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Title: Generation and analysis of blueberry transcriptome sequences from leaves, developing fruit, and flower buds from cold acclimation through deacclimation

Author
item Rowland, Lisa
item ALKHAROUF, NADIM - Towson University
item DARWISH, OMAR - Towson University
item Ogden, Elizabeth
item Polashock, James
item Bassil, Nahla
item MAIN, DORRIE - Washington State University

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2012
Publication Date: 4/2/2012
Citation: Rowland, L.J., Alkharouf, N., Darwish, O., Ogden, E.L., Polashock, J.J., Bassil, N.V., Main, D. 2012. Generation and analysis of blueberry transcriptome sequences from leaves, developing fruit, and flower buds from cold acclimation through deacclimation. Biomed Central (BMC) Plant Biology. 12:46.

Interpretive Summary: Increased consumption of blueberries in recent years has resulted in a need for improved varieties with enhanced nutritive value and yield potential. Unfortunately, scientists knowledge of genes that control these attributes in blueberry has been very limited. Here, using newer, more affordable DNA sequencing technology, we report approximately 139,000 new DNA sequences from genes that function in blueberry fruit at different stages of ripening, flower buds at different stages of development, and leaves. The gene sequences have been deposited in the public database known as GenBank and are accessible through the internet. The gene sequences have been used to develop molecular markers that will be useful for scientists in developing genetic maps of blueberry and for use by breeders in developing new varieties. This large collection of new sequences will also be a valuable resource for scientists attempting to identify genes that play important roles in flower bud development, winter hardiness, fruit ripening, and fruit quality in blueberry and related species.

Technical Abstract: There has been increased consumption of blueberries in recent years fueled in part because of their many recognized health benefits. Blueberry fruit is very high in anthocyanins, which have been linked to improved night vision, prevention of macular degeneration, anti-cancer activity, and reduced risk of heart disease. Very few genomic resources have been available for blueberry, however. Further development of genomic resources like expressed sequence tags (ESTs), molecular markers, and genetic linkage maps could lead to more rapid genetic improvement. Marker-assisted selection could be used to combine traits for climatic adaptation with fruit and nutritional quality traits. Efforts to sequence the transcriptome of the commercial highbush blueberry (Vaccinium corymbosum L.) and use the sequences to identify genes associated with cold acclimation and fruit development and develop SSR markers for mapping studies are presented here. Transcriptome sequences were generated from blueberry fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by next-generation Roche 454 sequencing. Over 600,000 reads were assembled into approximately 15,000 contigs and 124,000 singletons. The assembled sequences were annotated and functionally mapped to Gene Ontology (GO) terms. Frequency of the most abundant sequences in each of the libraries was compared across all libraries to identify genes that are potentially differentially expressed during cold acclimation and fruit development. Real-time PCR was performed to confirm their differential expression patterns. Overall, 14 out of 17 of the genes examined had differential expression patterns similar to what was predicted from their reads alone. The assembled sequences were also mined for SSRs. From these sequences, 15,886 blueberry EST-SSR loci were identified. Primers were designed from 7,705 of the SSR-containing sequences with adequate flanking sequence. One hundred primer pairs were tested for amplification and polymorphism among parents of two blueberry populations currently being used for genetic linkage map construction. The overall amplification rate was 68% and the polymorphism rate was 43%. These results indicate that this large collection of 454 ESTs will be a valuable resource for identifying genes that are potentially differentially expressed and play important roles in flower bud development, cold acclimation, chilling unit accumulation, and fruit development in blueberry and related species. In addition, the ESTs have already proved useful for the development of SSR and EST-PCR markers, and are currently being used for construction of genetic linkage maps in blueberry.