scholarly journals A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2229
Author(s):  
Ke Duan ◽  
Ying-Jie Zhao ◽  
Zi-Yi Li ◽  
Xiao-Hua Zou ◽  
Jing Yang ◽  
...  
Keyword(s):  
Agronomic Traits ◽  
Crop Improvement ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Sugar Transporter ◽  
Transporter Gene ◽  
Engineering Technology ◽  
Selectable Markers ◽  
Vector Backbone ◽  
First Time

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

scholarly journals Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

2019 ◽  
Author(s):  
Marta Puig ◽  
Jon Lerga-Jaso ◽  
Carla Giner-Delgado ◽  
Sarai Pacheco ◽  
David Izquierdo ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Genomic Variation ◽  
Factors Affecting ◽  
Genome Wide ◽  
Population Information ◽  
Genetic Length ◽  
The Impact ◽  
First Time ◽  
Inversion Breakpoints

ABSTRACTDespite the interest in characterizing all genomic variation, the presence of large repeats at the breakpoints of many structural variants hinders their analysis. This is especially problematic in the case of inversions, since they are balanced changes without gain or loss of DNA. Here we tested novel linkage-based droplet digital PCR (ddPCR) assays on 20 inversions ranging from 3.1 to 742 kb and flanked by long inverted repeats (IRs) of up to 134 kb. Among these, we validated 13 inversions predicted by different genome-wide techniques. In addition, we have generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions for which there were no high-throughput methods to determine directly the orientation, like the well-characterized 17q21 inversion. Through comparison with previous data, independent replicates and both inversion breakpoints, we have demonstrated that the technique is highly accurate and reproducible. Most of the studied inversions are frequent and widespread across continents, showing a negative correlation with genetic length. Moreover, all except two show clear signs of being recurrent, and the new data allowed us to define more clearly the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able to check inversion functional effects in multiple tissues, validating gene expression differences reported before for two inversions and finding new candidate associations. Our work therefore provides a tool to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

scholarly journals Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse

2020 ◽  
Vol 169 ◽  
pp. 115213 ◽  
Author(s):  
Michael A. Jahne ◽  
Nichole E. Brinkman ◽  
Scott P. Keely ◽  
Brian D. Zimmerman ◽  
Emily A. Wheaton ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

scholarly journals Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 813
Author(s):  
Michele Manganelli ◽  
Ilaria Grossi ◽  
Manuela Ferracin ◽  
Paola Guerriero ◽  
Massimo Negrini ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Healthy Individuals ◽  
Roc Curve Analysis ◽  
Multikinase Inhibitor ◽  
Sorafenib Treatment ◽  
The Third ◽  
Circulating Levels ◽  
Systemic Drug

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

scholarly journals Identification and semi-quantification of Atlantic salmon in processed and mixed seafood products using Droplet Digital PCR (ddPCR)

2021 ◽  
pp. 112329
Author(s):  
Dumas Deconinck ◽  
Kris Hostens ◽  
Isabel Taverniers ◽  
Filip A.M. Volckaert ◽  
Johan Robbens ◽  
...  
Keyword(s):  
Atlantic Salmon ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Seafood Products

scholarly journals Integration of high-density genetic mapping with transcriptome analysis uncovers numerous agronomic QTL and reveals candidate genes for the control of tillering in sorghum

2021 ◽  
Author(s):  
Rajanikanth Govindarajulu ◽  
Ashley N Hostetler ◽  
Yuguo Xiao ◽  
Srinivasa R Chaluvadi ◽  
Margarita Mauro-Herrera ◽  
...  
Keyword(s):  
Agronomic Traits ◽  
Interspecific Cross ◽  
Crop Improvement ◽  
Differentially Expressed ◽  
Plant Domestication ◽  
Sorghum Propinquum ◽  
Ril Population ◽  
Trait Locus ◽  
Genomic Regions ◽  
Low Coverage

Abstract Phenotypes such as branching, photoperiod sensitivity, and height were modified during plant domestication and crop improvement. Here, we perform quantitative trait locus (QTL) mapping of these and other agronomic traits in a recombinant inbred line (RIL) population derived from an interspecific cross between Sorghum propinquum and Sorghum bicolor inbred Tx7000. Using low-coverage Illumina sequencing and a bin-mapping approach, we generated ∼1920 bin markers spanning ∼875 cM. Phenotyping data were collected and analyzed from two field locations and one greenhouse experiment for six agronomic traits, thereby identifying a total of 30 QTL. Many of these QTL were penetrant across environments and co-mapped with major QTL identified in other studies. Other QTL uncovered new genomic regions associated with these traits, and some of these were environment-specific in their action. To further dissect the genetic underpinnings of tillering, we complemented QTL analysis with transcriptomics, identifying 6189 genes that were differentially expressed during tiller bud elongation. We identified genes such as Dormancy Associated Protein 1 (DRM1) in addition to various transcription factors that are differentially expressed in comparisons of dormant to elongating tiller buds and lie within tillering QTL, suggesting that these genes are key regulators of tiller elongation in sorghum. Our study demonstrates the usefulness of this RIL population in detecting domestication and improvement-associated genes in sorghum, thus providing a valuable resource for genetic investigation and improvement to the sorghum community.

Sign in / Sign up

Export Citation Format

Share Document