title: Comparative whole-genome analyses of selection marker-free rice-based cholera toxin B-subunit vaccine lines and wild-type lines.
abstract: BACKGROUND
  We have developed a rice-based oral cholera vaccine named MucoRice-CTB (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens-mediated co-transformation system. To assess the genome-wide effects of this system on the rice genome, we compared the genomes of three selection marker-free MucoRice-CTB lines with those of two wild-type rice lines (Oryza sativa L. cv. Nipponbare). Mutation profiles of the transgenic and wild-type genomes were examined by next-generation sequencing (NGS).
  
  
  RESULTS
  Using paired-end short-read sequencing, a total of more than 300 million reads for each line were obtained and mapped onto the rice reference genome. The number and distribution of variants were similar in all five lines: the numbers of line-specific variants ranged from 524 to 842 and corresponding mutation rates ranged from 1.41 × 10(-6) per site to 2.28 × 10(-6) per site. The frequency of guanine-to-thymine and cytosine-to-adenine transversions was higher in MucoRice-CTB lines than in WT lines. The transition-to-transversion ratio was 1.12 in MucoRice-CTB lines and 1.65 in WT lines. Analysis of variant-sharing profiles showed that the variants common to all five lines were the most abundant, and the numbers of line-specific variant for all lines were similar. The numbers of non-synonymous amino acid substitutions in MucoRice-CTB lines (15 to 21) were slightly higher than those in WT lines (7 or 8), whereas the numbers of frame shifts were similar in all five lines.
  
  
  CONCLUSIONS
  We conclude that MucoRice-CTB and WT are almost identical at the genomic level and that genome-wide effects caused by the Agrobacterium-mediated transformation system for marker-free MucoRice-CTB lines were slight. The comparative whole-genome analyses between MucoRice-CTB and WT lines using NGS provides a reliable estimate of genome-wide differences. A similar approach may be applicable to other transgenic rice plants generated by using this Agrobacterium-mediated transformation system.
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. koji-kashima@asahikogyosha.co.jp.|Asahi Kogyosha Co., Ltd., Tokyo, Japan. koji-kashima@asahikogyosha.co.jp.
authors: Koji
authors: K
authors: Kashima
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. m-meji@ims.u-tokyo.ac.jp.
authors: Mio
authors: M
authors: Mejima
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. shiho-k@ims.u-tokyo.ac.jp.
authors: Shiho
authors: S
authors: Kurokawa
authors: Crop Development Division, NARO Agriculture Research Center, Niigata, Japan. kurodama@affrc.go.jp.
authors: Masaharu
authors: M
authors: Kuroda
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. kiyono@ims.u-tokyo.ac.jp.|International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. kiyono@ims.u-tokyo.ac.jp.
authors: Hiroshi
authors: H
authors: Kiyono
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. yukiy@ims.u-tokyo.ac.jp.|International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. yukiy@ims.u-tokyo.ac.jp.
authors: Yoshikazu
authors: Y
authors: Yuki
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D002772:Cholera Toxin / Q000096:biosynthesis / Q000235:genetics*; D018745:Genome, Plant; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D002772:Cholera Toxin
references: Plant Cell Physiol. 2012 Jan;53(1):256-64
references: 22156226
references: Nat Rev Genet. 2011 Jul;12(7):499-510
references: 21681211
references: Mol Genet Genomics. 2014 Jun;289(3):303-15
references: 24414128
references: Science. 2002 Apr 5;296(5565):92-100
references: 11935018
references: Annu Rev Plant Biol. 2014;65:743-68
references: 24579993
references: Eukaryot Cell. 2010 Sep;9(9):1300-10
references: 20601439
references: Plant Biotechnol J. 2013 Sep;11(7):799-808
references: 23601492
references: Bioinformatics. 2009 Aug 15;25(16):2078-9
references: 19505943
references: Plant Cell Physiol. 2013 Feb;54(2):e6
references: 23299411
references: J Biol Chem. 1992 Jan 5;267(1):166-72
references: 1730583
references: Plant Cell Physiol. 2011 Feb;52(2):274-82
references: 21258067
references: EMBO Rep. 2005 Jul;6(7):593-9
references: 15995674
references: Trends Biotechnol. 2003 Dec;21(12):570-8
references: 14624867
references: Bioinformatics. 2010 Mar 15;26(6):841-2
references: 20110278
references: Biotechnol Lett. 2014 Dec;36(12):2367-79
references: 25048244
references: Transgenic Res. 1996 Mar;5(2):97-103
references: 8866891
references: J Biomed Biotechnol. 2006;2006(2):25376
references: 16883050
references: J Immunol. 2009 Nov 15;183(10):6538-44
references: 19880451
references: Fly (Austin). 2012 Apr-Jun;6(2):80-92
references: 22728672
references: Plant Cell. 2002 Aug;14(8):1691-704
references: 12172016
references: Science. 2010 Jan 1;327(5961):92-4
references: 20044577
references: Nat Rev Genet. 2012 Jan;13(1):36-46
references: 22124482
references: Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):10986-91
references: 17573530
references: Bioinformatics. 2009 Jul 15;25(14):1754-60
references: 19451168
references: Plant Biol (Stuttg). 2010 Nov;12(6):831-41
references: 21040298
references: Proc Natl Acad Sci U S A. 2010 May 11;107(19):8794-9
references: 20421480
references: DNA Res. 2013 Dec;20(6):623-31
references: 23956243
references: Biotechnol Appl Biochem. 2011 Jan-Feb;58(1):58-67
references: 21446960
references: Plant Biotechnol J. 2012 Aug;10(6):623-34
references: 22222031
references: BMC Genomics. 2010;11:267
references: 20423466