Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Joon Ki Hong,Yeon-Hee Lee,Eun Jung Suh,Jihee Park,Sang Ryeol Park

doi:10.3390/agriculture11121286

Joon Ki Hong, Yeon-Hee Lee + Show 3 more

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https://doi.org/10.3390/agriculture11121286

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Journal: Agriculture	Publication Date: Dec 17, 2021
Citations: 10	License type: CC BY 4.0

Affiliation: Rural Development Administration

Abstract

The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

Highlights

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop that contributes to human health as a source of beneficial dietary fiber, vitamins, and minerals [1]
Because the BrVRN1 homologs (BrVRN1s) GE2 mutants displayed late-flowering phenotypes, we considered the possibility that BrVRN1s may regulate the expression of other floral genes, such as FLC1 and FLOWERING LOCUS T (FT) [23]
BrVRN1a expression in mutants at the RNA level was similar to that of the wild type, occurring only after vernalization (Figure S8). These results revealed that the altered patterns of BrFLC1 and BrFT expression induced by BrVRN1 mutations could result in the late flowering time phenotypes of mutant plants

Summary

Introduction

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop that contributes to human health as a source of beneficial dietary fiber, vitamins, and minerals [1]. Flowering time has emerged as a key trait affecting yield in commercial agronomic and horticultural crops because certain biotic and abiotic stresses can be avoided altogether by inducing or delaying the transition to flowering. Multiple pathways, such as photoperiod sensation, vernalization, gibberellin signaling, and responses to autonomous signals, are involved in the timing of floral transition during specific seasons, and altered flowering time phenotypes have been generated in multiple cultivated species through targeted genetic modification [5,6,7,8,9].

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