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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

2021 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
This report documents progress for new project 2030-21000-054-000D, which started in December 2020 and replaces expired project 2030-21000-047-00D. The Molecular Biologist (Plants) SY position for this project is vacant and work in fiscal year (FY) 2021 focused on recruiting for this position. The position should be filled by the end of the FY. A research goal of this project is to develop lines for speed breeding in woody horticultural crops by expressing floral promoting gene FLOWERING LOCUS T (FT) in transgenic plants. For Objectives 1 and 2, public genome sequence databases were searched for amino acid sequences similar to well-known FT proteins (from the model plant Arabidopsis thaliana and the cereal crop rice) in five citrus species: Kumquat, Citron, Pomelo, Mandarin, and Meyer lemon. This effort identified two citrus FT proteins present in all five species that were highly similar amongst one another and to the known FT proteins. The conclusion was that these FT proteins, and the genes encoding them, are likely to have an important role in regulating flowering in citrus. Oligonucleotide primers with sequences from these FT genes were designed and purchased to amplify the FT transcript sequences. The same approach also was employed to identify genes encoding UBIQUITIN (UBQ) proteins. Expression of these UBQ genes are controls when monitoring FT gene expression in Objective 2. Leaf samples were taken from adult kumquat and Meyer lemon trees in the summer to provide a source of transcripts to amplify the citrus FT sequences for Objective 1. Samples were taken in the morning and evening in the event expression was time-of-day dependent. The next step for Objective 1 is to test these two citrus FT homologs for their capacity to functionally substitute for the Arabidopsis FT gene. The DNA sequences for the citrus FT transcripts will be put into a transgenic expression construct suitable for transformation of both Arabidopsis and citrus, which was identified in literature searches. For Objective 2, the morning and evening time point were used to evaluate how gene expression varies throughout the day and night. The levels of FT and UBQ transcripts were assessed by quantitative reverse transcriptase-polymerase chain reaction (qPCR). In the kumquat leaves, expression levels of the two FT genes were higher in the evening. In contrast, expression levels of the two FT genes in Meyer lemon leaves were higher in the morning. These observations indicate that while these two types of citrus have highly similar FT genes, their daily regulation is different. This indicates flowering regulatory networks in these closely related trees may be somewhat different. It is possible these differences may impact strategies for promotion of flowering.