients. In this study, DNA fingerprinting-based quality control markers [sequence characterized amplified region (SCAR) markers] were employed for authentication and discrimination of P. nigrum from its adulterant C. papaya. The 500- and 750-bp-sized SCAR markers of P. nigrum and C. papaya were amplified using their specific SCAR primer sets. Further, metabolic profiling was carried out using gas chromatography–mass spectrometry (GC-MS) on the fruits and seeds of P. nigrum and C. papaya, respectively. For P. nigrum and for C. papaya, 84 and 61 chemically diverse metabolites were observed, respectively, among which 44 and 33 metabolites were putatively selected, respectively. The detection efficiency of GC-MS was assessed by combining the powdered samples of both P. nigrum and C. papaya in different ratios and was able to detect as low as 20 mg/g (w/w) (20,000 ppm) of metabolites. Multivariate data analysis and clustering analysis was performed on the mixture samples of both the food ingredients. Thus, development of SCAR markers along with the phytochemical fingerprints can correlate DNA fingerprinting data with the quantity of active ingredient and/or phytochemical constituents associated with the specific spice/herb/(s).

SCAR Marker for the A Genome of Bananas (Musa spp. L.) Supports Lack of Differentiation between the A and B Genomes

Bananas (Musa spp. L.) are grouped on the basis of their genomic origins in relation to Musa acuminata (A genome) and M. balbisiana (B genome). The two ancestral wild seeded diploid species evolved in vastly different geographical areas and contributed several agronomic traits towards the present genetic composition of cultivated bananas. Most cultivated bananas are triploid (AAA, AAB and ABB), some are diploid (AA, BB and AB) and a few are tetraploids (AAAA, AAAB, AABB and ABBB). Limitations on the correct identification of the A and B genomes in Musa have generated need for the development of new and more reliable techniques. Distinguishing the A and the B genome remains practically and theoretically important for banana breeders. The aim of the research was to develop a DNA based A genome specific marker for the identification of the A genome in bananas. A putative marker (600 bp) specific to the A genome was identified by Random Amplified Polymorphic DNA (RAPD) technique. A sequence characterised amplified region (SCAR) marker was developed from the RAPD amplicon. The SCAR primers annealed a 500 bp fragment specific to the A genome in a sample of 22 randomly selected homo- and heterogenomic A genome containing accessions representing different genome combinations. The 500 bp SCAR marker is useful for the identification of the A genome. However an additional 700 bp fragment annealed in all M. balbisiana genotypes and in five of the eight heterogenomic accessions, suggesting lack of differentiation between the A and B genome. This study has provided a 500 bp A genome SCAR marker and recent evidence that the A and B genomes of banana may not be as differentiated as previously considered.

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

AbstractStripe 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.

Early Sex Identification in Date Palm by Male-Specific Sequence-Characterized Amplified Region (SCAR) Markers.

Date palm (Phoenix dactylifera L.) is a dioecious plant, and sex of the seedlings can be determined only at the time of first flowering which takes 4-5years. Female date palm plants are of economic importance as they bear the fruit. Therefore, sex identification at an early stage is highly desirable. DNA-based markers are useful for early sex detection. In this chapter, we describe male-specific sequence-characterized amplified region (SCAR) markers to identify sex in date palm at the seedling stage. Genomic DNA is isolated separately from both male and female date palm genotypes. Amplification of this genomic DNA isolated from male and female plants using the SCAR primers results in an amplicon of 406bp in both female and male samples and a unique amplicon of 354bp only in male samples. Based on this amplification pattern, the sex of date palm seedlings can be predicted.

Development of a SCAR marker associated with salt tolerance in durum wheat (Triticum turgidum ssp. durum) from a semi-arid region

Durum wheat (Triticum turgidum ssp. durum) is one of the main species of cultivated wheat. In arid and semi-arid areas, salinity stress reduces durum wheat productivity. This study used 26 durum wheat accessions from semi-arid regions in Tunisia to analyze plant tolerance to salt stress. Salt stress was experimentally applied by regularly submerging pots in NaCl solution. The salt tolerance trait index (STTI) and salt tolerance index (STI) of various growth parameters were used as criteria to select for salt tolerance. Analysis of genetic relationships was carried out to determine the genetic distance between durum wheat accessions. Based on simple sequence repeats analysis, a molecular marker for salt stress resistance in durum wheat was developed. Salt-treated plants had reduced morphological traits compared to control plants. Most STTIs in all genotypes were below 100 %. Based on STI, 8 accessions were found to be salt-resistant, 16 were salt-moderate, two were salt-susceptible. Analysis of the genetic relationships among 28 Tunisian durum wheat accessions revealed that landraces of the same nominal type are closely related. Of the 94 SSR primers investigated, three were selected and used to design sequence characterized amplified region (SCAR) primers. One SCAR primer pair, KUCMB_Xgwm403_2, produced a 207 bp band that was present in salt-resistant durum wheat lines but absent in salt-susceptible lines. The results suggest that KUCMB_Xgwm403_2 could be a potential genetic tag for salt-tolerant durum wheats.

Development of sequence-characterized amplified region (SCAR) markers as a quality standard of inoculants based on Azospirillum.

An attempt was made in this work to develop a strain-level molecular marker for unambiguous authentication of two Azospirillum inoculants, viz. A. lipoferum (strain Az204) and A. brasilense (strain Sp7). The sequence-characterized amplified region (SCAR) markers obtained from DNA fingerprints were designed for discrete detection of the strains. The SCAR primers could successfully amplify the target strain without cross-reaction with other Azospirillum strains, native isolates and other inoculants. The detection limit of SCAR primer for Az204 was 8.00 pg of DNA (approximately 10(5) cells per mL), and for Sp7, it was 0.49 pg of DNA (equal to 10(4) cells per mL). A simplified Sephadex G100-based crude DNA extraction protocol developed in this study was found suitable for SCAR marker-based strain authentication. Further, SCAR primers were assessed for simultaneous authentication as well as quantification of commercially prepared Azospirillum inoculants by quantitative real-time PCR (RT-PCR) and most-probable-number PCR (MPN-PCR). The RT-PCR assay can be able to quantify the commercial formulations as equal to culturable MPN method, while MPN-PCR failed for Az204. The SCAR marker-based strain authentication and presumptive quantification developed in the present work can contribute to improving the quality standard of commercial inoculants.

A two-step method for identification of the Chinese glutinous rice Suyunuo, based on ISSR-SCAR and allele-specific markers.

Suyunuo is a valuable glutinous rice variety cultivated mainly in the Lake Taihu area of China. Historically, Suyunuo was presented to emperors as a tribute, and, still today, enjoys a great reputation in China. This study aimed to develop a unique, specific molecular marker for the identification of Suyunuo rice. Polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) molecular markers was performed on Suyunuo and 11 other glutinous rice varieties that are mainly cultivated in the Yangtze River Delta region. A Suyunuo-specific band was detected in the PCR products generated from primer ISSR-807. A sequence characterized amplified region (SCAR) primer pair targeting a Suyunuo-specific band was subsequently designed. The SCAR primers amplified a target band in all individuals of Suyunuo and in four glutinous indica varieties, whereas no bands were found in the seven glutinous japonica varieties. Subsequently, sequences amplified by the SCAR primer pair were analyzed to facilitate the design of Suyunuo allele-specific primers. The allele-specific primer pair produced target bands in all individuals of Suyunuo rice but no bands in individuals of any of the other 11 rice varieties. This study provides a theoretical guideline for rice germplasm identification and innovation of other valuable rice landraces.