1a.Objectives (from AD-416)
Identify horticulturally useful genes and markers associated with important traits, with emphasis on increasing resistance to biotic and abiotic stresses that reduce the quality or the potential yield of blueberry, strawberry, and brambles, so that ultimately these genes and traits can be incorporated in small fruit cultivars using various biotechnological approaches. Important traits include tolerance to temperature extremes in blueberry and strawberry, disease resistance in strawberry, and repeat flowering in strawberry, blackberry, and raspberry.

1b.Approach (from AD-416)
Studies will focus on: .
1)developing reliable, stable blueberry transformation system using the Biolistic method of particle bombardment,.
2)developing and utilizing genomic tools, such as standard and subtracted/reverse subtracted cDNA libraries for the production of ESTs and custom microarrays, in blueberry and strawberry, for the identification and characterization of genes associated with increased production of fruit under stressful temperature conditions,.
3)identifying germplasm and developing molecular markers and genetic maps useful for conferring traits of horticultural value, such as cold tolerance in blueberry, disease resistance in strawberry, and repeat flowering in strawberry, raspberry, and blackberry, and.
4)evaluating somaclonal variants of strawberry for expression and stability of useful traits such as anthracnose resistance.

3.Progress Report
ARS scientists at Beltsville, Maryland and Chatsworth, New Jersey, together with scientists at Iowa State University, introduced the blueberry CBF gene (an important transcription factor involved in cold tolerance) into Arabidopsis for testing gene function. The plants carrying the blueberry gene were tested for cold tolerance and were found to be more cold tolerant than wild-type plants under non-acclimating conditions. The blueberry CBF gene was provided to Michigan State University, through a Material Transfer Agreement, for introduction into blueberry cultivars. Action Plan component 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement. Problem statement 1A, Advancing from model plants to crop plants.

ARS scientists at Beltsville, Maryland continued to use DNA markers to examine evolutionary relationships of the various blueberry species. The markers are being analyzed in about 50-60 different genotypes, including about 3 representatives of each species. Action Plan component 301 1, Plant and microbial genetic resource management. Problem statement 1B, Assess the systematic relationships and genetic diversity of crop genetic resources.

ARS scientists at Beltsville, Maryland and Chatsworth, New Jersey performed cold tolerance evaluations of flower buds for the second year on a group of blueberry cultivars to measure the rates of cold acclimation and deacclimation. Action Plan component 301 1, Plant and microbial genetic resource management. Problem statement 1B, Assess the systematic relationships and genetic diversity of crop genetic resources.

ARS scientists at Beltsville, Maryland analyzed 25 gene sequences from heat stressed diploid strawberries for differences (polymorphisms) that can be used to map these genes. Four genes were characterized by length polymorphisms in introns (regions within genes that do not encode protein and are spliced out of mRNA) between mapping parents, and several genes revealed other small differences in sequences that can be used for mapping. Action Plan component 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement. Problem statement 1A, Advancing from model plants to crop plants.

ARS scientists at Beltsville, Maryland analyzed 42,000 sequences from expressed genes (Expressed Sequence Tags or ESTs) obtained from strawberry under different stress conditions including cold, heat, drought, and salt stress. Genes were identified that are highly expressed, expressed specifically in response to one stress, and generally expressed in response to several stresses. Action Plan component 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement. Problem statement 1A, Advancing from model plants to crop plants.

ARS scientists at Beltsville, Maryland, in collaboration with scientists at the University of Arkansas, identified molecular markers linked to thorn production and repeat fruiting in blackberry. Action Plan component 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement. Problem statement 1B, Applying genomics to crop improvement.

4.Accomplishments
1. DNA markers developed from lowbush blueberry. There is a need to develop a reliable DNA marker system for lowbush blueberry. About two-thirds of commercial blueberry production is from improved cultivars of highbush blueberry, whereas about one-third is from managed, wild stands of lowbush blueberry, grown primarily in Maine. ARS scientists at Beltsville, MD together with scientists at the University of Maine demonstrated that DNA markers developed from highbush DNA sequences, termed EST-PCR markers, are effective at distinguishing individuals of lowbush blueberry plants called clones, even nearby clones within the same field. They also showed, using plants whose parentage was known, that these markers could accurately estimate their degree of relatedness. This work lays the foundation for use of these markers by other scientists in genetic studies on lowbush blueberry. Action Plan component 301 1, Plant and microbial genetic resource management. Problem statement 1B, Assess the systematic relationships and genetic diversity of crop genetic resources.

2. DNA markers developed for blackberry. There is need to develop reliable DNA markers for genetic mapping in blackberry. ARS scientists at Beltsville, Maryland, in collaboration with scientists at Clemson University sequenced 2,678 blackberry genes, from which 673 molecular markers were developed. These are the first blackberry gene sequences and markers to be made publicly available. This work lays the foundation for use of these markers by other scientists in genetic studies on blackberry. Action Plan component 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement. Problem statement 1B, Applying genomics to crop improvement.

3. Stress gene sequences obtained and analyzed for strawberry. There is a need to identify genes that help plants grow and produce fruit under stress conditions such as cold, heat, and drought. New sequences from approximately 10,000 genes expressed in strawberry under stress conditions were obtained this year and made publicly available by deposition into GenBank. Analysis of all expressed gene sequences from strawberry that are currently in the public domain identified 13,449 unique gene sequences. These sequences represent the foundation for the genomic study of environmental stress on production of flowers and fruit in strawberry and other members of the Rosaceae family. These sequences can be used by scientists for molecular marker development, for examining expression of genes in response to stress, and are for testing the function of genes identified as potentially useful for increasing plant tolerance to environmental stresses. Action Plan components: 302 1, Functional utilization of plant genomics: translating plant genomics into crop improvement, and 302 2, Biological processes that improve crop productivity and quality. Problem statements: 1A (Advancing from model plants to crop plants), 1B (Applying genomics to crop improvement), and 2B (Understanding plant interactions with their environment).

6.Technology Transfer

Number of the New MTAs (providing only)	3
Number of Web Sites Managed	1
Number of Non-Peer Reviewed Presentations and Proceedings	7
Number of Newspaper Articles and Other Presentations for Non-Science Audiences	2

Review Publications
Weebadde, C.K., Wang, D., Finn, C.E., Lewers, K.S., Luby, J.J., Bushakra, J., Sjulin, T.M., Hancock, J.F. 2008. Using a linkage mapping approach to identify QTL for day-neutrality in the octoploid strawberry. Plant Breeding. 127:94-101.