Sequencing and analysis of gerbera daisy leaf transcriptomes reveal disease resistance and susceptibility genes differentially expressed and associated with powdery mildew resistance

Abstract

Background: RNA sequencing has been widely used to profile genome-wide gene expression and identify candidate genes controlling disease resistance and other important traits in plants. Gerbera daisy is one of the most important flowers in the global floricultural trade, and powdery mildew (PM) is the most important disease of gerbera. Genetic improvement of gerbera PM resistance has become a crucial goal in gerbera breeding. A better understanding of the genetic control of gerbera resistance to PM can expedite the development of PM-resistant cultivars.

Results: The objectives of this study were to identify gerbera genotypes with contrasting phenotypes in PM resistance and sequence and analyze their leaf transcriptomes to identify disease resistance and susceptibility genes differentially expressed and associated with PM resistance. An additional objective was to identify SNPs and SSRs for use in future genetic studies. We identified two gerbera genotypes, UFGE 4033 and 06-245-03, that were resistant and susceptible to PM, respectively. De novo assembly of their leaf transcriptomes using four complementary pipelines resulted in 145,348 transcripts with a N50 of 1124 bp, of which 67,312 transcripts contained open reading frames and 48,268 were expressed in both genotypes. A total of 494 transcripts were likely involved in disease resistance, and 17 and 24 transcripts were up- and down-regulated, respectively, in UFGE 4033 compared to 06-245-03. These gerbera disease resistance transcripts were most similar to the NBS-LRR class of plant resistance genes conferring resistance to various pathogens in plants. Four disease susceptibility transcripts (MLO-like) were expressed only or highly expressed in 06-245-03, offering excellent candidate targets for gene editing for PM resistance in gerbera. A total of 449,897 SNPs and 19,393 SSRs were revealed in the gerbera transcriptomes, which can be a valuable resource for developing new molecular markers.

Conclusion: This study represents the first transcriptomic analysis of gerbera PM resistance, a highly important yet complex trait in a globally important floral crop. The differentially expressed disease resistance and susceptibility transcripts identified provide excellent targets for development of molecular markers and genetic maps, cloning of disease resistance genes, or targeted mutagenesis of disease susceptibility genes for PM resistance in gerbera.

Keywords: Differentially expressed genes; Disease resistance; Gerbera; Powdery mildew resistance; R-gene; Simple sequence repeats; Single nucleotide polymorphisms; Susceptibility gene.

Fig. 1

Screening of gerbera breeding lines for powdery mildew (PM) susceptibility. PM-resistant line UFGE 4033 was used as a control and a phenotypic rating scale of 1–10 where 1 indicates no or little visual presence of PM symptoms and 10 indicates the plant canopy completely covered with the disease. Data presented are means of three replicates. Error bars represent the standard error

Fig. 2

Volcano plot showing transcripts differentially expressed in gerbera breeding lines UFGE 4033 and 06–245-03. UFGE 4033 and 06–245-03 are resistant and susceptible to powdery mildew disease, respectively

Fig. 3

Venn diagram showing transcripts distribution in powdery mildew-resistant (UFGE 4033) and -susceptible (06–245-03) gerbera lines

Fig. 4

Heat map of relative expression level (FPKM values) of 17 upregulated and 24 downregulated transcripts. These transcripts were functionally annotated to confer disease resistance based on BLAST analysis in powdery mildew resistant (UFGE 4033) and susceptible (06–245-03) gerbera breeding lines. Samples R1-R3 and S1-S3 were three biological replicates of UFGE 4033 (R) and 06-245-03 (S) lines, respectively

Fig. 5

Quantitative RT-PCR validation of seven differentially expressed R-gene transcripts from gerbera transcriptome analysis. These transcripts were differentially upregulated in powdery mildew resistant (R) line (UFGE 4033) versus powdery mildew-susceptible (S) line (06–245-03). Expression of transcripts were normalized to the endogenous control Gerbera hybrida actin gene and the relative expression was calculated using 2{−Ct (gene of interest)-Ct (actin)}. The blue line shows the Log2 fold change expression of the transcripts from RNA-seq data. Error bars represent standard errors

Fig. 6

Venn diagram showing the distribution of SNPs in gerbera. SNPs were identified by GATK, Samtools and FreeBayes pipelines between powdery mildew resistant (UFGE 4033) and susceptible (06–245-03) gerbera breeding lines

Fig. 7

Distribution of simple sequence repeats (SSRs) with different motif unit sizes in gerbera transcriptome. SSRs with one – six motif sizes were identified using Misa tools. Bars represent the frequencies (log₁₀) of SSRs containing various lengths of nucleotide repeats