Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

Abstract

Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

Progresses, Challenges, and Prospects of Genome Editing in Soybean (Glycine max)

“…each of them in a separate plasmid as shown in previous reports (Jiang et al, 2013;Li et al, 2013;Brooks et al, 2014). In most cases in CRISPR system for soybean, the Streptococcus pyogenes Cas9 gene (Cai et al, 2015;Cheng et al, 2019), human codonoptimized Cas9 gene (Campbell et al, 2019;Do et al, 2019) or a soybean codon optimized Cas9 gene (Michno et al, 2015;Sun et al, 2015) was driven by commonly used promoters in soybean transformation, such as ubiquitin (soybean ubiquitin-3, Campbell et al, 2019), Arabidopsis egg-cell specific promoter (Zheng et al, 2020) and cauliflower mosaic virus 35S (CaMV 35S) promoters (Cai et al, 2015;Jacobs et al, 2015;Michno et al, 2015;Bao et al, 2019;Chen F. et al, 2019;Cheng et al, 2019;Do et al, 2019;Han et al, 2019). Some endogenous promoters, such as proGmSCREAM M4 (pM4), proGmSCREAM M8 (pM8) (Bai et al, 2020) and soybean ELONGATION FACTOR1 ALPHA2 (EF1A2) gene constitutive promoter (Li et al, 2015), were used as well.…”

“…Nuclear Localization Signal (NLS) sequences and Flag peptide sequences were normally inserted in each end of the Cas9 gene, which facilitate Cas9 protein entering the nucleus and protein identification. Because plant specific RNA polymerase III promoters such as AtU6 (Arabidopsis), TaU6 (wheat), and OsU6 or OsU3 (rice) are frequently used to drive gRNA in plant systems, dicotyledons U6 promoter such as Arabidopsis AtU6 (Cai et al, 2015;Michno et al, 2015;Do et al, 2019;Han et al, 2019), as well as Medicago truncatula MtU6 (Jacobs et al, 2015;Campbell et al, 2019), were frequently used to drive sgRNA for soybean. Soybean GmU6 promoters have been discovered and evaluated for their efficiency and used for GE (Li et al, 2015;Du et al, 2016;Di et al, 2019).…”

“…PCR/RE assay detection method was used frequently in soybean (Michno et al, 2015;Sun et al, 2015;Kanazashi et al, 2018;Di et al, 2019). T7EI assay was occasionally used (Cai et al, 2015;Du et al, 2016) and PCR/Sequencing assay is now the most popular method used to detect mutation in soybean (Haun et al, 2014;Campbell et al, 2019;Cheng et al, 2019;Do et al, 2019;Han et al, 2019;Li R. et al, 2019;Wang J. et al, 2020). PCR/RE and T7EI are cost efficient method when large number putative mutations need to be screened, but limitation of restriction enzyme cut sites can restrict design of sgRNA.…”

“…The quadruple mutant GmLCLa1a2b1b2 delays flowering and showed very short-period circadian rhythms. Early flowing mutations under natural longday (NLD) conditions was also created by knockout E1gene (Han et al, 2019) and GmPRR37 encoding a pseudo-response regulator protein which is related to photoperiod sensitivity using CRISPR system (Wang L. et al, 2020). These flowering time related mutations can be consistently inherited in next generation (Cai et al, 2018;Han et al, 2019;Cai et al, 2020a;Wang J. et al, 2020) and will be a useful resource for developing elite soybean varieties in the future.…”

Gene editing applications to modulate crop flowering time and seed dormancy

“…In addition to directly targeting FT homologs, other studies in soybean have targeted regulators of GmFT expression. For example, soybean E1 encodes a B3 domain transcription factor that suppresses GmFT; E1 truncated by CRISPR/Cas failed to inhibit GmFT2a/5a causing early flowering (Han et al 2019). Other studies have targeted Pseudo-Response Regulator (PRR) proteins, which play conserved roles in photoperiod responses in dicots and monocots (Wang et al 2020).…”