Sequence Characterized Amplified Region Primer Pair Research Articles

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.

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.

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.

Specific and Sensitive Detection of Venturia nashicola, the Scab Fungus of Asian Pears, by Nested PCR.

The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

AbstractAgrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.

The development of species-specific SCAR markers for delimitation of Calypogeia species

The genus Calypogeia belongs to one of the most difficult groups of leafy liverworts, with about 90 species. Nine species of the genus are currently known from Europe: C. azurea, C. integristipula, C. neesiana, C. suecica, C. muelleriana, C. sphagnicola, C. fissa, C. arguta, and C. azorica. Recently, another species, morphologically resembling C. muelleriana but genetically distinct from it, was detected using isozyme markers. In the present study, C. muelleriana and C. sp. nov. were further characterized by 13 and 14 specific inter-simple sequence repeat band patterns, respectively. Nei’s genetic distance between the two species was 0·61. Three species-specific bands were converted into sequence characterized amplified region (SCAR) markers. The first SCAR primer pair amplified a product of 360 and 600 bp in C. muelleriana and C. sp. nov., respectively. The two other SCAR markers were diagnostic for other European Calypogeia species.

느타리 버섯에서 수한 품종 특이 SCAR marker 개발

현재 70개 이상의 느타리 품종이 유통되어 재배되고 있다. 본 연구에서는 81개 품종을 수집하여 핵산지문법으로 분석하였다. 이중 수한과 그 유사품종에서 특이하게 나타나는 밴드를 이용하여 수한 품종에 특이적인 SCAR marker로 개발하였다. 수한 품종 특이 band에 대한 clone을 기존에 알려진 유전자 염기서열과 유사성이 있는지를 확인한 결과 POMFBO1 Pleurotus ostreatus cDNA clone MFB02-A05, mRNA sequence와 92%의 homology를 나타냈고, 등록된 아미노산 서열과의 유사성을 확인한 결과 큰졸각버섯인 Laccaria bicolor의 predicted protein과 가장 높은 sequence homology (BlastX score = 73.9, E value = <TEX>$5e^{-25}$</TEX>)를 보였다. 본 연구를 통하여 개발된 수한특이 마커는 정확한 품종구분이 요구되는 종균유통 과정에서 수한계통 품종을 구분하는 유용한 마커로 이용될 것으로 기대된다. In this study, 81 commercial strains of Pleurotus species cultivated in South Korea were analyzed with randomly amplified polymorphic DNA (RAPD) technique. Sequence characterized amplified region (SCAR) markers were developed by designing from one RAPD polymorhic band specific to Suhan strain. The SCAR primer pair 'S-OPA13-1' amplified a 590-bp fragment in the varieties originated from Suhan strain. The Blast search of S-OPA13-1 showed high homology to the POMFBO1 P. ostreatus cDNA clone MFB02-A05 and Laccaria bicolor S238N-H82. The results showed that this SCAR marker can clearly distinguish Suhan strains from Pleurotus spp.

Development of SCAR primers for the detection of Cadophora luteo-olivacea on kiwifruit and pome fruit and of Cadophora malorum on pome fruit.

800x600 Normal 0 14 false false false IT X-NONE X-NONE MicrosoftInternetExplorer4 In recent years a postharvest disease of kiwifruit, characterized by skin pitting appearing after 3 or more months of storage, and caused by C. luteo-olivacea , has been reported in most Italian packinghouses. Forty-four strains isolated from kiwifruit harvested in Italy during the period 2001-2009 were tested for their pathogenicity on kiwifruit, apple and pear. The pathogenicity tests showed that different isolates of C. luteo-olivacea were pathogenic on the three fruit species stored at 1°C for 120 days with various degree of virulence. A PCR-based method to identify the pathogen species in kiwifruit was developed. Sequencing of the internal transcribed spacer (ITS1, 5.8S gene and ITS2) region of the rDNA (ITS) showed high similarity with the species C. malorum , agent of side rot of apples and pears. Variation within the ITS was used to design one reverse primer common to both species and two species specific forward primers to specifically identify isolates of C. luteo-olivacea and C. malorum. Both SCAR (sequence characterized amplified region) primer pairs showed to be specific for either Cadophora species when cross-tested and assessed on other species of Cadophora or on species of typical postharvest pathogens of kiwifruit, apple and pear .

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.

Molecular Identification of Saccharum spp. × Erianthus fulvus Hybrids Using Sequence‐Characterized Amplified Region Markers

Identification of hybrids derived from crosses between cultivated sugarcane (Saccharum spp.) and its wild relatives, including Erianthus fulvus Nees ex Hack., is important for the development of new sugarcane cultivars. The aim of the present study was to identify true hybrids in 73 F1 progeny obtained from five crosses with cultivated sugarcane as female parents and E. fulvus as male parents. Three random 10mers were used as primers in random amplified polymorphic DNA (RAPD) reactions. A total of 10 E. fulvus–specific RAPD markers were cloned and sequenced, of which four were successfully applied as sequence‐characterized amplified region (SCAR) markers. Based on the four RAPD marker sequences, five SCAR primer pairs were designed for polymerase chain reaction amplifications. Of the 73 progeny examined, 58 produced E. fulvus–specific SCAR bands, thus representing true hybrids of cultivated sugarcane and E. fulvus These results demonstrate the first use of SCAR marker analysis as an effective tool for the identification of intergeneric sugarcane hybrids.

Morphological diversity and genomic DNA fingerprinting of the African rice gall midge Orseolia oryzivora (Diptera: Cecidomyiidae) and of two other species of African Orseolia

A morphological diversity study was conducted on African rice gall midge Orseolia oryzivora Harris & Gagne and two other African species, Orseolia bonzii Harris and Orseolia nwanzei Harris & Nwilene, sp. n. Morphological differences between adult and immature stages of the three species are slight and mainly evident in pupal characters. Due to limited differences in morphological characters, genomic DNA fingerprinting of these three insect species was carried out using random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) methods. Out of 90 operon primers, 15 showed polymorphism among the three species tested, generating 86 bands, 60 (70%) of which were polymorphic. Primer OPV6 produced three RAPD markers that distinguished the three Orseolia species. These markers were cloned and sequenced. Their sequence was then used to design six SCAR primer pairs. Each SCAR primer pair amplified and distinguished the three Orseolia species at genomic DNA level. Both SCAR and RAPD genomic DNA fingerprinting revealed that O. oryzivora and O. bonzii are most closely related and O. nwanzei is distinct. Each of the six SCAR primer pairs produced a specific DNA fragment size specific for O. nwanzei, O. oryzivora and O. bonzii. The DNA fingerprints will be useful for entomological survey, identification of new species and differentiating aggressive from non-aggressive species, aimed at the effective development of rice cultivars with durable resistance to African rice gall midge.

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers forLentinula edodes

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.

Construction of an oilseed rape (Brassica napus L.) genetic map with SSR markers

We constructed a Brassica napus genetic map with 240 simple sequence repeats (SSR) primer pairs from private and public origins. SSR, or microsatellites, are highly polymorphic and efficient markers for the analysis of plant genomes. Our selection of primer pairs corresponded to 305 genetic loci that we were able to map. In addition, we also used 52 sequence-characterized amplified region primer pairs corresponding to 58 loci that were developed in our lab. Genotyping was performed on six F2 populations, corresponding to a total of 574 F2 individual plants, obtained according to an unbalanced diallel cross design involving six parental lines. The resulting consensus map presented 19 linkage groups ranging from 46.2 to 276.5 cM, which we were able to name after the B. napus map available at http://ukcrop.net/perl/ace/search/BrassicaDB , thus enabling the identification of the A genome linkage groups originating from the B. rapa ancestor and the C genome linkage groups originating from the B. oleracea ancestor in the amphidiploid genome of B. napus. Some homologous regions were identified between the A and the C genomes. This map could be used to identify more markers, which would eventually be linked to genes controlling important agronomic characters in rapeseed. Furthermore, considering the good genome coverage we obtained, together with an observed homogenous distribution of the loci across the genome, this map is a powerful tool to be used in marker-assisted breeding.