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1 Supplementary information Plant phosphomannose isomerase as a selectable marker for rice transformation Lei Hu, Hao Li, Ruiying Qin, Rongfang Xu, Juan Li, Li Li, Pengcheng Wei & Jianbo Yang
2 Figure S1. Homology analysis of plant PMI amino acid sequences with w typical type I PMIs. Alignments of the amino acid sequences off the plant PMIs and otherr PMIs of various species were produced using the BioEdit program. The conserved YXDXNHKPE motif is boxed in red. Four zinc-ligand residues, including Gln-111, Glu-138, His-113 and His-285, are labeled with green arrows.
3 Figure S2. Predicted 3D structures of plant PMIs and typical type I PMIs. P The amino acid sequences were used to predict the 3D structure of the PMIs withh a SWISS-MODEL online tool. The images were captured using Cn3DD software.
4 Figure S3. The transgenic Kasalath carrying the PMI vectors grew and a developedd normally in the greenhouse. The plants were grown for 10 weeks after being transferred to soil. From F left to right: the untransformed Kasalath plant,, the transgenic Kasalath carrying EcPMI, CvPMI, AtPMI2 and OsPMI1.
5 Figure S4. The germination of untransformed Nipponbare and transgenic lines carrying CvPMI, AtPMI2 and OsPMI1 expression vectors onn Man selection medium. Approximately 15 seeds of untransformed Nipponbare and the transgenic plants expressing CvPMI, AtPMI2 and OsPMI1 were germinated in MS plus 12.5 g/l Man mediumm for 10 days. The yellow arrows indicate the failed germination of the transgenic plants.
6 Table S1. Detailed information regarding the Nipponbare transformations using EcPMI and the plant PMIs as SMGs. SMG a Incubated calli b Resistant Regenerated Positive Low copy,d Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 EcPMI CvPMI AtPMI OsPMI a, The transformation of each PMI vector was separately examined in two independent experiments; b, the data represent the number of calli incubated by agrobacteria in each individual experiment; c, the data represent the number of independent events (resistant calli or regenerated plants produced from one original agrobacteria-incubated callus were recognized as an independent event); d, low-copy events (harboring one or two copy) were determined by Real-time PCR by the Taqman probe on the 35S promoter as previously described.
7 Table S2. Detailed information regarding the Kasalath transformations using EcPMI and the plant PMIs as SMGs. SMG a Incubated calli b Resistant Regenerated Positive Low copy Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 EcPMI CvPMI AtPMI OsPMI a, The transformation of each PMI vector was separately examined in two independent experiments; b, the data represent the number of calli incubated by agrobacteria in each individual experiment; c, the data represent the number of independent transformation events.
8 c Table S3. Regeneration of EcPMI and the plant PMIs selection events under different Man pressures. Sugar composition Regeneration events b Positive events in medium a EcPMI CvPMI AtPMI2 OsPMI1 EcPMI CvPMI AtPMI2 OsPMI1 30 Suc Exp Exp Man+20 Suc Exp Exp Man+15 Suc Exp Exp Man+10 Suc Exp Exp Man Exp Exp a, The effect of sugar composition in the regeneration medium were examined in two separate experiments. b, the data represent the number of regenerated events produced from 60 Man-resistant calli; c, the data represent the number of PCR-positive regenerated events. - : not tested.
9 Table S4. GOI transformations using the plant PMIs as SMGs in rice. SMG a Incubated calli b Resistant Regenerated Positive SMG d GOI e Both f CvPMI Exp Exp AtPMI2 Exp Exp OsPMI1 Exp Exp a, The Ubq-driven plant PMIs were inserted into a HPT-contained pcambia1300 vector separately, then the Man-selected transformation of derivative constructs were examined individually; b, the data represent the number of calli incubated by agrobacteria in each individual experiment; c, the data represent the number of independent transformation events; d, positive events determined by sequence specific PCR on the Ubq promoter; e, positive events determined by sequence specific PCR on the HPT gene; f, the regenerated events positive on both of Ubq promoter and HPT gene.
10 Table S5. Segregation of T 1 progenies of the transgenic plants harboring different PMI-HPT vectors. Line Positive Hygromycin Negative Mendelian ratio Positive Man Negative Mendelian ratio CvPMI : : : : : : : : : : : :1 AtPMI : : : : : : : : : : : :1 OsPMI : : N 50 0 N : : : : : : : :1 3:1 and 15:1 indicate one T-DNA copy and two independent copies, respectively. N suggested the line is not the low-copy line or the segregation of T 1 seeds does not follow a standard Mendelian laws. The chi-square test was used to test goodness-of-fit to the expected segregation ratio.
11 Table S6. The parallel rice transformation of the PMI-HPT vectors using hygromycin selection. Vector a Incubated calli b Resistant Regenerated Positive, d TFs e CvPMI HPT Exp % Exp % AtPMI2 HPT Exp % Exp % OsPMI1 HPT Exp % Exp % a, The Ubq-driven plant PMIs were inserted into a HPT-contained pcambia1300 vector separately, then the Hygromycin-selected transformation of derivative constructs were examined individually; b, the data represent the number of calli incubated by agrobacteria in each individual experiment; c, the data represent the number of independent transformation events; d, positive events determined by sequence specific PCR on the Ubq promoter; e, The ratio of PCR-positive events to agrobacteria-incubated calli.
12 Table S7. Primers used in this study. 1. Primers used to amplify the PMIs Primer Primer sequence (5 to 3 ) OsPMI1 FP TTCGCCTCCCTCCTCCCTCCCA OsPMI1 RP CACTGACCTTTTACCTACAGCA OsPMI2 FP: ATGGAGGACCCGCCGCCGCCGC OsPMI2 RP: TTAACTGAAGAATCTGCTGTTT OsPMI3 FP: CTTTTACTAATTTAATGACAGC OsPMI3 RP: CTCGCAATAACCTTTAATCCCT CvPMI FP: ATGGCTGGAACGGCGACAGAGA CvPMI RP: CTCAAAGGCCATTCCGTTG AtPMI2 FP: ATGGGAGCAGACGCAATCCAAA AtPMI2 RP: CAATGTTTGGAAGAATCTGCTG 2. Primers used to construct vectors Primer Primer sequence (5 to 3, sequences Restriction underlined to show endonuclease site recognition site) Experiment EcPMI-GST FP: GGATCCATGCAAAAACTCATTAACTCAG BamHI EcPMI-GST RP: CTCGAGCAGCTTGTTGTAAACACGC XhoI GST-EcPMI expression CvPMI-GST FP: GGATCCATGGCTGGAACGGCGACAGAGA BamHI CvPMI-GST RP: CTCGAGCTCAAAGGCCATTCCGTTG XhoI GST-CvPMI expression AtPMI2-GST FP: GGATCCATGGGAGCAGACGCAATCCAAA BamHI AtPMI2-GST RP: CTCGAGCAATGTTTGGAAGAATCTGCTG XhoI GST-AtPMI2 expression OsPMI1-GST FP: GGATCCATGGCCGGCCCTACTCCTCCTC BamHI OsPMI1-GST RP: CTCGAGATTGAAGAATCTGCTATTGACC XhoI GST-OsPMI1 expression OsPMI2-GST FP: CTCGAGATGGAGGACCCGCCGCCGCCGC XhoI OsPMI2-GST RP: CTCGAGACTGAAGAATCTGCTGTTTACC XhoI GST-OsPMI2 expression OsPMI3-GST FP: GAATTCATGACAGCAAGCAAAGAAACAG BamHI OsPMI3-GST RP: CTCGAGGCTGAAGAATCTGCTGTTCACC XhoI GST-OsPMI3 expression EcPMI-1381 FP: CTCGAGATGCAAAAACTCATTAACTCAG XhoI Rice transformation by EcPMI-1381 RP: CTCGAGTTACAGCTTGTTGTAAACACGC XhoI EcPMI CvPMI-1381 FP: CTCGAGATGGCTGGAACGGCGACAGAGA XhoI Rice transformation by CvPMI-1381 RP: CTCGAGTCACTCAAAGGCCATTCCGTTG XhoI CvPMI AtPMI FP: CTCGAGATGGGAGCAGACGCAATCCAAA XhoI Rice transformation by AtPMI RP: CTCGAGCTACAATGTTTGGAAGAATCTG XhoI AtPMI2 OsPMI FP: CTCGAGATGGCCGGCCCTACTCCTCCTC XhoI Rice transformation by OsPMI RP: CTCGAGTTAATTGAAGAATCTGCTATTG XhoI OsPMI1 3. Primers used for semi-quantitative RT-PCR assays Primer Primer sequence (5 to 3 ) ACTIN-SemiF: TCAGCAACTGGGATGATATGGAG ACTIN-SemiR: GCCGTTGTGGTGAATGAGTAAC EcPMI-SemiF: TTGACTGAACTTTATGGTATGG EcPMI-SemiR: TCCGGCTTGTGGTTAGGATCTT
13 CvPMI-SemiF: CGCTCAAAACTATGCCTGGGG CvPMI-SemiR: GCTTGTGGTTGGCGTCCTTGTA AtPMI2-SemiF: TGGTCACGATCAAGCCAAGT AtPMI2-SemiR: CGACAATGCTTTCGTTACTG OsPMI1-SemiF: ACGCCGAGCTGTGGATGG OsPMI1-SemiR: GGCAAAGCCGCAGAGGAC 4. Primers used for Real-Time PCR assays on the identification of copy numbers SPS-FP TCTCCTCGTCCAGTGCTTCTC SPS-RP TTGGTGGACGCGCTTCTAG SPS-Probe HPT-FP HPT-RP HPT-Probe 35S-FP 35S-RP 35S-probe TET-TCCTCGCAACCGAAC-TAM CTATTTCTTTGCCCTCGGACGA GGACCGATGGCTGTGTAGAAG FAM-CGCCGATAGTGGAAACCGACGCCC-TAM CGACAGTGGTCCCAAAGA AAGACGTGGTTGGAACGTCTTC FAM-TGGACCCCCACCCACGAGGAGCATC-TAM
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