On February 22, 2020, the David Jackson research group of the Cold Spring Harbor Laboratory (CSHL) in the United States published a paper entitled Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes in Nature Plants. The first author of the paper). The study used the CRISPR/Cas9 system to edit the promoter of the maize CLE gene and created a high-yielding allele of maize.
Breeding for genetic improvement of crop yield has undergone several changes. The first-generation breeding strategy is based on the collection, evaluation and selection of germplasm sources, retaining high-yield and high-quality germplasm resources for planting; the landmark event of the second-generation breeding strategy is the proposal and application of the concept of cross breeding, through inter-inbred crosses , To obtain hybrids, and the yield of hybrids is significantly higher than that of their parents due to the effect of heterosis. At the same time, the application of fertilizers and pesticides, and the application of dwarf genes in multiple crops have increased the yield of crops; The development of technology, through genetic modification technology, molecular marker-assisted selection, whole genome selection, etc., has further increased the yield of crops. However, with the rapid growth of the global population, the adverse effects of climate change on crop yields, and the increase in demand for bioenergy based on crops such as corn, higher requirements have been placed on crop yields. The domestication and improvement of modern maize originated about 10,000 years ago. After a long period of domestication and selection, the wild corn species Zea maysssp. Parviglumis evolved into a landrace of maize. After about 150 years of genetic improvement, Furthermore, excellent corn lines with good adaptability and high grain yield have been developed. In this process, maize has undergone extremely significant changes in its adaptability to photoperiod, plant morphology, ear size, and grain yield (Duvick 2005; Doebley et al 2006). Among them, the yield traits of maize are strongly selected. The ears of wild zeagrass have only dozens of kernels, while the ears of modern corn have as many as hundreds. Increasing the number of kernels per ear of corn can significantly increase the yield of corn.
Maize is a dioecious plant. The ears (female ears) of corn are female inflorescences, which develop female flowers, and by receiving the pollen from the male flowers of the male ears, they eventually develop into grains after fertilization. The number of kernels in a maize ear is formed and determined during the early development of the female inflorescence (Vollbrecht and Schmidt 2009). The female inflorescences of maize generally begin to develop at the 9-10 leaf stage, at which time the axillary meristem (Axillary Mersitem, AM) located in the leaf axil transforms into the Inflorescence Meristem (IM) (Vollbrecht and Schmidt 2009). Subsequently, multiple rows of neatly arranged meristems are formed on IM, namely Spikelet-Pair Meristem (SPM). Each SPM then differentiates into two short-branched meristems: Spikelet Meristem (SMs). Each SM eventually undergoes differentiation of floret meristems and floral organs, and develops into mature grains after pollination (Vollbrecht and Schmidt 2009). Therefore, the ability of IM to continuously differentiate into SPM and the ability of SPM to differentiate into SM during the development of maize ears determines the yield traits such as the number of rows per ear and the number of grains per ear on the final corn ear (Vollbrecht and Schmidt 2009). Therefore, increasing the activity of maize inflorescence meristem can theoretically increase the number of ears and grains and increase the yield.
The establishment and maintenance of maize inflorescence meristem (IM) is the basis for the normal development of maize inflorescence. Similar to other meristems, IM mainly plays two roles: through the differentiation of plant stem cells (Stem cells) to initiate the development of other tissues and organs; through cell division to proliferate itself (Vollbrecht and Schmidt 2009). In plants, the classic way to maintain the growth and differentiation of inflorescence meristem is the CLAVATA-WUSCHEL feedback loop, which includes WUSCHEL (WUS) gene specifically expressed in the middle of IM, CLAVATA1 (CLV1) and CLAVATA2 (CLV2) specifically expressed in IM. ), and the CLAVATA3 (CLV3) signaling molecule responsible for WUS's signal transmission to CLV1 and CLV2 (Williams and Fletcher 2005). The product of the WUS gene in Arabidopsis can promote the growth of meristems and the expression of CLV3 signaling molecules. CLV3 can interact with the CLV1-CLV2 complex to further inhibit the expression of WUS, thus forming a dynamically balanced negative feedback loop pathway. Mutants that maintain the differentiation activity of meristems (Williams and Fletcher 2005), and break the balance of this pathway, show excessive proliferation of meristems (Williams and Fletcher 2005). The CLV-WUS pathway is relatively conserved in plants. The thick tassel dwarf1 (td1) (Bommert et al 2005), fasciated ear2 (fea2) (Taguchi- Shiobara et al 2001; Bommert et al 2013a) and Zmcle7, Zmfcp1 (Rodriguez-Leal et al 2019). The phenotypes of these mutations are similar, and they are all manifested as the increase of IM, the thickening of male ears and the increase of ear rows. However, in these mutants, due to the excessive proliferation of meristems, the development of ears is abnormal, and the yield is significantly reduced. The weak mutant of fea2 created by the David Jackson research group of the Cold Spring Harbor Laboratory in the United States using EMS and other technologies can ensure the normal development of ears, and increase the activity of meristems, thereby increasing the number of ear rows and other yield traits. The artificial modification of these genes to finely optimize their biological activity has important potential value for optimizing the activity of corn meristems and increasing corn yield. Rodríguez-Leal et al. used the CRISPR-Cas gene editing technology to edit the regulatory sequence of the tomato CLV3 gene, and obtained new alleles that can regulate the variation of tomato fruit size and quantity, and realized the use of genome editing technology to target genes. Artificial design (Rodríguez-Leal et al 2017). Therefore, the application of genome editing technology can further create excellent allele resources, which can be quickly applied to the genetic improvement of maize, and has broad application prospects in maize breeding (Wolter et al. 2019).
Corn CLAVATA3 (ZmCLE7 and ZmFCP1) is a kind of short peptide signal molecule, which transmits signals to CLAVATA1 (TD1), CLAVATA2 (FEA2) and FEA3, and through them inhibits the expression of WUSHEL gene, thereby forming a feedback-regulated signaling pathway , Precisely regulate the development of inflorescence meristem. In order to obtain its weakly mutant alleles, this latest study explores the use of CRISPR-Cas9 genome editing technology to target its promoter region to obtain weak mutants that affect its expression. For the ZmCLE7 and ZmFCP1 promoter regions, the researchers used ATAC-seq and MNase-seq data to analyze the accessible chromatin regions, and at the same time used evolutionary analysis to predict possible conservative regulatory sites in the promoter region. And design 9 sgRNAs respectively to target their promoter regions. Through screening, ZmCLE7 and ZmFCP1 promoter regions were screened for several large fragment deletion and inversion editing events, respectively. These editing events can significantly change the expression level of genes, and editing alleles of several promoters can reduce the number of expression levels by ~45%-~69%. Among them, the alleles edited by the ZmCLE7 promoter inverted, but the ZmCLE7 expression site expanded into the IM.
Through further phenotypic analysis, it is found that the deletion of edited alleles in some promoters can increase the size of the inflorescence meristem to a certain extent, thereby significantly increasing several yield-related traits and the grain yield of a single panicle. They can significantly increase the yield in the background of inbred lines and hybrids. The increase in yield of these alleles is mainly reflected in the thickening of the ear, the increase in the number of ear rows, and the increase in the number of grains per ear. Compared with the control, the increase in the number of ear rows is very significant, with an increase of ~6 rows, and the number of grains per ear is increased. The weight did not change significantly, and the final yield per ear increased by approximately up to ~26%. The study also found that the editing allele of one of the promoter inversions expanded the expression site of ZmCLE7, which resulted in smaller ears and reduced yield. These results indicate that the editing of ZmCLE7 and ZmFCP1 promoters can quantitatively regulate gene expression, affect its influence on the activity of maize inflorescence meristem, affect the quantitative variation of maize yield traits, and significantly increase the yield per ear of maize.
There are 49 homologous genes of CLAVATA3 (ZmCLE7 and ZmFCP1) in maize. The researchers found that CLAVATA3 and homologous genes often have functional redundancy in multiple species. For example, in Zmcle7 mutants, ZmFCP1 can be up-regulated by expression to make up for a certain function of ZmCLE7. The researchers further analyzed the transcriptome of the Zmcle7 mutant and screened another CLAVATA3 homologous gene, ZmCLE1E5, which also showed up-regulation in the expression of the Zmcle7 mutant. The researchers edited its coding region and obtained two null alleles mutants with different frameshift mutations. The inflorescence meristem development of the Zmcle1e5 mutant is relatively normal, but it is significantly enlarged, the ears are significantly enlarged, the number of ears increases, the ear morphology is normal, and the yield is significantly improved. Zmcle1e5 can significantly enhance the Zmcle7 mutant phenotype. Therefore, ZmCLE1E5 can partially Complement the function of ZmCLE7. Furthermore, it is shown that the same gene editing of ZmCLE7 complementary factor can also create high-yield alleles similar to ZmCLE7 promoter editing.
Maize yield is an extremely complex quantitative trait. The predecessors used methods such as genome-wide association analysis and linkage analysis to identify hundreds of QTL loci (www.maizegdb.org/qtl) for yield-related traits, most of which were minor effects. QTL sites, and only a few sites have been cloned, such as the QTL site KRN4 for the number of rows of ears, QTL qHKW1, which controls 100-kernel weight, and KNR6, which controls the number of kernels, etc. (Liu et al 2015; Yang et al 2019; Jia et al. al 2020). These QTL loci often only show greater genetic effects under a certain genetic background. For example, the KRN4 locus can increase the number of ear rows by 2.2 rows in the H21 background, while in the Mo17 background, its ear row number effect Only 0.7 lines (Liu et al 2015). In addition, the superior alleles of these QTL loci are often enriched in superior inbred populations, which further limits the application of these loci in breeding. On the other hand, some key genes that control the development of yield-related traits do not have natural mutation sites that can lead to changes in the number of phenotypes. On the one hand, it may be due to the fact that the excellent natural mutation sites of these genes do not exist or are in domestication. And the improvement is lost due to the bottleneck effect. For example, the two CLAVATA3 homologous genes ZmCLE7 and ZmFCP1 involved in this study did not detect QTLs or associated loci that control related traits in the NAM population with extensive genetic variation (Brown et al 2011). Therefore, this study uses the superior alleles of ZmCLE7 and ZmFCP1 created by genome editing technology as new genetic resources. The excellent alleles edited in the regulatory region of ZmCLE7 and ZmFCP1 finely regulate the expression level of candidate genes, balance their functions in inflorescence development, and ultimately enhance the performance of corn yield traits, increase corn grain yield at a quantitative level, and improve their yield-related traits The effect is greater than any of the currently cloned QTL sites (Liu et al 2015; Yang et al 2019; Jia et al 2020). In addition to the promoter editing strategy, the research also proposes a strategy for editing complementary genes of key genes in the development of meristems, which can also optimize the activity of meristems and create high-yielding alleles. In summary, on the one hand, this research has created excellent allele resources, on the other hand, it has provided new ideas for the reuse of important genes.
references:
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Paper link:
www.nature.com/articles/s41477-021-00858-5
On March 2, 2021, "Gene Editing Webinar is Coming", welcome to discuss it together~
