1a.Objectives (from AD-416):
Characterize the fungal transcriptome of Colletotrichum acutatum in response to treatment with blueberry flower extracts from resistant and susceptible varieties and to Characterize the fungal and plant transcriptomes in the mummy berry (Monilinia vaccinii-corymbosi)- blueberry flower style interaction using resistant and susceptible blueberry varieties.
1b.Approach (from AD-416):
Six RNAseq libraries [water control, ‘Duke’ (resistant) flower extract treated and ‘Bluecrop’ (susceptible) flower extract treated, all at four hours and 12 hours post-treatment] will be constructed. Time points may be adjusted as more preliminary data are collected. Libraries will be prepared for 300 bp inserts and paired-end sequenced on the Illumina GAIIX platform. We will identify the specific C. acutatum genes for which expression is impacted by exposure to blueberry flower extracts. We will further determine the differences and similarities in fungal gene expression when using flower extracts from resistant versus susceptible blueberry cultivars. Data will be assembled and analyzed using appropriate software and procedures. C. acutatum gene annotation will be facilitated by comparison with an in-house genome database we have constructed. 2)Monilinia vaccinii-corymbosi conidia will be obtained from blueberry plants with foliar infections. Flowers of ‘Bluejay’ (resistant) and ‘Berkeley’ (susceptible) will be tagged at anthesis. Treatments will be. 1)untreated control,. 2)pollinated only,. 3)inoculated only, and. 4)pollinated and inoculated. ‘Bluecrop’ pollen will be applied to flowers one day after anthesis (treatments 2 and 4). Flowers will also be inoculated as appropriate (treatments 3 and. 4)with conidia of M. vaccinii-corymbosi on the same day. Untreated flowers at the same stage will simply be tagged. There will be 80 flowers/treatment/cultivar (320/cultivar total). Flower styles will be harvested two days post-treatment and stored in RNAlater until processed. RNA will be isolated and eight RNAseq libraries will be constructed (two cultivars, four treatments). Libraries will be designed for paired-end reads and run on the Illumina HiSeqII platform. Data will be assembled and analyzed using appropriate software and procedures. Blueberry sequences will be identified by comparison with available blueberry expressed sequence tag (EST) database (BBGD; http://bioinformatics.towson.edu/BBGD/), which contains sequences from flowers. Digital gene expression profiles will be used for pair-wise comparisons (e.g., inoculated or pollinated vs. control, inoculated vs. pollinated, resistant vs. susceptible, etc.) to identify differentially expressed genes. Differential expression will be confirmed for selected candidate genes using real-time PCR.
The six libraries were generated and sequenced on the Illumina platform. The resulting sequence data were assembled into six transcriptomes. The transcriptomes are being annotated using appropriate procedures. When annotation is complete, digital gene expression profiles will be used for pair-wise comparisons to identify differentially expressed genes. Differential expression will be confirmed for selected candidate genes using real-time PCR.