Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

<div><p><i>Cucumber green mottle mosaic virus</i> (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (<i>Cucumis sativus</i>). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including <i>Actin</i>, <i>Tubulin</i>, <i>EF-1α</i>, <i>18S rRNA</i>, <i>Ubiquitin</i>, <i>GAPDH</i> and <i>Cyclophilin</i>, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that <i>EF-1α</i> and <i>GAPDH</i> were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while <i>Ubiquitin</i> and <i>EF-1α</i> were the most suitable combination overall.</p></div

RT-qPCR cycle threshold values of seven reference genes in cucumber leaf, stem and root samples at different time points post-inoculation with CGMMV.

<p>Error bars represent the mean of three technical replicates ± SD.</p

Primers used to amplify partial sequences of 7 reference genes and miR159 from cucumber samples.

<p>Primers used to amplify partial sequences of 7 reference genes and miR159 from cucumber samples.</p

Expression stability of five reference genes in cucumber analyzed using the BestKeeper algorithm.

<p>Expression stability of five reference genes in cucumber analyzed using the BestKeeper algorithm.</p

Data_Sheet_1_Analysis of the coupling coordination of livestock production, residential consumption, and resource and environmental carrying capacity in China.DOCX

Increasing demands for livestock products have stimulated rapid increases in the number of livestock and the scale of farming, thus increasing pressure on resources and the environment. Coordinating the development of livestock production (LP) with residential consumption (RC), resources and the environmental carrying capacity (RECC) is important to ensure sustainable development. In this study, the entropy weight method and the improved-coupling coordination degree (CCD) model were used to identify the spatial–temporal coordination development characteristics of livestock production-residential consumption-resource and environmental carrying capacity (LRRE). Furthermore, the spatial autocorrelation model (SAM) and gray model (GM) were used to analyze the spatial aggregation characteristics and future development trends of the CCDs of the LRRE in China. The findings show that the CCDs of the LRRE values in 31 Chinese provinces increased from 2005 to 2020, but no provinces reached a high coordination level. Specifically, the coordinated development level of LRRE is relatively high in the central, eastern and northeastern regions and relatively low in the western region. The spatial autocorrelation analysis confirmed that the high-high (H-H) aggregation areas were mainly distributed in the northeastern, eastern, and central regions, while low-high (L-H) agglomeration was distributed in the western region. This phenomenon is mainly attributed to the continuous expansion of the scale of livestock production in western China. Regions with relatively developed economies have more funds to invest in environmental protection projects. Using GM method, we find that the CCDs of LRRE in 31 provinces in China will increase from 2021 to 2030, and all provinces will reach the basic coordination level. However, most of the western regions will barely reach the basic coordination level. This result indicates that the low level of LRRE development in western China may be difficult to change in the short term. The level of coordinated LRRE development in the relatively developed eastern region is increasing. The spatial layout of China’s livestock industry should be appropriately adjusted, its expansion rate in the western region should be decreased, and the ecological environment of the areas bordering the western and central regions should be improved. These findings have practical implications for other large livestock production countries. Promoting the coordinated development of LRRE is also an important condition for agricultural transformation in developing countries, especially for improving the environment in key areas of the livestock industry.</p

Expression stability of seven reference genes in cucumber analyzed using the NormFinder algorithm.

<p>Expression stability of seven reference genes in cucumber analyzed using the NormFinder algorithm.</p

Expression profile of miR159 and validation of selected reference genes in different cucumber tissues infected with CGMMV.

<p>Error bars represent the mean of three technical replicates ± SD.</p

GeNorm data for 5 cucumber reference genes in cucumber leaf, stem and root samples at different time points post-inoculation with CGMMV.

<p>(A) to (D) Average expression stability values (M) of cucumber reference genes during stepwise exclusion of the least stable reference gene in different tissue samples. (A) leaf, (B) stem, (C) root, and (D) overall. (E) to (H) Optimal number of reference genes required for accurate normalization of expression based on pairwise variation (V_n/n+1) analysis of the normalization factors of the reference genes in different tissue samples. (E) leaf, (F) stem, (G) root, and (H) overall.</p

Mycelial growth, conidial production and spore germination of the recovered pathogens growing on a fresh plate transferred from treated cultures.

<p>x and y: expressed as mean value ± SD.</p><p>+: Viable. TNTC = too numerous to count. NA = not applicable. ND = not determinable.</p><p>* indicates a significant difference between the control and recovered treatment (P < 0.05).</p><p>Mycelial growth, conidial production and spore germination of the recovered pathogens growing on a fresh plate transferred from treated cultures.</p

Most suitable combination of reference genes for normalizing expression in cucumber according to NormFinder analysis.

<p>Most suitable combination of reference genes for normalizing expression in cucumber according to NormFinder analysis.</p