Polymerase Chain Reaction Primer Pairs Research Articles

Quantitative detection of <i>Pythium porphyrae</i> and <i>Pythium chondricola</i> (Oomycota), the causative agents of red rot disease in <i>Pyropia farms</i> in China

Red rot disease is one of the notorious algal diseases that threaten the cultivation of <i>Pyropia</i> in China, and two <i>Pythium pathogens</i>, i.e., <i>Pythium porphyrae</i> and <i>P. chondricola</i>, have been reported as causative agents. To monitor the pathogens, a fluorescent quantitative polymerase chain reaction (PCR) method was developed to quantitatively detect their abundance. Using overlapping PCR and pathogen-specific primer pairs, two pathogen-specific fragments were concatenated to construct an internal standard plasmid, which was used for quantification. For zoospores of known numbers, the results showed that this method can detect as less as 100 and 10 zoospores mL<sup>-1</sup> in a 200 mL solution for <i>P. porphyrae</i> and <i>P. chondricola</i>, respectively. Using monthly collected seawater at 10 sites in Haizhou Bay, a typical aquaculture farm in China, a significantly higher temperature and a significantly lower salinity were determined in December 2021. <i>P. porphyrae</i> was determined to be more abundant than <i>P. chondricola</i>, though with similar temporal distribution patterns from December 2021 to February 2022. When a red rot disease occurred in December 2021, the two pathogens were significantly more abundant at two infected sub-sites than the uninfected sub-site within both seawater and sediment, though they were all significantly more enriched in sediment than in seawater. The present method provides the capability to quantify and compare the abundance of two pathogens and also has the potential to forecast the occurrence of red rot disease, which is of much significance in managing and controlling the disease.

Cross-pathogenicity of Phytophthora palmivora associated with bud rot disease of oil palm and development of biomarkers for detection.

Phytophthora palmivora has caused disease in many crops including oil palm in the South America region. The pathogen has had a significant economic impact on oil palm cultivation in Colombia, and therefore poses a threat to oil palm cultivation in other regions of the World, especially in Southeast Asia, the largest producer of the crop. This study aimed to look at the ability of isolates from Malaysia, Colombia, and other regions to cross-infect Malaysian oil palm, durian, and cocoa and to develop specific biomarkers and assays for identification, detection, and diagnosis of P. palmivora as a key component for the oil palm biosecurity continuum in order to contain the disease especially at the ports of entry. We have developed specific molecular biomarkers to identify and detect Phytophthora palmivora using polymerase chain reaction (PCR) and real-time loop mediated isothermal amplification (rt-LAMP) in various sample types such as soil and plants. The limit of detection (DNA template, pure culture assay) for the PCR assay is 5.94 × 10-2 ng µl-1 and for rt-LAMP is 9.28 × 10-4 ng µl-1. Diagnosis using rt-LAMP can be achieved within 30min of incubation. In addition, PCR primer pair AV3F/AV3R developed successfully distinguished the Colombian and Malaysian P. palmivora isolates.

Comprehensive genomic analysis of the potential limitations of several published PCR primers targeting prfA-virulence gene cluster in Listeria species.

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. The present study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl and 176 hlyA unique gene sequences. Only hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB and mpl genes showed weak (less than 50%) matching results. In addition, nucleotide variations were observed at the 3' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible, to minimize the false-negative risk and reach the aim of low tolerable limits of detection.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Development of a new PCR assay and a recombinase-aided amplification based isothermal amplification coupled with lateral flow dipstick assay for potato late blight detection

Late blight caused by Phytophthora infestans is the most devastating potato disease, yet leaf infections are not reliably detected by current assays during the incubation phase of the pathogen and asymptomatic plants. In this study, the genome of P. infestans was queried against the NCBI nucleic acid database and a 1050 bp species-specific DNA fragment was identified. Using this fragment, polymerase chain reaction (PCR) and recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assays were developed for the detection of potato late blight pathogen. Both assays effectively distinguished P. infestans from related species (e.g., P. capsici). The detection limits of the three PCR primer pairs under optimized conditions (59.3 °C, 35 PCR cycles) were 1.31 × 10−1 fg, 1.18 × 10−2 fg, and 1.24 × 10−1 fg, corresponding to 27.31 copies, 2.49 copies, and 25.51 copies, respectively. While the detection limit of the RAA-LFD assay at 37 °C for 25 min was 5 × 10−4 fg (0.10 copies), which was better than all reported molecular detection assays for potato late blight. Both PCR (59.3 °C, 35 PCR cycles) and RAA-LFD (37 °C for 10 min) positively detected all symptomatic leaves. When extending the amplification time to 20 min, the RAA-LFD assay detected most of the leaves with incubation phase of the disease. The molecular detection assays developed in this study were species-specific and are expected to be used in detecting potato late blight in the incubation phase in leaves and asymptomatic plants, which will aid in accurate monitoring and early warning of potato late blight.

Development of the LCPDb-MET database facilitating selection of PCR primers for the detection of metal metabolism and resistance genes in bacteria

Bacterial metal metabolism and resistance genes are important indicators for microbial ecology, biogeochemistry, and biotechnology. Their investigation enables understanding how bacteria influence the geochemical cycles of elements and how bacteria can be employed in the bioremediation of polluted areas. For environmental screening, the polymerase chain reaction (PCR) technique has been used as the method of choice for the detection of metal metabolism and resistance genes, and subsequently, many different PCR primer pairs have been developed for such screening. The aim of this study was the development of a database of PCR primers suitable for the screening of metal metabolism and resistance genes in environmental samples. We conducted an in silico benchmark of 291 previously published PCR primer pairs designed to amplify genes involved in bacterial metabolism and resistance to arsenic, cadmium, chromium, cobalt, copper, gold, iron, lead, mercury, nickel, silver, and zinc. Our analysis showed that only 16 PCR primer pairs can be considered well designed and suitable for reliable screening in an environmental setup. Furthermore, 55 primer pairs delivered ambiguous results, while the remaining 220 pairs showed no in silico PCR product within their expected product size range. Based on the obtained results, we developed a ranked database (LCPDb-MET; http://lcpdb.ddlemb.com/met/) of primers suitable for the screening of bacterial metal metabolism and resistance genes in various environmental settings. Moreover, since in the course of the study we recognized a serious problem with the classification of metal metabolism and resistance reference sequences, we developed also the METGeneDb database (http://lcpdb.ddlemb.com/downloads). This database covers 106 genes with 7,624 unique sequences divided into 137 potential gene subclusters and it can be used as the reference database in the genomic and metagenomic studies.

Detection of A2058G and A2059G on the 23S rRNA Gene by Multiplex Nested PCR to Identify Treponema pallidum Resistance to Azithromycin in Indonesia.

Azithromycin is an antibiotic used to treat syphilis, especially in the context of penicillin allergy. Although resistance to azithromycin has been widely reported to be associated with one- and/or two-point mutations on the 23S rRNA gene, it has yet to be described in Indonesia. Specimens were collected from 220 patients diagnosed with secondary syphilis. A multiplex nested polymerase chain reaction (PCR) testing system using the 23S rRNA target gene of Treponema pallidum was designed using three primer pairs. The first step involved the use of PCR primer pairs to detect T. pallidum. In the second step, two PCR primer pairs were constructed to identify azithromycin-resistant T. pallidum based on A2058G and A2059G point mutations. T. pallidum detected in samples from Jakarta or Bandung were not resistant to azithromycin. However, azithromycin-resistant T. pallidum were found in samples from Makassar, Medan, and Bali. Specimens from heterosexual males and patients with HIV accounted for the majority of azithromycin resistance noted in the study. This study demonstrated that the azithromycin-resistant T. pallidum detected in Indonesia appear to be a novel variant of resistance, containing both the A2058G and A2059G mutations found in Medan and Makassar.

Molecular detection of cacao swollen shoot badnavirus species by amplification with four PCR primer pairs, and evidence that Cacao swollen shoot Togo B virus-like isolates are highly prevalent in Côte d’Ivoire

Badnaviruses are plant pararetroviruses that infect economically important crops worldwide, and several badnaviral species have been reported causing significant economic losses in cacao plantations in West Africa. Based on the available full-length genome sequences of cacao swollen shoot disease (CSSD)-associated badnaviruses (n = 66), four primer pairs (CSSD1-CSSD4) were designed to amplify a fragment of the 5′ region of open reading frame (ORF) 3, which comprises the movement protein, by polymerase chain reaction (PCR). Primers were evaluated for their ability to amplify a badnaviral fragment(s) from symptomatic and asymptomatic cacao leaf samples collected in Cote d’Ivoire during 2017–2019. The PCR products obtained by amplification with the CSSD1 primers showed high sequence variability and were phylogenetically related to one of three different badnaviral species, Cacao swollen shoot Togo B virus, Cacao swollen shoot CD virus, and Cacao swollen shoot CE virus, while the CSSD2 and CSSD4 amplicon sequences grouped exclusively with either cacao swollen shoot Togo B virus (CSSTBV) and Cacao swollen shoot Ghana M virus isolates, respectively. The majority of the isolates obtained here were most closely phylogenetically related to CSSTBV, with which they shared 81.0–98.0% nucleotide identity, making the CSSTBV-like isolates the predominant species associated with badnavirus-infected cacao trees tested in Cote d’Ivoire.

Salt-Tolerant Phenomena, Sequencing and Characterization of a Glyoxalase I (Jojo-Gly I) Gene from Jojoba in Comparison with Other Glyoxalase I Genes.

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of Glyoxalase I (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

Analysis of Published PCR Primers to Determine Sex of Industrial Hemp Seedlings

Industrial hemp refers to non‐intoxicating strains of Cannabis sativa that produce high levels of cannabidiolic acid (CBDA), the precursor to cannabidiol (CBD). CBD has known health benefits and is being tested for a wide range of potential medicinal uses. When growing hemp for CBD, unfertilized female plants are desired as they produce high amounts of CBDA in their flower organs. Therefore, early identification and elimination of male hemp plants is imperative and allows cultivators to spend fewer resources on plants that won’t produce flower and could possibly pollinate female plants. It has been reported that male cannabis plants can be identified weeks before they show any visual sex features using the polymerase chain reaction (PCR), however the results from reported studies have been inconsistent in identifying male plants. Here, genomic DNA from thirteen hemp seedlings was analyzed using male‐DNA‐specific PCR primer pairs described in the research literature. Three different primer pairs (SCAR323, SCAR119, and MADC2) were used in the analyses. SCAR323 primers have been reported to amplify a male‐specific amplicon of 323 base pairs (bp); SCAR119 have been reported to amplify a male‐associated amplicon of 119 bp; and MADC2 primers have been reported to amplify a male‐associated amplicon of 300 bp and a female‐associated amplicon of 450 bp. PCR analyses showed DNA from all 13 of our tested hemp seedlings produced a single SCAR323 amplicon of around 320 bp, suggesting this primer set might not be useful for sex determination. A SCAR119 amplicon of the expected size was seen using DNA from 6 of the 13 seedlings. The DNA from the same 6 plants produced an amplicon of about 400 bp using the MADC2 primers, while DNA from plants that lacked SCAR119 amplicon showed a MADC2 amplicon of about 550 bp. After seven weeks of growth, the hemp plants revealed the presence of male or female reproductive organs. The presence of male pollen sacs correlated with the SCAR119 amplicon and the MADC2 amplicon of 400 bp, indicating these primer sets could be a useful tools for determining plant sex at the seedling stage.

Translation elongation factor 1-alpha gene as a marker for diagnosing of candidal onychomycosis.

Background and Purpose:Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species.Materials and Methods:For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens.Results:The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively.Conclusion:Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

Development of a genotyping method for onion (Allium cepa L.) male-fertility based on multiplex PCR

ABSTRACTThe most widely used source of onion cytoplasmic male-sterility (CMS) is controlled by a single nuclear gene and a cytoplasmic factor, and the sterility trait can be recovered by the dominant nuclear gene (Ms). Marker-assisted selection (MAS) has a great potential in the selection of male sterility lines and maintainer lines. In this study, two markers at the Ms loci and one cytoplasm marker were used to a develop multiplexed polymerase chain reaction (PCR) system for MAS of fertility traits in onion. Seven pairs of PCR primers were designed on the basis of the flanking sequences (F1890 and S1887) of the male-fertility restoration (Ms) locus and a mitochondrial sequence (mt472) in onion. One set of compatible primers were selected to develop a multiplex-PCR molecular marker (AcCN), which was established in forty-eight lines. The fertility types of cytoplasm and nuclear were distinguished by one-time PCR detection. The accuracy of the AcCN marker was verified in practical backcross breeding population. The AcCN marker types perfectly matched with their genotypes and phenotypes. It can be concluded that this marker is of great practical significance for MAS in the selection of onion maintainer lines.

Herbalogical studies of Ranae oviductus (Hamayou) and its species identification using DNA barcoding method

<p indent=0mm>Ranae oviductus is a rare and precious Chinese medicine; however, there are many challenges connected with identifying this medicine, such as presence of many adulterants, the controversy over its origin, and the complexity of its classification history. First, we performed textual criticism of herbal literature and modern research data, constructed a phylogenetic relationship using whole mitochondrial genomes, and created a spatial overlay for the traditional production areas of Ranae oviductus and animal geographical distribution. The results obtained confirm that <italic>Rana chensinensis</italic>, <italic>R. dybowskii</italic>, and <italic>R. amurensis</italic> can be considered as a valid species for Ranae oviductus and that the <italic>Chinese Pharmacopoeia’s </italic>use of <italic>Rana temporaria chensinensis</italic> David. as the scientific name for Chinese brown frog is inappropriate. We further believe that the original species for Ranae oviductus could be <italic>R. dybowskii</italic>. Second, a debonding solution was prepared and used to pretreat medicinal materials to overcome the harmful effects of high expansion properties of Ranae oviductus during its DNA extraction. A new polymerase chain reaction primer pair was designed to resolve nonspecific amplification of cytochrome c oxidase subunit I (<italic>COI</italic>) sequences of Ranae oviductus using universal primers. A DNA barcoding reference library was established that contained 259 <italic>COI</italic> barcodes for 43 species covering Ranae oviductus and most of its adulterants. Finally, the DNA barcoding procedure for the identification of Ranae oviductus was verified based on simulation of real identification practices using commercially available medicinal materials. The results indicate that there are several adulterants, such as frog oil (<italic>Pelophylax nigromaculatus</italic>) and bullfrog oil (<italic>R. catesbeiana</italic>), in the herbal market. This study shows that DNA barcoding can successfully distinguish Ranae oviductus and its adulterants. In addition, the procedure established in this study can provide strong support for the safety of Ranae oviductus and protect the interests of consumers.

Amplification of bacterial genomic DNA from all ascitic fluids with a highly sensitive polymerase chain reaction.

Due to varying positive rates of polymerase chain reaction (PCR) amplification, interpretation of conventional PCR results for non-infectious ascites remains problematic. The present study developed a highly sensitive PCR protocol and investigated the positive rate of PCR for the 16S ribosomal (r)RNA gene in non-infectious ascites. Following the design of a new PCR primer pair for the 16S rRNA gene (800F and 1400R), the sequences of PCR products were analyzed and the lower limit for bacterial DNA detection evaluated. The positive rate of PCR for 16S rRNA gene in non-infectious ascites was also evaluated. PCR with the primer pair amplified the genomic DNA of 16S rRNA genes of major disease-causing bacterial strains. Additionally, PCR with this primer pair provided highly sensitive detection of bacterial genomic DNA (lower limit, 0.1 pg of template DNA). When DNA samples isolated from ascites were used, the 16S rRNA gene was amplified independently of the presence of bacterial infection. PCR products contained the genomic DNA fragments of multiple bacterial species. Bacterial genomic DNA can be amplified from all ascitic fluids using a highly sensitive PCR protocol. Careful attention is required to interpret the results based on simple amplification of 16S rRNA gene with conventional PCR.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

Simple sequence repeat (SSR) markers for New Zealand mānuka (Leptospermum scoparium) and transferability to kānuka (Kunzea spp.)

ABSTRACTA multi-allelic genetic marker resource for future genetics studies in mānuka (Leptospermum scoparium) and its close relatives has been developed by mining simple sequence repeats (SSR) from a preliminary draft genome assembly of L. scoparium ‘Crimson Glory’. In total, 469 and 169 trinucleotide and dinucleotide repeats, respectively, were detected in a set of 89,619 predicted gene models. Polymerase chain reaction primer pairs were designed for 32 loci exhibiting high sequence similarity to Eucalyptus grandis. These 32 primer pairs were screened over a population of mānuka and 3 accessions of sympatric, related kānuka (genus Kunzea). The 15 polymorphic SSR markers that were successfully developed are now available for genetic analysis in Myrtaceae.

Development of retrotransposon-based molecular markers and their application in genetic mapping in chokecherry (Prunus virginiana L.)

Retrotransposons are the largest group of transposable elements (TEs) that are ubiquitous and well dispersed in plant genomes. Transposition/insertion of TEs on chromosomes often generates unique repeat junctions (RJs) between TEs and their flanking sequences. Long terminal repeats (LTR) are well conserved and abundant in plant genomes, making LTR retrotransposons valuable for development of TE junction-based markers. In this study, LTR retrotransposons and their RJs were detected from chokecherry genome sequences generated by Roche 454 sequencing. A total of 1246 LTR retrotransposons were identified, and 338 polymerase chain reaction primer pairs were designed. Of those, 336 were used to amplify DNA from chokecherry and other rosaceous species. An average of 283 of 336 (84.2 %) LTR primer pairs effectively amplified DNA from chokecherries. One hundred and seventeen chokecherry LTR primers also produced amplification in other Prunus (99) or rosaceous species (19). A total of 59 of 78 polymorphic LTR markers were qualified for linkage map construction according to the segregation distortion Chi-square (χ 2) test. Forty-eight LTR markers were successfully located on a previously constructed chokecherry map. The majority of the LTR markers were mapped on LG XI of the chokecherry map. Our results suggest that LTR marker development using random genome sequences is rapid and cost-efficient. Confirmed applicability of LTR markers in map construction and genetic mapping will facilitate genetic research in chokecherry and other rosaceous species.

Molecular diagnosis of the brown root rot disease agent Phellinus noxius on trees and in soil by rDNA ITS analysis

Phellinus noxius (P. noxius) is an important pathogen that causes brown root rot of trees in tropical and sub-tropical areas and has led to severe damage to trees. A quick and accurate diagnostic technique is essential to the timely confirmation of the pathogen and possible treatment. In this study, a fast, sensitive and accurate approach of molecular technique was used to diagnose the brown root rot pathogen on trees and in soils of subtropical Hong Kong. Two pairs of specific PCR primers were used to amplify the target rDNA internal transcribed spacer (ITS) region through polymerase chain reaction (PCR) for both tree tissues and soil samples. The amplified ITS fragments were then sequenced and analyzed phylogenetically for the diagnostic identification of the pathogen P. noxius. The results showed that 13 of 38 suspected trees in Hong Kong were infected with P. noxius through molecular detection. The pathogen showed no specific preference to any particular tree species. Quantitative PCR was applied to soils grown with trees identified both positive and negative for P. noxius, but the soils with healthy trees were also found positive for P. noxius. For the first time, P. noxius was reported to infect a wide range of tree species in Hong Kong and widely presented in soils, probably serving as a reservoir for the pathogen. Through this study, it is proposed that P. noxius is a soil-borne pathogen, which increases its infectivity when trees start to grow in the soil as a means in addition to the previously proposed root-to-root contact.

Scanning for α-Hemoglobin Variants by High-Resolution Melting Analysis.

Definitive detection of hemoglobin (Hb) variants requires DNA sequencing. High-resolution melting (HRM) analysis of polymerase chain reaction (PCR) amplicons was applied to detect and discriminate among uncommon α-Hb variants found in Thailand. Uncommon suspected α-Hb variants observed in Hb typing were identified by sequencing of DNA from whole blood samples. Three pairs of PCR primers covering the mutation regions in the three α-globin exons then were used for PCR coupled with difference in HRM analysis to subtract out the concomitant melting profile of the normal allele in the heterozygous state. DNA sequencing identified six heterozygous α-Hb variants, namely, Hb G-Waimanalo (HBA2: exon 2, codon 64 G>A), Hb J-Buda (HBA1: exon 2, codon 61 G>T), Hb Kurosaki (HBA2: exon 1; codon 7 A>G), Hb O-Indonesia (HBA1: exon 3 codon 116 G>A), Hb Q-India (HBA1:exon 2, codon 64 G>C), and Hb Q-Thailand (HBA1: exon 2 codon 74 G>C). Difference HRM analysis showed one temperature melting profile using exon 1 primer pair, four different profiles with exon 2 primer pair, and one profile with exon 3 primer pair. PCR-HRM analysis was effective in detecting and discriminating among single point mutations causing six uncommon α-Hb variants in heterozygous individuals. The method can be applied for routine screening due to its simplicity and relatively low cost.

Isolation and characterization of eight polymorphic microsatellites for the spotted spiny lobster, Panulirus guttatus.

Microsatellite sequences were isolated from enriched genomic libraries of the spotted spiny lobster, Panulirus guttatus using 454 pyrosequencing. Twenty-nine previously developed polymerase chain reaction primer pairs of Panulirus argus microsatellite loci were also tested for cross-species amplification in Panulirus guttatus. In total, eight consistently amplifying, and polymorphic loci were characterized for 57 individuals collected in the Florida Keys and Bermuda. The number of alleles per locus ranged from 8 to 20 and observed heterozygosities ranged from 0.409 to 0.958. Significant deviations from Hardy–Weinberg equilibrium were found in one locus from Florida and three loci from Bermuda. Quality control testing indicated that all loci were easy to score, highly polymorphic and showed no evidence of linkage disequilibrium. Null alleles were detected in three loci with moderate frequencies ranging from (20% to 22%). These eight microsatellites provide novel molecular markers for future conservation genetics research of P. guttatus.