Study of Cold Tolerance in Blueberry Using EST Libraries, cDNA Microarrays, and Subtractive Hybridization

Abstract

<jats:p>To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (<jats:italic>Vaccinium corymbosum</jats:italic> L.) and ultimately use this information to develop more freeze-tolerant cultivars, a genomics approach based on the analysis of expressed sequence tags (ESTs) and microarrays was undertaken. Initially, two standard cDNA libraries, constructed using RNA from cold-acclimated (CA) and nonacclimated (NA) floral buds of the blueberry cultivar Bluecrop, were used for the generation of ≈2400 ESTs, half from each library. Putative functions were assigned to cDNAs based on homology to other genes/ESTs from GenBank. From contig analyses, 796 and 865 unique transcripts were identified from the CA and NA libraries, respectively. The most highly abundant cDNAs, that were picked many more times from one library than from the other, were identified as representing potentially differentially expressed transcripts. A cDNA microarray was constructed and used to study gene expression under cold-acclimating conditions in the field and cold room. Results indicated that the abundance of transcripts of numerous blueberry genes change during cold acclimation, including genes not found previously to be cold-responsive in <jats:italic>Arabidopsis</jats:italic>, and, interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Finally, forward and reverse subtracted cDNA libraries were prepared from ‘Bluecrop’ RNA to enrich for transcripts that are expressed at higher levels in floral buds at 400 h and at 0 h of low-temperature exposure, respectively. Many genes encoding putative transcription factors and other proteins related to signal transduction were identified from both libraries.</jats:p>

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

“…These results were very similar to what we found previously using a small microarray to identify differentially expressed transcripts during cold acclimation/deacclimation [ 9 ]. Although the microarray was based on only about 1,500 unique genes [ 9 , 15 ], many of the transcripts identified as differentially expressed in that study were also identified as potentially differentially expressed in this study. Some of the transcripts identified as potentially downregulated during cold acclimation in both studies encoded dehydration-induced RD22-like protein/BURP domain-containing protein, certain heat shock proteins, certain dehydrins, beta-glucosidase, UDP-glucose dehydrogenase, and ascorbate peroxidase (Additional file 1 and [ 9 ]).…”

“…Current mapping studies are focused on identifying quantitative trait loci (QTL) associated with chilling requirement, freezing tolerance, and fruit quality traits in blueberry. The first microarray experiments have been carried out in blueberry and have successfully identified many transcripts whose abundances increase with cold acclimation [ 9 , 15 ]. More gene expression studies need to be undertaken, however, that are based on a larger collection of gene sequences in order to sort out genes that are expressed in response to various stimuli and during development.…”

Molecular footprints of selection effects and whole genome duplication (WGD) events in three blueberry species: detected by transcriptome dataset

“…For example, the genome draft of a northern high-bush blueberry has been published [48,49]. The transcriptome analyses have been used for blueberry studies about characteristics of gene expression in the cold environment [50][51][52], the metabolics related genes of blueberry antioxidant substances [53], the changes of gene expression profiles in blueberry after infection with Colletotrichum acutatum [54], the gene expression dynamics during five blueberry fruit development stages [48], and detection of molecular markers [55]. However, the reports about the genome evolution of blueberry are limited.…”

Breeding blueberries for a changing global environment: a review

“…This change was positively correlated with the total amount of soluble solids in the wood, which likely served as osmoprotectants able to reduce the freezing point. Inter-species differences in the level of expression of β-amylase genes in northern and southern highbush were described by Rowland et al ( 2008 ).…”