Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA

Abstract

Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T and T generations indicates that putative mutation induced in the T plant is transmitted to the T generation. The inheritable mutation induced in the T plant was also detected. This result indicates that continuous induction of mutations during T plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

Generation of a multiplex mutagenesis population via pooled CRISPR‐Cas9 in soya bean

“…Multiplex mutations have been induced by CRISPR‐Cas9 in several crops, such as hexaploid wheat (Gil‐Humanes et al ., ; Sánchez‐León et al ., ), paleopolyploid maize (Char et al ., ) and allotetraploid rapeseed (Li et al ., ). In soya bean, the CRISPR‐Cas9 system has been successfully utilized to create nonheritable somatic mutations in hairy root cultures and/or whole‐plant heritable mutations (Cai et al ., , ,b; Curtin et al ., ; Du et al ., ; Jacobs et al ., ; Kanazashi et al ., ; Li et al ., ; Michno et al ., ; Sun et al ., ). It has been repeatedly shown that a single sgRNA can induce simultaneous mutations on endogenous genes in soya bean (Du et al ., ; Jacobs et al ., ; Kanazashi et al ., ; Sun et al ., ).…”

SlKIX8 and SlKIX9 are negative regulators of leaf and fruit growth in tomato

“…In multiple eudicot species that belong to the rosid order of Fabales, orthologs of the KIX and PPD proteins have been reported to negatively regulate seed pod size (Ge et al, 2016;Kanazashi et al, 2018;Li et al, 2019;Naito et al, 2017). To examine whether the KIX proteins might also have a role in determining tomato fruit size in the asterid model species tomato, we investigated whether the development of reproductive organs was affected in tomato kix8 kix9 mutants.…”

“…In Arabidopsis, the asymmetric cell division of meristemoids, which are precursor cells of the stomatal lineage, positively affects final leaf size and is restricted by a transcriptional repressor complex in which the co-repressor TPL is recruited to PPD2 by KIX8/KIX9 (Gonzalez et al, 2015;White, 2006). Members of this repressor complex were shown to regulate leaf size and shape in a variety of species that belong to different orders of the rosids (Ge et al, 2016;Gonzalez et al, 2015;Kanazashi et al, 2018;Li et al, 2019;Naito et al, 2017), suggesting that the repressor complex is a conserved regulator of leaf growth among rosid eudicots. Here, we demonstrate that the tomato orthologs of KIX8 and KIX9 act as TPL adaptors for PPD proteins as well and, thereby, regulate leaf growth in tomato, a model species of the asterid clade that also includes tobacco (Nicotiana tabacum), carrot (Daucus carota) and sunflower (Helianthus annuus).…”

“…Several eudicot members, in which orthologs of the PPD or KIX genes were mutated or downregulated, including Medicago truncatula, soybean (Glycine max), blackgram (Vigna mungo), and pea (Pisum sativum), produced enlarged leaves (Ge et al, 2016;Kanazashi et al, 2018;Li et al, 2019;Naito et al, 2017). Moreover, overexpression of SAP orthologs in poplar (Populus trichocarpa) and cucumber (Cucumis sativus L.) also increased leaf size (Townsley et al, 2015;Yang et al, 2018;Yordanov et al, 2017).…”

Impacts of genomic research on soybean improvement in East Asia

“…A protocol for whole plant transformation has been well documented (Liu et al 2019). Successful stable lines have been generated to disable the GmFT2a family, the GmSPL9 family, and two copies of PEAPOD genes to alter flowering time, root nodule number, and seed pod morphology, respectively (Bao et al 2019;Cai et al 2018aCai et al , 2019Kanazashi et al 2018). Nonetheless, the generation of stably edited lines largely relies on the soybean transformation efficiency, editing efficiency, and inheritability.…”