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 Innovative Fruit Production, Improvement, and Protection: Kearneysville, WV
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ARS Home Northeast Area Kearneysville, West Virginia Appalachian Fruit Research Laboratory Innovative Fruit Production, Improvement, and Protection Research





- Dardick, Christopher - Chris
- Liu, Zongrang
- Gottschalk, Christopher
- Artlip, Timothy - Tim
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Superior Fruit Tree Cultivars for Orchard Resilience, Sustainability, and Consumer Appeal **Location:
                            Innovative Fruit Production, Improvement, and Protection** 2023 Annual Report **Objectives**
                                

Objective 1: Discover the genetic and molecular mechanisms that underlie tree architectural traits in fruit crops.

Sub-objective 1A: 
Evaluate transgenic TAC1-silenced and LAZY1-silenced plum germplasm with different architectures for their potential use in novel growing systems. (Hypothesis) - Trees having more upright (TAC1-silenced) or horizontal (LAZY1-silenced) branch angles will be amenable to novel growing systems and enable ,high-density orchard systems.

Sub-objective 1B:
Integration of a narrow leaf (NL) trait into diverse canopy types to study the role of leaf size/shape in tree productivity. (Hypothesis) -The NL trait will improve light penetration and productivity, particularly within peach tree architectures having dense canopies. 

Objective 2: Identify genes for pitless and robust flavor traits and incorporate into stone fruits breeding.
(Hypothesis)- The naturally occurring stoneless trait in plum is conferred by a single dominant mutation. 

Objective 3: Identify and characterize genes associated with resilience to climate change, spring frost injury, and associated abiotic stresses.

Sub-objective 3A:
Study the role of individual DAM genes within a set of specially designed plum transgenic lines. (Hypothesis) DAM genes have sub-functionalized in Prunus to precisely couple chilling and heat requirements to flowering time in flower or vegetative buds. 

Sub-objective 3B: 
Create a set of Apple transgenic lines with altered DAM gene expression to compare to DAM gene sub-functionalization in Prunus. (Hypothesis) DAM genes function differently in Malus compared to Prunus regarding chilling and heat requirements for flowering time. 

Objective 4: Breed improved pome and stone fruit cultivars that combine disease resistance, enhanced production, and high fruit quality traits. 
The Unit has active conventional breeding programs in stone and pome fruits that have multiple long-term goals. Specific efforts that will be accomplished within the proposed 5-year project plan are detailed below.

Sub-objective 4A: 
Generate high-quality super sweet nectarine varieties and phenotype segregating populations for genetic mapping.

Sub-objective 4B: 
Develop late-flowering peach/nectarine cultivars, characterized parental germplasm for breeding late flowering traits, and associated mapping populations and genomic information to facilitate future breeding efforts.

Sub-objective 4C: 
Develop new pre-breeding apple lines and varieties with stacked traits related to fruit quality, productivity, and abiotic stress-resilience into disease-resistant backgrounds. 

Sub-objective 4D: 
Develop pear varieties with improved fruit quality, storage, and supply-chain resilience traits in disease-resistant backgrounds. **Approach**
                                

This project leverages plant breeding, genomics, genetics, molecular biology, and biotechnology strategies to address fundamental problems facing tree fruit production. The variety development and basic research activities are synergistic as the germplasm developed through the breeding efforts serve as a critical resource for identifying the genetic basis for important production traits. Many of the objectives will leverage the unique plant transformation capabilities of the unit coupled with available genome sequences for several tree fruit species. For Obj 1, tree architecture will be studied by evaluating horticultural characteristics of plum tree mis-expressing the TAC1 and LAZY1 genes. Collectively, these data will provide important practical information about how IGT genes contributes to tree shape. For Obj 2,technology to breed or engineer stoneless fruits will be developed by identifying the gene responsible for a naturally occurring stoneless trait in plum. In Obj 3, regulation of flowering time will be investigated using genetic, molecular, biotechnology, and next generation sequencing-based strategies. The breeding efforts in Obj 4 will include: 1) a novel super sweet trait in peach/nectarine that confers extremely high brix will be bred to develop commercial quality super sweet varieties; 2) peaches with delayed bloom will be developed by introgressing a naturally occurring late blooming trait, 3) Apple breeding will leverage the existing rapid cycle breeding system to intogress traits from wild germplasm and them stack key traits into parental germplasm including fire blight and scab resistance along with crisp fruit texture and superior flavor, and 4) in pear, we will leverage our unique germplasm to integrate fire blight resistance with important fruit quality characteristics. Collectively, these efforts will fill in key knowledge gaps about fundamental fruit tree developmental processes, provide new technologies for developing fruit tree germplasm with economically important traits, and lead to the development of new fruit varieties with superior traits. **Progress Report**
                                

Horticultural characteristics of plum trees silenced for TAC1 and LAZY1 were characterized in a randomized block for fruit yield, quality, and leaf photosynthetic status. The results showed that fruit from TAC1 and LAZY1 trees were similar to parental commercial plum types. This establishes that these genes can be readily manipulated or bred for without significant negative agronomic impacts. In addition, crosses were made between TAC1 and LAZY1 silenced plums and approximately 40 hybrid trees were obtained. These trees will be analyzed for phenotypes in the next reporting cycle.

Narrow leaf trees were self-hybridized, and the seedlings were evaluated in 2023. Each individual was scored for leaf width. The population displayed clear segregation for the NL trait and DNA was extracted for genetic mapping. 

The genome of the stoneless plum parental type ‘Ramming stoneless’ was fully sequenced and assembled. A combination of Pacbio, Illumina, and Nanopore sequencing was used along with HiC to enable chromosome scale assembly. The result was a fully assembled phased genome with the six haplotigs resolved for each of the 8 chromosomes. The genome was annotated using existing RNAseq datasets. In addition, seedlings from all stoneless segregating populations were scored for the stoneless trait. DNA was extracted from all seedlings and bulk sequenced via Illumina sequencing platform.  

Besides characterization of the DAM function in transgenic plants as described in 029 annual report, we also work on analysis of transcriptome profile in the late-flower peach, plum and EVEGROWING and Johnboy peach, respectively.  The aims of these studies are to: 1) identify key genes that are associated with dormancy exit, bud break and late flowering trait; 2) understand gene regulation and potential regulators of chilling requirement in plum; 3) address potential DAM targets by comparing transcriptome profiles between EVERGROWING and Johnboy; and 4) separate the DAM-targeted genes expressed specifically in floral and scale tissues, respectively. We have collected the shoot apical meristem/buds and floral buds at various time points during dormancy induction and chilling treatment. We isolated over 300 RNA samples and are in process of RNA-seq. We are currently analyzing the RNA-seq data collected from late-flower peach, Johnboy and analyze it and prepare manuscripts. In addition, we have initiated and designed over 20 gRNA cassettes that target various DAMs in apple, plum and cherry, aiming at creation of new cultivars with shorter or longer chilling requirement, early flowering and late flowering traits, respectively, to address potential threats resulted from climate change in near future.  

Nine crosses of advanced transgenic lines with novel germplasm were made in 2022/2023. The currently populations are nearing 400 total individuals and include a small progeny of a Malus fusca x T1190 (native interspecific hybrid carrying transgenic early flower gene) for disease and abiotic resistances. Attempts to hybridize T1190 with M. angustifolia, another native Malus sp., has been unsuccessful but another attempt is planned for spring 2024. Moreover, two crosses conducted in 2023 with M. sylvestris and M. sieversii to generate additional disease (post-harvest) and abiotic resistance germplasm of interspecific origins. An ARS SY's contribution to Obj. 4D, is leading the pear breeding program to develop high fruit-quality, storage, and supply-chain resilient pear varieties. Here, the ARS SY has generated eight new populations of elite × elite lines for development of new varieties, a new mapping population that aims to segregate for chilling independent fruit ripening (e.g., storage requirements), and seven populations for perry/processing pear varieties. Moreover, two new variety trial plantings have had trees propagated for evaluation of perry pear genotypes in collaboration with Cornell University, and a trial planting of advanced pear breeding lines from AFRS including the prized prospect US 79439-004. They are expected to be planting in Spring of 2024 and Fall of 2023, respectively. 

In addition to apple crosses, we sequenced and assembled the M.26 apple rootstock genome.  Sequencing the M.26 apple genome was necessary to properly design CRISPR silencing constructs to investigate apple dormancy per the CRIS plan (Sub-Objective 3b).  The genome is nearly ready, requiring corroborating RNAseq data.  An experiment was devised to supplement the M.26 genome by investigating why regeneration and transformation of M.26 is relatively easier than other rootstock and scion cultivars that are of greater interest for eventual transformation or gene editing.  The experiment focused on collecting regenerating tissue over a time course of weeks for evaluation of gene expression via RNAseq.  In addition, regeneration experiments were conducted on various Geneva rootstocks that may be used in future collaborative research with PGRU, Geneva, New York, to test potential candidate genes responsible for dwarfing rootstock traits that are critical for current apple orchard systems. **Accomplishments**
                                

1. Chromosome-scale assembly of the fungal plant pathogen Fusarium avenaceum. We assembled a new genome of the fungal plant pathogen, Fusarium avenaceum. We used the latest sequencing approaches and software to achieve a near-complete genome for this fungi. Additionally, we conducted extensive gene annotation. As a result, we identified many novel secondary metabolic gene clusters. Some of these gene clusters are responsible for the production of mycotoxins. Mycotoxins are a food safety concern as they are dangerous to animals and humans if consumed. This genomic resource is available publicly and has 15 downloads from a repository. The publication has been downloaded 285 times. The dataset is also being used to develop a new gene annotation software. Additionally, the genome is listed as the NCBI reference genome for the species. This accomplishment has had a significant impact on many different stakeholders including, post-harvest plant researchers who have new resource for investigating the virulence of this pathogen, food safety researchers who have a new resource for mycotoxin diversity and production, and the publication provides a model for rapid and thorough genome assembly and annotation of fungal species.


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