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Mei (Prunus mume) is widely cultivated in eastern Asia owing to its favored ornamental characteristics and its tolerance for low temperatures. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for gene expression analysis, requiring carefully selected reference genes to ensure data reliability. The aim of this study was to identify and evaluate reference genes for qRT-PCR in mei. Ten candidate reference genes were chosen, and their expression levels were assessed by qRT-PCR in four sample sets: 1) flowering mei; 2) mei undergoing abiotic stress; 3) different genotypes of Prunus species; and 4) all mei samples. The stability and suitability of the candidate reference genes were validated using commercially available software. We found that protein phosphatase 2A-1 (PP2A-1) and PP2A-2 were suitable reference genes for flowering with ubiquitin-conjugating enzyme E2 (UBC) also being suitable for different genotypes of Prunus species. UBC and actin (ACT) were most stably expressed under abiotic stress. Finally, the expression of an AGAMOUS homolog of Arabidopsis thaliana (PmAG) and a putative homolog of Group 2 late embryogenesis abundant protein gene in A. thaliana (PmLEA) were assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.

Lignin is the main component of stone cells, and stone cell content is one of the crucial factors for fruit quality in chinese white pear (Pyrus ×bretschneideri). The lignin biosynthesis pathway is complex and involves many enzymatic reactions. Cinnamate-4-hydroxylase [C4H (EC.1.14.13.11)] is an essential enzyme in lignin metabolism. This study was conducted to investigate the effect of bagging on lignin metabolism during fruit development in chinese white pear. The study showed that bagging had little effect on stone cell content, lignin content, C4H activity, and C4H gene expression and that there was a positive correlation between C4H gene expression and lignin content as well as stone cell content. Moreover, a full-length complementary DNA (cDNA) encoding C4H (PbrC4H, GenBank accession number KJ577541.1) was isolated from chinese white pear fruit. The cDNA is 1515 bp long and encodes a protein of 504 amino acids. Sequence alignment suggested that the deduced protein belongs to the P450 gene family and that C4H might be located subcellularly in the cell membrane. The results indicate that bagging cannot change the lignin and stone cell content significantly and that C4H catalyzes a step in lignin biosynthesis. These findings provide certain theoretical references and practical criteria for improving the quality of chinese white pear.

Cytosine methylation plays important roles in regulating gene expression and modulating agronomic traits. In this study, the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique was used to study variation in cytosine methylation among seven pecan (Carya illinoinensis) cultivars at four developmental stages. In addition, phenotypic variations in the leaves of these seven cultivars were investigated. Using eight primer sets, 22,796 bands and 950 sites were detected in the pecan cultivars at four stages. Variation in cytosine methylation was observed among the pecan cultivars, with total methylation levels ranging from 51.18% to 56.58% and polymorphism rates of 82.29%, 81.73%, 78.64%, and 79.09% being recorded at the four stages. Sufficiently accompanying the polymorphism data, significant differences in phenotypic traits were also observed among the pecan cultivars, suggesting that cytosine methylation may be an important factor underlying phenotypic variation. Hypermethylation was the dominant type of methylation among the four types observed, and full methylation occurred at higher levels than did hemimethylation in the pecan genomes. Cluster analysis and principal coordinate analysis (PCoA) identified Dice coefficients ranging from 0.698 to 0.778, with an average coefficient of 0.735, and the variance contribution rates of the previous three principal coordinates were 19.6%, 19.0%, and 18.2%, respectively. Among the seven pecan cultivars, four groups were clearly classified based on a Dice coefficient of 0.75 and the previous three principal coordinates. Tracing dynamic changes in methylation status across stages revealed that methylation patterns changed at a larger proportion of CCGG sites from the 30% of final fruit-size (30%-FFS) stage to the 70%-FFS stage, with general decreases in the total methylation level, the rate of polymorphism, and specific sites being observed in each cultivar. These results demonstrated that the F-MSAP technique is a powerful tool for quantitatively detecting cytosine methylation in pecan genomes and provide a new perspective for studying many important life processes in pecan.