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      Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development

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          Summary

          Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis ×  urophylla wild‐type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

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          MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.

          We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
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            The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors.

            Mutations in the homeotic gene agamous of the plant Arabidopsis cause the transformation of the floral sex organs. Cloning and sequence analysis of agamous suggest that it encodes a protein with a high degree of sequence similarity to the DNA-binding region of transcription factors from yeast and humans and to the product of a homeotic gene from Antirrhinum. The agamous gene therefore probably encodes a transcription factor that regulates genes determining stamen and carpel development in wild-type flowers.
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              CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees.

              Forest trees display a perennial growth behavior characterized by a multiple-year delay in flowering and, in temperate regions, an annual cycling between growth and dormancy. We show here that the CO/FT regulatory module, which controls flowering time in response to variations in daylength in annual plants, controls flowering in aspen trees. Unexpectedly, however, it also controls the short-day-induced growth cessation and bud set occurring in the fall. This regulatory mechanism can explain the ecogenetic variation in a highly adaptive trait: the critical daylength for growth cessation displayed by aspen trees sampled across a latitudinal gradient spanning northern Europe.
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                Author and article information

                Contributors
                Steve.Strauss@Oregonstate.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
                04 May 2021
                September 2021
                : 19
                : 9 ( doiID: 10.1111/pbi.v19.9 )
                : 1743-1755
                Affiliations
                [ 1 ] Department of Forest Ecosystems and Society Oregon State University Corvallis OR USA
                [ 2 ] Department of Biology University of Colorado Colorado Springs Colorado Springs CO USA
                [ 3 ] Department of Zoology and Entomology University of Pretoria Pretoria South Africa
                [ 4 ] Beijing Advanced Innovation Center for Tree Breeding by Molecular Design National Engineering Laboratory for Tree Breeding College of Biological Sciences and Biotechnology Beijing Forestry University Beijing China
                [ 5 ] Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI) University of Pretoria Pretoria South Africa
                [ 6 ]Present address: Department of Molecular and Structural Biochemistry North Carolina State University Raleigh NC USA
                Author notes
                [*] [* ] * Correspondence (Tel +1 541‐737‐6578; fax +1 541‐737‐1393; email: Steve.Strauss@ 123456OregonState.Edu )

                Article
                PBI13588
                10.1111/pbi.13588
                8428835
                33774917
                1d10cf05-2088-435d-af26-ca3c6d4b6c53
                © 2021 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/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, Tables: 1, Pages: 13, Words: 11274
                Product
                Funding
                Funded by: U.S.A. National Science Foundation
                Funded by: Tree Biosafety and Genomics Research Cooperative
                Funded by: U.S. Department of Agriculture , doi 10.13039/100000199;
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                September 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.7 mode:remove_FC converted:09.09.2021

                Biotechnology
                leafy,eucalyptus,flowering,crispr,containment
                Biotechnology
                leafy, eucalyptus, flowering, crispr, containment

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