Table 1.
Evaluation of selected reference genes using RNA-Seq data.
Table 2.
Description of candidate reference genes and primer sequences for qRT-PCR.
Fig 1.
Cycle threshold (Ct) values of the fifteen tested genes across all samples.
(A) Abiotic stresses treated root tissues of cultivar ‘Gala’; (B) P. ultimum infected #58 roots; (C) P. ultimum infected #75 roots; (D) R. solani infected #115 roots. For each box, the upper and lower edges indicate the 25th and 75th percentiles, while whisker caps represent the maximum and minimum values. The line across the box depicts the median.
Fig 2.
Gene expression stability (M) of candidate genes calculated by geNorm.
(A) Abiotic stresses treated root tissues from cultivar ‘Gala’; (B) P. ultimum infected #58 roots; (C) P. ultimum infected #75 roots; (D) R. solani infected #115 roots. The least stable genes are on the left, while the most stable genes are on the right.
Fig 3.
Determination of the optimal number of reference genes.
The pairwise variation (Vn/Vn+1) was calculated between the normalization factors NFn and NFn+1 by geNorm. If the Vn/Vn+1 value is less than 0.15, there is no need to add an additional gene for normalization.
Table 3.
Stability ranking of 15 candidate reference genes of four treatments.
Fig 4.
The frequency of appearance in top five spots by individual tested genes.
Base on the ranking order by four different analytic methods and comprehensive ranking (see detail in Table 3), the values of Y axis indicate the times for an individual gene being ranked in the top five spots from all four tissue types used. The distribution of the top-rated candidate genes from evaluation by various methods were combined to indicate their overall suitability as the most reliable reference genes for gene expression analysis in apple root tissues.
Fig 5.
Normalized expression patterns of MdLecRLK5 and MdMAPK3 using selected reference genes.
(A) MdLecRLK5 and (B) MdMAPK3 in “#58 Pu” root tissues at 24, 48 and 72 hpi; (C) MdLecRLK5 and (D) MdMAPK3 in “#115 Rs” root tissues at 24, 48 and 72 hpi. The error bars are standard errors. T-test statistics were generated by ANOVA among relative expression levels in the same sample. * P<0.05, ** P<0.01.