prism v.8.4.2 statistical software  (GraphPad Software Inc)

Journal: Communications Biology

Article Title: Natural epialleles of Arabidopsis SUPERMAN display superwoman phenotypes

doi: 10.1038/s42003-020-01525-9

Figure Lengend Snippet: a , b 5-Azacytidine treatment of Wa-1 plants does not suppress the silencing of lol-1 phenotypes. The Wa-1 ( lol-1 ) floral phenotypes (from ~50-day old plants) are identical both in the untreated control ( a ) and 50 µm treated 5-Azacytidine plants ( b ). c Chop PCR assay with McrBC to confirm the SUP locus’s unchanged methylated state in 5-Azacytidine-treated plants . d , e Mutations in the histone methyltransferase KYP and DNA methyltransferase CMT-3 restore the WT phenotypes in lol-1 plants. Representative homozygous double mutant combinations of lol-1;kyp-2 ( d ) and lol-1;cmt3-7 ( e ) showing WT inflorescence phenotype in comparison to lol-1 plant. f – i Trans -chromosome methylation analysis in the F2 segregating population and the complemented tetraploid Wa1 plants. f Chop PCR(C) assay reveals the absence of trans chromosomal methylation in complemented tetraploid Wa-1 plants. Control 4 x Wa-1 plants are C and dCAPS(D) assay negative, whereas 4 x Wa-1 complemented plant with Col-0 SUP genomic locus is PCR positive only for Col-0 allele after C + D assay. The D assay alone on 4 x Wa-1 complemented plant reveal heterozygosity with strong PCR signal for 4 x Wa-1 allele consistent with its abundance (as indicated by asterisks) compared to the weak signal of ectopically inserted Col-0 clone (as indicated by arrowhead). g C and D assay in the patch-1 of SUP locus from representative F2 progeny from Wa-1 × Col-0 segregating population. Plants(#9,#23) displaying WT phenotype show Col-0 unmethylated allele both in D and C + D assay (190 bp fragment), indicating they are homozygous for Col-0 allele. Plants (#18, #63) displaying the Wa-1 inflorescence phenotype show Wa-1 unmethylated allele both in D and C + D assay (223 bp fragment), indicating they are homozygous for Wa-1 allele. Plants (#19, #27), display WT phenotype, but heterozygous in D (both 223 and 190 bp), and PCR positive only for uncut, unmethylated Col-0 allele after McrBC digestion. h , i C and D assay in the patch-2 of SUP locus from representative F2 progeny from Wa-1 × Sorbo F2 segregating population. Plants (#12, #28) display WT phenotype confirmed by D assay and C positive with McrBC digested template. Plants (#33,#66) display Wa-1 inflorescence phenotype, confirmed by D assay showing homozygosity for Wa-1 allele and C assay negative. Plants (#3,#20) display WT phenotypes, are heterozygous as revealed by D assay and C positive only for Col-0 allele. j A dot plot of Δ ct values representing SUP mRNA abundance, with respect to ACTIN2 as internal control in hypermethylated Wa-1 and Geg-14 accessions, compared to unmethylated WT Col-0 control (unpaired t -test (two-tailed) p = 0.76 (Col-0 and Wa-1), p = 0.09 (Col-0 and Geg1), ns non-significant, ɑ = 0.05). Six and four biological replicates for Wa-1 and Geg-14, respectively, were analyzed. k Semi-quantitative RT-PCR showing the abundance SUP mRNA and alternative splice variant in the control Col-0, and representative lol accessions. ACTIN2 is used as an internal control. Error bar = Standard deviation (SD) of biological replicates. DNA markers used in the gels is 50 bp NEB ladder. Scale bars: a , b = 2 cm; d , e = 1 cm.

Article Snippet: All the statistical analysis described in the study: the chi-square test for goodness of fit for genetic crosses, Kruskal–Wallis (KW) test for seed set data, two-tailed unpaired t -test for SUP mRNA quantitation was performed using GraphPad PRISM (v 8.4.2) statistical software.

Techniques: Control, Methylation, Mutagenesis, Comparison, Two Tailed Test, Quantitative RT-PCR, Variant Assay, Standard Deviation