Sequence Characterized Amplified Region Primers Research Articles

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.

Development and detection efficiency of sequence characterized amplified region markers for authentication of medicinal plant Ruta graveolens and its adulterant Euphorbia dracunculoides

Background: With the increase in demand of herbal medicines, adulteration in these drugs is also gaining momentum and remains an indispensable problem in domestic and export markets. Correct identification is the first step toward assuring quality, safety, and efficacy of indigenous herbal medicines. Materials and Methods: In this study, sequence characterized amplified region (SCAR) markers were developed to discriminate Ruta graveolens from its adulterant Euphorbia dracunculoides . Random amplified polymorphic DNA (RAPD) was performed and subsequently converted into SCAR markers. Results: After performing RAPD, SCAR primers were designed from the selected unique RAPD amplicons of the genuine drug as well as its adulterant. These primers produced 670 bp and 750 bp SCAR markers with genomic DNA sample of R. graveolens and E. dracunculoides , respectively. Conclusion: Development of these markers will help in the quality control of herbal drugs and monitoring widespread adulteration of these drugs by pharmaceutical industries and government agencies.

Development of a thematic collection of Musa spp accessions using SCAR markers for preventive breeding against Fusarium oxysporum f. sp cubense tropical race 4.

Bananas are one of the most consumed fruits worldwide, but are affected by many pests and diseases. One of the most devastating diseases is Fusarium wilt, caused by Fusarium oxysporum f. sp cubense (Foc). Recently, Fusarium tropical race 4 (Foc TR4) has been causing irreparable damage, especially in Asia and Africa where it has devastated entire plantations, including areas with Cavendish, which is known to be resistant to Foc race 1. Although this race is not yet present in Brazil, results obtained by Embrapa in partnership with the University of Wageningen, The Netherlands, indicate that 100% of the cultivars used by Brazilian growers are susceptible to Foc TR 4. In our study, 276 banana accessions were screened with sequence characterized amplified region (SCAR) markers that have been linked to the resistance of Foc TR 4. Two SCAR primers were tested and the results revealed that SCAR ScaU1001 was efficient at discriminating accessions with possible resistance in 36.6% of the evaluated accessions. This is the first attempt to develop a thematic collection of possible Foc TR 4 resistant banana accessions in Brazil, which could be tested in Asian or African countries to validate marker-assisted selection (MAS), and for use in the preventive breeding of the crop to safeguard our banana plantations against Foc TR 4. We believe that this is an important step towards the prevention of this devastating disease, especially considering that our banana plantations are at risk.

English

While random amplification of polymorphic DNA (RAPD) markers linked to disease resistance genes have been widely used in plant breeding programs, they generally lack reproducibility. To overcome this major disadvantage and other drawbacks, RAPD markers can be converted into sequence characterized amplified region (SCAR) markers, which are genetically defined loci amplified by polymerase chain reaction (PCR) using specific primers. Thus, SCAR markers are typically more reproducible than RAPD markers, due to specific amplification of genomic regions. In this study, a previously identified RAPD marker AT9/917 that is linked to the Puccinia psidii Winter (rust) resistance gene 1 (Ppr1) in Eucalyptus grandis was successfully converted into a specific SCAR marker. Seven specific SCAR primers were designed based on cloning and sequencing of the RAPD marker AT9/917. Different pairs of SCAR primers were tested in an E. grandis family from a crossing between a resistant and a susceptible E. grandis. Prime pair SCAR AT99151L and AT9915914R produced amplicons of expected size. Restriction enzyme digestion of the amplicon revealed polymorphisms between the resistant and susceptible parents. Association analysis between phenotype (rust resistance) and SCAR genotypes in the E. grandis family suggests that this specific SCAR is useful for marker-assisted selection of E. grandis trees resistance to P. psidii Winter. Key words: Plant breeding, molecular markers, random amplified polymorphic DNA (RAPD), Mark-assisted selection, sequence characterized amplified region (SCAR).

Molecular authentication of Gynostemma pentaphyllum through development and application of random amplification polymorphic DNA sequence-characterized amplified region marker.

Due to the morphological similarities of aerial parts, it is difficult to distinguish Gynostemma pentaphyllum from Cayratia japonica, which is usually an adulterant of the former. To develop a reliable method for the identification and authentication of G. pentaphyllum, a combination of random amplification polymorphic DNA (RAPD) technique with sequence-characterized amplified region (SCAR) markers was studied. Twenty-five samples of G. pentaphyllum and two samples of C. japonica were collected from different regions in Guangxi or bought from different provinces in China. Through the RAPD analysis, significant genetic polymorphism was observed among the intraspecies samples of G. pentaphyllum. Furthermore, a specific marker, J-750, was obtained for authentication. Therefore, the SCAR marker for G. pentaphyllum (359 bp) was developed from the RAPD amplicon. With PCR amplification using the SCAR primers, a specific band of 359 bp was distinctly visible for all tested samples of G. pentaphyllum, but was absent in the samples of C. japonica. Furthermore, the results revealed that the SCAR marker was useful for the identification and authentication of G. pentaphyllum irrespective of whether samples were fresh, dry, or of commercial origin. The SCAR marker obtained in this study successfully authenticated G. pentaphyllum through an integrated PCR system containing SCAR and control primer combinations of two pairs. In addition, it was also used for simultaneous discrimination of G. pentaphyllum from C. japonica.

Development of a SCAR marker associated with male sex determination in Garcinia indica Choisy

SummaryGarcinia indica is an endemic fruit-yielding tree which is naturally distributed in the Western Ghats regions of India. It is also cultivated as a plantation crop. It is dioecious tree with a long vegetative phase (10 - 12 years). There are no morphological or biochemical markers to differentiate the sex of individual trees before they reach maturity. In the present study, a randomly amplified polymorphic DNA (RAPD) marker was identified, using a pooled DNA approach, which was able to identify male trees. PCR screening of bulked DNA samples from male and female trees, using 150 decamer primers, revealed one primer (OPL-05) that produced a unique band of 1,501 bp in male tree DNA samples only. OPL-05 was then used to screen individual male and female trees in different collections. The 1.5 kbp band was produced in all male samples, but was absent in all female samples. This male-specific PCR fragment was cloned and sequenced and sequence-characterised amplified region (SCAR) primers were designed from the sequence of this unique fragment.The SCAR primers produced a 1,501 bp fragment in all male tree samples, but not in any female tree DNA sample.The SCAR primers were also used to investigate the sex of unknown trees before maturity.

Development of an ISSR-derived SCAR marker linked to apospory in buffel grass (Cenchrus ciliarisL.)

Cenchrus ciliaris (buffel grass) is a valuable forage grass, reproducing through aposporous apomixis. Inter Simple Sequence Repeat (ISSR) markers converted to SCAR marker linked to apospory in this crop is reported. Out of twenty-five ISSR primers tested in DNA bulks from apomictic and sexual progenies, five primers detected polymorphism. The 1.1 kb amplicon derived from the primer ISSR-856 showed co-segregation with apomictic mode of reproduction. Accordingly, a pair of SCAR (Sequence Characterized Amplified Region) primers were designed which also generated a 1.1kb amplicon (Apo-856) only in the apomictic plants. Importance of such molecular markers in genetics and breeding of C. ciliaris is discussed.

Development of SCAR Markers Linked to Cold Resistance for Marker-assisted Selection of Grapevines

포도 신품종 육성에서 내한성 계통의 조기 선발을 위하여, 다양한 품종을 대상으로 randomly amplified polymorphic DNA(RAPD)를 수행하고, 그 결과를 바탕으로‘Campbell Early’x ‘Kaiji’, ‘Golden Muscat’x‘Tamnara’,‘Campbell Early’x‘Neo Muscat’와‘Alden’x‘Kaiji’의 포도교배조합 실생계통을 이용하여 내한성 관련 sequence characterized amplified region(SCAR) 분자표지를 개발하였다. 400여개의 primer를 사용하여 bulk집단에서의 RAPD 분석을 통해서 6개의 UBC random primer를 최종적으로 선발하였고, RAPD 산물을 클로닝한 후 염기서열을 분석하였다. 6개의 SCAR primer (UBC123-428, UBC196-353, UBC221-503, UBC317-800, UBC342-500, UBC344-451)를 제작하고 교배조합 실생에 재검정한 결과 내한성과 관련한 특이밴드를 확인할 수 있었다. 포도나무의 내한성과 관련한 특이적인 밴드출현양상은 수피의 탄수화물함량, 전해질전도도, 포장에서의 신초생존율과 유사한 양상을 보였다. 본 연구에서 개발한 SCAR marker는 포도의 염색체상에서 위치를 확인할 수 있었으며, 그 주변에는 주로 cytochrome P450, glutathione S-transferase, leucine-rich repeat, pleiotropic drug resistance 12, ribosomal protein 등의 유전자가 분포하였다. 본 연구에서 개발된 포도 내한성 관련 SCAR marker는 내한성 포도 품종의 조기선발과 육종효율 증진에 기여할 수 있을 것으로 기대된다.

Development of SCAR marker associated with downy mildew disease resistance in pearl millet (Pennisetum glaucum L.).

Pearl Millet is an important crop coarse grain cereal crop in the semi arid tropics which is extremely susceptible to oomycete plant pathogen Sclerospora graminicola causing downy mildew (DM) disease. The aim of the current study is to breed resistance against downy mildew disease into high yielding cultivars of pearl millet. Hence, in the present work a sequence characterized amplified region (SCAR) marker was developed as a molecular screening tool to identify DM resistance source and presented here. Of the 27 inter simple sequence repeats (ISSR) decamer primers used to identify polymorphism amongst pearl millet genotypes ICMR-01007 (P1) and ICMR-01004 (P2) and their populations (F1 and F2), only one primer pair ISSR-22 produced polymorphic bands on ICMR-01004 producing 1.4 kb size. The PCR amplification of 1.4 kb band was found tightly linked to the resistant line of ICMR-01004 and also in F2 segregation population was in the ratio 3:1. This band was cloned, sequenced and candidate SCAR primer (SCAR ISSR 863 ) was designed. Segregant analysis of their F2 progeny revealed that the SCAR ISSR 863 marker was linked to downy mildew resistance linkage group (χ(2) 3:1 = 0.86, P = 0.22) with a genetic distance of 0.72 cM. This SCAR marker was further validated using diverse pearl millet lines of India and Africa. Results indicated that the SCAR ISSR 863 band was amplified in all the seven resistant lines and were absent in five susceptible lines. The confirmation of the ISSR-derived SCAR marker in different genetic backgrounds of pearl millet lines suggests that this marker can be exploited for DM resistance screening in pearl millet breeding programs.

Development of a SCAR Marker for Sex Identification in Asparagus

A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers were used for the RAPD analysis. Among them, the primer UBC347 amplified one female-specific 400 base pair DNA. Subsequently, the amplified RAPD fragment was cloned and sequenced. The fragment was abundant in AT and shared sequence homology with retrotransposon elements. On the basis of the sequence obtained, a pair of SCAR primer was designed. The amplification product, named F400, was the same size as the respective RAPD fragment from which it was derived. The F400 SCAR marker resulted to be female-specific in the three asparagus varieties tested in this study. This SCAR marker can be used for an early and rapid identification of female and male plants during breeding programs of asparagus.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis (Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplified fragment length polymorphism (AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplification primers were used in this study. The primer combination E-TG/M-CCA detected a specific band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplified region (SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identification of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Role of Overwintering Forms of Erysiphe necator in Epidemiology of Grapevine Powdery Mildew in Palestinian Vineyards

The overwintering modes of E. necator were studied on Palestinian vineyards, through observations on the differentiation and maturation of cleistothecia and on the occurrence of flag-shoots (deriving from overwintering mycelium) in vineyards. Field surveys were carried out in 17 vineyards for the presence of Flag shoots and cleistothecia, both forms were not observed. Genetic structure and composition of E. necator populations were investigated by application of already available SCAR (Sequence Characterized Amplified Region) primers specific for the "flag-shoot" and "ascospore" biotypes. These primers were used to evaluate the dynamics of the spatial and temporal distribution of the two biotypes, into fungal populations present in 8 vineyards, with different cultivars and spray histories, in various Palestinian districts (Hebron, Bethlehem, Jerusalem, Ramallah, Jericho, Nablus, Jenin, Tulkarm). 397 samples were analyzed by the uses of the primer pairs UnE-UnF in PCR reactions. All samples were found to be of the ascospore biotypes. This finding shows that the “flag shoot” biotype, appears soon after bud breaking and disappears later, while the “ascospore” biotype is more frequently associated to later infections and bunches damages. Such information would be helpful to understand the reasons underlying possible temporal evolution of the pathogen's populations in vineyards, and can have important implications for powdery mildew rationale control strategies.

Role of Overwintering Forms of Erysiphe necator in Epidemiology of Grapevine Powdery Mildew in Palestinian Vineyards

The overwintering modes of E. necator were studied on Palestinian vineyards, through observations on the differentiation and maturation of cleistothecia and on the occurrence of flag-shoots (deriving from overwintering mycelium) in vineyards. Field surveys were carried out in 17 vineyards for the presence of Flag shoots and cleistothecia, both forms were not observed. Genetic structure and composition of E. necator populations were investigated by application of already available SCAR (Sequence Characterized Amplified Region) primers specific for the "flag-shoot" and "ascospore" biotypes. These primers were used to evaluate the dynamics of the spatial and temporal distribution of the two biotypes, into fungal populations present in 8 vineyards, with different cultivars and spray histories, in various Palestinian districts (Hebron, Bethlehem, Jerusalem, Ramallah, Jericho, Nablus, Jenin, Tulkarm). 397 samples were analyzed by the uses of the primer pairs UnE-UnF in PCR reactions. All samples were found to be of the ascospore biotypes. This finding shows that the “flag shoot” biotype, appears soon after bud breaking and disappears later, while the “ascospore” biotype is more frequently associated to later infections and bunches damages. Such information would be helpful to understand the reasons underlying possible temporal evolution of the pathogen's populations in vineyards, and can have important implications for powdery mildew rationale control strategies.

Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R2 of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.

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.

당근(Daucus carota var. sativa) 종자모 형질 관련 RAPD-SCAR 분자표지 개발

당근의 기계적인 제모 시 발생하는 종자의 손실과 발아 시의 문제점을 개선한, 고품질의 당근 종자 생산을 위한 단모종자 당근 품종 육성에 이용할 분자표지를 개발하기 위하여 본 연구를 수행하였다. 본 연구에서는 2008년도부터 2013년도까지 단모종자 표현형 CT-SMR 616 OP 389-1 개체와 장모종자 표현형 CT-SMR 616-33 개체를 자가수분하여 세대 진전된 당근 계통들을 종자모 형질 관련 RAPD-SCAR 분자표지를 개발하는데 이용하였다. 이들 계통의 종자모 길이를 현미경을 이용하여 분석하였으며, 분석된 결과를 바탕으로 계통을 세대진전 시켜, 분자표지 다형성과도 비교분석하였다. 분자 표지 개발을 위하여 세대가 고정되었다고 판단되는 2011년 계통을 대상으로 80개의 random primer를 이용한 RAPD 분석을 통해 12개의 개체간 뚜렷한 차이를 보이는 종자모 형질 관련 특이적 band를 확인하였다. RAPD-SCAR 분자표지 개발을 위해 확인된 이들 특이적 band의 염기서열 분석을 통해 SCAR primer를 작성하였으며 각 SCAR primer는 24-28mer 크기로 3조합 이상 작성하였다. 분석 결과 작성된 SCAR primer 중 <TEX>$SCA2_{1.2}$</TEX>가 단모종자 표현형 계통에서만 특이적으로 증폭되는 것을 확인하였다. 이 <TEX>$SCA2_{1.2}$</TEX> 분자표지의 정확성을 검증하기 위해 2012년도 계통과 2013년도 계통을 이용하여 재검증하였으며, 그 결과 개발된 SCAR 분자표지는 단모종자와 장모종자 계통을 구분할 수 있는 충분한 다형성을 제공하였다. 따라서 본 연구에서 개발된 SCAR 분자표지, <TEX>$SCA2_{1.2}$</TEX>는 당근의 단모종자 품종 육성 연구에 충분히 활용가능 할 것으로 기대된다. Mechanical hair removal of carrot seed causes seed injuries and suppresses the germination in carrot cultivation. This study was performed to develop molecular markers for breeding high quality cultivars with short-hair seed. To meet this objective, random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) markers specifically linked to seed-hair characteristic were identified using CT-SMR 616 OP 389-1 line with short-haired seed and CT-SMR 616 OP 616-33 line with long-haired seed, bred by self-pollination for 6 years from 2008 to 2013, as parents. After seed hair lengths of these lines were analyzed using microscope, next generations were advanced and compared with the molecular markers polymorphism. From RAPD analysis using fixed lines in 2011, twelve RAPD primers showing polymorphic bands specific between the two lines were identified from 80 random primers. To develop RAPD-SACR marker, SCAR primers were designed based on sequence analysis of these specific RAPD bands and more than three combinations of primers were tested. As a result, it was found that the <TEX>$SCA2_{1.2}$</TEX> amplified single polymorphic band from short-haired seed line. To confirm this result, <TEX>$SCA2_{1.2}$</TEX> marker was retested by applying to the 2012 and 2013 progenies. Finally, it was concluded that the developed <TEX>$SCA2_{1.2}$</TEX> marker distinguished short-haired line from long-haired seed line. Therefore, SCAR marker, <TEX>$SCA2_{1.2}$</TEX> is expected to be utilized for breeding of the short-haired seed cultivars.

English

Wet root rot caused by Rhizoctonia solani is one of the important diseases in chickpea worldwide. In the present study, 10 random amplified polymorphic DNA (RAPD) primers were used to assess the molecular diversity of 50 chickpea isolates of R. solani. There was a great diversity among the isolates studied and was in the range of 52 to 93%. The isolates were highly variable in aggressiveness and caused up to 100% wet root rot incidence in chickpea. Accurate detection and identification of plant pathogens are fundamental to plant pathogen diagnostics and management. Therefore, a polymerase chain reaction (PCR) assay was developed for accurate and sensitive detection of R. solani from mycelial DNA and infected chickpea plants. RAPD primer OPA 11 consistently amplified &asymp;1700 base pairs (bp) product in PCR only from the DNA of R. solani isolated from chickpea. The common DNA fragment was sequenced and used to design a pair of oligonucleotide primers amplifying 285 bp sequence characterized amplified region (SCAR). The specificity of the SCAR primers was evaluated. The detection sensitivity of R. solani was 0.5 ng for the genomic DNA and 5 ng for the DNA extracted from infected chickpea root samples. Also, SCAR primer was validated with Q-PCR to detect and quantify R. solani upto 1 pg from infected chickpea root samples. These new SCAR marker are useful for early detection and quantification of wet root rot pathogen in chickpea. Key words: &nbsp;Anastomosis grouping, chickpea, wet root rot, quantitative-PCR (Q-PCR), random amplified polymorphic DNA (RAPD), sequences characterized amplified region (SCAR).

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.

Molecular cloning and development of RAPD-SCAR markers for Dimocarpus longan variety authentication

As an edible fruit and source of traditional medicine, D. longan is grown in most areas of Southern China. Identification of D. longan cultivars by using molecular markers is important genetically. In this study, we cloned fragments from improved randomly amplified polymorphic DNA (RAPD), and developed stably diagnostic sequence-characterized amplified region (SCAR) markers. The specific RAPD bands of D. longan cultivars from Guangxi, with size ranging from 500 bp to 900 bp were gel-purified, cloned and sequenced. Four clones named LY2-1, LY4-7, LY4-8 and LY5-2 were identified. In order to investigate whether the fragments were specific for the species, four pairs of SCAR primers were then designed. PCR amplifications were conducted to analyze 18 samples including different D. longan cultivars and other species. The specific bands with expected sizes were amplified in five D. longan samples but not in others. To identify and characterize the difference between D. longan and D. confinis, PCR amplifications were performed again. The specific bands with expected sizes were found in D. longan but not in D. confinis by SCAR markers LY2-1, LY4-7 and LY5-2, respectively. These results showed that our developed SCAR markers could be very useful as a specific D. longan variety authentication. Therefore, our study provides an effective and precise PCR-based diagnostic method and markers to identify D. longan species.