Clonejet vector

A previously published annotated sequence of MiFT from mango cv. Irwin (AB671587.1), and two annotated sequences of MiTFL1s from mango cv. Alphonso (KF258590 and KU206290), were utilized in this study. We also made use of an additional sequence annotation, putatively encoding for an FT-like gene (mango_rep_c5502), which was identified in a search conducted against ‘Tommy Atkins’ and ‘Keitt’ mango transcriptomes [71 (link)]. The four corresponding ‘Shelly’ cDNAs were isolated by real-time qPCR using pairs of end-to-end primers, designed within the 5′ and 3′ UTRs of each sequence annotation (see Table S1), and cDNA synthesized from ‘Shelly’ tissues as the template (a mixture of samples collected from leaves and buds at different time points). The obtained PCR products were ligated into the CloneJET vector (Fermentas, Vilnius, Lithuania), sequenced (Hy-labs Laboratories, Rehovot, Israel), and further used as templates to generate constructs for Arabidopsis transformation purposes.

To identify mango sequences encoding proteins similar to the product of IDA genes, a nucleotide BLAST search was conducted against a ‘Kent’ mango transcriptome database generated by our group (Rai et al., unpublished), with conserved regions of AtIDA and CiIDA3 RNA serving as query. This search led to identifying two expressed sequence tags (ESTs) putatively encoding IDA proteins. The two EST sequences were next verified by RT-PCR, using specific pairs of end-to-end primers designed within their 5’ and 3’ UTRs, and cDNA synthesized from ‘Kent’ fruitlet AZs (a mixture of samples collected at different intervals) as a template (Supplementary Table S1). The obtained PCR products were ligated into the CloneJET vector (Fermentas, Vilinius, Lithuania), sequenced (Hy-labs Laboratories, Rehovot, Israel), and further used as templates to generate constructs for Arabidopsis transformation purposes (see below).

RNA extracts were reverse-transcribed by a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) using random primers according to the manufacturer’s instructions. Subsequent PCR analysis (Table S3) was performed with Phire Hot Start II DNA Polymerase (Thermo Fisher) or PPP Master Mix (Top-Bio, Vestec, Czech Republic). The quality of the cDNA was tested by amplifying a part of the Malus domestica actin gene. The PCR products were evaluated by gel electrophoresis. PCR products intended for Sanger sequencing were amplified with Q5 DNA polymerase (New England Biolabs, UK) and then cut and purified from the agarose gel using a GeneJET Gel Extraction Kit (Thermo Fisher). The products were Sanger-sequenced directly or cloned into the CloneJET vector (Thermo Fisher). Sequences of the PCR products or the cloned viruses were deposited in NCBI GenBank (Table S4).