Additional file 1: of GhFAD2–3 is required for anther development in Gossypium hirsutum
Creators: name: Liu, Feng
name: Ma, Lihong
name: Wang, Youwu
name: Li, Yanjun
name: Zhang, Xinyu
name: Xue, Fei
name: Nie, Xinhui
name: Zhu, Qianhao
name: Sun, Jie
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Evolutionary Biology
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Virology

description: Figure S1. The coding sequences and phylogenetic analysis of GhFAD2. A. The coding sequences of GhFAD2 genes. The fragment in GhFAD2–3D and GhFAD2–3A targeted for RNAi is highlighted in red color. The underlined sequence in GhFAD2–4A was filled up in this study, which is a gap in the TM-1 genome (Zhang et al. 2015). B. Putative members of the GhFAD2 family in the TM-1 genome identified based on blastp search using the protein sequence of the published GhFAD2–1 (X97016). Gh_D13G2237 contains three indels (50, 14 and 21 aa, respectively) compared to other proteins, and its 3rd deletion contains the 3rd conserved histidine-cluster observed in all FAD2 protein, this gene was thus considered as a non-functional FAD2 and not analysed further in this study. The three conserved histidine-clusters are highlighted in red. The annotated Gh_A01G2091 was incomplete due to gap in the genomic sequence, and the missing sequence was filled up by sequence cloning in this study. C. Phylogenetic analysis of the cotton FAD2 family. The tree was generated based on protein sequences of cotton FAD2 using the Maximum likelihood module of the MEGA6 software. Figure S2 Generation and molecular analysis of transgenic plants. A. Schematic representation of the pBIAP-dsGhFAD2 expression cassettes used for cotton transformation. Promoter, Anther-specific promoter; NPT II, neomycin phosphotransferase II gene; GhFAD2, The partial fragment GhFAD2–3D coding sequence; RB, right border; LB, left border; B. Generation of transgenic plants. a, induction of embryonic calli; b, calli produced from explants; c, embryoid produced from calli; d, regeneration of kanamycin resistant plantlets. C. Detection of the GhFAD2-gus linker fusion fragment in non-transformed control and transgenic plants by PCR. A 1113-bp fusion fragment was amplified and 18 s rDNA was served as a control. The primers used in amplification were 5′-CTGTACAGCGAAGAGGCAGTC-3′ and 5′-CGTTGTAGATAGGACCGTAT-3′ for GhFAD2-gus linker fusion fragment, and 5′-GAGTCTGGTAATTGGAATGAG-3′ and 5′-TTCGCAGTTGTTCGTCTT-3′ for 18S rDNA. Lane M, DNA marker III; Lane 1–4; 18 s rDNA sequence was amplified from transformants line; Lane 5, 18 s rDNA sequence was amplified from untransformed control; Lane 6–9, The fusion fragment was amplified from transformants line; Lane 10, The fusion fragment was amplified from untransformed control. Table S1. Representative differentially expressed genes involved in anther lipid metabolism. Table S2. Primers of selected genes used for qRT-PCR analysis. (DOCX 1056 kb)