Transcriptome assembly and expression analysis in Colletotrichum gloeosporioides-tolerant Rubus glaucus Benth

Juliana Arias1, Juan C. Rincón1*, Ana M. López1, and Marta L. Marulanda1
Andean blackberry (Rubus glaucus Benth.) is an important crop of the Andean region affected by Colletotrichum gloeosporioides. In Colombia, tolerant plant material has been detected, but it has not been completely characterized. The objective of this research was oriented to analyze de novo transcriptome assembly of R. glaucus, and the comparison of the assembly with different reference genomes to further complete differential expression analysis of R. glaucus tolerant to C. gloespoiorides attack. To achieve this, three groups were used: infected tolerant material, infected susceptible material, and a susceptible group without inoculation. The RNA-seq sequencing was achieved through Illumina Hi-seq 2000. De novo assembly (Trinity, CD-HIT, TopHat) and functional annotation of sequences were carried out, additionally, mapping with reference genomes belonging to Rosaceae families was conducted (Bowtie2, TopHat). Subsequently, the differential expression was quantified (Cuffdiff) and analyzed through EdgeR. Variant analysis was made using MISA and SAMtools. After editing and assembly, 43579 consensus sequences were obtained (N50 = 489 bp; GC = 44.6%), annotation detected 35824 and 35602 sequences in Nt (partially non-redundant nucleotide sequences) and Nr (non-redundant protein sequences) databases, respectively. The 85% of Nr sequences was linked to members of Rosaceae family, mainly strawberry (67.6%). A total of 3570 simple sequence repeat (SSR) markers and 38791 single nucleotide polymorphisms (SNP) were found. The transcriptome of tolerant plants exhibited less SNPs. Finally, differentially expressed genes were found, including RPM1, MAPKBP1, CKX2. This research represents a contribution for future understanding of R. glaucus transcriptome, since there is little information available, and it would help further tolerance-related analysis.
Keywords: Blackberry, fungal tolerance, molecular markers, RNA-seq, tolerance-related genes.
1Universidad Tecnológica de Pereira, Grupo de Investigación en Biodiversidad y Biotecnología, Carrera 27 No. 10-02 Pereira, Risaralda, Colombia. *Corresponding author (rincon.juan@utp.edu.co).