A Low-Cost High-Throughput Method for Plant Genomic DNA Isolation

Gupta, Prahlad; Salava, Hymavathi; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

doi:10.1007/978-1-4939-9865-4_1

Cited by 5 publications

(6 citation statements)

References 12 publications

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Section: Methodsmentioning

confidence: 99%

“…Leaf samples for genomic DNA were collected from 132 randomly selected M 2 M 4 plants. Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen) and in-house lab protocol(Gupta et al, 2020b, Sreelakshmi et al, 2010.DNA Sequencing, Read Mapping, and Variant CallingWhole Genome Sequencing was performed on the HiSeqX sequencing system (Illumina) by GeneWiz Inc., NJ, following the manufacturer's protocol. For each mutant line, a minimum of ~200 million reads (30 Gb/sample) with a target of a minimum of 25 Gb > Q30 were generated.The raw reads were filtered using fastp software (v0.19.5) using parameters -M 30 -3 -5.…”

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confidence: 99%

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Whole-Genome Profiling of Ethyl Methanesulfonate Mutagenesis in Tomato

Gupta¹,

Dholaniya

Princy³

et al. 2022

Preprint

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The induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but having an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. We sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected ca. 41 million SNPs and 5.5 million short-INDELs. We found a very high average density of mutations 1/3.05 Kb compared to other species. About 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than 1/3rd of genes in the mutagenized population had one or more deleterious mutations predicted by SIFT. Nearly 1/4th of deleterious genes mapped on tomato metabolic pathways modulating multiple pathway steps. Contrary to the reported GC>AT transition bias for EMS, we found EMS also produced nearly equal AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is least mutagenic towards EMS. The reduction in carotenoids in ζ-carotene isomerase mutant fruits and chloroplasts relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our study makes a large repertoire of mutations accessible to genetic studies and the breeding of tomato.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Whole-Genome Profiling of Ethyl Methanesulfonate Mutagenesis in Tomato

Gupta¹,

Dholaniya

Princy³

et al. 2022

Preprint

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“…The chloroplast genomic DNA of Populus yunnanensis was extracted from 25 mg silica-gel-dried leaves with Ezup Plant Genomic DNA Prep Kit Sangon Biotech, Shanghai, China under the manufactures instructions. The quality and quantity of the chloroplast genomic DNA was measured with Agarose gel electrophoresis by measuring A260/A280 15,16 .…”

Section: Plant Dna Isolationmentioning

confidence: 99%

Anti-osteosarcoma Biological Activity Evaluation and Complete Chloroplast Genome Sequencing of <i>Populus yunnanensis</i>

et al. 2021

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IntroductionCancer is one of the most dreaded diseases of the 21th century, which is also a leading cause of death all over the world, seriously influence the economic development and quality of people s life. The risk of cancer is still increasing because of the increased population aging and adopt lifestyle behaviors 1, 2 . Osteosarcoma is one of the most common high-grade malignant tumors that occurs in middle-aged and young people. As molecular biology techniques are widely used in the study of tumor etiology, we have a better understanding of the evolutionary characteristics of cancer. Exploring the molecular biological mechanism of osteosarcoma has positive significance for the diagnosis and treatment of the disease. Surgery, radiation, chemotherapy and immunotherapy were the traditionally the cancer treatment commonly used in clinic 3 . Improper operation of malignant tumor surgery can lead to tumor dissemination. The large-dose and long-term radiotherapy and chemotherapy are very harmful to the immune system of the patients 4 . The immunotherapy would sometime cause the over-immune, as well as off-target effects 5, 6 . Al- § Authors are contributed equally to this work.

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“…Prior to resuspension of DNA pellet in TE buffer, DNA pellet extracted using methods of Dellaporta et al (1983), Edwards et al (1991) and Taylor and Powell (1982) were observed to result in transparent pellets while others result in white opaque DNA pellet (results not shown). This observation implies that the isolated DNA is free from interfering contaminants such as polyphenols and polysaccharides (Ali et al, 2019;Gupta et al, 2020). The absorbance data taken from spectrophotometer provide an indication of sample purity and also their availability for PCR amplification and downstream sequencing.…”

Section: Dna Quantificationmentioning

confidence: 99%

“…Furthermore, phenol-using protocols are typically go to denaturing step twice with phenol, then chloroform: isoamyl alcohol. This additional move will, therefore, result in further depletion of DNA yield (Gupta et al, 2020).…”

Section: Dna Visualisation By Agarose Gel Electrophoresis and Pcr Promentioning

confidence: 99%