Plant Genomic DNA Research Articles

Genetic Diversity in Tomato Genotypes (<i>Solanum lycopersicum</i>) Based on Salinity Responsive Candidate Gene Using Simple Sequence Repeats

Abstract Salinity inhibition of plant growth is the result of osmotic and ionic effect and different plant species have developed different mechanisms to cope with those effects. With the discovery of molecular markers and marker assisted selection technology, it is possible to develop markers that identify salt tolerance. The genetic diversity of tomato genotypes were analyzed using SSRs polymorphic markers and Unweighted Pair Group Method with Arithmetic Mean. Leaves of the twenty tomato genotypes (landraces/accessions in Nigeria) were used to isolate their DNA using Bioland Plant Genomic DNA protocols. Primers were designed from 15 different salt responsive candidate genes, using Vector NTI and the sequence genes were obtained from ncbi genomice web site. All 15 primers sets generated shows clear distinct polymorphic profiles as evident from the 6% agarose gel profile. Dendrogram generated shows three groups, none of the panel intermixed in a subgroup. The genetic distance information reported in this study might be used by breeders when planning future crosses among tomato genotypes. From the result obtained UC82B recorded the highest vegetative and yield parameters, therefore, adoption of this genotype could be help increasing the tomato production in Sokoto agro-climatic area. Abstract Salinity inhibition of plant growth is the result of osmotic and ionic effect and different plant species have developed different mechanisms to cope with those effects. With the discovery of molecular markers and marker assisted selection technology, it is possible to develop markers that identify salt tolerance. The genetic diversity of tomato genotypes were analyzed using SSRs polymorphic markers and Unweighted Pair Group Method with Arithmetic Mean. Leaves of the twenty tomato genotypes (landraces/accessions in Nigeria) were used to isolate their DNA using Bioland Plant Genomic DNA protocols. Primers were designed from 15 different salt responsive candidate genes, using Vector NTI and the sequence genes were obtained from ncbi genomice web site. All 15 primers sets generated shows clear distinct polymorphic profiles as evident from the 6% agarose gel profile. Dendrogram generated shows three groups, none of the panel intermixed in a subgroup. The genetic distance information reported in this study might be used by breeders when planning future crosses among tomato genotypes. From the result obtained UC82B recorded the highest vegetative and yield parameters, therefore, adoption of this genotype could be help increasing the tomato production in Sokoto agro-climatic area.

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6–$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384–576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Assessment of sampling and DNA extraction methods for identification of grapevine trunk microorganisms using metabarcoding

For a deeper understanding of grapevine trunk disease (GTD) in New Zealand, a cheap, rapid, sensitive method for identifying within-vine microbial communities is required. Wood tissue from grapevine trunks was collected and three different DNA extraction methods were compared: a cetyltrimethylammonium bromide (CTAB) method, the Geneaid Plant Genomic DNA Mini Kit and the Qiagen DNeasy Plant Mini Kit. DNA samples from the CTAB and Geneaid methods were used for MiSeq DNA metabarcoding targeting the ribosomal internal transcribed spacer 1 (ITS1) region. DNA produced by the CTAB method was of a greater quantity and quality than for the other two methods, although the majority of the DNA samples provided polymerase chain reaction (PCR) amplification of fungal DNA sequences. Fungal metabarcoding profiles from the CTAB and Geneaid samples indicated the presence of fungi normally associated with GTD in New Zealand. The CTAB method was chosen for subsequent work due to its low-cost, simplicity and effective detection of typical GTD fungi. The complete process of sampling through to metabarcoding is now used annually as part of a wider ecological study, screening more than 600 vines at 12 Marlborough vineyards.

Development and application of polymorphic microsatellite markers in Saxifraga genus

In order to develop genomic-SSR markers for species of Saxifraga genus, a mixed plant genomic DNA sample was sequenced based on high-throughput Illumina MiSeq platform. According to genomic sequencing data, SSR loci were identified with MISA software, and then primers were designed with Primer 3 software. A total of 120 pairs of primers were randomly synthesized and amplified in genomic DNA of a few plant samples. Those primers who have yielded polymorphic bands and were considered easy to amplify were identified. After that, transferability of these primers was evaluated, and phylogenetic relationship of 25 species of Saxifraga genus was analyzed with UPGMA (unweighted pair group method analysis). In our results, 587 256 sequences containing SSRs were identified from a total of 1 881 979 combined read pairs obtained in genomic sequencing. Primers were designated to amplify SSRs containing two to six nucleotide repeat units, screened in a small portion of species. Finally, 17 pairs of primers which have produced abundant of polymorphic bands with little problem were amplified in 25 species of Saxifraga genus. A total of 2 687 polymorphic bands were obtained, the average polymorphic rate was 158 bands per pairs of primers. The transferability rate was ranging from 88.0% to 100% across 25 species of Saxifraga. In phylogenetic analysis, the clustering of 25 species based on 17 pairs of SSR primers was different from morphological classification. Our analysis has provided molecular data for genetic relationship of Saxifraga genus, and the transferable and polymorphic SSRs have provided information for genetic diversity research.

Plant Genomic DNA Isolation-the Past and the Present, a Review

Successful isolation of a good quality of plant genomic DNA in sufficient quantity is a necessary prerequisite for its downstream applications, that are aimed to examine and analyze the molecular intricacies of the plant kingdom. The quantity and quality of isolated genomic DNA often determines the accuracy and robustness of results derived from many DNA based applications. The choice of the method adopted for DNA isolation depends on a large number of factors like the amount, nature and age of the starting plant tissue, the infrastructure available in terms of laboratory facilities, ready availability of required laboratory chemicals, time at hand and the downstream applications of the obtained DNA. A large number of modifications have been suggested to optimize DNA isolation from a particular plant species or tissue. There cannot be a universal protocol for DNA isolation across all plant species or tissues owing to the highly heterogeneous nature of plant cells. Hence, efforts need to be made to carry out suitable variations in the extraction method, so as to develop a protocol which is best suited to a particular plant species or tissue or application. So far, a large number of DNA isolation protocols from plants have been published, but there is no comprehensive report on suitability of these methods over each other and the different modifications which have been attempted to optimize the yield and quality of DNA. Many review papers have described various DNA isolation protocols and their underlying principles, that have been adopted in order to optimize DNA isolation from different plant species and tissues (Varma et al., 2007; Tan and Yiap 2009; Kumari et al., 2012). However, there is no comprehensive study which gives details of modifications at different steps of the plant genomic DNA isolation protocol, that have been attempted by researchers to overcome the specie and tissue specific limitations. This review gives a detailed account of the steps involved in genomic DNA isolation from various plant species and tissues and how each of these steps have been modified to overcome and eliminate the problem of contaminants in the extracted DNA so as to obtain a good quality of DNA amenable to downstream molecular biology applications.

SCAR MARKER DEVELOPMENT FOR THE CORRECT IDENTIFICATION OF IRIS ENSATA

Objective: The objective of this research was to develop the RAPD based SCAR marker for the correct identification of the Iris ensata Thunb. (I. ensata) plant from its adulterants.Methods: Five samples of I. ensata. from the different geographical area were used in this study. The plant genomic DNA was isolated with the CTAB method with some modification (as dried samples were also used). After that, polymorphism was checked with the help of the 10-mer random primers of OPAA and BG series. Then, the bands of interest were eluted and cloned into pGEMT easy vector for the sequencing. Finally, the sequence is used to develop the SCAR primers (Ir-f andIr-R) specific for I. ensata and the developed primers also validated with respect to the market samples.Results: A putative 580 bp sequence specific for Iris ensata was identified from the randomly amplified polymorphic DNA (RAPD) analysis. To overcome the main limitation of RAPD it has been converted into SCAR markers. So that, this specific band was then eluted, cloned and sequenced. After that, SCAR primers (Ir-F and Ir-R) were synthesized by using this sequence. For the validation of the synthesized SCAR primers, they were tested with respect to the market samples. The amplicon of 260 bp was produced by the SCAR primers in the authentic I. ensata but market samples did not produce any bands with the synthesized SCAR primers.Conclusion: The results of this study show a high level of polymorphism in the RAPD pattern of the different accessions of the plant. Furthermore, this study results in the successful development of the RAPD based SCAR marker for the identification of the I. ensata.

The repetitive landscape of the 5100 Mbp barley genome

BackgroundWhile transposable elements (TEs) comprise the bulk of plant genomic DNA, how they contribute to genome structure and organization is still poorly understood. Especially in large genomes where TEs make the majority of genomic DNA, it is still unclear whether TEs target specific chromosomal regions or whether they simply accumulate where they are best tolerated.ResultsHere, we present an analysis of the repetitive fraction of the 5100 Mb barley genome, the largest angiosperm genome to have a near-complete sequence assembly. Genes make only about 2% of the genome, while over 80% is derived from TEs. The TE fraction is composed of at least 350 different families. However, 50% of the genome is comprised of only 15 high-copy TE families, while all other TE families are present in moderate or low copy numbers. We found that the barley genome is highly compartmentalized with different types of TEs occupying different chromosomal “niches”, such as distal, interstitial, or proximal regions of chromosome arms. Furthermore, gene space represents its own distinct genomic compartment that is enriched in small non-autonomous DNA transposons, suggesting that these TEs specifically target promoters and downstream regions. Furthermore, their presence in gene promoters is associated with decreased methylation levels.ConclusionsOur data show that TEs are major determinants of overall chromosome structure. We hypothesize that many of the the various chromosomal distribution patterns are the result of TE families targeting specific niches, rather than them accumulating where they have the least deleterious effects.

First Report of Daylily Flower Rot Caused by Fusarium proliferatum in China

Daylily (Hemerocallis citrina Baroni) is an important plant whose flowers are commonly used as a vegetable and in medicine in China. From June 2015 to July 2016, brown and rotted flowers, some of which were covered with white mycelia, were observed in a 0.8-acre commercial daylily field in Yanqing District, Beijing, China. Disease incidence was ∼40%. A total of 24 diseased flowers were collected, cut into 0.5 cm diameter sections from the edge of the rot/healthy tissues, surface-sterilized with 1% NaClO for 1 min followed by 75% ethanol for 30 s, and then rinsed twice with sterilized distilled water. Five surface-sterilized sections per flower were placed on potato sucrose agar (PSA) and carnation leaf agar (CLA) (Leslie and Summerell 2006) and incubated for 5 days at 25°C with a 12-h light-dark cycle in a chamber. Seventy-eight singe-spore isolates with similar morphological features as determined by a microscope were obtained (Song et al. 2017), and 24 were used as the research materials and incubated on PSA and CLA. The colonies on PSA were initially white, becoming purple, with rose to dark purple pigment on the surface. The macroconidia were abundant on CLA, hyaline, straight to slightly curved, 3 to 5 septate, 21.4 to 49.3 × 2.3 to 4.9 µm. Microconidia were oval, obovoid, or ellipsoidal, 0 to 1 septate, 4.5 to 15.1 × 2.2 to 4.3 µm. The cultural and morphological characteristics of the fungus were similar with previous reports of Fusarium proliferatum (Ritieni et al. 1995). Genomic DNA of all 24 isolates were extracted using the plant genomic DNA kit (Tiangen, China). The small subunit ribosomal RNA (mtSSU), elongation factor 1 alpha (EF-1α), and beta-tubulin (Tub) regions were amplified using the primers NSM1/NSM2 (Li et al. 1994), EF-1Ha/EF-2Tb (Alastruey-Izquierdo et al. 2008), and Beta1/Beta2 (Geiser et al. 2004). The amplicon sizes were ∼534, 691, and 745 bp, respectively. Three sequences of isolate HYC1410080201 were submitted to GenBank (accessions MF442355, MF448528, and MF442354, respectively). Isolates had 100% similarity with sequences of several isolates of F. proliferatum in GenBank (KX218406, KY081546, and KR861512, respectively). Pathogenicity was tested and Koch’s postulates satisfied in a growth chamber by inoculating daylily flowers. Briefly, spores were collected from 7-day-old PDA cultures of each isolate (n = 24) maintained at 25°C and suspended in sterilized water at 1 × 10⁶ conidia/ml. Spore suspension, 5 ml from each isolate, was sprayed onto the flowers of a plant with 10 plants inoculated per fungal isolate. Control plants were sprayed with 5 ml of sterile water. Each isolate was replicated three times. Five days after inoculation, small, pale yellow, water-soaked spots were observed on flowers of all inoculated plants. Under high humidity conditions, inoculated flowers were covered with a layer of white mycelia. Infected flowers wilted and dropped off, similar to symptoms observed on plants in the field. No symptoms were observed on control plants. Isolates obtained from inoculated plants had identical morphological features to the original isolates. Thus, Koch’s postulates were fulfilled and showed that F. proliferatum was the causal agent. To our knowledge, this is the first report of F. proliferatum causing daylily flower rot worldwide. Since this disease occurs during flowering and seriously affects edible and medicinal value, its presence has economic implications for daylily production.

A rapid and cost effective protocol for plant genomic DNA isolation using regenerated silica columns in combination with CTAB extraction

AbstractIsolation of high quality DNA from multiple samples can be both time-consuming and expensive. We have developed a combined protocol to reduce the time component of the hexadecyltrimethylammonium bromide (CTAB) extraction method and reduced costs by regenerating the silica columns used to purify genomic DNA. We present data that shows, by increasing the temperature used during the CTAB method, the time required to extract crude genomic DNA can be reduced. We show that silica columns can be regenerated using HCl and still maintain their DNA-binding capacity. Furthermore, we show both spectrophotometrically, and by restriction enzyme cutting, that the quality of the eluted DNA is high. Critically, using both genomic DNA from pea and perennial ryegrass we demonstrate, using species-specific PCR primers, that there is no carry-over of DNA from repeated use of a single column. The main advantages of the method are high yield, high quality, cost effectiveness and time-saving. This method could satisfy demand when large numbers of plant genomic DNA samples are required, for example from targeting induced local lesions in genomes (TILLING) populations.

Identification of Heading Date Six (Hd6) Gene Derived from Rice Mutant Varieties

<p>Genes which were associated with flowering time to indicate the early maturity is known as heading date (<em>Hd</em>). Heading date six (<em>Hd6)</em> gene<em> </em>was identified from rice mutant varieties were Atomita 2, Atomita 3, Atomita 4, Bestari, Cilosari, Diah Suci, Sidenuk, Kahayan, Mayang, Meraoke, Mira-1, Pandan Putri, Superwin, Suluttan Unsrat 1, Suluttan Unsrat 2, Winongo, Woyla, Yuwono, while the rice var. Nipponbare was used as a positive control. All of rice mutant varieties derived from mutation induction by the dose of 0.2 kGy. The aim of this experiment was to find out the data base of mutant varieties which could be used as parent material with earlier maturity trait genetically. To obtain the DNA of plants, young leaves of each variety were extracted by liquid nitrogen, and then lysis and extracted by Kit Plant Genomic DNA. The amplification of DNA with 7 primers of <em>Hd6</em> conducted of 40 cycles by PCR and were continues to separated by 1 % agarose. The results were shown that the rice Mira-1 and Bestari varieties obtained from mutation of Cisantana highly different from one to another on 7 primers of <em>Hd6</em> used. Mayang variety from mutation of cross breeding between Cilosari and IR64, Pandan putri from Pandan wangi and Woyla from mutation of cross breeding from Atomita 2 and IR64 were highly different with those of their parents. Identification of <em>Hd6</em> gene on Sidenuk variety was shown the same bands pattern with Nipponbare as control positive toward all primers used, this variety would be better for earlier maturity parent material compared to others. The information could be useful for breeding programs aiming to develop early maturing widely adaptive and high yielding rice cultivars.</p>

First Report of the Association of the 16SrII-D Phytoplasma Subgroup With Little Leaf Disease of Crotalaria in Karnataka, India

Smooth rattle pods plants (Crotalaria pallida Aiton. [Fabaceae]) are commonly grown as fodder and green manure in tropical and subtropical regions. In India, they are commonly used as nitrogen fixer in various agro-systems and as a source of various organic residues for soil nutrition enrichment. C. pallida is also used in folk medicine to treat urinary infections and as an anti-inflammatory agent. The extracted oil from the leaves of C. pallida is known for its antimicrobial properties. In January 2016, C. pallida grown in Mysuru area, Karnataka (India) was found associated with a characteristic little leaf disease, which was suggestive of phytoplasma infection. Symptoms also included small, yellow leaves with reduced lamina, stunting, and phyllody. The disease was noticed in the wild as well as in field-grown green manure crop. Approximately 30 to 50 plants from seven fields; that is, 10 to 15 plants randomly selected in 3 to 5 rows, were visually recorded for disease symptoms. Thirty-two out of 282 plants in different fields were estimated with phyllody symptoms, resulting in disease incidence of 11%. Leaf samples from five symptom-bearing plants along with two healthy leaf samples were collected and the DNA was manually extracted using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). PCR was performed to amplify the phytoplasma 16S rRNA gene using P1/P7 primers (Smart et al. 1996). The expected 1.8-kb amplicons were detected from symptomatic Crotalaria samples and none from the asymptomatic ones. The PCR products were purified, sequenced, and the consensus sequence was deposited in GenBank with accession number KX013260 (1,566 bp). It showed a 99% sequence identity with phytoplasma members of the group 16SrII ‘Candidatus Phytoplasma aurantifolia.’ The iPhyClassifier analysis (http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) revealed that the C. pallida phytoplasma (KX013260) had a coefficient of similarity of 1.0 and shared identical RFLP profiles to those of Ca. P. australasia (Y10097), which belongs to the subgroup 16SrII-D. Phytoplasmas of group 16SrII-D have been found affecting various vegetables, crops, and weeds in India, including tomato, brinjal, sesame, and parthenium. Crotalaria species are the reported hosts of several groups of phytoplasma which include 16SrVII-D (C. juncea: shoot proliferation) from Brazil (Flores et al. 2013); 16SrII-A (C. szemaoensis, C. zanzibarica, C. juncea: witches’-broom) from China (Wang et al. 2007), and 16SrI-B (C. spectabilis, C. tetragona: ‘fasciation’ and witches’-broom) from India (Kumar et al. 2010). To our knowledge, this is the first report of the association of the phytoplasma subgroup 16SrII-D with a disease on C. pallida in India. Recent disease reports and nucleotide sequence submissions revealed that the 16SrII phytoplasmas possess a wide plant host range; particularly economically important Fabaceae pulse crops (Thorat et al. 2016). Understanding the etiology and management of little leaf disease of C. pallida is critical as this may be a potential reservoir for 16SrII phytoplasmas.

Plant Genomic DNA Extraction for Selected Herbs and Sequencing their Internal Transcribed Spacer Regions Amplified by Specific Primers

This study was focused on plant genomic DNA extraction and sequencing from five commonly used medicinal herbs, namely Impatiens balsamina, Ficus deltoidea, Centella asiatica, Andrographis paniculata and Orthosiphon aristatus. This molecular technique is another highly reliable alternative for plant species identification besides phytochemical profiling. Three cetyl hexadecyltrimethylammonium bromide (CTAB) based methods with slight modification on incubation time, salt content and other additives were used for DNA extraction. The CTAB method of Doyle and Doyle produced higher DNA concentration from L balsamina, most probably due to the presence of ammonium acetate in the washing buffer and longer incubation time (2 h). The CTAB based method was suitable for A. paniculata because a high DNA concentration of acceptable quality was obtained for all the modified methods. However, Ο. aristatus was likely to have a lower DNA concentration (33-87 μg/g) and quality, probably due to the high concentration of phenolic compounds, particularly rosmarinic acid. The extracted genomic DNA was effectively amplified by a polymerase chain reaction using a universal primer of internal transcribed spacer (ITS), particularly AB 101 and AB 102 at the optimum annealing temperature of 48°C. The DNA sequences were analyzed by phenetic analysis and it was found that they have high similarity with the nucleotide sequences of ITS regions for similar plant species in the GenBank database of the National Center for Biotechnology Information.

FIRST REPORT OF FIG MOSAIC VIRUS AND FIG BADNAVIRUS 1 ON COMMON FIG TREES IN CROATIA

Common fig (Ficus carica L.) is one of the most common fruit trees in the Croatian coastal region. Fig mosaic symp- toms, first reported more than a half century ago (Perisic , 1952) are found on most cultivars. In 2014, leaf samples were taken from 30 autochthonous fig trees from two locations of the Istrian peninsula for laboratory analyses. Twenty six trees exhibited typical symptoms of foliar mosaic, one tree exhibited also fasciations, while the remaining four trees were asymptomatic. Extraction of total RNA was done us- ing an RNeasy plant mini kit (Qiagen, Germany) and ex- traction of DNA using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). Plants were tested by PCR for the presence of Fig mosaic virus (FMV) and Fig badnavirus 1 (FBV-1), applying previously described condi- tions and diagnostic primers (Elbeaino et al., 2009; Laney et al., 2012). A symptomless fig tree was used as negative control. The infection with FMV was confirmed in 26 symp- tomatic out of 30 sampled trees (87%), while all 30 plants were positive for FBV-1. One amplicon each of FMV and FBV-1 (isolates FMV-Cro-19S and FBV-1-Cro-17S, respec- tively), were sequenced in both directions (GenBank acces- sion Nos KT312843 and KT312844). A BLASTN program search showed 97% identity at the nucleotide level of the FMV-Cro-19S RdRp gene with the comparable sequence of FMV SB2-2 isolate from Serbia (AB697838), while the poly- protein gene from FBV-1-Cro-17S had a sequence identical to that of several FBV-1 isolates from the USA (JN050864, JN050875, JN112365). To the best of our knowledge, this is the first report of FMV and FBV-1 in Croatia.

Preparation of Rice Plant Genomic DNA for Various Applications.

Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA samples. Here we describe protocols for preparation of rice plant gDNAs for various applications including: (1) maxipreps and (2) minipreps of gDNAs for restriction analysis, gene cloning, PCR amplification, genomic sequencing, and genotyping; (3) preparation of megabase-sized nuclear DNA for construction of large-insert genomic libraries and long-range physical mapping; and (4) 96-well-format high-throughput gDNA preparation for PCR-based genotyping. The methods are also suitable for other plant species including dicots. © 2016 by John Wiley & Sons, Inc.

Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BackgroundWhile DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng).MethodsGenomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke’s Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA–trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0.ResultsThe accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA–trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA–trnH was higher than the corresponding intraspecific distance. Additionally, psbA–trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method.ConclusionWhile psbA–trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA–trnH was a more suitable barcode for authentication of Uncaria macrophylla.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0072-7) contains supplementary material, which is available to authorized users.

First Report of Botryosphaeria dothidea Causing Canker of Acer platanoides in China

HomePlant DiseaseVol. 99, No. 12First Report of Botryosphaeria dothidea Causing Canker of Acer platanoides in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Botryosphaeria dothidea Causing Canker of Acer platanoides in ChinaX. Wang, Y. X. Li, H. X. Dong, X. Z. Jia, and X. Y. ZhangX. WangSearch for more papers by this author, Y. X. LiSearch for more papers by this author, H. X. DongSearch for more papers by this author, X. Z. JiaSearch for more papers by this author, and X. Y. ZhangSearch for more papers by this authorAffiliationsAuthors and Affiliations X. Wang Y. X. Li H. X. Dong X. Z. Jia X. Y. Zhang , Key Laboratory of Forest Protection, State Forestry Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, P.R. China 100091; Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing Jiangsu, China 210037. Published Online:13 Nov 2015https://doi.org/10.1094/PDIS-03-15-0265-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In November of 2014, elongated oval-shaped cankers were observed on Norway maple (Acer platanoides) in a gardening nursery located in Henan Province of east-central China. The cankers were concentrated on the trunk and extended up to branches as disease developed. Bark on cankers was necrotic with black liquid visible. Cross sections of cankers revealed abnormally dark-brown tissues below the bark. Healthy tissues and tissue at the margins of cankers were cut into 1-mm2 pieces, sterilized in 75% ethanol for 30 s, then soaked in 1% sodium hypochlorite for 30 s, washed with sterile water, and finally placed onto potato dextrose agar (PDA). After 1 day of incubation at room temperature, white fluffy mycelia grew from diseased tissues. After 2 to 3 days, the centers of fungal colonies were dark gray. By 5 days, mycelia extended to the edges of 9-cm plates. After 7 days, entire colonies were dark gray and some pycnidia developed on the dark-gray PDA culture after about 30 days. Pycnidia were globose, and the hyaline cylindrical conidiophores or conidiogenous cells were attached to the inner walls of pycnidia. Conidia were hyaline, fusoid to ellipsoid, aseptate, with a subtruncate base, and measured 20 to 28 × 5.5 to 8.5 μm (mean dimensions of 50 conidia: 23 × 7 μm). DNA was extracted from the mycelia of one colony and carried out as specified by the manufacturer (plant genomic DNA kit, Tiangen, Beijing, China). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 and the ITS region of rDNA was sequenced and deposited in GenBank (Accession No. KP749828). The 574-bp region showed 100% identity to ITS sequence reference isolates of Botryosphaeria dothidea (KJ801792) (Zhou et al. 2015). The pathogen was identified as B. dothidea (Moug.) Ces. & De Not. on the basis of morphology and ITS gene sequence. The strain was deposited in the culture collection of Chinese Academy of Forestry and marked as CXY1813. Pathogenicity tests were done by inoculating each of five branches on one 3-year-old Norway maple tree with a mycelial plug (0.5-cm diameter) harvested from the margin of a 7-day-old colony. Five branches inoculated with plugs of PDA served as the negative control. After incubation for 20 days at 25°C, inoculated branches turned black and necrotic, while no signs or symptoms were observed on plants inoculated only with PDA. A fungus matching the description above was isolated from the diseased branches but not from the negative control. From these experiments, we infer that the cause of Norway maple canker is B. dothidea. This fungus was previously reported to cause canker on sycamore (Acer pseudoplatanus) in Italy (Turco et al. 2006). To our knowledge, this is the first report of B. dothidea causing canker of Acer platanoides in China.

First Report on the Occurrence of Virescence of Chrysanthemum Associated with 16SrII-A Group Phytoplasma in India

Chrysanthemum (Chrysanthemum indicum L.) is an important floriculture crop grown in different parts of Karnataka, India for its beautiful cut flowers. Recently, there has been a decline in flower quality due to major pests and diseases. A field survey conducted during 2012-2013 in chrysanthemum-growing areas revealed the occurrence of virescence with an incidence of 5 to 10% in nearly 6 to 8 ha of chrysanthemum fields in Mandya and Mysore districts. Symptoms including malformed flowers with greenish florets and reduced flower size and floral virescence with sepaloidy were observed. Diseased leaves and flower parts were collected and used for identification of the causative agent. Ten samples (6 symptomatic, 2 healthy, and 2 phytoplasma-associated with phyllody of Catheranthus roseus as positive control) were used for total genomic DNA isolation using GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) and the DNA was used as template to amplify the phytoplasma-specific 16SrDNA gene using P1/P7 primers followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected only with DNA from symptomatic and positive control samples. Nested PCR products of all six phytoplasma-associated samples were purified and sequenced from both the directions using a ABIx370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The 16SrDNA sequences were aligned using CodonCode Aligner (Centerville, MA) and analyzed by BLASTn. The analysis revealed that all six sequences shared 100% identity with Cassava witches broom (KM280680.1) and Peanut witches broom (JX403944.1) phytoplasma. The in-silico RFLP pattern of F2n/R2 primed region of 16SrDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 99.8% nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup A (Peanut witches broom phytoplasma; Accession No. L33765.1). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chrysanthemum virescence was identified as a member of 16SrII-A group. The representative sequence was deposited in GenBank with Accession Number KP027534.1. Phytoplasma associated with little leaf and witches broom of chrysanthemum have been reported earlier from India and were assigned to groups 16SrI-A and 16SrII, respectively (Kumar et al. 2012; Raj et al. 2007). Chrysanthemum virescence phytoplasma belonging to Candidatus phytoplasma aurantifolia has been reported from Japan and has not been assigned to any group (Naito et al. 2007). The occurrence of virescence is of great importance as it affects the overall productivity and quality of flowers, which reduces the market value of chrysanthemum. To our knowledge, this is the first report of a group 16SrII-A phytoplasma associated with chrysanthemum virescence in India.

Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

First report of Clerondendron yellow mosaic virus on golden dewdrop (Duranta erecta) in India

Figure 2 Golden dewdrop (Duranta erecta,Verbenaceae) is a species of flowering shrub and is commonly grown as an ornamental hedge plant in gardens. During a survey in August 2013, yellow mosaic disease was observed on D. erecta growing in several public gardens/parks at Lucknow, India (Latitude: 26° 55' N; Longitude: 80° 59' E) with disease incidence 80-90%. Infected plants exhibited severe yellow mosaic accompanied by upward leaf curl symptoms (Fig. 1A, B) and stunting in severely infected plants as compared to healthy plants (Fig. 1C). These symptoms reminiscent of a begomovirus infection reported previously on D. erecta in Pakistan (Iram et al., 2004) and hence a similar infection was suspected. Twenty leaf samples from infected and four from healthy plants were collected from Goldmohar Park at Jankipuram, Lucknow, India and total DNA was isolated using a plant genomic DNA isolation kit (Sigma, USA). PCR amplifications were performed with begomovirus-specific degenerate primers (Rojas et al., 1993) which resulted in the generation of amplicons of the anticipated size of ~1.2 kb in all samples from leaves showing disease symptoms and no amplicons from any of the four healthy samples suggestive of begomovirus infection in D. erecta. To identify the begomovirus responsible for the infection, total DNA isolated from a representative sample was subjected to rolling circle amplification (RCA, TempliPhi kit, GE healthcare, USA) and the product was then digested with a range of restriction enzymes including EcoRI. Electrophoresis of the EcoRI digested product revealed a DNA fragment ~2.7 kb which was cloned into pCAMBIA1300 vector, sequenced and deposited in GenBank (Accession No. KR869857, MJGD1). Sequence analysis of isolate MJGD1 revealed 93-95% nucleotide identities with DNA-A of Clerodendron yellow mosaic virus (ClYMV): RKAS1 (KF704391) of Bougainvillea peruviana and IARI (EF408037) of Clerodendron spp. from India, and SA23 (HE863667) of Croton spp. from Pakistan. It also showed close phylogenetic relationships with these ClYMV isolates (Fig. 2) suggesting that begomovirus isolated from D. erecta is a new strain or species of ClYMV designated as ClYMV

First report of natural occurence of Papaya leaf crumple virus on soyabean in India

Soybean (Glycine max, family Fabaceae) is a most important nutrient crop. It is native to East Asia and is grown widely as a pulse rich in protein, and also has numerous other uses. Symptoms of leaf crumple disease at Lalitpur (24.6° N, 78.4° E), India and yellow mosaic disease at Lucknow (26.8° N, 80.9° E), India, were noticed on a number of soybean plants in 2013. The naturally infected soybean plants exhibited yellow mosaic on leaves (Fig. 1a) and leaf crumpling and distortion symptoms (Fig. 1b). Severely infected plants were stunted and bore less number of flowers and pods as compared to the healthy ones. Based on leaf crumple and yellow mosaic symptoms, begomovirus infection was suspected. Twenty-two leaf samples of infected soybean plants were collected from each location and total DNA was isolated using a plant genomic DNA isolation kit (Sigma, USA). The PCRs were performed with begomovirus degenerate primers (Rojas et al., 1) which resulted in expected size amplicon of ~1.2 kb from 16/22 samples of yellow mosaic diseased plants collected from Lucknow and 12/22 samples of leaf crumple diseased plants collected from Lalitpur, but not from the healthy samples (Fig. 2). This indicated the presence of begomovirus in the soybean plants that had been sampled. To identify the complete genome sequence of these begomoviruses, total DNAs isolated from a representative leaf sample exhibiting yellow mosaic and leaf crumple symptoms were subjected separately to rolling circle amplification (RCA, TempliPhi kit, GE Healthcare, USA) followed by digestion with EcoRI, BamHI, HindIII, Sal1, XhoI, PstI and XbaI enzymes. Electrophoresis of products digested only with BamHI resulted in a DNA fragment of ~2.7 kb in both the samples. The products obtained ~2.7 kb were cloned separately into the pCAMBIA1300 vector, sequenced and deposited in GenBank with Accession Nos. KR052025 (MJS-1 isolate from yellow mosaic sample of Lucknow) and KR071789 (MJS-2 isolate from crumpled leaf sample of Lalitpur). The sequence analyses of MJS-1 and MJS-2 isolates showed the typical genome of begomovirus containing six open reading frames (V2 and V1 in sense strand and C1-C4 in antisense strand). BLASTn analysis of nucleotide sequence of begomovirus isolate MJS-1 under study showed 98-99% nucleotide sequence identity with Mungbean yellow mosaic India virus (MYMIV) isolates of Vigna unguiculata (DQ389153) and of G. max (EU523045, KC852204) reported from India. However, begomovirus isolate MJS-2 shared 94-97% identity with isolates of Papaya leaf crumple virus (PaLCrV) from Carica papaya (HM140367, HM140368, HM140369) and Solanum nigrum (KJ028210) both from India, and C. papaya (HE580236) from Pakistan. During phylogenetic analysis by MEGA v6.0 program (Tamura et al., 6) using nucleotide sequences of MYMIV and PaLCrV and some other begomoviruses available in GenBank database, MJS-1 and MJS-2 isolates showed close relationships with MYMIV and PaLCrV isolates, respectively (Fig. 3), hence the begomovirus isolated from soybean were identified as MYMIV and PaLCrV. A literature survey revealed the natural occurrence of MYMIV (Girish and Usha 2), Tomato leaf curl Karnatka virus (Raj et al., 3) and Cotton leaf curl Kokhran virus (Raj et al., 4) on soybean in India. The existence of PaLCrV is reported on C. papaya (Singh-Pant et al., 5), S. nigrum and Andrographis paniculata from India. However, this is the first report of natural occurrence of PaLCrV in soybean from India.