Fig 1: Foot rot tolerant transgenic rough lemon rootstock. (a) Map of gene cassette carrying 1764-bp ß-1,3-glucanase catalytic domain (KJ603460). (b) In vitro-raised seedling. (c) Epicotyl segment. (d) Direct regeneration. (e) Root formation. (f) T0 plants. (g) Semi-quantitative RT-PCR using catalytic domain primers 5?-ATGTTGAAGCTCACGGCGCTCGTTG-3? and 5?-ACTCGATTGCAGGGAAAGGCGGA-3? (top panel), RL-5 to RL-165 denote transgenic plants; cDNA integrity using 26SrRNA primers 5?-CACAATGATAGGAGGAGCCGAC-3? and 5?-CAAGGGAACGGGCTTGGCAGAATC-3? (AY283368) [bottom panel]. (h) Phytophthora mycelial growth inhibition by transgenic plant proteins; growth inhibition (%) = [(growth in NT ? growth in RL)/growth in NT] × 100; the assay was performed in triplicate; results are presented as mean ± SE. (i) Swollen hyphae; numbers indicate hyphal thickness. (j) Hyphal cytoplasmic constrictions with beaded appearance (solid arrows, bracket) and termination of mycelial branches (broken arrow). (k) Hyphal fragmentation; arrows indicate break points. (l) Normal unswollen hyphae. (m) Phytophthora-inoculated NT plant displaying gumming symptoms. (n) Inoculated transgenic plant RL-78 with the absence of gumming. (o) Estimation of foot rot incidence and plant morphological parameters: †Gumming scale 0–4, where 0 = nil, 1 = slight gumming, 2 = moderate gumming, 3 = severe gumming, 4 = tree girdled; ¶leaf chlorosis scale 0–3, where 0 = no symptoms, 1 = chlorotic leaves (light symptoms), 2 = necrotic leaves (moderate symptoms), 3 = wilt and defoliation (severe symptoms); ‡feeder root rot scale 1–5, where 1 = no visible symptoms, 2 = a few roots (1–25%) with rotting symptoms, 3 = majority of roots (26–50%) with rotting symptoms, 4 = all roots infected (51–75% rotting), major roots dead, 5 = majority of roots (>76% rotting) dead or missing; §propagule count was mean of three replicates; ††trunk girth was assessed at 20 cm above ground; ¶¶plant height was measured from ground to highest point; ‡‡canopy volume = 0.5236 × plant height × canopy spread (north to south) × canopy spread (east to west); §§feeder root volume = mean feeder root volume/total soil volume, where total soil volume = 141.14 cc [? × (radius of cylinder 1.59 cm)2 × height of cylinder 17.78 cm]; plant morphological parameters were recorded thrice, and P < 0.05 was statistically significant. (p) Quantitative RT-PCR in leaves, roots of transgenic plants before and after Phytophthora inoculation using qRT-PCR ß-1,3-glucanase primers 5?-CTCTCCAGAATGCTATCACCAC-3? and 5?-GGTATATGTTCCCGGAGGAATG-3?; 18SrRNA reference gene primers 5?-TCGGGTGTTTTCACGTCTCA-3? and 5?-TGGATGCCGCTGGGAAGC-3? (FJ356261.1); error bars represent range of change in ß-1,3-glucanase expression determined by 2-??C T method. (q) Transverse root section of susceptible NT plant revealing the occurrence of ligni-tubers (solid arrows) and callose deposition (broken arrows) in cortex cells. (r) The absence of ligni-tubers and callose deposition in cortex cells of tolerant plant RL-78. (s) The occurrence of zoospores on side wall (solid arrow) and end plate (broken arrow) of root xylem cell from susceptible NT plant. (t) The absence of zoospores in xylem cells of tolerant plant RL-78. (u) Endophytic microbial cell count in roots of transgenic rough lemon plants inoculated with Phytophthora; the microbial cell count (103 cfu/mL) was recorded in triplicate, and results are presented as mean ± SE. (v) Allelic pattern using CCSM-17 (5?-ACATGGACAGGACAACTAAG-3? and 5?-GTTATGATACGTCTGTGTTCC-3?); arrows indicate the presence of additional allele, RL-5 to RL-78 denotes transgenic plants, M refers to mother plant, B represents blank lane, L denotes 100-bp DNA ladder (Promega, Cat. No. G2101), and N indicates nucellar plant. (w) CCSM-6 (5?-ATCTGTGTGAGGACTGAA-3? and 5?-CCTCTATTAATGTGCCTG-3?); arrows depict the absence of allele. (x) CCSM-13 (5?-CTAGAGCCGAATTCACC-3? and 5?-AACAGCTACCAAGACACC-3?); arrows show the absence of allele. (y) CCSM-18 (5?-GTGATTGCTGGTGTCGTT-3? and 5?-AACAGTTGATGAAGAGGAAG-3?); arrows specify the absence of alleles.
Fig 2: Molecular genetic findings for subject 2. A, Sanger sequencing of genomic DNA from subject 2 (S2), father (F), mother (M) and control (C) show maternal transmission of the c.1029_1030delinsCA dinucleotide variant that is absent in her father and healthy control. B, Schematic illustrating the maternally transmitted c.1029_1030delinsCA allele, where X indicates a sequence variant (sample abbreviations as before). C, Agarose electrophoresis of PCR amplified fibroblast-derived cDNA demonstrates an aberrantly spliced product in subject 2 (S2) relative to an age and tissue matched control cDNA sample (C). N: no template control. DNA marker is 100 bp size standard (Promega cat G2101). Sanger sequencing of the aberrantly spliced amplicon confirms skipping of exon 6 (C, lower panel) and shows only wildtype sequence (GG) at nucleotides c.1029_1030 (denoted by an asterisk) confirming in trans variation. D, Agarose electrophoresis of genomic DNA amplified by long-range PCR shows a smaller amplicon of size ~1.3 kb in the Subject (S2) relative to the control amplicon (C) with the anticipated size (5.7 kb), consistent with a genomic deletion. N: no template control. Promega 1 kb ladder was used as a size standard. E, Sanger sequencing of the ~1.3 kb amplicon from subject 2 confirmed a ~4.5 kb deletion, c.730+608_c.854-25del, GRCh38: g.226978131_226982653del. F, BLAT search and IGV visualization of subject 2's deletion (dashed line) involving intron 5, exon 6 and intron 6
Fig 3: Gel electrophoresis of amplicons generated for the molecular barcodes with the genomic DNA of M. coriacea.(A) Amplification of rbcL (rbcLA_F/ rbcLA_R), matK (matK_3F_KIM f/matK_1R_KIM R). (B) Amplification of ITS1 (5a_F/ITS 4_R), and ITS2 (S2f/S3R). Numbers (1–3 and 4–6) indicate three technical replicates of DNA. + is the positive control. - is the negative control. M is the 100 bp DNA Ladder (Cat. # G2101; Promega, Madison, WI, USA).
Fig 4: Association of stem growth habit with DNA polymorphism of Dt1. A. Alignment of nucleotide sequences of PCR products amplified by a pair of primer Dt1Int showing 6-bp length polymorphism between cultivars with Dt1 (Ohsuzu and Tachinagaha) and dt1 (Athow and Stressland). B. PCR products amplified by a pair of primer Dt1Int (separated on a 7.5% polyacrylamide gel). Stem termination was evaluated by visual inspection of main stem growth after flowering. Analyzed plants are as follows: 1, ‘Stressland’; 2, ‘Tachinagaha’; 3–16, recombinant inbred lines derived from the cross between ‘Stressland’ and ‘Tachinagaha’. S indicates 100-bp DNA ladder (Promega G2101). IND: indeterminate growth plant, DET: determinate growth plant.
Fig 5: Agarose (1.8%) gel electrophoresis of two-step PCR DNA amplicons. DNA ladder (DL; 100-bp, Promega Cat# G2101), Akkermansia muciniphila (AM, 327 bp; Lanes 1–2), Bacteroides fragilis (BF; 265 bp; Lanes 3–4), Faecalibacterium prausnitzii (FP; 198 bp; Lanes 5–6), Desulfovibrio vulgaris (DV, 196 bp; Lanes 7–8) 16s rRNA amplicons coming from the two-step PCR protocol [Low (odd lanes)/high (even lanes) astringent conditions (? vs. ? °Tm)]. See Figure 1 and text for details.
Supplier Page from Promega for 100bp DNA Ladder
Fragment Sizes:100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 bp