scholarly journals Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection

Agronomy ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1368
Author(s):  
Carmen Arlotta ◽  
Angelo Ciacciulli ◽  
Maria Concetta Strano ◽  
Valeria Cafaro ◽  
Fabrizio Salonia ◽  
...  
Keyword(s):  
High Resolution ◽  
Nested Pcr ◽  
Marker Assisted Selection ◽  
High Resolution Melting ◽  
Brown Spot ◽  
Restriction Enzyme Digestion ◽  
Resistance Locus ◽  
Citrus Breeding ◽  
Alternaria Brown Spot ◽  
Young Leaves

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.

Differentiation of five enterohepaticHelicobacterspecies by nested PCR with high-resolution melting curve analysis

Helicobacter ◽  
2016 ◽  
Vol 22 (2) ◽  
pp. e12362 ◽  
Author(s):  
Miaoli Wu ◽  
Dan Rao ◽  
Yujun Zhu ◽  
Jing Wang ◽  
Wen Yuan ◽  
...  
Keyword(s):  
High Resolution ◽  
Nested Pcr ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
High Resolution Melting Curve

scholarly journals Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto

2016 ◽  
Vol 106 (11) ◽  
pp. 1366-1375 ◽  
Author(s):  
Xianzhou Nie ◽  
Darcy Sutherland ◽  
Virginia Dickison ◽  
Mathuresh Singh ◽  
Agnes M. Murphy ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Marker Assisted Selection ◽  
High Resolution Melting ◽  
Chromosome Region ◽  
Sts Markers ◽  
Reverse Primer ◽  
Selection For ◽  
Development And Validation ◽  
Selection Of

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato ‘Barbara’ (Rrrr) and ‘AC Chaleur’ (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

scholarly journals Efficacy of Pre- and Postinoculation Application of Fungicides to Expanding Young Citrus Leaves for Control of Melanose, Scab, and Alternaria Brown Spot

Plant Disease ◽  
2007 ◽  
Vol 91 (12) ◽  
pp. 1600-1606 ◽  
Author(s):  
S. N. Mondal ◽  
A. Vicent ◽  
R. F. Reis ◽  
L. W. Timmer
Keyword(s):  
Disease Control ◽  
Leaf Area ◽  
Quadratic Equation ◽  
Brown Spot ◽  
Copper Hydroxide ◽  
Fungicide Application ◽  
Potted Plants ◽  
Rough Lemon ◽  
Alternaria Brown Spot ◽  
Young Leaves

In greenhouse trials, copper hydroxide, pyraclostrobin, and famoxadone were applied to actively growing young citrus seedlings to determine the duration of protection of young leaves provided by these fungicides against melanose, caused by Diaporthe citri, citrus scab, caused by Elsinoe fawcettii, and Alternaria brown spot, caused by Alternaria alternata. Fungicides were applied to different sets of potted plants of grapefruit for control of melanose, of rough lemon for control of scab, and of Dancy tangerine for control of Alternaria brown spot 1 to 6 days prior to inoculation, as well as on the day of inoculation. Leaf area of treated shoots was estimated on the day of fungicide application and the day of inoculation and disease severity evaluated subsequently. In most cases, copper hydroxide and famoxadone provided at least 50% control of all three diseases for only about 2 days after application. Generally, there was little or no disease control when the products were applied 4 or more days before inoculation. In contrast, pyraclostrobin usually provided a high level of control of all three diseases when applied up to 5 days prior to inoculation. The level of disease control decreased as the interval between a fungicide application and inoculation increased and the relationship between disease control and leaf expansion best fit a quadratic equation. Effective disease control was observed with copper hydroxide and famoxadone until leaf area had increased by 100 to 200%, whereas control with pyraclostrobin was observed up to 400 to 500% increase in leaf area. In postinoculation tests with scab and melanose, pyraclostrobin provided high levels of disease control (>75%) when applied up to 2 days after inoculation, whereas copper hydroxide and famoxadone had minimal postinoculation activity. Applications of pyraclostrobin to the spring flush growth of citrus trees are much more likely to provide control of melanose, scab, and Alternaria brown spot than those of famoxadone or copper hydroxide.

Marker-assisted selection for rice blast resistance genesPi2andPi9through high-resolution melting of a gene-targeted amplicon

2016 ◽  
Vol 136 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Wenlong Luo ◽  
Ming Huang ◽  
Tao Guo ◽  
Wuming Xiao ◽  
Jiafeng Wang ◽  
...  
Keyword(s):  
High Resolution ◽  
Marker Assisted Selection ◽  
Rice Blast ◽  
High Resolution Melting ◽  
Blast Resistance ◽  
Rice Blast Resistance ◽  
Selection For

scholarly journals A Panel of High Resolution Melting (HRM) Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

2009 ◽  
Vol 31 (5) ◽  
pp. 329-333
Author(s):  
D. A. M. Heideman ◽  
F. B. Thunnissen ◽  
M. Doeleman ◽  
D. Kramer ◽  
H. M. Verheul ◽  
...  
Keyword(s):  
High Resolution ◽  
Targeted Therapies ◽  
Nested Pcr ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Tumour Response ◽  
Egfr Mutations ◽  
Cycle Sequencing ◽  
Ras Genes

Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.Methods: In this study, we describe the development of high resolution melting (HRM) technology-based assays with direct sequencing confirmation possibility for mutation screening of the EGFR gene (exons 19, 20 and 21) in routine diagnostic specimens, and compared assay findings to those of conventional nested-PCR following cycle-sequencing.Results: In reconstruction experiments, each HRM assay following sequencing demonstrated a sensitivity of ≤5% of mutated DNA in a background of wild-type DNA. The panel of EGFR HRM assays following sequencing applied to a series of genomic DNA samples isolated from 68 FFPE NSCLC specimens correctly identified all EGFR mutations that were previously found by nested-PCR following cycle-sequencing. The HRM approach additionally scored two mutations not detected by the conventional assay.Complementary HRM following sequencing for K-ras revealed three mutations. EGFR and K-ras mutations were mutually exclusive.Conclusions: The panel of designed HRM assays with direct reflex sequencing possibility provides an effective method for mutation screening of EGFR and K-ras genes in routine diagnostic specimens, thereby allowing the selection of the treatment of choice in clinical practice.

scholarly journals First Report of Alternaria Brown Spot of Citrus in Spain

Plant Disease ◽  
2000 ◽  
Vol 84 (9) ◽  
pp. 1044-1044 ◽  
Author(s):  
A. Vicent ◽  
J. Armengol ◽  
R. Sales ◽  
J. García-Jiménez ◽  
F. Alfaro-Lassala
Keyword(s):  
External Surface ◽  
Morphological Characteristics ◽  
Brown Spot ◽  
Economic Losses ◽  
Sterile Water ◽  
Eastern Province ◽  
First Report ◽  
Alternaria Brown Spot ◽  
Young Leaves ◽  
Pathogenicity Tests

In 1998, a new disease of Fortune mandarin trees was detected in orchards in the eastern province of Valencia. This is one of the most important late-maturing cultivars grown in Spain. Symptoms were typical of Alternaria brown spot of citrus (2). Young leaves showed brown necrotic and irregular blighted areas with characteristic yellow halos. The necrosis had a tendency to follow the veins. On fruits, symptoms included light brown, slightly depressed spots to circular and dark brown areas on the external surface. Infected young fruits and leaves often fell and the mature fruits were unmarketable due to lesions, resulting in important economic losses. Isolations on potato dextrose agar supplemented with 0.5 mg/ml of streptomycin sulfate (PDAS) from affected leaves and fruits consistently yielded Alternaria alternata (Fr.:Fr.) Keissl., which was identified based on conidial morphological characteristics. Pathogenicity tests were conducted using 15 isolates from fruit and leaves by inoculating detached immature Fortune leaves with a sterile water suspension of 5 × 105 conidia per ml. Drops of this suspension (40 μl each) were placed on the lower surfaces of each leaflet using four leaves per isolate. Leaves were incubated in a moist chamber in the dark at 27°C (1). After 48 h, most of these isolates caused necrotic lesions on the leaves similar to those observed in the field, and the fungus was reisolated, confirming Koch's postulates. In 1999, the fungus spread to other citrus-growing areas, and to date the disease has been detected affecting Fortune and Nova mandarins and Minneola tangelo. This is the first report of Alternaria brown spot of citrus in Spain. References: (1) K. Kohmoto et al. Phytopathology 81:719, 1991. (2) J. O. Whiteside. Plant Dis. Rep. 60:326, 1976.

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