Sequence Characterized Amplified Region Markers Research Articles

Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers

Dried parts of different plant species often look alike, especially in powdered form, making them very difficult to identify. Ruta graveolens, sold as a dried medicinal herb, can be adulterated with Euphorbia dracunculoides. The genomic DNA was isolated from the leaf powder (100 mg each) using the modified CTAB method. Internal transcribed spacer sequences of nuclear ribosomal DNA (nrDNA-ITS), and chloroplast spacer sequences (rpoB and rpoC1) are regarded as potential genes for plant DNA barcoding. We amplified and sequenced these spacer sequences and confirmed the sequences with a BLAST search. Sequence alignment was performed using ClustalX to look for differences in the sequences. A DNA marker was developed based on rpoB and rpoC1 of the nrDNA-ITS for the identification of the adulterant E. dracunculoides in samples of R. graveolens that are sold in local herbal markets. Sequence-characterized amplified region markers of 289 and 264 bp for R. graveolens and 424 bp for E. dracunculoides were developed from dissimilar sequences of this nrDNA-ITS to speed up the authentication process. This marker successfully distinguished these species in extracted samples with as little as 5 ng DNA/μL extract.

Development of Molecular Markers Linked to the Allele Associated with the Non-astringent Trait of the Chinese Persimmon (Diospyros kaki Thunb.)

Persimmon cultivars are classified into four types depending on the nature of astringency loss of the fruit, namely pollination constant non-astringent (PCNA), pollination variant non-astringent (PVNA), pollination variant astringent (PVA), and pollination constant astringent (PCA). Among these four types, PCNA is the most important cultivar for persimmon breeding due to the stable loss of natural astringency on the tree. The trait of natural astringency loss is recessive in Japanese PCNA cultivars, while that in the Chinese PCNA cultivar, ‘Luo tian tian shi’, is dominant and the latter locus, termed CPCNA, is different from that in Japanese cultivars. In order to develop a molecular marker for the selection of the CPCNA-type cultivar, we performed amplified fragment length polymorphism (AFLP) in combination with bulked segregant analysis for F1 offspring derived from ‘Luo tian tian shi’, which was used as the maternal parent. A total of 384 primer combinations were tested, and three AFLP markers, namely EACT-MCCC-222 (RO1), EGGC-MCTC-309 (RO2), and EGCC-MCGA-105 (RO3), linked to the CPCNA dominant allele were obtained. Among these markers, EGGC/MCTC-309 (RO2) was converted into a sequence-characterized amplified region (SCAR) marker. PCR analysis using F1 offspring (n = 264) revealed that the relevance ratio of the SCAR marker was 94%. The polymorphism of the RO2 marker, which was strongly linked to the CPCNA dominant allele, was detected in only two Chinese PCNA cultivars, namely ‘Luo tian tian shi’ and ‘Tian bao gai’, among the Chinese, Korean, and Japanese cultivars tested. These results indicate that the RO2 marker contributes to marker-assisted selection for breeding programs of new PCNA cultivars having the CPCNA trait.

Development of SCAR marker in Casuarina equisetifolia for species authentication

Casuarina equisetifolia is one of the most extensively introduced tree species outside its natural range and is a true multipurpose species, providing a range of services and products for industrial and local end users. Natural hybrids of C. equisetifolia have been reported and a putative hybrid of C. junghuhniana and C. equisetifolia is commercially cultivated in Thailand and India. In Taiwan, studies have also revealed that most plants of Casuarina are the result of introgressive hybridization involving C. equisetifolia, C. glauca, and C. cunninghamiana causing difficulty in species identification. The present study was aimed at developing DNA markers for the identification of C. equisetifolia. Seven ISSR primers were amplified in 120 individuals belonging to three species of Casuarina (C. equisetifolia, C. glauca and C. junghuhniana) and two Allocasuarina species (A. littoralis and A. huegeliana). One species-specific amplicon at 650 bp amplified in all individuals of C. equisetifolia was cloned, sequenced and primer pairs were developed and designated as IFGTBCE01. The sequence characterized amplified region (SCAR) marker was multiplexed with internal SSR primer pair (positive control) and amplified in ten randomly selected individuals of C. equisetifolia, C. junghuhniana, C. glauca, A. littoralis and A. huegeliana. The species-specific band amplified only in C. equisetifolia at 500 bp. Subsequently, the SCAR marker was validated in 30 individuals collected from 10 provenances belonging to 9 countries and in 10 locally selected clones. The marker was validated in the natural male hybrid of C. equisetifolia × C. junghuhniana which is widely planted in India for fast growth, wide adaptability and good form. Hence, this marker can be used for accurate and rapid identification of the species during certification and determination of putative hybrids in breeding programs.

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

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

Development of SCAR Markers for Species Identification in the Genus Shorea (Dipterocarpaceae)

Abstract The development of molecular markers unambiguously distinguishing groups at different taxonomic levels has numerous forensic applications. The identification of tropical timber is of particular interest in this context. We describe the development of SCAR (Sequence Characterized Amplified Region) markers for forensic applications taking the example of two closely related species of the tropical tree family Shorea (Dipterocarpaceae). Two AFLP (Amplified Fragment Length Polymorphism) fragments have been described earlier showing strong differentiation between S. leprosula and S. parvifolia. The AFLP markers were isolated from a gel, re-amplified, cloned and sequenced. Primer sets were designed from these sequences and AFLP fragments were converted into SCAR markers. The SCAR markers and PCR-RFLP markers of the chloroplast region trnLF digested with HinfI were used to screen in total 557 samples of S. parvifolia and S. leprosula from nineteen widely separated populations in Indonesia. Complete genetic differentiation between species was observed based on the putatively nuclear SCAR marker and the PCR-RFLP of the cpDNA region. We found a good agreement between leaf morphological variation and species identification based on both marker types and no indication for interspecific hybridization.

The development of a new system of genetic markers for Kamchatka mykiss Parasalmo (Onchorhynchus) mykiss

Two new SCAR (Sequence Characterized Amplified Region) and ISSR (Inter Simple Sequence Repeats or Inter-Microsatellite-PCR) gene marker systems were developed for genetic analysis of populations of Kamchatka mykiss Parasalmo (O.) mykiss and other salmonids. Both systems demonstrated a high level of variability between newborn populations of mykiss, and, despite the initial species specificity, can be used for analysis of other salmonids with a complex population structure.

Development and validation of molecular markers for characterization of Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima

BackgroundThe root of Boehmeria spp (ramie) is a hepatoprotective Chinese herbal medicine. Medicinal properties vary between Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima, which are local species found in Taiwan. As commercial preparations may use either species, there is a need for a rapid and simple assay to identify variants for quality control.MethodsFour methods were developed and tested for their applicability in differentiating the two species. These methods were random amplified polymorphic DNA (RAPD); sequence characterized amplified regions (SCAR); single nucleotide polymorphisms (SNP) and cleaved amplified polymorphic sequences (CAPS).ResultsThree RAPD markers were developed that produced unique bands in B. nivea var. tenacissima and B. nivea var. nivea. Based on sequenced RAPD bands, one SCAR marker was developed that produced a single DNA band in B. nivea var. nivea. Two SNP markers differentiated between B. nivea var. nivea and B. nivea var. tenacissima based on single nucleotide substitutions. A pair of CAPS oligonucleotides was developed by amplifying a 0.55-kb DNA fragment that exhibited species-specific digestion patterns with restriction enzymes Alf III and Nde I. Consistent results were obtained with all the four markers on all tested Boehmeria lines.ConclusionThe present study demonstrates the use of the RAPD, SCAR, SNP and CAPS markers for rapid identification of two closely related Boehmeria species.

Development of Y‐chromosome‐Specific SCAR Markers Conserved in Taurine, Zebu and Bubaline Cattle

Sex pre-selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR-specific primers are derived from the few single-copy Y-chromosome-specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male-specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male-specific markers (OPC16(323) and OPF10(1168)) by means of RAPD. These markers were successfully converted into SCARs (OPC16(726) and OPF10(984)) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16(323) marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16(323) is also highly similar to a bubaline Y-chromosome-specific sequence. The primers derived from the two Y-chromosome-specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.

Molecular Fingerprinting and Population Dynamics of Brevipalpus Mites on Texas Citrus

Abstract Three species of Brevipalpus mites are known potential vectors of the citrus leprosis virus (family Rhabdoviridae, genus Cyto- and Nucleorhabdovirus, CiLV). Species identification of these mites is difficult because of their small size and morphological similarities. The objective of this study was to develop an accurate and rapid molecular fingerprinting method for identifying B. phoenicis and B. californicus on Texas citrus. Iso-colonies of the two Brevipalpus species were cultured on immature citrus fruit and identified using a dichotomous key. Whole genome amplification was used to produce DNA template from single mites to identify the two species by amplified fragment length polymorphism and sequence-characterized amplified region (SCAR). Population dynamics of Brevipalpus were monitored in four citrus orchards during the 2007 growing season. Subsamples of mites were identified morphologically or by species-specific SCAR markers. Molecular fingerprinting was very efficient in identifying the two species of mites in subsamples, even in cases where the morphological identification did not discriminate between species. Brevipalpus spp. density per fruit was significantly affected by the host plant species, with sweet orange fruit hosting significantly higher density than grapefruit, Citrus x paradisi Macfad. The percentage of fruit infested with Brevipalpus mites varied significantly with time but not with host plant and the host plant by sampling date interaction. Comparison of molecular fingerprinting and morphological identification provided a high percentage of match >85% between the two methods, suggesting that molecular fingerprinting could facilitate studies on population dynamics of Brevipalpus in citrus.

Analysis of SCAR marker nucleotide sequences in maize ( Zea mays L.) somaclones

SCAR (sequence characterized amplified region) markers allow the reliable identification of unique somaclonal variations. Six SCAR markers were developed previously and were thought to be exclusively characteristic of eight maize somaclones. However, we detected two of these markers in maize lines and a cultivar unrelated to the progenitor line of the somaclones. Therefore, we sequenced these markers and performed bioinformatic searches to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat (LTR) transposon(s), precise miniature inverted repeat transposable element (MITE) excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. For example, the SCAR marker QR is represented by the two variants QR-A and QR-2. The sequences of the two variants were similar, except for a 457-bp fragment found only in QR-A; this region was denoted as Q. Region Q was flanked by the direct 3-bp repeat 5′-TAA-3′ (target site duplication; TSD) and the inverted 14-bp repeat 5′-GGGCCTGTTTGGAA-3′ (terminal inverted repeats; TIRs). These features confer the Q region with similarity to the nonautonomic Tourist-like MITE. In two groups of independently produced somaclones, the same features (morphological, molecular) were variable, which confirms the theory of ‘hot spots’ occurring in the genome. The distribution of one of the SCAR markers was confirmed using Southern blot hybridization. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases, the same mechanisms determine both in vitro and in vivo variability and that cell culture enhances the rate of heritable genomic changes that naturally occur in living organisms.

Detecting Satureja montana L. and Origanum majorana L. by means of SCAR–PCR in commercial samples of Mediterranean oregano

A recent pharmacognostic survey of the European market highlighted a high frequency of adulteration of commercial Mediterranean oregano with species of the Lamiaceae family ( Satureja montana L. and Origanum majorana L.) having a similar appearance and a superimposing essential oil profile. When performed by skilled adulterators, this dilution can only be detected by pharmacognostic assays or phytochemical analyses, which can be either extremely laborious, expensive and time consuming when multiple batches of oregano have to be evaluated, or can produce inconclusive results. Sequence-Characterized Amplified Regions markers (SCARs) specific for these adulterants were developed from Random Amplified Polymorphic DNA (RAPD) markers, in order to support results of pharmacognostic or chemical analyses and/or to speed up the primary screening of oregano batches and allow the pre-emptive rejection of suspect samples, thus narrowing the number of samples to be subjected to further tests. The SCAR primers gave rise to the amplification of specific bands of expected sizes which allowed the detection of down to 0.5% of adulterating plants, despite the small genetic distance between the species involved. A specific protocol for DNA extraction was set up. In addiction, the relatively small size of these amplicons is suitable for the analysis of potentially degraded DNA obtained from dried and stored commercial material.

Mapping of the oat crown rust resistance gene Pc91

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Crown rust is efficiently and effectively managed through the development of resistant oat varieties. Pc91 is a seedling crown rust resistance gene that is highly effective against the current P. coronata population in North America. The primary objective of this study was to develop DNA markers linked to Pc91 for purposes of marker-assisted selection in oat breeding programs. The Pc91 locus was mapped using a population of F7-derived recombinant inbred lines developed from the cross 'CDC Sol-Fi'/'HiFi' made at the Crop Development Centre, University of Saskatchewan. The population was evaluated for reaction to P. coronata in field nurseries in 2008 and 2009. Pc91 mapped to a linkage group consisting of 44 Diversity Array Technology (DArT) markers. DArTs were successfully converted to sequence characterized amplified region (SCAR) markers. Five robust SCARs were developed from three non-redundant DArTs that co-segregated with Pc91. SCAR markers were developed for different assay systems, such that SCARs are available for agarose gel electrophoresis, capillary electrophoresis, and Taqman single nucleotide polymorphism detection. The SCAR markers accurately postulated the Pc91 status of 23 North American oat breeding lines.

Using a sequence characterized amplified region (SCAR) marker for detection of Bacillus strain TS02 sprayed on strawberry plants to bio-control powdery mildew in fields

This work used sequence characterized amplified region (SCAR) marker to detect the Bacillus cereus strain in strawberry fields. The purpose was to develop an effective molecular method for detecting the functional target microorganisms applied in agricultural fields. A 3×10 9 CFU/ml vegetative cell suspension based on the functional B. cereus strain TS02 was sprayed on strawberry plants to control powdery mildew. Primer pair LS400: 5’-TCC AAC TAC TTC TCC AT-3' and 3’-TTT GCC ATT ACA TAG AGT-5’ was used to amplify the 400 bp SCAR marker TSS 1 . TSS 1 could detect TS02 specifically, in relation to other 7 Bacillus species and 6 B. cereus strains. Sensitivity detection of TSS 1 to the lowest DNA concentration of TS02 was 78 pg/μl, which corresponded to a density of 8×10 5 CFU/ml cells of strain TS02. TS02 was specifically detected by TSS1 from the collected mixture of microorganisms on leaves during the days 1 - 12 after TS02 was sprayed in strawberry fields. TS02 DNA concentration reached about 2500 pg/μl during the days 5 - 7 after spraying and 156 pg/μl in the final day 12, which corresponded to a density of about 2.5×10 7 and 1.6×10 6 CFU/ml cells of TS02 respectively. These results showed that SCAR marker TSS1 is an effective method for the detection of strain TS02 after applied in fields. This research provided a simple and convenient method of detection of target microorganisms in fields, It could especially help monitor strain TS02 more effectively when widely been applied in agriculture. Key words : Bacillus cereus strain, biocontrol, sequence characterized amplified region marker, strawberry, Sphaerotheca macularis.

An ISSR-based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat, Caused by Tilletia controversa Kühn

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is an important international quarantine disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from intersimple sequence repeat (ISSR) for rapid identification of T. controversa. A total of 60 primers were tested by ISSR to detect DNA polymorphisms between T. controversa and related species. The primer ISSR818 generated a polymorphic pattern displaying a 952- bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF2/TCKSR2) were designed for use in a PCR detection assay. Its detection limit was 1 ng of DNA, which could be yielded by 1.1 μg of teliospores in a 25- μl PCR. Conclusively, a method to distinguish T. controversa from similar pathogenic fungi has been successfully developed based on the use of a SCAR marker.

Identification of SCAR marker linking to longer frond length of Saccharina japonica (Laminariales, Phaeophyta) using bulked-segregant analysis

To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F2-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F2-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA), out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that 80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569 will be beneficial to MAS in S. japonica breeding.

Molecular tagging and candidate gene analysis of the high gamma-tocopherol trait in safflower (Carthamus tinctorius L.)

Genetic control of the synthesis of high gamma-tocopherol (gamma-T) content in the seed oil of safflower (Carthamus tinctorius L.) and development of highly reliable molecular markers for this trait were determined through molecular tagging and candidate gene approaches. An F2 population was developed by crossing the high gamma-T natural mutant IASC-1 with the CL-1 line (standard, high alpha-T profile). This population segregated for the partially recessive gene Tph2. Bulked segregant analysis with random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers revealed linkage of eight RAPD and one SSR marker loci to the Tph2 gene and allowed the construction of a Tph2 linkage map. RAPD fragments closest to the Tph2 gene were transformed into sequence-characterized amplified region markers. A gamma-T methyltransferase (gamma-TMT) locus was found to co-segregate with Tph2. The locus/band was isolated, cloned and sequenced and it was confirmed as a gamma-TMT gene. A longer partial genomic DNA sequence from this gene was obtained. IASC-1 and CL-1 sequence alignment showed one non-synonymous and two synonymous nucleotide mutations. Intron fragment length polymorphism and insertion-deletion markers based on the gamma-TMT sequence diagnostic for the Tph2 mutation were developed and tested across 22 safflower accessions, cultivars, and breeding lines. The results from this study provide strong support for the role of the gamma-TMT gene in determining high gamma-T content in safflower and will assist introgression of thp2 alleles into elite safflower lines to develop varieties with improved tocopherol composition for specific market niches.

MELON BREEDING FOR RESISTANCE TO FUSARIUM WILT USING MOLECULAR MARKERS AND DIHAPLOIDIZATION

In this breeding program aimed at developing resistant varieties to Fusarium wilt in Turkish melon type, molecular markers and dihaploidization techniques were used. Cum334 was used as the donor parent in a backcross program and the haploid plants were obtained via pollination with the irradiated pollen from the BC3 population to shorten the breeding period. Four different embryo rescue methods were tested to extract haploid embryos. According to the results, the best methods were found to be inspecting seeds on the light and sowing seeds directly to nutrient media, and a total of the 122 haploid plants were obtained. The haploid plants were screened by molecular markers for fom1 and fom2 genes. A Simple Sequence Repeat (SSR) and a Randomly Amplified Polymorphic DNA (RAPD) marker were used for fom2 gene and a Cleaved Amplified Polymorphic Sequences (CAPS) and three Sequence Characterized Amplified Region (SCAR) markers were used for fom1 gene. While SSR (SSR180) did not segregate the haploid plants the RAPD marker (OPG17) segregated those haploid plants for fom2 gene. Haploid plants were screened for fom1 by a SCAR marker (SB17). Among the tested haploids, 41% of plants were found to be resistant and the rest of 59% were susceptible to the Fusarium wilt races 0 and 2. On the other hand while 34% of plants were resistant 66% were susceptible to the races 0 and 1 of the Fusarium oxysporum f.sp. melonis.

Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis

Accurate and early diagnosis of tuberculosis (TB) is of major importance in the management and control of TB. Because the conventional bacteriological diagnosis of TB has several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. A potential problem with NAA tests is that some strains lack a target, which may be the one of main reasons for the much lower and highly variable accuracy in diagnosis. A possible solution may be to use more valid and applicable targets to increase detection accuracy. In this paper, we designed a two-step program to obtain NAA test targets. Inter-simple sequence repeats (ISSR) based on oligonucleotide (GTG)(5) were first constructed to genotype Mycobacterium strains to obtain Mycobacterium tuberculosis (MTB)-specific fragment. Second, sequence characterized amplified region (SCAR) markers were developed from these species-specific sequences to identify MTB. Some 312 Mycobacterium strains were used to evaluate the efficacy of the SCAR markers, IS6110 element [specific identification of Mycobacterium tuberculosis complex (MTC)] and 16SrRNA gene (specific identification of Mycobacterium) amplification, together with traditional bacteriology testing was used as a control. MTB-specific sequences located in a gene coding for Rv1508c, as a new NAA test target, were obtained using ISSR-PCR genotyping. Based on these sequences, the SCAR primer pairs MISP1 and MISP2 were designed. All 312 strains from Mycobacterium accurately produced the genus-specific 16SrRNA amplicon. 271 MTB strains and M. africanum were positive. However, all nontuberculous mycobacteria (NTM) strains and 1 MTB strain named 1143 were negative in both SCAR and IS6110 PCR amplification. M. bovis, bacille Calmette-Guérin (BCG) were IS6110-PCR positive, while SCAR-PCR was negative. Strain 1143 was defined as M. arupense with 99% identity by 16SrRNA gene sequencing identification, despite being diagnosed as MTB using traditional testing. SCAR markers developed with this two-step program can be used as a new NAA test target to correctly detect MTB.

Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat (ISSR) and sequence-characterized amplified region (SCAR) melocular markers and to analyze their gene difference and genetic relationship. Methods Five carrion fly species were collected from 12 cities and regions in China, including Musca domestica (M. domestica), Lucilia sericata (L. sericata), Chrysomya megacephala (C. megacephala), Helicophagella melanura (H. melanura), Boethcherisca peregrina, and they were studied using ISSR and SCAR markers. Results Eight ISSR primers were used for amplification of 121 samples. 679 clear and stable bands were identified, of which 516 bands were polymorphic. Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers. Using M. domestica SCAR specific primers, SCAR-PCR amplification was performed for 8 M. domestca population sample DNA from different regions in China as well as L. sericata, C. megacephala, H. melanura and Lucillia cupirina. The result showed only M. domestica produced specificalty 600 bp fragment, but L. sericata, C. megacephala, H. melanura and Lucillia cupirina did not produce the same specific fragment. Clustering analysis showed clustering of most flies of M. domestica, C. megacephala and L. sericata. M. domestica samples from different regions in China yielded different banding patterns. Conclusions Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported. ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.