To gain a better understanding of changes in gene expression associated with cold stress in the woody perennial blueberry (Vaccinium spp.), a genomics approach based on the analysis of expressed sequence tags (ESTs) was undertaken. Two cDNA libraries were constructed using RNA from cold acclimated (mid winter conditions when the plants are cold stressed) and non-acclimated (before they received any chilling) floral buds of the blueberry cultivar Bluecrop. About 600 5'-end ESTs were generated from each of the libraries. Putative functions were assigned to 57% of the cDNAs that yielded high quality sequences based on homology to other genes/ESTs from Genbank, and these were classified into 14 functional categories. From a contig analysis, which clustered sequences derived from the same or very similar genes, 430 and 483 unique transcripts were identified from the cold acclimated and non-acclimated libraries, respectively. Of the total unique transcripts, only 4.3% were shared between the libraries, suggesting marked differences in the genes expressed under the two conditions. 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. Northern analyses were performed to examine expression of eight selected transcripts and seven of these were confirmed to be preferentially expressed under either cold acclimating or non-acclimating conditions. Only one of the seven transcripts, encoding a dehydrin, had been found previously to be up-regulated during cold stress of blueberry. This study demonstrates that analysis of ESTs is an effective strategy to identify candidate cold-responsive transcripts in blueberry.
Anik L. Dhanaraj*, Janet P. Slovin, and Lisa J. Rowland
Lisa J. Rowland, Anik L. Dhanaraj, Dhananjay Naik, Nadim Alkharouf, Ben Matthews, and Rajeev Arora
To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium corymbosum 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 Arabidopsis, 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.
Lisa J. Rowland, Anik L. Dhanaraj, James J. Polashock, and Rajeev Arora
Expressed sequence tag-polymerase chain reaction (EST-PCR) markers for DNA fingerprinting and mapping in blueberry (Vaccinium sp.) had previously been developed from expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants. Because EST-PCR markers are derived from gene coding regions, they are more likely to be conserved across populations and species than markers derived from random regions of DNA, such as randomly amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers. In this study, we tested whether many of the EST-PCR primer pairs developed for blueberry are capable of amplifying DNA fragments in other members of the family Ericaceae. In addition, we cloned and sequenced a selection of 13 EST-PCR fragments to determine if they showed homology to the original blueberry cDNA clones from which the EST-PCR primer pairs were derived. Closely related cranberry genotypes (two wild selections of V. oxycoccus L. and two cultivars of V. macrocarpon Aiton, `Early Black' and `Stevens') and more distantly related rhododendron genotypes (one wild selection each of Rhododendron arboreum Marsh, R. maximum L., and R. ponticum L. and three complex species hybrids, `Sonata', `Grumpy Yellow', and `Roseum elegans') were used. Of 26 primer pairs tested in cranberry, 23 (89%) resulted in successful amplification and eight of those (35%) amplified polymorphic fragments among the cranberry genotypes. Of 39 primer pairs tested in rhododendron, 29 (74%) resulted in successful amplification and 21 of those (72%) amplified polymorphic fragments among the rhododendron genotypes. Approximately 50% of the 13 sequenced EST-PCR fragments were found to be homologous to the original blueberry cDNA clones. These markers should be useful for DNA fingerprinting, mapping, and assessing genetic diversity within cranberry and rhododendron species. The markers which are shown to be homologous to the blueberry cDNA clones by DNA sequencing should also be useful for comparative mapping and genetic diversity studies between some genera of the family Ericaceae.
Rajeev Arora, Lisa J. Rowland, Elizabeth L. Ogden, Anik L. Dhanaraj, Calin O. Marian, Mark K. Ehlenfeldt, and Bryan Vinyard
Loss of freeze tolerance, or deacclimation, is an integral part of winter survival in woody perennials because untimely mid-winter or spring thaws followed by a hard freeze can cause severe injury to dehardened tissues. This study was undertaken to investigate deacclimation kinetics, particularly the timing and speed, of five blueberry (Vaccinium L.) cultivars (`Bluecrop', `Weymouth', `Ozarkblue', `Tifblue', and `Legacy'), with different germplasm compositions and mid-winter bud hardiness levels, in response to an environmentally controlled temperature regime. Based upon bud cold hardiness evaluations in 2000 and 2001, `Tifblue', a Vaccinium ashei Reade cultivar, was one of the least hardy and the fastest to deacclimate; `Bluecrop', a predominantly V. corymbosum L. cultivar, was the most hardy and the slowest to deacclimate; and `Ozarkblue', a predominantly V. corymbosum cultivar but including southern species V. darrowi Camp. and V. ashei, was intermediate in speed of deacclimation. `Weymouth' (predominantly V. corymbosum) and `Legacy' (73.4% V. corymbosum and 25% V. darrowi) were slow to intermediate deacclimators. Deacclimation rates did not correlate strictly with mid-winter bud hardiness. Data suggest that the southern germplasm component V. ashei may be responsible for the observed faster deacclimation whereas both southern species, V. darrowi and V. ashei, may contribute genes for cold sensitivity. Strong positive correlations between stage of bud opening and bud cold hardiness existed in both years (r = 0.90 and 0.82 in 2000 and 2001 study, respectively). Previously identified major blueberry dehydrins, 65-, 60-, and 14-kDa, progressively decreased in their abundance during incremental dehardening in `Bluecrop', `Weymouth', and `Tifblue'. However, down-regulation of the 14-kDa dehydrin most closely mirrored the loss in cold hardiness during deacclimation, and, therefore, may be involved in regulation of bud dehardening. Because differences in deacclimation rate were clearly evident among the genotypes studied, rate of deacclimation of the flower buds of blueberry should be an important consideration in breeding to improve winter survival.
Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin, and Mark K. Ehlenfeldt
Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.