Specific Sequence Characterized Amplified Region Markers Research Articles

Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

A rapid identification assay for Waitea circinata (anamorph: Rhizoctonia spp.) varieties zeae and circinata causing patch diseases on turfgrasses was developed based on the universally primed PCR (UP-PCR) products cross-blot hybridization. Tester isolates belonging to the two varieties of W. circinata were amplified with a single UP primer L21, which generated multiple DNA fragments for each variety. Probes were prepared with UP-PCR products of each tester isolate by labeling with digoxigenin. Fieldcollected W. circinata isolates and representative isolates of different R. solani anastomosis groups (AG) and AG subgroups were amplified with L21, immobilized on nylon membrane and cross hybridized with the two probes. Isolates within a W. circinata variety cross-hybridized strongly, while non-homologous isolates did not cross-hybridize or did so weakly. Closely related W. circinata varieties zeae and circinata were clearly distinguished with this assay. Sequence-characterized amplified region (SCAR) markers also were developed from UP-PCR products to identify isolates of Thanatephorus cucumeris (anamorph: R. solani) AG 1-IB and AG 2-2IIIB. These two AGs are commonly isolated from diseased, cool-season turfgrasses. The specific SCAR markers that were developed could differentiate isolates of AG 1-IB or AG 2-2IIIB groups. These SCAR markers did not amplify a product from genomic DNA of nontarget isolates of Rhizoctonia. The specificities and sensitivities of the SCAR primers were tested on total DNA extracted from several field-grown, cool-season turf species having severe brown-patch symptoms. First, the leaf samples from diseased turf species were tested for the anastomosis groups of the causal pathogen, and thereafter the total DNA was amplified with the specific primers. The specific primers were sensitive and unique enough to produce a band from total DNA of diseased turfgrasses infected with either AG 1-IB or AG 2-2IIIB.

오미자 (五味子) 종 감별을 위한 RAPD 유래 SCAR Marker 및 Multiplex-PCR 기법 개발

The fruits of Schisandra chinensis have been used as an edible ingredient and traditional medicine in Korea. Due to morphological similarities of dried mature fruits, the correct identification of S. chinensis from other closely related Schisandrae species is very difficult. Therefore, molecular biological tools based on genetic analysis are required to identify authentic Schisandrae Fructus. Random amplifed polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop an easy, reliable and reproducible method for the authentication of these four species. In this paper, we developed several RAPD-derived species specific SCAR markers and established a multiplex-PCR condi-tion suitable to discriminate each species. These genetic markers will be useful to distinguish and authenticate Schisandrae Fructus and four medicinal plants, S. chinensis, S. sphenanthera, S. repanda and K. japonica, in species level.

A new genotype of Miscanthus sacchariflorus Geodae-Uksae 1, identified by growth characteristics and a specific SCAR marker

Miscanthus is referred to as an ideal lignocellulosic bioenergy crop, which can be used to generate heat, power, and fuel, as well as to reduce carbon dioxide emissions. The new Miscanthus sacchariflorus genotype named Geodae-Uksae 1 was recently collected from damp land in southern Korea. This study investigated the growth characteristics of Miscanthus genotypes, and developed a specific, sensitive, and reproducible sequence characterized amplified region (SCAR) marker to distinguish new M. sacchariflorus genotype Geodae-Uksae 1 from other native Miscanthus species in Korea. Growth characteristics such as stem length, stem diameter, and dry weight of Geodae-Uksae 1 were greater than those of normal M. sacchariflorus. The genotypes within Geodae-Uksae 1 were had the highest genetic similarity. A putative 1,800-bp polymorphic sequence specific to Geodae-Uksae 1 was identified with the random amplified polymorphic DNA (RAPD) N8018 primer. The sequence-characterized amplified region (SCAR) primers Geodae 1-F and Geodae 1-R were designed based on the unique RAPD amplicon. The SCAR primers produced a specific 1,799-bp amplicon in authentic Geodae-Uksae 1, whereas no amplification was observed in other Miscanthus species. The SCAR marker could contribute to identify Geodae-Uksae 1 among native Miscanthus species. The new Miscanthus genotype Geodae-Uksae 1 has great potential as an alternative lignocellulosic biomass feedstock for bioenergy productions.

Identification of a specific SCAR marker for detection of Tilletia foetida (Wall) Liro pathogen of wheat

Common bunt is one of the most important destructive diseases of wheat worldwide and is a domestic quarantined disease in China. However, a rapid and efficient method to identify the corresponding pathogens is currently limited. The objective of the present study was to develop a diagnostic molecular marker specific towards Tilletia foetida (Wall) Liro, a causal agent of the bunt disease. One specific DNA fragment for T. foetida (286 bp in length) was amplified using an Amplified Fragment Length Polymorphism (AFLP) assay and, this fragment was cloned and sequenced. One pair of specific primers (SC(286-1)/SC(286-2)), which was designed according to the sequence, could specifically amplify the corresponding fragment in all of the T. foetida isolates employed from both the People's Republic of China and United States, whereas this fragment could not be amplified by the other fungal species tested. Therefore, a specific Sequence Characterized Amplified Region (SCAR) marker was developed. This SCAR marker could distinguish T. foetida from related pathogenic fungi efficiently and could be used for the early diagnosis of the common bunt of wheat in the field, and provide an efficient way for disease surveillance and disease forecasting in cereal crop.

Development of Specific Sequence-Characterized Amplified Region Markers for Detecting Histoplasma capsulatum in Clinical and Environmental Samples

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283(220) and 1281-1283(230). The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283(220) and 1281-1283(230) as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283(220) SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283(230) SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283(220) marker can be used to detect and identify H. capsulatum in samples from different sources.

An Isaria fumosorosea SCAR marker for evaluation of soil, insect, and airborne samples

Molecular detection markers are needed for ecological studies of entomopathogenic fungi. In this study, a sequence-characterized amplified region (SCAR) marker of Isaria fumosorosea was used to detect the fungus in soil, insects, and airborne samples. These were artificially added with different fungal conidial concentrations. Specificity and sensitivity were tested with semi-nested PCR using oligonucleotides E-AA/M-CTA124 F and E-AA/M-CTA124 R for the first amplification and E-AA/M-CTA124 F and E-AA/M-CTA103 R for the second amplification. Specificity assays showed a specific band of 103 bp for DNA samples from 10 I. fumosorosea strains used. Negative results were observed for DNA samples from other species of Isaria, including I. amoene-rosea, I. farinosa, and Paecilomyces carneus as well as with other entomopathogens such as Metarhizium acridum, M. anisopliae, M. majus, M. flavoviride Type E, and Lecanicillium lecanii. Sensitivity assays showed that the specific SCAR marker detected 104 conidia from I. fumosorosea EH-511/3 that were artificially mixed with soil, from 1 to 104 conidia artificially mixed with Galleria mellonella (Lepidoptera: Pyralidae), and from 10 to 105 conidia in Melinex tape for airborne samples. The marker was also able to detect conidia in airborne samples from cotton wicks in a mini wind tunnel. These SCAR markers for I. fumosorosea had excellent specificity and sensitivity and are relevant tools for ecological studies of this fungus.

Sequence characterized amplified region markers: A reliable tool for adulterant detection in turmeric powder

Turmeric powder ( Curcuma longa L.), an important medicinal spice product traded internationally, is subjected to adulteration by design or default with powders of related curcumin containing wild species like Curcuma zedoaria and Curcuma malabarica leading to toxicity and poor quality of the produce. The present study aims at development of specific, sensitive and reproducible Sequence Characterized Amplified Region (SCAR) markers to detect these adulterants in traded turmeric powder. Two putative RAPD markers, ‘Cur 01’ and ‘Cur 02’, generated by random primers OPA 01 and OPE 18 were identified as C. zedoaria/ C. malabarica specific by comparative RAPD analysis of genuine turmeric and market samples of turmeric powder, C. zedoaria and C. malabarica. These specific RAPD markers were cloned and sequenced. Two pairs of SCAR primers were designed from the RAPD markers ‘Cur 01’ and ‘Cur 02’, respectively. Six market samples of turmeric powder and four simulated standards besides the genuine samples were analyzed using the specific SCAR markers. Both the SCAR markers detected the presence of C. zedoaria/ C. malabarica adulteration in four market samples and all the simulated standards prepared in different concentrations. The two SCAR markers developed in the study would be potentially useful for the regulatory agencies to detect C. zedoaria/ C. malabarica adulteration in traded turmeric powder. The analytical strategy being very simple could be used for large scale screening of turmeric powder samples intended for export and domestic uses.

Molecular characterization of cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots.

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.

Development and Application of SCAR Marker for the Detection of Papaya Seed Adulteration in Traded Black Pepper Powder

The present study reports the development of a specific, sensitive, and reproducible Sequence Characterized Amplified Region (SCAR) marker to detect papaya seed powder adulteration in traded black pepper powder. A putative RAPD marker (449 bp) specific to papaya seed was identified, cloned, and sequenced to design the SCAR primers. This specific SCAR marker could detect the presence of papaya seed in all the analyzed simulated standards and in one of five branded market samples of black pepper powder tested. The analytical strategy being very simple could be used for large scale screening of powdered black pepper market samples intended for export and domestic uses.

RAPD-based SCAR marker SCA 12 linked to recessive gene conferring resistance to anthracnose in sorghum [Sorghum bicolor (L.) Moench

Anthracnose, caused by Colletotrichum graminicola, infects all aerial parts of sorghum, Sorghum bicolor (L.) Moench, plants and causes loss of as much as 70%. F(1) and F(2) plants inoculated with local isolates of C. graminicola indicated that resistance to anthracnose in sorghum accession G 73 segregated as a recessive trait in a cross with susceptible cultivar HC 136. To facilitate the use of marker-assisted selection in sorghum breeding programs, a PCR-based specific sequence characterized amplified region (SCAR) marker was developed. A total of 29 resistant and 20 susceptible recombinant inbred lines (RILs) derived from a HC 136 x G 73 cross was used for bulked segregant analysis to identify a RAPD marker closely linked to a gene for resistance to anthracnose. The polymorphism between the parents HC 136 and G 73 was evaluated using 84 random sequence decamer primers. Among these, only 24 primers generated polymorphism. On bulked segregant analysis, primer OPA 12 amplified a unique band of 383 bp only in the resistant parent G 73 and resistant bulk. Segregation analysis of individual RILs showed the marker OPA 12(383) was 6.03 cM from the locus governing resistance to anthracnose. The marker OPA 12(383) was cloned and sequenced. Based on the sequence of cloned RAPD product, a pair of SCAR markers SCA 12-1 and SCA 12-2 was designed using the MacVector program, which specifically amplified this RAPD fragment in resistant parent G 73, resistant bulk and respective RILs. Therefore, it was confirmed that SCAR marker SCA 12 is at the same locus as RAPD marker OPA 12(383) and hence, is linked to the gene for resistance to anthracnose.

Molecular markers detecting an ectomycorrhizal Suillus collinitus strain on Pinus halepensis roots suggest successful inoculation and persistence in Mediterranean nursery and plantation

Survival of the ectomycorrhizal fungal strain Suillus collinitus Sc-32 on Pinus halepensis after inoculation and outplanting was monitored in a Mediterranean plantation. Three molecular fingerprints were developed: RFLP of the internal transcribed spacer ribosomal DNA, intersimple sequence repeat, and a specific sequence-characterized amplified region marker. The inoculant was demonstrated to survive on inoculated seedlings 4 years after outplanting (56 months after inoculation), although S. collinitus was not fruiting. The designed markers set allows reliable and inexpensive monitoring of inoculated seedlings and suggests that S. collinitus is suitable for inoculation of Mediterranean Pinus. These data are discussed in the framework of suilloid population ecology.

DEVELOPMENT OF SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS FROM INTERSIMPLE SEQUENCE REPEAT FINGERPRINTS FOR THE MOLECULAR DETECTION OF TOXIC PHYTOPLANKTON ALEXANDRIUM CATENELLA (DINOPHYCEAE) AND PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA (BACILLARIOPHYCEAE) FROM FRENCH COASTAL WATERS1

Harmful algal blooms are a serious threat to shellfish farming and human health all over the world. The monitoring of harmful algae in coastal waters originally involved morphological identification through microscopic examinations, which was often difficult unless performed by specialists and even then often did not permit identification of toxic species. More recently, specific molecular markers have been used to identify specific phytoplankton species or strains. Here we report on the use of the intersimple sequence repeat (ISSR) technique to develop specific sequence characterized amplified region markers (SCAR) and to identify with these tools two toxic species in French coastal waters, the diatom Pseudo‐nitzschia pseudodelicatissima (Hasle) Hasle and the dinoflagellate Alexandrium catenella (Whedon and Kofoid 1936), Balech 1985. Six polymorphic ISSR regions were selected among amplified fingerprints of a representative sample of phytoplankton species. After cloning and sequencing the selected polymorphic ISSR regions, pairs of internal primers were designed to amplify a unique and specific sequence designed as a SCAR marker. Of the six selected SCAR markers, three were specific to P. pseudodelicatissima and one for A. catenella. The SCAR marker specificity was confirmed by using basic local alignment search tool comparison, by experimental assays on different strains from 11 countries, and by checking that the sequence amplified was the expected one. When tested on water samples collected along the French shores, the four specific SCAR markers proved to be efficient tools for fast and low‐cost detection of toxic phytoplankton species.

DNA markers for sex: Molecular evidence for gender dimorphism in dioecious Mercurialis annua L.

Male specific Random Amplified Polymorphic DNA (RAPD) markers, OPB01-1562 and OPC07-303, were identified and sequenced in dioecious Mercurialis annua. Sequence Characterized Amplified Region (SCAR) primers were designed. Several internal segments of OPB01-1562 were amplified as male specific SCAR markers. These markers were PCR amplified from strong, intermediate and weak male subtypes selected according to their resistance to feminization by cytokinin. Nucleotide sequence of OPB01-1562 isolated from three male subtypes were near identical. The OPB01-1562 and derived SCAR markers were absent in females as well as hexaploid Mercurialis male and monoecious individuals. The gender relationship of the markers was maintained in all ecotypes tested. There were 2 internal fragments of OPB01-1562, which were PCR amplified from all genotypes of diploid and hexaploid Mercurialis. It is argued that identification of gender specific DNA suggests a dimorphic differentiation of the genome of dioecious Mercurialis annua.

Development of SCAR and CAPS Markers Linked to the Beta Gene in Tomato

Two previously identified random amplified polymorphic DNA (RAPD) markers, OPAR181100 and UBC792830, linked to the Beta (B) locus in tomato (Lycopersicon esculentum Mill.) were cloned and sequenced. Their end sequences were used to design allele specific sequence characterized amplified region (SCAR) primer pairs, SCAR18f/r and SCBC792f/r. Each of these primer pairs amplified a single product of the same size as their respective progenitor RAPD markers, but did not differentiate the two parental genotypes under a variety of polymerase chain reaction (PCR) conditions. Sequence analysis of cloned RAPD and SCAR products revealed an RsaI site mutation in the allele of the high beta‐carotene parent in the SCAR18f/r amplified DNA product which was used to develop the codominant SCAR181067 cleaved amplified polymorphic sequence (CAPS) marker. Similarly, a HinfI site mutation in the allele of the high beta‐carotene parent in SCBC792f/r amplified DNA product was used to develop the codominant SCBC792779 CAPS marker. Sequences from SCBC792f/r amplified DNA products revealed additional single nucleotide polymorphisms between the two parental genotypes. Three such polymorphisms were used to design the nested primer SCBC792f1 located 97 bp internal to the SCBC792f sequence. The SCBC792f1/r primer pair amplified the dominant SCB792682 SCAR marker present only in the high beta‐carotene parent. Linkage of the CAPS and SCAR markers to B was confirmed by means of a population of 144 F2 individuals segregating for B Using introgression line analysis, we mapped the two CAPS and the SCAR markers to the long arm of chromosome 6, consistent with the location of B on the classical linkage map of tomato.

049 Development of Molecular Markers Linked to High Pigment (hp) and Dark Green (dg) Loci in Tomato

The carotenoids have an important influence on tomato fruit quality and enhance the fruit contribution to human nutrition. Expression of the high pigment (hp) locus in tomato results in increased total carotenoids and increased efficiency of utilization of the polyenes. A similar mutant, dark green (dg), contains higher level of chlorophyll in immature fruit and results in darker red pigmentation, both externally and internally in ripe fruit. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses were performed using two pairs of near isogenic lines (NILs) designed to be isogenic at the hp and dg loci. Sixty-four AFLP primer pairs and more than 1000 RAPD 10-mer primers were screened for polymorphism between each pair of the NILs. One RAPD marker was identified to be linked to the hp gene, and two AFLP primer pairs showed polymorphic fragments which distinguished the dg NILs. The markers identified in this study will be converted to allele specific SCAR (sequence characterized amplified region) markers, which are more useful in marker-assisted selection breeding programs.