Supplementary file 3

1. MusaSAP1 copy number determination using real time quantitative PCR 100 Fluorescence 50 gDNA SD1 SD3 SD4 SD5 SD2 10 10 20 30 35 Cycle A 4289 bp plasmid containing a single copy of 1403 bp 5’ upstream genomic region of MusaSAP1 (isolated using TAIL-PCR) was used to make five serial dilutions starting from 1/1000 dilution to 1/10000000 dilution. These five serial dilutions namely SD1, SD2, SD3, SD4 and SD5 were used as template (in triplicates) in real time quantitative PCR reactions with 2X SYBR Green Extract-N-Amp PCR ReadyMix (Sigma, USA) and primers specific for MusaSAP1 5’ UTR sequence. The 12 μl PCR reaction mixture contained 6 μl 2X SYBR Green mix, 1 μl of each primer (2 picomoles) and 4 μl of plasmid template. For genomic DNA template, the 12 μl PCR reaction mixture contained 6 μl 2X SYBR Green mix, 1 μl of each primer (2 picomoles) and 4 μl of 1/15 diluted genomic DNA template (~5.86 ng). Cycling parameters for these reactions were 94°C for 5 min initially followed by 35 cycles, each comprising of 94°C for 25 s, 56°C for 25 s and 72°C for 25 s. Ct values obtained using rotor gene software (Qiagen, Germany) were plotted against log10 of the plasmid copy number calculated based on the total base pairs in the plasmid and its concentration determined based on the absorbance at 260 nm. The mean Ct value for the genomic DNA was then plotted on this standard curve to obtain the copy number of MusaSAP1 gene in ~5.86 ng of genomic DNA. The total no. of genomes of banana cv. Karibale Monthan (ABB group) in ~5.86 ng genomic DNA was calculated by assuming A genome to be 550 Mbp and the B genome to be 610 Mbp. Copy number of MusaSAP1 gene in ~5.86 ng of genomic DNA was then correlated with the number of genomes present in ~5.86 ng genomic DNA. This value was approximately 3 and since each genome of banana cv. Karibale Monthan contains three chromosome sets, one copy of MusaSAP1 is estimated to be present in each of these three chromosome sets. SD5 SD4 SD3 Ct SD2 SD1 Log10 plasmid copy number Real time quantitative amplification of MusaSAP1 partial 5’ UTR from five serial dilutions (SD1, SD2, SD3, SD4 and SD5) of the plasmid and banana cv. Karibale Monthan genomic DNA (gDNA) Standard curve between Log10 plasmid copy number and Ct values obtained for the various serial dilutions. The equation for the straight line obtained is also displayed. Concentration of the plasmid containing a single copy of MusaSAP1 5’ UTR : 50 ng/μl Total length of the plasmid containing a single copy of MusaSAP1 5’ UTR : 4289 bp Total weight (Daltons) of the plasmid containing a single copy of MusaSAP1 5’ UTR : 2.83 x 106 Total weight (grams) of the plasmid containing a single copy of MusaSAP1 5’ UTR : 4.69 x 10-18 No. of copies of the plasmid in SD1 mix (4 μ l of 1/1000 diluted plasmid added as template) : 42.64 x 106 No. of copies of the plasmid in SD2 mix (4 μ l of 1/10000 diluted plasmid added as template) : 42.64 x 105 No. of copies of the plasmid in SD3 mix (4 μ l of 1/100000 diluted plasmid added as template) : 42.64 x 104 No. of copies of the plasmid in SD4 mix (4 μ l of 1/1000000 diluted plasmid added as template) : 42.64 x 103 No. of copies of the plasmid in SD5 mix (4 μ l of 1/10000000 diluted plasmid added as template) : 42.64 x 102 Concentration of banana cv. Karibale Monthan genomic DNA : 22 ng/μl Total estimated length of one genome of banana cv. Karibale Monthan : 1710 Mbp Total weight (grams) of one genome of banana cv. Karibale Monthan : 1.8734 x 10-12 No. of copies of MusaSAP1 gene in the genomic DNA (gDNA) mix (4 μ l of 1/15 diluted gDNA added as template) : 9333 No. of genomes of banana cv. Karibale Monthan in the genomic DNA (gDNA) mix : ~3140 No of copies of MusaSAP1 per genome of banana cv. Karibale Monthan : 3 No of copies of MusaSAP1 per chromosome set of banana cv. Karibale Monthan (a triploid cultivar) : 1 Supplementary file 3