Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria.

Chenhua Zhang,Kelei Han,Heng Xu,Yuwen Lu,Hongying Zheng,Xiaohua Wu,Lin Lin,Jiejun Peng,Xinyang Wu,Guojing Li,Pei Xu,Jianping Chen,Fei Yan

doi:10.7717/peerj.5642

Abstract

Lagenaria siceraria is an economically important cucurbitaceous crop, but suitable reference genes (RGs) to use when the plants are infected by cucumber green mottle mosaic virus (CGMMV) have not been determined. Sixteen candidate RGs of both leaf and fruit and 18 candidate RGs mostly from separate RNA-Seq datasets of bottle gourd leaf or fruit were screened and assessed by RT-qPCR. The expression stability of these genes was determined and ranked using geNorm, NormFinder, BestKeeper and RefFinder. Comprehensive analysis resulted in the selection of LsCYP, LsH3, and LsTBP as the optimal RGs for bottle gourd leaves, and LsP4H, LsADP, and LsTBP for fruits. LsWD, LsGAPDH, and LsH3 were optimal for use in both leaves and fruits under the infection of CGMMV. Isopentenyl transferase (IPT) and DNA-directed RNA polymerase (DdRP) were used to validate the applicability of the most stable identified RGs from bottle gourd in response to CGMMV. All the candidate RGs performed in RT-qPCR consistently with the data from the transcriptome database. The results demonstrated that LsWD, LsGAPDH and LsH3 were the most suitable internal RGs for the leaf, and LsH3, LsGAPDH, LsP4H and LsCYP for the fruit.

Highlights

Lagenaria siceraria (Molina) Standl. is a specie belongs to Cucurbitaceae family, which was cultivated widely in tropical and temperate regions of the world, it is commonly known as bottle gourd that has good edible, medicinal and horticultural value (Wang et al, 2018; Decker-Walters et al, 2004)
Because there is little information on reference genes (RGs) that can be used to normalize gene expression data in cucumber green mottle mosaic virus (CGMMV)-infected bottle gourd, we evaluated the selected candidate genes by RT-qPCR, focusing on novel reference gene selection and analysis in CGMMV-infected leaves and fruits
Bottle gourd leaves and fruits infected by CGMMV were collected from three replicate virus-inoculated plants, and the presence of CGMMV in each sample was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, and the contamination of ZYMV and WMV was excluded by RT-PCR (Fig. S1)

Summary

Introduction

Lagenaria siceraria (Molina) Standl. is a specie belongs to Cucurbitaceae family, which was cultivated widely in tropical and temperate regions of the world, it is commonly known as bottle gourd that has good edible, medicinal and horticultural value (Wang et al, 2018; Decker-Walters et al, 2004). Is a specie belongs to Cucurbitaceae family, which was cultivated widely in tropical and temperate regions of the world, it is commonly known as bottle gourd that has good edible, medicinal and horticultural value (Wang et al, 2018; Decker-Walters et al, 2004) It could be routinely used as one rootstock source for watermelon and other cucurbit crops in both Japan and Korea in order to reduce the incidence of soil-borne diseases and promote the vigor of the root system of the crops in low temperature conditions (Yetisir & Sari, 2013; Cho et al, 2017; Spalholz & Kubota, How to cite this article Zhang et al (2018), Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria. Since CGMMV could pose a great threaten to bottle gourd production, it attracted our attention

Methods

Results

Discussion

Conclusion