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

Abstract

BackgroundThere 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.ResultsEfforts to sequence the transcriptome of the commercial highbush blueberry (Vaccinium corymbosum) cultivar Bluecrop 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 tetraploid mapping population was based on a cross between the highbush cultivars Draper and Jewel (V. darrowii is also in the background of 'Jewel'). The diploid mapping population was based on a cross between an F1 hybrid of V. darrowii and diploid V. corymbosum and another diploid V. corymbosum. The overall amplification rate of the SSR primers was 68% and the polymorphism rate was 43%.ConclusionsThese 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 bl...

Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor

“…We have done numerous attempts for characterizing MYBA-type regulators involved in anthocyanin biosynthesis from the Vaccinium species starting with degenerated primers and going through cDNA libraries and different databases, but so far we have not been able to find potential candidates neither in our earlier bilberry EST nor recent bilberry transcriptome libraries (data not shown). Interestingly, the careful search by using sequence homology and available bioinformatics tools of the other currently available Vaccinium EST and transcriptome databases (Rowland et al 2012;Zifkin et al 2012), recently published genome database of American cranberry (V. macrocarpon) (Polashock et al 2014) and draft genome of blueberry (Gupta et al 2015) did either not reveal potential transcripts/sequences for anthocyanin biosynthesis associated MYBA-type R2R3 MYB genes. The difficulty to find MYBA-type R2R3 MYB transcripts in the Vaccinium transcriptome databases is noteworthy since Vaccinium species are among the best natural sources of anthocyanins.…”

Enriching Genomic Resources and Marker Development from Transcript Sequences ofJatropha curcasfor Microgravity Studies

“…The de novo assembled UAS set with an average length of 1,381 bp, which was longer than those in previous jatropha transcriptome studies, 916 bp, [17] and 789 bp [22], and transcriptome studies of other plants using the 454 sequencing platform ( Panax ginseng , 559 bp [64]; blueberry, 933 bp [65]; Japanese larch, 667 bp [66]; switchgrass, 535 bp [67]; and sheepgrass, 607 bp [68]). In total, 18,391 (89.2%) contigs in our dataset were longer than 500 bp, and 12,008 (58.2%) contigs were longer than 1000 bp.…”

Comparative Transcriptional Analysis of Loquat Fruit Identifies Major Signal Networks Involved in Fruit Development and Ripening Process

“…Data evaluation was performed according to the description of Müller-Herbst et al [63]. To annotate isotigs and singletons, the non-redundant sequences were searched against the protein databases obtained from NCBI with a search threshold of E -value cut-off 10 −5 [18]. Based on the BlastX top hit genes [24], the abundant sequences were used to obtain further information on the function and motif through the InterPro member databases.…”

“…Instead, it has been more useful to obtain unigene information through RNA-Seq. Transcriptome analysis provides an insight into functional genes and signal pathways involved in fruit ripening without the corresponding genome information as a reference [18,19,20,21,22]. These data were used to investigate allele-specific expression and splice junction variation.…”