An Actinidia chinensis (kiwifruit) eFP browser and network analysis of transcription factors Lara Brian Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Ben Warren Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Peter McAtee Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Jessica Rodrigues Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Niels Nieuwenhuizen Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Karine M. David School of biological sciences: The University of Auckland Annette Richardson Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Andrew C. Allan Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Erika Varkonyi-Gasic Plant and Food Research Ltd: New Zealand Institute for Plant and Food Research Ltd Robert J Schaffer ( [email protected] ) Plant and Food Research Ltd https://orcid.org/0000-0003-1272-667X Research article Keywords: Actinidia, eFP Browser, Transcription factors Posted Date: November 10th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-101514/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Version of Record: A version of this preprint was published on February 27th, 2021. See the published version at https://doi.org/10.1186/s12870-021-02894-x. Page 2/21 Abstract Background Transcriptomic studies combined with a well annotated genome have laid the foundations for new understanding of molecular processes. Tools which visualise gene expression patterns have further added to these resources. The manual annotation of the Actinidia chinensis (kiwifruit) genome has resulted in a high quality set of 33,044 genes. Here we investigate gene expression patterns in diverse tissues, visualised in an Electronic Fluorescent Pictograph (eFP) browser, to study the relationship of transcription factor (TF) expression using network analysis. Results Sixty-one samples covering diverse tissues at different developmental time points were selected for RNAseq analysis and an eFP browser was generated to visualise this dataset. 2,839 TFs representing 57 different classes were identied and named. Network analysis of the TF expression patterns separated TFs into 14 different modules. Two modules consisting of 237 TFs were correlated with oral bud and ower development, a further two modules containing 160 TFs were associated with fruit development and maturation. A single module of 480 TFs was associated with ethylene-induced fruit ripening. Three “hub” genes correlated with ower and fruit development consisted of a HAF-like gene central to gynoecium development, an ERF and a DOF gene. Maturing and ripening hub genes included a KNOX gene that was associated with seed maturation, and a GRAS-like TF. Conclusions This study provides an insight into the complexity of the transcriptional control of ower and fruit development, as well as providing a new resource to the plant community. The eFP browser is provided in an accessible format that allows researchers to download and work internally. Background Global transcriptomic approaches are a common tool used to obtain a better understanding of gene function and regulation. The composition of the transcriptome is the result of a dynamic balance between chromatin state, the activation of gene expression by transcription factors (TFs) and the speed of transcript degradation. The combination of good genomic information and robust gene models paves the way for systematic and consistent gene and gene family naming. This combined with other genomics tools, such as Electronic Fluorescent Pictograph (eFP) browsers[1] to help visualise where a gene is expressed, allows faster identication of gene function in different species. To date, eFP browsers have been successfully developed in plants such as Arabidopsis[1], tomato[2], strawberry[3], and pineapple[4]. TFs are one of the largest groups of genes in a genome; in Arabidopsis there are over 1500 TFs described, belonging to a number of different classes representing 5% of all genes[5]. In other species TFs represent Page 3/21 3–5% of coding genes, with function often conserved across species[6]. TFs have been grouped into 57 different classes[5] with some classes having multiple types of DNA binding domains. Each class of TF is represented by a gene family. These gene families vary in size from species to species depending on events such as individual gene and genome duplications, leading to expansions of certain or most families[6]. In higher plants the MYB, bHLH and Zinc nger classes of TF contain many hundreds of members[6]. There are numerous examples demonstrating the strong evolutionary maintenance of TF primary protein structure across species, with the homologous genes having a similar gene function. This allows researchers to predict function by homology[7]. The MADS-box containing TFs form arguably one of the best understood classes of TF. Members of the MADS-box gene family, including the well-known oral organ structure ABCE TFs, determine many aspects of plant development[8, 9]. Even though the fruiting bodies of Angiosperms are homoplasious, with eshy fruit evolving numerous times within many plant families the function of these genes appear conserved[10]. Angiosperm ower structure and fruiting bodies are remarkably conserved, with whorls of sepals, petals, stamens and carpels[8]. The MADS protein sequence is also conserved with many examples within plants demonstrating similar control mechanisms across many species[7, 11]. Kiwifruit are part of the Actinidiaceae which is a basal family within Ericales[12], and contains the genus Actinidia comprising of a number of economically important fruit species such as Actinidia chinensis var. deliciosa (green kiwifruit), A. chinensis var. chinensis (gold and red kiwifruit) and A. arguta (hardy kiwifruit or kiwiberries). The green ‘Hayward’ kiwifruit is hexaploid, while a commercially released yellow eshed variety A. chinensis var. chinensis, ‘Hort16A’, and the red eshed A. chinensis var. chinensis ‘Hongyang’ are large fruiting diploid genotypes making them ideal for understanding molecular processes in Actinidiaceae. More recently a new Pseudomonas syringae pv. actinidiae (Psa) tolerant tetraploid gold variety, ‘Zesy002’, has replaced ‘Hort16A’ in the markets. The two diploid cultivars have been used to understand the molecular control of many aspects of development including owering, fruit ripening, colour and avour development[13–16]. Genomics tools such as CRISPR gene editing have been successfully used to edit the oral repressors in ‘Hort16A’ to create a small fruiting plant that can be used to rapidly test gene function in fruit, further building on their utility[17]. The rst draft kiwifruit genome was of A. chinensis ‘Hongyang’, published in 2013[18], paving the way for genomics in Actinidiaceae. More recently a second A. chinensis genome of a more inbred related genotype, Red5, further improved the construction and importantly manual annotation of gene models[19]. The manual annotation of the kiwifruit genome improved the quality of the published computer predicted gene models, and provided a quality resource for future gene mining. Here we build on these data by identifying TF genes, analysing their expression over a number of tissues and providing an eFP Browser tool to analyse gene expression. Results Mining of transcription factor gene families Page 4/21 Using the kiwifruit manually annotated gene models[19], protein translations containing InterPro DNA binding domains (http://www.ebi.ac.uk/interpro) were identied. These were manually checked, resulting in 2,839 gene models with at least one TF domain in 61 TF classes in 32 global classes (Fig. 1, Table 1, Additional data 1). The most abundant global class of TFs in kiwifruit was the Zinc nger class, represented by 571 genes in 11 different gene classes, followed by 428 genes with MYB domains within four different classes of genes, and 333 bHLH genes within two different classes of genes (Table 1, Additional data 1). In total the 2,839 TFs represented 8.6% of the annotated genes in the kiwifruit genome. Table 1 Summary of transcription factors. Major classes total class class class class Zn nger domain 571 C2H2 177 CO 16 DOF 53 C3H 130 SQBP 32 GATA 50 VOZ 4 DBB 32 LSD 9 NF 58 SRS 10 MYB domain 428 MYBR 126 MYB 219 ARR 19 GARP 64 BHLH domain 333 BHLH 293 TCP 40 AP2 domain 270 ERF 227 AP2 43 HB domain 176 HD-ZIP 79 WOX 18 HD-PHD 3 TALE 44 HB 16 ZFHD 16 B3 domain 111 ARF 42 RAV 12 LAV 57 MADS domain 79 TYPE 1 23 MICK 56 HMG type 50 HMGB 22 ARID 17 YABBY 11 Other groups NAC 147 WRKY 116 Trihelix 67 FAR 53 BZIP 100 GRAS 12 BCP 17 BES 12 LFY 11 EIL 8 CATMA 10 E2F 14 CPP 12 GFR 25 GeBP 7 HST 36 LBD 59 NZZ 3 NIN 18 S1Fa 8 SAP 12 HRT 13 WHIRLY 4 PLATZ 17 STAT 0 Using the DNA binding domains, each TF class was aligned in a phylogenetic tree and named using the following criteria. Firstly, if the gene had been previously published in the literature, this name was given. For genes not previously published, a sequential naming down an initial phylogenetic tree was used. This naming method allows genes within subclades to have numbers which are close to each other. An Page 5/21 exception was made for the ARF genes where the whole family has been well characterised in a number of species [20–22], so a naming convention related to the closest Arabidopsis and tomato homologues[22] was taken. An example cluster of the MADS TF Type 1 and MICK cluster is shown in Fig. 2 and full phylogenies can be found in additional data 2.