Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.

Yang Liu,Shui-Zhang Fei,Bing Yang,Christopher Currey,Weiling Wang

doi:10.3389/fpls.2020.572193

Abstract

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

Highlights

Switchgrass (Panicum virgatum), a C4 perennial grass with demonstrated high biomass yield is native to North America and is well adapted to marginal land not suitable for food crops (Mitchell et al, 2008; Narasimhamoorthy et al, 2008)
We showed that double biallelic mutant for Pvtb1a and 1b genes enhanced tiller production and increased biomass yield, indicating that Pvtb1 genes negatively regulate tillering in switchgrass
To determine the allelic compositions of Pvtb1a and Pvtb1b genes in 10 randomly selected micropropagated plants including 9 plants generated from primary mutants and 1 plant from the wild-type, sequencing libraries for the tb1a amplicons with an insert size of 250 bp and tb1b amplicons with an insert size of 288 bp were constructed for each individual plant

Summary

Introduction

Switchgrass (Panicum virgatum), a C4 perennial grass with demonstrated high biomass yield is native to North America and is well adapted to marginal land not suitable for food crops (Mitchell et al, 2008; Narasimhamoorthy et al, 2008). The low production cost and high lignocellulose-based biofuel potential make it well-suited for bioenergy crop (McLaughlin and Kszos, 2005). It was Switchgrass Pvtb Genes Regulate Tillering named the model species for herbaceous bioenergy crop by the U.S Department of Energy in 1991 following more than a decade of research (Wright and Turhollow, 2010). Most of the low-tillering lowland ecotypes are tetraploid (2n=4x=36), while the high-tillering upland ecotypes contain both tetraploids and octoploids (2n=8x=72) with hexaploids (2n=6x=54) being reported rarely (Zhang et al, 2011). Unlike the lowland ecotype, which has a caespitose growth habit and poor freezing tolerance, upland ecotypes are highly rhizomatous and cold hardy (Mitchell and Schmer, 2012)

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