Specific Sequence Characterized Amplified Region Markers Research Articles

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance

Identification of SCAR markers for genetic authentication of Dendrobium nobile Lindl.

Dendrobium nobile Lindl. is an orcid plant with important medicinal values. This is a colourful houseplant, and also a popular herb in traditional Chinese medicine (TCM). The variants of this plant from different geographic regions might be high, and in this study, we aimed to develop specific sequence characterized amplified region (SCAR) markers for the identification of specific variant of this plant. Different cultivars of D. nobile were collected from nine different places of China, and one cultivar from Myanmar. DNA materials were extracted from the plant samples, random amplified polymorphic DNA (RAPD) were developed, cloned and sequenced for the development of SCAR markers. We have developed four SCAR markers, which are specific to the cultivar from Luzhou China, and clearly distinguishable (genetically) from other cultivars. These SCAR markers are deposited in GenBank (accession number MZ417502, MZ484089, MZ417504 and MZ417505). Four SCAR markers for D. nobile are effective molecular technique to genetically identify the different cultivars or species, and this method is applicable for genetic characterization and identification of other plant species too.

Registration of ‘White Pearl’ great northern common bean cultivar with upright plant architecture and high yield

AbstractGreat northern common bean (Phaseolus vulgaris L.) cultivar ‘White Pearl’ (Reg. no. CV‐343, PI 698189) was developed by the dry bean breeding program at the University of Nebraska and released in 2018. It was bred specifically for adaptation to western Nebraska growing conditions and for upright plant architecture, high yield, and enhanced resistance to bean rust, common bacterial blight (CBB), and Bean common mosaic virus (BCMV). Seed yield of White Pearl was comparable and slightly higher than other great northern cultivars in on‐station trials conducted in Nebraska and did not differ significantly in yield from ‘Aries’ and ‘Matterhorn’ in regional trials. Seed size (100‐seed weight) of White Pearl was larger than ‘Marquis’, ‘Orion’, ‘Gemini’, ‘Beryl‐R’, and Aries in trials grown in Nebraska. White Pearl carries the sequence characterized amplified region (SCAR) marker SAP6 linked to a major quantitative trait locus (QTL) for resistance to CBB. White Pearl had a CBB disease rating of 6.0 (intermediate) under field conditions at the Scottsbluff and Mitchell Ag Labs in 2020. White Pearl also has the Ur‐3 gene for resistance to bean rust and carries the SS68 Kompetitive Allele Specific polymerase chain reaction (KASP) and SK14 SCAR markers linked to the Ur‐3 locus. It also contains the single dominant hypersensitive I gene that confers resistance to all non‐necrotic strains of BCMV and the SW13 SCAR marker linked to the I gene. White Pearl has bright white seed, blooms 46 d after planting, and is a midseason bean, maturing 89 d after planting.

Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD

BackgroundLeishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD.MethodsRAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics.ResultsThe cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein.ConclusionsThe RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.

South American species <i>Solanum alandiae</i> Card. and <i>S. okadae</i> Hawkes et Hjerting as potential sources of genes for potato late blight resistance

For several decades, wild species of Solanum L. section Petota Dumort. have been involved in potato cultivar breeding for robust resistance to pests and diseases. Potato late blight (LB) is caused by oomycete Phytophthora infestans (Mont.) de Bary, and the genes for race-specific resistance to P. infestans (Rpi genes) have been introgressed into cultivated potatoes by remote crosses and trans- or cisgenesis, first from S. demissum Buk. and, more recently, from other wild species, such as S. bulbocastanum Dun., S. stoloniferum Schlechtd. et Bché, and S. venturii Hawkes et Hjerting (according to the nomenclature by Hawkes, 1990). Most wild species already involved in breeding for LB resistance came from North and Central Americas: series Bulbocastana (Rydb.) Hawkes, Demissa Buk. and Longipedicellata Buk., and some Rpi genes of these species have been already characterized in much detail. Rpi genes of South American species, including the series Tuberosa (Rydb.) Hawkes, have not been sufficiently investigated. Among the latter, this study focuses on the Rpi genes of S. alandiae Card. and S. okadae Hawkes et Hjerting. Four accessions of S. alandiae, one accession of S. okadae and 11 clones of interspecific potato hybrids comprising S. alandiae germplasm from the VIR collection were PCR-screened using specific SCAR (Sequence Characterized Amplified Region) markers for eight Rpi genes. SCAR amplicons of five Rpi genes registered in this study were validated by comparing their sequences with those of prototype genes deposited in the NCBI Genbank. Among the structural homologues of Rpi genes found in S. alandiae and S. okadae, of special interest are homologues of CC-NB-LRR resistance genes with broad specificity towards P. infestans races, in particular R2=Rpi-blb3, R8, R9a, Rpi-vnt1 and Rpi-blb2 (94–99, 94–99, 86–89, 92–98 and 91% identity with the prototype genes, respectively). Our data may help to better understand the process of Rpi gene divergence along with the evolution of tuberbearing Solanum species, particularly in the series Tuberosa.

Development of the sequence-characterized amplified region (SCAR) marker for distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries.

Distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries is important for the development of new intergeneric hybrids. The aim of this study was to develop a simple and cost-effective DNA marker to distinguish between a large number of putative hybrids of A. frutescens × R. gayanum during primary selection. We designed sequence-characterized amplified region (SCAR) markers based on the genetic sequences of the internal transcribed spacer (ITS) region for A. frutescens and R. gayanum. Specific SCAR markers for A. frutescens amplified bands in A. frutescens and hybrids but not in R. gayanum. In contrast, specific SCAR markers for R. gayanum amplified bands in R. gayanum and hybrids but not in A. frutescens. Additionally, we confirmed the reproducibility of the SCAR marker using 31 cultivars. The findings demonstrated that these SCAR markers could be used to distinguish putative hybrids between A. frutescens and R. gayanum. This is the first report of the development of a SCAR marker to facilitate distinction of these hybrids.

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

Introduction: For the rapid and accurate genetic identification and authentication of living organisms, improved random amplified polymorphic DNA (RAPD) fragment based development of sequence-characterized amplified region (SCAR) markers is an important genetic technique. Objective: This study aimed to develop SCAR markers for perennial herb Eclipta prostrate (E. prostrate). Methods: Here the RAPD fragments by improved RAPD amplification with primers A11 and N-7 for E. prostrate were cloned into pGEX-T vector, and PCR amplification identified the positive clones. After the enzymatic digestion, they were sequenced with Sanger sequencing. Results: Two SCAR markers were developed, which were very specific to E. prostrate, not found in Penthorum chinense Pursh (P. chinense). The nucleotide sequence search by BLAST GenBank database showed that they are novel in E. prostrate, therefore they were deposited in Genbank with accession number KX671034, KX671035. The markers did not show any identity to other species. Conclusions: Thus, in this study two specific SCAR markers were developed for genetically distinguishing and identifying the plant species E. prostrate from herb P. chinense and others.

Polymorphisms and Evolution of Solanum bulbocastanum Genes for Broad-Spectrum Resistance to Phytophthora infestans

For two decades, Solanum bulbocastanum Dun. has been challenging potato geneticists with its durable resistance to Phytophthora infestans Mont. de Bary, the causal agent of late blight, the most devastating potato disease. In this study, highly specific SCAR (sequence characterized amplified region) markers for three S. bulbocastanum genes of broad-spectrum resistance to P. infestans,RB/Rpi-blb1,Rpi-blb2 and Rpi-blb3, were employed to screen a vast collection of wild tuber-bearing species from several series of Solanum L. section Petota Dumort. and also potato hybrids and varieties. In S. bulbocastanum and S. stoloniferum Schlechtd. et Bche, the sequences of the markers Rpi-blb1-820 and Rpi-sto1-890, which recognize the LRR and CC regions respectively differed inconsiderably in the orthologous genes RB/Rpi-blb1 and Rpi-sto1. These two markers were tightly linked in diverse wild Solanum genotypes. These markers as well as RB-226, another marker recognizing the LRR region of the Rpi-blb1 gene, were obviously related to high resistance to P. infestans. One more marker recognizing the CC region of the gene, RB-629, was found in much wider range of Solanum genotypes; it was not related to resistance to P. infestans and is therefore considered non-specific. In addition to Rpi-blb1 = Rpi-sto1, the Rpi-blb2 and Rpi-blb3 genes were registered in both S. bulbocastanum and S. stoloniferum accessions. In most cases, three bulbocastanum genes were not linked together. The markers specific for Rpi-blb1 = Rpi-sto1 genes were found in the Solanum series Bulbocastana (Rydb.) Hawkes (genome designation BB) and Longipedicellata Buk. (genome designation AABB) and were absent from five Pinnatisecta Rydb. species also comprising genome B. The marker for Rpi-blb2 was found in the series Bulbocastana and Longipedicellata and also in three accessions of Pinnatisecta, while the Rpi-blb3 gene, the orthologue of widely spread R2 gene for resistance to P. infestans, was present in all three Solanum series. In contrast with Rpi-blb1 = Rpi-sto1, two other functional RB homologues, Rpi-bt1 in S. bulbocastanum and RBver in S. verrucosum Schlechtd. (genome AA), apparently diverged earlier in the evolution of tuber-bearing Solanum species. Our evidence on functional Rpi-blb1 = Rpi-sto1 genes in three Petota series substantiates the assumption that the B genome of tetraploid Longipedicellata evolved from diploid Bulbocastana rather than from diploid Pinnatisecta progenitors. Similar to wild Solanum species, the markers Rpi-blb1-820 and Rpi-sto1-890 were tightly linked in advanced potato hybrids and established varieties bred from S. stoloniferum as the conventional source of bulbocastanum genes for late blight resistance, but did not always match the markers for the Rpi-blb2 and Rpi-blb3 genes and the marker for the Rysto gene as an independent indicator of stoloniferum germplasm.

Registration of Great Northern Common Bean Cultivar ‘Panhandle Pride’ with Enhanced Disease Resistance to Bean Rust and Common Bacterial Blight

‘Panhandle Pride’ (Reg. no. CV‐325, PI 691461) great northern common bean (Phaseolus vulgaris L.) cultivar was developed by the dry bean breeding program at the University of Nebraska and released in 2016. It was bred specifically for adaptation to western Nebraska growing conditions and for enhanced resistance to bean rust and common bacterial blight (CBB). Seed yield of Panhandle Pride was comparable to the other cultivars in the on‐station and on‐farm trials conducted in Nebraska and was greater than ‘Orion’ and lower than ‘Matterhorn’ in regional trials. Seed size (100‐seed weight) for Panhandle Pride was larger than Marquis and Beryl‐R in trials grown in Nebraska, and larger than Orion and Matterhorn in regional trials. Panhandle Pride carries the sequence characterized amplified region (SCAR) markers SAP6 and SU91 linked to a major CBB resistance quantitative trait locus, and its reaction to the CBB pathogen under field conditions was consistent across 3 yr at the West Central Research and Extension Center, North Platte, NE, with disease ratings of 6.0, 4.3, and 5.2. Panhandle Pride has the Ur‐3 and Ur‐6 genes for resistance to bean rust and carries the SS68 Kompetitive Allele Specific polymerase chain reaction (KASP) and SK14 SCAR markers linked the Ur‐3 bean rust resistance locus. It also carries the single dominant hypersensitive I gene that provides resistance to all non‐necrotic strains of the Bean common mosaic virus (BCMV) and the SW13 SCAR marker linked to the I gene for resistance to BCMV. Panhandle Pride has bright white seed, blooms 44 d after planting, and is a midseason bean, maturing 87 d after planting.

Analysis of Genetic Diversity and Development of a SCAR Marker for Green Tea (Camellia sinensis) Cultivars in Zhejiang Province: The Most Famous Green Tea-Producing Area in China

Camellia sinensis (L.) O. Kuntze is one of the most important non-alcoholic beverage crops in Asian and African countries. In recent years, many green tea cultivars have been released and played an important role in improving the production and quality of tea trees. The objectives of this study were to assess the genetic diversity of the eighteen main green tea cultivars in Zhejiang Province-the most famous green tea-producing area of China-using start codon-targeted (SCoT) markers and to develop a specific sequence-characterized amplified region (SCAR) marker for application in cultivar diagnosis. Thirty-one SCoT primers produced 264 loci, 226 of which were polymorphic. The genetic similarity coefficients among these green tea cultivars ranged from 0.587 to 0.814, indicating that a high level of genetic diversity was present. Both a UPGMA dendrogram and a PCoA plot grouped the tea cultivars into three groups. The partitioning of groups in the UPGMA and PCoA was similar, and much of the clustering was highly consistent with the classification of tea cultivars according to their genetic backgrounds. A unique SCoT band, SCoT4-1649, specific to the tea cultivar 'Yingshuang,' was transformed into a SCAR marker. This SCAR marker is highly useful for the identification and germplasm conservation of green tea cultivars.

Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method.

A rapid and sensitive loop-mediated isothermal amplification (LAMP) method was developed for the specific detection of Pseudocercospora eumusae. The LAMP primers designed based on the specific SCAR (Sequence Characterized Amplified Region) marker sequence of P. eumusae proved highly specific to P. eumusae and there was no cross reactivity with closely related Pseudocercospora spp. (P. fijiensis and P. musicola) and 17 other leaf spot causing fungal pathogens of banana. The developed LAMP method exhibited greater sensitivity as the minimum detectable concentration of P. eumusae genomic DNA was 10 ƿg/µl which was 100 times lower than that of conventional PCR (1ng/µl).This method also detected the target pathogen from crude DNA of the mycelium and single leaf spot tissues which eliminates laborious purification steps in DNA isolation and requires less operational time. To the best of our knowledge, this is the first report on the development of this LAMP method to specifically detect and diagnose P. eumusae pathogen from pure cultures and leaf spot tissues.

Development of CACTA transposon derived SCAR markers and their use in population structure analysis in Zea mays.

Molecular marker technologies have proven to be an important breakthrough for genetic studies, construction of linkage maps and population genetics analysis. Transposable elements (TEs) constitute major fractions of repetitive sequences in plants and offer a wide range of possible areas to be explored as molecular markers. Sequence characterized amplified region (SCAR) marker development provides us with a simple and time saving alternative approach for marker development. We employed the CACTA-TD to develop SCARs and then integrated them into linkage map and used them for population structure and genetic diversity analysis of corn inbred population. A total of 108 dominant SCAR markers were designed out of which, 32 were successfully integrated in to the linkage map of maize RIL population and the remaining were added to a physical map for references to check the distribution throughout all chromosomes. Moreover, 76 polymorphic SCARs were used for diversity analysis of corn accessions being used in Korean corn breeding program. The overall average polymorphic information content (PIC) was 0.34, expected heterozygosity was 0.324 and Shannon's information index was 0.491 with a percentage of polymorphism of 98.67%. Further analysis by associating with desirable traits may also provide some accurate trait specific tagged SCAR markers. TE linked SCARs can provide an added level of polymorphism as well as improved discriminating ability and therefore can be useful in further breeding programs to develop high yielding germplasm.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

Mei Z, Khan MA, Zhang X, Fu J. 2017. Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers. Biodiversitas 18: 1243-1249. For the genetic identification and authentication of living organisms, development of Sequence-Characterized Amplified Region (SCAR) markers from Random Amplified Polymorphic DNA (RAPD) fragments is a valuable molecular approach. By using SCAR markers, molecular analysis is simplified to a Polymerase Chain Reaction (PCR) analysis using PCR primers designed from specific sequences of the RAPD amplicons. In this study, RAPD fragments from improved RAPD amplification of a perennial herb Penthorum chinense Pursh from China were cloned into a T-vector, and positive clones were identified by PCR amplification, and sequenced with the Sanger sequencing method for the SCAR marker development. Five SCAR markers were developed that were very specific to P. chinense, and deposited in GenBank (accession numbers: KX671029, KX671030, KX671031, KX671032 and KX671033). BLAST searches of these five nucleotide sequences in the GenBank database showed no identity with markers from other species. This study was developing five specific SCAR markers, has enabled reliable genetic identification of the herbal plant species Penthorum chinense Pursh, useful for authenticating future samples of this important medicinal herb.

Development of specific SCAR markers identifying recessive genic male sterile gene in sesame (Sesamum indicum L.)

To develop a simple and convenient marker for genetic linkage of sesame male sterile gene,based on an AFLP marker,7 SCAR( sequence characterized amplified region) markers were developed using recessive genic male sterile two- type line 95ms- 5AB as fertile and sterile gene bulks. These 7 markers could detect bands from fertile bulk with 806 bp to 1 638 bp in size,and could not from sterile bulk. They were designated as PMC- 1to PMC- 7. These markers were used to detect 1 184 fertile and sterile plants of 95ms- 5AB with 0. 25% exchange rate. 4 F2 populations obtained from 95ms- 5A and different genotypes and 2 recessive genic male sterile lines were identified using these 7 SCAR markers. Result indicated that the SCAR markers were specific for 95ms- 5.

Analysis of genetic diversity of Brassica rapa var. chinensis using ISSR markers and development of SCAR marker specific for Fragrant Bok Choy, a product of geographic indication.

Non-heading Chinese cabbage [Brassica rapa var. chinensis (Linnaeus) Kitamura] is a popular vegetable and is also used as a medicinal plant in traditional Chinese medicine. Fragrant Bok Choy is a unique accession of non-heading Chinese cabbage and a product of geographic indication certified by the Ministry of Agriculture of China, which is noted for its rich aromatic flavor. However, transitional and overlapping morphological traits can make it difficult to distinguish this accession from other non-heading Chinese cabbages. This study aimed to develop a molecular method for efficient identification of Fragrant Bok Choy. Genetic diversity analysis, based on inter-simple sequence repeat molecular markers, was conducted for 11 non-heading Chinese cabbage accessions grown in the Yangtze River Delta region. Genetic similarity coefficients between the 11 accessions ranged from 0.5455 to 0.8961, and the genetic distance ranged from 0.0755 to 0.4475. Cluster analysis divided the 11 accessions into two major groups. The primer ISSR-840 amplified a fragment specific for Fragrant Bok Choy. A pair of specific sequence-characterized amplified region (SCAR) primers based on this fragment amplified a target band in Fragrant Bok Choy individuals, but no band was detected in individuals of other accessions. In conclusion, this study has developed an efficient strategy for authentication of Fragrant Bok Choy. The SCAR marker described here will facilitate the conservation and utilization of this unique non-heading Chinese cabbage germplasm resource.

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst.

Development of sequence-characterized amplified region (SCAR) markers from random-amplified polymorphic DNA (RAPD) fragments is a valuable molecular approach for the genetic identification of different species. By using SCAR markers, molecular analysis is reduced to a simple polymerase chain reaction (PCR) analysis using primers designed from the amplicon sequence of RAPD. In this study, the DNA fragments from an improved RAPD amplification of Ganoderma species were cloned into a pGM-T vector; positive clones were identified by PCR amplification and enzymatic digestion, and finally, DNA fragments were sequenced using the Sanger sequencing method for developing the SCAR markers. Two SCAR markers, named LZ4-1 with 534 nucleotides, and LZ5-2 with 337 nucleotides were identified, which are specific to Ganoderma lucidium (Leysser: Fr) Karst species. BLAST of these two nucleotide sequences in the GenBank database showed no identity to other species. We deposited these sequences into the GenBank database (LZ4-1 accession No. KM391933, LZ5-2 accession No. KM391934). PCR assays confirmed them as novel molecular markers for G. lucidium (Leysser: Fr) Karst, which might be used for genetic authentication of adulterant samples. Thus, our study developed two specific SCAR markers for identifying and distinguishing the medicinal mushroom G. lucidium (Leysser: Fr) Karst from other Ganoderma species.

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).

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Development of a specific SCAR marker for the Ns genome of Psathyrostachys huashanica Keng

Wang, J., Du, W., Wu, J., Chen, X., Liu, C., Zhao, J., Yang, Q. and Li, F. 2014. Development of a specific SCAR marker for the Ns genome of Psathyrostachys huashanica Keng. Can. J. Plant Sci. 94: 1441–1447. Psathyrostachys huashanica Keng (2n=2x=14, NsNs) possesses many agronomically desirable traits that could be used in wheat improvement. We have previously produced a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n=44=22 II). To track the addition of P. huashanica chromatin in wheat rapidly and effectively, a repetitive sequence of 1665 base pairs, designated pHs8, was isolated based on 21 different Triticeae species, including the parents’ common wheat cv. 7182 and P. huashanica, by RAPD analysis. The diagnostic fragments of the RAPD marker OPF151665 were cloned, sequenced, and converted into a sequence-characterized amplified region (SCAR) marker, known as RHS12. Southern hybridization using labeled pHs8 as probe showed intense hybridization signals on P. huashanica, but not on the other 20 species at all. RHS12 was validated using 21 different plant species and a complete set of wheat–P. huashanica disomic addition lines. Our results indicated that the SCAR marker targeted the Ns genome of P. huashanica and it was present in all seven P. huashanica chromosomes. The newly developed SCAR marker should help wheat breeders to screen for genotypes containing P. huashanica chromatin with low costs and high throughput.

Development of a specific SCAR marker for the Ns genome of Psathyrostachys huashanica Keng

Wang, J., Du, W., Wu, J., Chen, X., Liu, C., Zhao, J., Yang, Q. and Li, F. 2014. Development of a specific SCAR marker for the Ns genome of Psathyrostachys huashanica Keng. Can. J. Plant Sci. 94: 1441-1447. Psathyrostachys huashanica Keng (2n=2×=14, NsNs) possesses many agronomically desirable traits that could be used in wheat improvement. We have previously produced a complete set of wheat-P. huashanica disomic addition lines (1Ns-7Ns, 2n=44=22 II). To track the addition of P. huashanica chromatin in wheat rapidly and effectively, a repetitive sequence of 1665 base pairs, designated pHs8, was isolated based on 21 different Triticeae species, including the parents' common wheat cv. 7182 and P. huashanica, by RAPD analysis. The diagnostic fragments of the RAPD marker OPF151665 were cloned, sequenced, and converted into a sequence-characterized amplified region (SCAR) marker, known as RHS12. Southern hybridization using labeled pHs8 as probe showed intense hybridization signals on P. huashanica, but not on...