Zhihong Gao

Selection of reliable reference genes for gene expression studies in peach using real-time PCR

BackgroundRT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.ResultsIn this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.ConclusionOur study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.

High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot

BackgroundMicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in high plants. However, the diversity of miRNAs and their roles in floral development in Japanese apricot (Prunus mume Sieb. et Zucc) remains largely unexplored. Imperfect flowers with pistil abortion seriously decrease production yields. To understand the role of miRNAs in pistil development, pistil development-related miRNAs were identified by Solexa sequencing in Japanese apricot.ResultsSolexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22,561,972 and 24,952,690 reads were sequenced from two small RNA libraries constructed from perfect and imperfect flower buds, respectively. Sixty-one known miRNAs, belonging to 24 families, were identified. Comparative profiling revealed that seven known miRNAs exhibited significant differential expression between perfect and imperfect flower buds. A total of 61 potentially novel miRNAs/new members of known miRNA families were also identified by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that six potentially novel miRNAs were differentially expressed between perfect and imperfect flower buds. Target predictions of the 13 differentially expressed miRNAs resulted in 212 target genes. Gene ontology (GO) annotation revealed that high-ranking miRNA target genes are those implicated in the developmental process, the regulation of transcription and response to stress.ConclusionsThis study represents the first comparative identification of miRNAomes between perfect and imperfect Japanese apricot flowers. Seven known miRNAs and six potentially novel miRNAs associated with pistil development were identified, using high-throughput sequencing of small RNAs. The findings, both computationally and experimentally, provide valuable information for further functional characterisation of miRNAs associated with pistil development in plants.

Comparative proteomic and transcriptomic approaches to address the active role of GA4 in Japanese apricot flower bud dormancy release

Hormones are closely associated with dormancy in deciduous fruit trees, and gibberellins (GAs) are known to be particularly important. In this study, we observed that GA4 treatment led to earlier bud break in Japanese apricot. To understand better the promoting effect of GA4 on the dormancy release of Japanese apricot flower buds, proteomic and transcriptomic approaches were used to analyse the mechanisms of dormancy release following GA4 treatment, based on two-dimensional gel electrophoresis (2-DE) and digital gene expression (DGE) profiling, respectively. More than 600 highly reproducible protein spots (P<0.05) were detected and, following GA4 treatment, 38 protein spots showed more than a 2-fold difference in expression, and 32 protein spots were confidently identified according to the databases. Compared with water treatment, many proteins that were associated with energy metabolism and oxidation–reduction showed significant changes after GA4 treatment, which might promote dormancy release. We observed that genes at the mRNA level associated with energy metabolism and oxidation–reduction also played an important role in this process. Analysis of the functions of the identified proteins and genes and the related metabolic pathways would provide a comprehensive proteomic and transcriptomic view of the coordination of dormancy release after GA4 treatment in Japanese apricot flower buds.

Genome-wide expression profiles of seasonal bud dormancy at four critical stages in Japanese apricot.

Dormancy is one of the most important adaptive mechanisms developed by perennial plants. To reveal the comprehensive mechanism of seasonal bud dormancy at four critical stages in Japanese apricot (Prunus persica), we applied Illumina sequencing to study differentially expressed genes (DEGs) at the transcriptional level. As a result, 19,759, 16,375, 19,749 and 20,800 tag-mapped genes were sequenced from libraries of paradormancy (R1), endodormancy (R2), ecodormancy (R3) and dormancy release (R4) stages based on the P. persica genome. Moreover, 6,199, 5,539, and 5,317 genes were differentially expressed in R1 versus R2, R2 versus R3, and R3 versus R4, respectively. Gene Ontology analysis of dormancy-related genes showed that these were mainly related to the cytoplasm, cytoplasmic part metabolism, intracellular metabolism and membrane-bound organelle metabolism. Pathway-enrichment annotation revealed that highly ranked genes were involved in ribosome pathways and protein processing in the endoplasmic reticulum. The results demonstrated that hormone response genes such as auxin, abscisic acid, ethylene and jasmonic acid, as well as zinc finger family protein genes are possibly involved in seasonal bud dormancy in Japanese apricot. The expression patterns of DEGs were verified using real-time quantitative RT-PCR. These results contribute to further understanding of the mechanism of bud dormancy in Japanese apricot.

Differential expression of proteins associated with seasonal bud dormancy at four critical stages in Japanese apricot.

Dormancy is of great significance in the growth and development of deciduous fruit trees. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) to identify the differentially expressed proteome of Japanese apricot flower buds at four critical stages, from paradormancy before leaf fall to dormancy release. More than 400 highly reproducible protein spots (P < 0.05) were detected: 34 protein spots showed a greater than twofold difference in expression values, of which 32 protein spots were confidently identified from databases. Identified proteins were classified into six functional categories: stress response and defence (11), energy metabolism (ten), protein metabolism (five), cell structure (three), transcription (one) and unclassified (two). The glyoxalase I homologue could help Japanese apricot survival under various abiotic and biotic stresses, greatly contributing to its dormancy. Enolase, thioredoxin family proteins and triose phosphate isomerase provide adequate energy to complete consecutive dormancy release and bud break in Japanese apricot. Cinnamyl alcohol dehydrogenase 9 and arginase enhance the resilience of plants, enabling them to complete dormancy safely. Analysis of functions of identified proteins and related metabolic pathways will increase our knowledge of dormancy in woody plants.

Identification of Differentially-Expressed Genes Associated with Pistil Abortion in Japanese Apricot by Genome-Wide Transcriptional Analysis

The phenomenon of pistil abortion widely occurs in Japanese apricot, and imperfect flowers with pistil abortion seriously decrease the yield in production. Although transcriptome analyses have been extensively studied in the past, a systematic study of differential gene expression has not been performed in Japanese apricot. To investigate genes related to the pistil development of Japanese apricot, high-throughput sequencing technology (Illumina) was employed to survey gene expression profiles from perfect and imperfect Japanese apricot flower buds. 3,476,249 and 3,580,677 tags were sequenced from two libraries constructed from perfect and imperfect flower buds of Japanese apricot, respectively. There were 689 significant differentially-expressed genes between the two libraries. GO annotation revealed that highly ranked genes were those implicated in small molecule metabolism, cellular component organisation or biogenesis at the cellular level and fatty acid metabolism. According to the results, we assumed that late embryogenesis abundant protein (LEA), Dicer-like 3 (DCL3) Xyloglucan endotransglucosylase/hydrolase 2 (XTH2), Pectin lyase-like superfamily protein (PPME1), Lipid transfer protein 3 (LTP3), Fatty acid biosynthesis 1 (FAB1) and Fatty acid desaturase 5 (FAD5) might have relationships with the pistil abortion in Japanese apricot. The expression patterns of 36 differentially expressed genes were confirmed by real-time (RT)-PCR. This is the first report of the Illumina RNA-seq technique being used for the analysis of differentially-expressed gene profiles related to pistil abortion that both computationally and experimentally provides valuable information for the further functional characterisation of genes associated with pistil development in woody plants.

Isolation and Characterization of an AGAMOUS Homologue PmAG from the Japanese Apricot (Prunus mume Sieb. et Zucc.)

An AGAMOUS homologue, named PmAG (GenBank accession number EU068730), was isolated from the Japanese apricot cultivar ‘Shirokaga’ (Prunus mume) via the homology cloning method. The cDNA was 812-base pairs long with an open reading frame of 732 base pairs, and encoded for a putative protein of 243 amino acid residues. Sequence alignment showed that the sequence of PmAG was 98% identical to that of the peach (Prunus persica). Genomic DNA analysis revealed an intron just before the stop codon of the ORF, and PmAG had AG motifs I and II in its C-terminal region, which suggested that PmAG was an AGAMOUS homologue. Real-time quantitative reverse transcription polymerase chain reaction and in situ hybridization showed that the PmAG mRNA was highly expressed in the sepals, carpel and stamens, and a weak signal was detected in the seed and nutlet. No expression was detected in the leaves or petals. Overexpression of PmAG in transgenic tobacco exhibited no effect on the phenotype of the flower organs, but changes in the floral color from pink to white.

Identification of miRNAs and Their Target Genes in Peach (Prunus persica L.) Using High-Throughput Sequencing and Degradome Analysis

MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development.

Evaluation of different kinds of organic acids and their antibacterial activity in Japanese Apricot fruits

The fruit of Japanese apricot is rich in organic acids, which have strong antibacterial activities. The types and contents of organic acids in six different cultivars of Japanese apricot fruit were evaluated by reverse-phase high performance liquid chromatography (HPLC). The antibacterial activity against Escherichia coli, Bacillus subtilis and Streptococcus suis was also determined. The results revealed that there are nine types of organic acids in the presence of the extracts of Japanese apricot fruit, including oxalic, tartaric, malic, ascorbic, acetic, citric, maleic, fumaric and succinic acids. The total organic acid content of ‘Zhonghong’ was the highest among the studied cultivars, and the main organic acids present were citric and malic acids. The antibacterial activity on the growth of E. coli and B. subtilis was higher than on the growth of S. suis. The antibacterial effect of acetic acid against bacteria was the best, and the minimum antibacterial concentration (MIC) against E. coli and B. subtilis was 0.417 mg/mL. The citric and maleic acids were also against these three strains of bacteria. The results suggest that the antibacterial activity is related to the organic acid composition and content of Japanese apricot fruit.

Genomic variants of genes associated with three horticultural traits in apple revealed by genome re-sequencing.

The apple (Malus × domestica Borkh.) cultivar ‘Su Shuai’ exhibits greater disease resistance, shorter internodes and lighter fruit flavor compared with its parents ‘Golden Delicious’ and ‘Indo’. To obtain a comprehensive overview of the sequence variation in these three horticultural traits, the genomes of ‘Su Shuai’ and ‘Indo’ were resequenced using next-generation sequencing and compared to the genome of ‘Golden Delicious’. A wide range of genetic variations were detected, including 2 454 406 and 18 749 349 single nucleotide polymorphism (SNP) and 59 547 and 50 143 structural variants (SVs) in the ‘Indo’ and ‘Su Shuai’ genomes, respectively. Among the SVs in ‘Su Shuai’, 17 genes related to disease resistance, 10 genes related to Gibberellin (GA) and 19 genes associated with fruit flavor were identified. The expression patterns of eight of the SV genes were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of this study illustrate the genomic variation in these cultivars and provide evidence for a genetic basis for the horticultural traits of disease resistance, short internodes and lighter flavor exhibited in these cultivars. These results provide a genetic basis for the phenotypic characteristics of ‘Su Shuai’ and, as such, these SVs could serve as gene-specific molecular markers in maker-assisted breeding of apples.