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      Horizontally transferred genes as RNA interference targets for aphid and whitefly control

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          Summary

          RNA interference (RNAi)‐based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi or plants, are attractive targets for RNAi‐mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco‐adapted isolate of Myzus persicae (green peach aphid) and bioinformatically identified 30 HTGs. We then used plant‐mediated virus‐induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal‐origin HTGs did not affect aphid survivorship but decreased aphid reproduction. Importantly, although there was uptake of plant‐expressed RNA by Coccinella septempunctata (seven‐spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid‐targeted VIGS constructs. To demonstrate that this approach is more broadly applicable, we also targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant‐mediated RNA interference.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Fast and accurate short read alignment with Burrows–Wheeler transform

            Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk
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              BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs.

              Genomics has revolutionized biological research, but quality assessment of the resulting assembled sequences is complicated and remains mostly limited to technical measures like N50.
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                Author and article information

                Contributors
                gj32@cornell.edu
                Journal
                Plant Biotechnol J
                Plant Biotechnol J
                10.1111/(ISSN)1467-7652
                PBI
                Plant Biotechnology Journal
                John Wiley and Sons Inc. (Hoboken )
                1467-7644
                1467-7652
                25 January 2023
                April 2023
                : 21
                : 4 ( doiID: 10.1111/pbi.v21.4 )
                : 754-768
                Affiliations
                [ 1 ] Boyce Thompson Institute Ithaca NY USA
                [ 2 ] Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology Zhejiang University Hangzhou China
                [ 3 ] Department of Entomology Cornell University Ithaca NY USA
                [ 4 ]Present address: National Institute for Biotechnology and Genetic Engineering College Pakistan Institute of Engineering and Applied Sciences Faisalabad Pakistan
                [ 5 ]Present address: Gembloux Agro‐Bio Tech Institute The University of Liege Gembloux Belgium
                [ 6 ]Present address: Jacob Blaustein Institutes for Desert Research Ben‐Gurion University of the Negev Sede Boqer Israel
                [ 7 ]Present address: Drexel University College of Medicine Philadelphia PA USA
                Author notes
                [*] [* ] Correspondence (Tel 607 254 1365; fax 607 254 1502; email gj32@ 123456cornell.edu )
                Author information
                https://orcid.org/0000-0001-9684-1450
                https://orcid.org/0000-0002-9675-934X
                Article
                PBI13992 PBI-01165-2022.R1
                10.1111/pbi.13992
                10037149
                36577653
                a6e46dfa-4e29-4c30-8dcd-50884b3eb806
                © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                History
                : 14 December 2022
                : 07 October 2022
                : 22 December 2022
                Page count
                Figures: 7, Tables: 1, Pages: 768, Words: 13842
                Funding
                Funded by: BARD , doi 10.13039/100006031;
                Award ID: FI‐471‐2012
                Funded by: Defense Advanced Research Projects Agency , doi 10.13039/100000185;
                Award ID: HR0011‐17‐2‐0053
                Funded by: National Institute of Food and Agriculture , doi 10.13039/100005825;
                Award ID: 2021‐67013‐33565
                Award ID: 2021‐67014‐342357
                Funded by: Pakistan Higher Education Commission
                Award ID: 1‐8/HEC/HRD/2020/10897
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                April 2023
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.2.6 mode:remove_FC converted:24.03.2023

                Biotechnology
                horizontally transferred genes,green peach aphid,whitefly,seven‐spotted ladybug,rna interference,virus‐induced gene silencing

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