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ARS Home » Pacific West Area » Albany, California » Plant Gene Expression Center » Research » Research Project #439498

Research Project: Developing Tools to Accelerate Genetic Improvement for Woody Horticultural Crops

Location: Plant Gene Expression Center

2022 Annual Report

A major bottleneck to research progress and the rate of crop genetic improvement is lengthy periods (months to years) of vegetative growth preceding initiation of reproductive maturity, thereby extending generational times. Methods for controlling and accelerating the transition to flowering, and reducing dormancy time, can dramatically compress this time scale, giving breeders and researchers a significant time advantage for identifying and introgressing desirable alleles for crop traits. Such accelerated crop breeding methods are widely used in annual crops but remain to be established for most woody crop species. A promising opportunity for compressing the generational time lies in manipulation of Flowering locus T (FT) gene expression or by mutating the homologous gene, Terminal Flower (TFL), which is a floral repressor. Manipulation of FT to reduce flowering time has been achieved through genetic engineering, grafting, and viral induction-based approaches. TFL can be edited to achieve a similar early flowering. Controlling the time to flowering and reproductive maturity might also be achieved by decreasing juvenility through manipulation of evolutionarily conserved microRNA (miRNAs). This project will integrate cutting-edge technologies, such as new transformation strategies, genome editing, grafting, and computational biology to develop and implement accelerated crop breeding strategies for California woody horticultural crops such as pistachio, almonds, walnuts, and/or grapes. The deliverables include research and breeding tools and information for controlling flowering, reproductive maturity, and decreasing generation time for woody horticultural crops. Specific Objectives of this project are: Objective 1: Develop a suite of research and breeding tools to accelerate time to flowering and reproductive maturity in woody horticultural crops through modification of florigen or miR156 activity. Objective 2: Enlist and apply the approaches of transcriptomics, network analysis and comparative genomics to discover novel genetic mechanisms that operate across woody horticultural crops that control initiation and maturation of the reproductive phase.

Objective 1, Hypothesis: Expression of FT in the desired crop will shorten time to flowering. The first step is to identify FT orthologs in the selected woody horticultural crop by sequence homology-based searches and domain-based gene phylogenetic approaches, using existing genome sequences for related tree species, published RNA-seq transcriptome assemblies, and resources from Objective 2. Next, FT gene activity will be assessed by testing for complementation of the late flowering phenotype of the Arabidopsis ft-10 mutant. Flowering time of Arabidopsis transformants overexpressing the candidate FT will be assayed under long day conditions, where the ft-10 phenotype is most severe, based on rosette leaf number. FT ortholog(s) found to accelerate flowering will be moved to the next phase to test these FT constructs in the crop of interest as first generation proof-of-principle transgenics. Initial transformation attemps will employ epicotyl transformation with Agrobacterium. Whole genome sequencing will confirm transgene location and verify that transformation caused no other genome changes. Long term goals will be identifying inducible promoters and possible virus-inducible systems to control FT expression. Data from Objective 2 is expected to help guide the decision of which FT to use in the final round of tree transformation. Objective 2, Hypothesis: Discover novel genetic mechanisms that control initiation and maturation of the reproductive phase. A quantitative RT-PCR (qPCR) experiment will document the 24-hour, or diurnal, expression pattern for the FT genes identified in Objective 1. Testing samples taken during a summer month (April-June) and a winter month (November-February) will reveal where seasonal regulation occurs for these FT genes. This effort is expected to give insight into potential functions of each FT paralog. Transcriptional profiling by RNA-seq of the same temporal and seasonal samples will identify differentially expressed genes by pairwise comparisons (leaf vs flower, summer vs winter, etc.) and assess temporal expression pattern (diurnal change over 72 hours, season). This analysis will determine what other genes are expressed in dynamic, coordinated networks with FT genes. To catalogue the transcript splice forms that exist across seasons and tissues and to further refine available reference transcriptomes of the selected tree species, a full-length transcriptome will be constructed using Pacific Biosciences Single Molecule Real-Time sequencing. This full-length transcriptome will help to identify the total number and sequence similarity of FT orthologues, design primers for cDNA cloning or qPCR, and profile isoform-level gene expression in RNA-seq projects. Also, this resource will be of use to future genomics research and breeding efforts in the woody crop species of interest.

Progress Report
ARS researchers in Albany, California, made progress toward the goal of Objective 1 to design and develop constructs for testing FLOWERING LOCUS T (FT) gene homologs for promoting flowering in Arabidopsis thaliana (Arabidopsis) and woody crops. One of the unknowns associated with this work is which promoter DNA sequence will produce the best results, given the diversity of woody plant species that will be transformed, the potential variation in each FT gene’s effectiveness at promoting flowering, and the degree to which promoting flowering alters other important plant traits. A modular system that makes it easy to test different promoters in combination with different FT homologs was developed to streamline these tests. To this end, the Golden Gate method of combining DNA sequences was used to rapidly test promoters in diverse transformation systems. In these experiments, the coding sequence of the FT1 gene from poplar (Populus tremula x alba) and two different strong, constitutive promoter sequences (double 35S and Arabidopsis UBI10) were combined with the octaline synthase terminator to transform wildtype Arabidopsis. Both promoters were able to induce early flowering, with the 35S promoter generating earlier bolting than the UBI10 promoter. These promoter-FT1 DNA sequence combinations were tested with two different DNA sequences for selection of transgenic plants based on antibiotic resistance. These successful experiments made a system that will enable planned experimental tests of citrus FT homologs in a citrus transformation system. Additionally, the researchers cloned several other promoters into the Golden Gate system to allow fine tuning of the expression level and tissue-specific expression of FT homologs. For Objective 2, which aims to identify genetic mechanisms controlling initiation and maturation of the reproductive phase, progress was made toward the goal of generating gene models from woody crops. The researchers took advantage of the many recent publicly available transcriptome data sets for this purpose. A data set from mature shoot apices of Key Lime that was generated to examine the developmental mechanisms of thorn production by comparing thornless and thorned varieties, and also includes samples of young leaf tissue and apical meristems, was used to generate de novo transcriptomes assemblies. This effort identified 5,310 unique expressed genes across the thornless and thorned datasets, and 62 genes differentially expressed between varieties. Going forward, this pipeline will be used to identify genes differentially expressed between citrus samples differing in maturity or across floral initiation to identify novel factors controlling these processes.