查看更多>>摘要：Abstract The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean.
查看更多>>摘要：Abstract Native promoters that can drive high and stable transgene expression are important tools for modifying plant traits. Although several such promoters have been reported in soybean (Glycine max), few of them function at multiple growth and development stages and during nodule development. Here, we report that the promoters of 40S RIBOSOMAL PROTEIN SMALL SUBUNIT S28 (RPS28) and EUKARYOTIC TRANSLATION INITIATION FACTOR 1 (EIF1) are ideal for high expression of transgene. Through bioinformatic analysis, we determined that RPS28 and EIF1 were highly expressed during soybean growth and development, nodule development, and various biotic and abiotic stresses. Fusion of both RPS28 and EIF1 promoters, with or without their first intron, with the reporter gene β-GLUCURONIDASE (uidA) in transgenic soybean, resulted in high GUS activity in seedlings, seeds, and nodules. Fluorimetric GUS assays showed that the RPS28 promoter and the EIF1 promoter yielded high expression, comparable to the soybean Ubiquitin (GmUbi) promoter. RPS28 and EIF1 promoters were also highly expressed in Arabidopsis thaliana and Nicotiana benthamiana. Our results indicate the potential of RPS28 and EIF1 promoters to facilitate future genetic engineering and breeding to improve the quality and yield of soybean, as well as in a wide variety of other plant species.
查看更多>>摘要：Abstract Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T2 progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.
查看更多>>摘要：Abstract Genetic diversity is a cornerstone of crop improvement, However, cultivated soybean (Glycine max) has undergone several genetic bottlenecks, including domestication in China, the introduction of landraces to other areas of the world and, latterly, selective breeding, leading to low genetic diversity the poses a major obstacle to soybean improvement. By contrast, there remains a relatively high level of genetic diversity in soybean’s wild relatives, especially the perennial soybeans (Glycine subgenus Glycine), which could serve as potential gene pools for improving soybean cultivars. Wild soybeans are phylogenetically diversified and adapted to various habitats, harboring resistance to various biotic and abiotic stresses. Advances in genome and transcriptome sequencing enable alleles associated with desirable traits that were lost during domestication of soybean to be discovered in wild soybean. The collection and conservation of soybean wild relatives and the dissection of their genomic features will accelerate soybean breeding and facilitate sustainable agriculture and food production.
查看更多>>摘要：Abstract Modern crops were created through the domestication and genetic introgression of wild relatives and adaptive differentiation in new environments. Identifying the domestication-related genes and unveiling their molecular diversity provide clues for understanding how the domesticated variants were selected by ancient people, elucidating how and where these crops were domesticated. Molecular genetics and genomics have explored some domestication-related genes in soybean (Glycine max). Here, we summarize recent studies about the quantitative trait locus (QTL) and genes involved in the domestication traits, introduce the functions of these genes, clarify which alleles of domesticated genes were selected during domestication. A deeper understanding of soybean domestication could help to break the bottleneck of modern breeding by highlighting unused genetic diversity not selected in the original domestication process, as well as highlighting promising new avenues for the identification and research of important agronomic traits among different crop species.
查看更多>>摘要：Abstract Weeds are a major biotic constraint that can cause dramatic crop production losses. Herbicide technology has been widely used by farmers as the most cost-effective weed control measure, and development of new strategy to improve herbicide tolerance in plants is urgently needed. The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to agricultural technology for crop improvement. Here we identified three polyamine uptake transporter (PUT) genes in rice that are homologous to the Arabidopsis AtRMV1. We successfully demonstrate that CRISPR/Cas9-targeted mutagenesis of OsPUT1/2/3 greatly improves paraquat resistance in rice without obvious yield penalty. Therefore, manipulation of these loci could be valuable for producing transgene-free rice with improved herbicide resistance in future.
查看更多>>摘要：Abstract Brassica species are a global source of nutrients and edible vegetable oil for humans. However, all commercially important Brassica crops underwent a whole-genome triplication event, hindering the development of functional genomics and breeding programs. Fortunately, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies, by allowing multiplex and precise genome engineering, have become valuable genome-editing tools and opened up new avenues for biotechnology. Here, we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing. We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements. Finally, we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops. Ongoing advancement in CRISPR/Cas technologies, in combination with other achievements, will play a significant role in the genetic improvement and molecular breeding of Brassica crops.
NSTL
Springer Nature