Table 1.
Primers used to amplify coding sequences of cassava eIF4E and its homologues.
Fig 1.
RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers.
Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1. Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.
Fig 2.
Phylogenetic analysis of eIF4E superfamily proteins.
The phylogenetic tree was inferred by using the Maximum Likelihood method based on the Jones-Taylor-Thornton matrix-based model with gamma-distributed rate variation (JTT+G) as implemented in MEGA version 7.0 [25]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (6 categories, +G, parameter = 1.7002). The tree with the highest log likelihood (-2897.9607) is shown. The tree is drawn to scale, and the scale bar represents 0.2 substitution per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The analysis involved 42 protein sequences. All positions containing gaps and missing data were eliminated. Protein sequences of additional eIF4E family proteins were retrieved from NCBI. Proteins are identified with two letters representing the initials of genus and species, followed by NCBI accession number. At, Arabidopsis thaliana; Ca: Capsicum annuum; Cl, Citrullus lanatus; Cm, Cucumis melo; Cs, Cucumis sativus; Gm, Glycine max; Jc, Jatropha curcas; Ps, Pisum sativum; Pt, Populus trichocarpal; Pv, Phaseolus vulgaris; Rc, Ricinus communis; St, Solanum tuberosum; and Vv, Vitis vinifera. Clustering of three families of eIF4E proteins are indicated and labeled: eIF4E, eIF(iso)4E, and novel cap binding protein (nCBP).
Table 2.
Response of selected cassava accessions to CBSV/UCBSV infection*.
Table 3.
Distribution and characteristics of SNP sites in five eIF4E genes and neighboring regions in 14 cassava accessions*.
Table 4.
Association of eIF4E non-synonymous SNPs with CBSD-susceptible and tolerant phenotypes.
Fig 3.
Manhattan scatter plots of probabilities of 1358 SNPs in eIF4E genes and surrounding regions (+/- 5 kb) associated with CBSV responses.
Informative SNPs within +/- 5 kb regions of each eIF4E genes were tested for their likelihood to be associated with the susceptible or resistant responses to CBSD. Negative log-transformed probability scores are plotted again the relative distances of each SNP in each chromosomes for each of the SNPs. Horizontal axis represents relative distances of each SNP from each other. SNPs from each eIF4E gene are color-coded. The orange and red lines indicate p-value thresholds of 0.01 and 0.005, respectively. Schematic of the gene model in each locus is depicted below the graph and color-coded. eIF4E gene in each locus in indicated by red rectangles. For each eIF4E gene, exons are represented by rectangle boxes while intros are indicated by lines.
Fig 4.
Expression levels of cassava eIF4E genes in the susceptible Albert and the tolerant Kaleso lines.
Illimina reads were obtained from the NCBI SRA depository (accessions SRR1213744- SRR1213747) [28]. Cleaned reads were aligned to the cassava genome and differential gene expression were analyzed using the Cuffdiff program [31]. Expression levels of each gene were represented with sequence reads per kilobase per million reads (FPKM). Error bars indicate the 95% confidence interval of the FPKM of each gene. Samples include healthy Albert and Kaleso cassava accessions and CBSV infected Albert (Albert_CBSV) and Kaleso (Kaleso_CBSV).
Fig 5.
Boxplot of expression levels of eIF4E genes in six cassava lines.
The 454 sequencing data were obtained from six cassava accessions (AR 37–80, AR40-6, Mkombozi, Kiroba, Nachinyaya, and Namikonga) [29]. Reads mapped to each gene were normalized to total read counts and lengths of each gene in kilobases (FPKM) and subsequently used as a measurement of expression levels. Statistical analyses and boxplotting were performed with MATLAB version R2017a.
Fig 6.
Boxplot of eIF4E gene expression levels of CDSD-susceptible and tolerant cassava accessions.
The 454 sequencing data were obtained from three CBSD-susceptible accessions (AR 37–80, AR40-6, and Mkombozi) and three CBSD-tolerant accessions (Kiroba, Nachinyaya, and Namikonga) [29]. Reads mapped to each gene were normalized to total read counts and lengths of each gene in kilobases (FPKM) and subsequently used as a measurement of expression levels. Data from the susceptible and tolerant accessions were grouped together for statistical analysis. Each sample in the susceptible and tolerant group was considered as an independent sample. Statistical analyses and boxplotting were performed with MATLAB version R2017a.