Research Project: Strawberry Crop Improvement through Genomics, Genetics, and Breeding

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2019 Annual Report

Objectives
Objective 1: Generate once-fruiting strawberry selections and varieties for the Mid-Atlantic and surrounding region, for use in traditional matted-row and/or annual plasticulture production systems, with emphasis on high yield; excellent fruit quality; long shelf life; and resistance to Colletotrichum, Botrytis, and foliar and fruit-rot diseases. [NP301, C1, PS1A, PS1B] Objective 2: Generate repeat-fruiting strawberry breeding selections with an open plant architecture; adequate runner production; high continuous yield; large fruit with excellent quality; and resistance to Colletotrichum, Botrytis, and foliar and fruit-rot diseases for use in developing varieties for extended-season production systems. [NP301, C1, PS1A, PS1B] Objective 3: Dissect the molecular, genetic, and environmental factors affecting strawberry production-efficiency traits, especially disease resistance and control of plant architecture, through initiation and development of plant organs such as stolons, branch crowns, and inflorescence structures. [NP301, C3, PS3A] Objective 4: Identify or generate new strawberry mutant genetic stocks for determining the functions or regulation of genes affecting disease resistance. [NP301, C3, PS3A]

Approach
Standard plant breeding methods will be used to generate superior strawberry cultivars for traditional production practices and fruiting for the traditional short spring season. Novel evaluation practices for fruit quality and flavor will be developed and incorporated into the annual breeding cycle. A seedling screen for resistance to anthracnose crown rot, an emerging disease of worldwide importance, will be developed to identify resistant strawberry plants and increase the breeding population’s average resistance to the disease. New cultivars resulting from selection based on increased disease resistance, fruit quality, yield, and shelf life will be released. To help satisfy demand for year-round availability, similar methods will be used to generate improved strawberry plants that fruit for an extended season from April through December. Because the longer-fruiting plants will face weather and pest challenges that are not problems during the traditional fruiting season, new comparison methods will be developed to facilitate identification of plants that produce fruit within the traditional season, and produce equally well outside the traditional strawberry season. Additional research will be done to optimize the season-extending “low-tunnel” production system developed in the previous Project Plan to better evaluate advanced breeding selections. Inheritance of the strawberry’s capacity for continuous fruiting will be studied with both classical and molecular genetics. Characterization of novel mutant diploid lines with microscopy, hormone physiology and analysis, genetics, and genomics will illuminate genetic control and regulation of stolon production, a trait of vital importance to strawberry nurseries and growers.

Progress Report
In support of Objective 1, ARS scientists at Beltsville, Maryland, patented B1806 as ‘Keepsake’ and directed distribution of plants to nine U.S. and Canadian nurseries for propagation and licensing for sale. Taste-panel evaluation of ten cultivars identified ARS-Beltsville, Maryland, cultivars as having superior eating quality. ‘Flavorfest’ received top ratings for several quality traits. Grower demand for ‘Flavorfest’ continues to exceed nursery production as acreage expands. A second year of brix (sugar content) and pH (tartness) data were collected from replicated plots of 13 breeding selections and 9 regionally important cultivars, allowing the determination of relative impact of genotypic, environmental, and interaction effects on these key flavor components. A Felix Instruments F-750 analyzer for non-destructive fruit quality testing is being calibrated for non-destructive pH estimates. When calibration for strawberry pH is complete, the F-750 can be used to breed strawberries that retain desired brix and pH levels in refrigerated storage for eventual enjoyment by consumers. An APHIS permit was acquired to allow import of leaves from strawberry plants infected with viruses not present in Maryland. Viral RNA extracted from these leaves will be used as positive controls for testing plants imported from the National Clonal Germplasm Repository at Corvallis, Oregon, as the NCGR does not test germplasm for viruses on site or before shipping. The Beltsville breeding program tests germplasm to be used as parents before growing in a protected screenhouse. In support of Objective 2a, ARS scientists at Beltsville, Maryland, established observation plots of 68 repeat-fruiting strawberries selected in 2018 for comparison with 4 cultivars, all grown under low tunnels. A field of 6,277 seedlings was established, from which 60-80 additional breeding selections will be made. In support of Objective 2b, ARS scientists at Beltsville, Maryland, will analyze (Summer 2019) strawberry production levels and quality under different types of plastic covering low tunnels to determine which plastic type should be used to cover low tunnels in the breeding program and for commercial production of non-local cultivars in the Mid-Atlantic. In support of Objective 2c, ARS scientists at Beltsville, Maryland, have been unable to identify collaborators willing to address the problem of epistasis (the effect of one gene on another gene) in mapping inheritance to repeat fruiting in strawberry. In support of Objective 3, ARS scientists at Beltsville, Maryland, conducted bulked-sequencing analysis of the nru2 population and RNA seq of the ywr population to better understand genetic control of runner production. ARS scientists at Beltsville, Maryland, developed and scored an F2 population of an inbred line of the diploid strawberry Fragaria vesca segregating for aberrant anther and pollen development. This trait segregates as a single gene mutation. ARS scientists at Beltsville, Maryland, developed an F2 population of an inbred line of F. vesca segregating for highly elongated and narrow fruit shape. This trait segregates as a single gene mutation. ARS scientists at Beltsville, Maryland, tested flower buds at various developmental stages from two subspecies of F. vesca for efficiency of differentiation of shoots from culture, and compared efficiency of transformation of these bud explants using Agrobacterium tumefasciens with previously published transformation efficiencies of leaf explants. ARS scientists at Beltsville, Maryland, also participated as part of a collaboration with Rutgers University in developing VARMAP, a publicly available pipeline for identification of candidate gene variants in larger genomes. And ARS scientists at Beltsville, Maryland, participated as part of a collaboration with the University of Minnesota in determining that the major IAA-conjugates in strawberry are IAA-aspartate and IAA-glutamate. This work used definitive technology to show for the first time and in contrast to what is in the literature, that these amino acid conjugates are hydrolyzed by a plant to release free auxin for plant growth and development. In support of Objective 4, ARS scientists at Beltsville, Maryland, have organized a collaborative multi-state effort to track the movement of Colletotrichum gloeosporioides on strawberry plants shipped from North Carolina nurseries to growers in Maryland and other states, and to determine persistence of these isolates in Maryland. Isolates collected in Maryland can be used for testing to meet the goals of this objective. In addition, the ARS collaborator at Poplarville began developing a screen using pathogen extracts instead of live spores. Both these approaches have reduced risk to the Maryland strawberry industry, compared with using isolates from the ARS collaborator at Poplarville, Mississippi. Meanwhile, an APHIS permit was acquired to allow import of C. gloeosporioides isolates from the ARS collaborators at Poplarville, Mississippi.

Accomplishments
1. ‘Keepsake’ strawberry cultivar was released (PP30578). Strawberries are a very valuable crop, so loss of a small percentage to disease or physical degradation in the field or in storage after harvest is costly to growers and consumers. ARS researchers at Beltsville, Maryland, released and patented ‘Keepsake’, a mid-season, “spring-bearing” strawberry with fewer rotted or physiologically degraded fruits in the field or after refrigerated storage. The fruits are very sweet with outstanding flavor, and are firm and tough enough for commercial handling. ‘Keepsake’ is expected to be of greatest value to growers in the Mid-Atlantic, Northeastern U.S., and adjacent areas, especially to those who must store harvested fruit for delivery to market. Plants of ‘Keepsake’ were directed to nine U.S. and Canadian nurseries for propagation and licensing for sale. Nursery and grower demand for ‘Keepsake’ already exceeds supply.

Review Publications
Dong, J., Tu, M., Feng, Y., Zdepski, A., Ge, F., Kumar, D., Slovin, J.P., Messing, J. 2018. Candidate gene identification of existing or induced mutations with pipelines applicable to large genomes. Plant Journal. 97:673-682. https://doi.org/10.1111/tpj.14153.
Lewers, K.S., Enns, J.M., Castro, P.R. 2019. ‘Keepsake’ strawberry. HortScience. 54(2):362-367.
Tang, Q., Yu, P., Tillman, M., Cohen, J., Slovin, J.P. 2018. Indole-3-acetyl-aspartate and indole-3-acetyl-glutamate, the IAA-amide conjugates in the diploid strawberry achene, are hydrolyzed in growing seedlings. Planta. 249(4):1073-1085. https://doi.org/10.1007/s00425-018-3061-0.