Phylogenetic, structural and functional analysis of the PEBP-sequences in C. pallens
There is no published genome for Chionochloa. All 23 New ZealandChionochloa species have the same chromosome number (n=7) and are regarded as ancient hexaploids (2n = 6x = 42) which have undergone diploidization (Linder & Barker, 2014; Murray, De Lange, & Ferguson, 2005). The reference ‘C. pallens’ transcriptome assembly published in Samarth et al. (2019) was used to identify the potential homologues of the PEBP protein family (Table 2). The identified homologous PEBP sequences were further clustered into their respective sub-families using a phylogenetic approach. The PEBP-phylogenetic tree consisting of 49 protein sequences (Table S1) was reconstructed using the maximum likelihood method with Jukes Cantor substitution matrix and 1000 bootstrap replication using the PhyML package. Homologous PEBP protein sequences were also modelled using a homology modelling approach at the SWISS-PROT server and visualised using Pymol (student version).
For functional analysis, the homologous PEBP-gene sequences (CpFT1, CpFT2, CpFT3, CpFT4, CpFT5 and CpTFL1 ) were transformed into Arabidopsis accession Landsberg erecta ft-1mutant plants. The cDNA sequences were cloned into the binary vector (pB2GW7), downstream of the AtSUC2 promoter, and transformed intoAgrobacterium tumefaciens GV3101 by electroporation. Arabidopsisft-1  mutants were transformed using the method of Martinez-Trujillo et al., (2004) as follows: A. tumefaciensharbouring the binary vector were streaked onto LB media plates with selective antibiotics and incubated at 28 ºC for two days. The bacteria were then scraped off the plate and resuspended in 10 mL of infiltration media (0.5x MS (Duchefa), 0.05% Silwett L-77 (Lehle Seeds), 5% sucrose) to an OD600 = 2 and drops of this solution were pipetted onto unopened flower buds of Arabidopsis. This was repeated three times at 2–3-day intervals. The resulting seed was sown on seed-raising mix and transformants selected by spraying the young seedlings with Basta herbicide (glufosinate ammonium; Bayer) at 120 mg/ L. The number of rosette leaves was counted at the time of flowering. Wild-type Arabidopsis Landsberg erecta plants flower after producing ~ 8-10 leaves whereas ft-1 is a late-flowering mutant, flowering after ~20 leaves (Martinezzapater & Somerville, 1990).