Systematic Identification and Validation of Housekeeping and Tissue-Specific Genes in Allotetraploid Chenopodium quinoa

Bing He,Fengqin Hu,Wenjing Song,Lin Meng,Pibiao Shi,Yuanda Lv,Hui Chen

doi:10.3390/horticulturae7080235

Abstract

Quinoa is a gluten-free food crop that contains all the essential amino acids and vitamins. The selection of proper housekeeping and tissue-specific genes is the crucial prerequisite for gene expression analysis using the common approach, real-time quantitative PCR (RT-qPCR). In this study, we identified 40 novel candidate housekeeping genes by the minimum transcript per million (TPM), coefficient of variation (CV) and maximum fold change (MFC) methods and 19 candidate tissue-specific genes by the co-expression network method based on an RNA-seq dataset that included 53 stem, leaf, flower and seed samples, as well as additional shoot and root samples under different stresses. The expression stability of 12 housekeeping and tissue-specific genes, as well as that of another two traditionally used housekeeping genes, was further evaluated using qPCR and ranked using NormFinder, BestKeeper and the comparative delta-Ct method. The results demonstrated that MIF, RGGA, VATE and UBA2B were ranked as the top four most stable candidate housekeeping genes. qPCR analysis also revealed three leaf-specific genes and five root-specific genes, but no stem-specific gene was identified. Gene Ontology (GO) enrichment analysis identified that housekeeping genes were mainly enriched in the small molecule metabolic process, organonitrogen compound metabolic process, NAD binding and ligase activity. In addition, tissue-specific genes are closely associated with the major functions of a specific tissue. Specifically, GO terms “photosynthesis” and “thylakoid” were most significantly overrepresented in candidate leaf-specific genes. The novel housekeeping and tissue-specific genes in our study will enable better normalization and quantification of transcript levels in quinoa.

Highlights

Quinoa (Chenopodium quinoa Wild., 2n = 4x = 36) is an ancient seed crop originating from the Andean region of South America, where it has developed tolerance to various environmental stresses [1,2,3]
Three biological replicates were collected for each tissue to validate housekeeping and tissue-specific gene candidates with quantitative real-time PCR
There was no maximum fold change (MFC) value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) because GAPDH was not expressed in quinoa roots under heat stress. These results suggested that candidate housekeeping genes were more stable than traditional reference genes and were not abnormally expressed in any of the samples

Summary

Introduction

Quinoa (Chenopodium quinoa Wild., 2n = 4x = 36) is an ancient seed crop originating from the Andean region of South America, where it has developed tolerance to various environmental stresses [1,2,3]. Quinoa can be grown on marginal lands unsuitable for other main crops but is used as a highly nutritional food source. Quinoa seeds are gluten-free and contain an excellent balance of essential amino acids, dietary fiber, lipids, carbohydrates, vitamins and minerals [4]. The genome of quinoa has been sequenced and assembled [5], which enables the genetic improvement of quinoa. Considerable attention has been focused on defining the biological significance and functional characteristics of the quinoa genes

Objectives

Methods

Results

Conclusion