scholarly journals Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Agriculture ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1286
Author(s):  
Joon Ki Hong ◽  
Eun Jung Suh ◽  
Sang Ryeol Park ◽  
Jihee Park ◽  
Yeon-Hee Lee
Keyword(s):  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Crop Improvement ◽  
Sequencing Analysis ◽  
Vrn1 Gene ◽  
Floral Repressor ◽  
Dna Sequencing Analysis ◽  
Delayed Flowering ◽  
Golden Gate Cloning

The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

scholarly journals BBX32 Interacts with AGL24 Involved in Flowering Time Control in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

2018 ◽  
Vol 47 (1) ◽  
pp. 34-45
Author(s):  
Guan-Peng MA ◽  
Da-Qin ZHAO ◽  
Tian-Wen WANG ◽  
Lin-Bi ZHOU ◽  
Gui-Lian LI
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Clock ◽  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Time Control ◽  
Flowering Time Control ◽  
Molecular Regulatory Mechanisms

B-box (BBX) zinc finger proteins play critical roles in both vegetative and reproductive development in plants. Many BBX proteins have been identified in Arabidopsis thaliana as floral transition regulatory factors, such as CO, BBX7 (COL9), BBX19, and BBX32. BBX32 is involved in flowering time control through repression of COL3 in Arabidopsis thaliana, but it is still elusive that whether and how BBX32 directly interacts with flowering signal integrators of AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Chinese cabbage (Brassica rapa L. ssp. pekinensis) or other plants. In this study, B-box-32(BBX32), a transcription factor in this family with one B-box motif was cloned from B. rapa, acted as a circadian clock protein, showing expression changes during the circadian period. Additional experiments using GST pull-down and yeast two-hybrid assays indicated that BrBBX32 interacts with BrAGL24 and does not interact with BrSOC1, while BrAGL24 does interact with BrSOC1. To investigate the domains involved in these protein-protein interactions, we tested three regions of BrBBX32. Only the N-terminus interacted with BrAGL24, indicating that the B-box domain may be the key region for protein interaction. Based on these data, we propose that BrBBX32 may act in the circadian clock pathway and relate to the mechanism of flowering time regulation by binding to BrAGL24 through the B-box domain. This study will provide valuable information for unraveling the molecular regulatory mechanisms of BrBBX32 in flowering time of B. rapa.

scholarly journals Selection on BrFLC1 Is Related to Intraspecific Diversity of Brassica rapa Vegetables

Horticulturae ◽  
2021 ◽  
Vol 7 (8) ◽  
pp. 247
Author(s):  
Jiahe Liu ◽  
Xu Cai ◽  
Yufang Li ◽  
Yue Chen ◽  
Baozhen Gao ◽  
...  
Keyword(s):  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Allelic Diversity ◽  
Intraspecific Diversity ◽  
Site Mutation ◽  
Nonsynonymous Mutation ◽  
Yield And Quality ◽  
Significant Difference ◽  
Almost All

Flowering time is important for Brassica rapa vegetables because premature bolting before harvest can lower yield and quality. FLOWERING LOCUS C (FLC) acts as a key repressor of flowering. In this study, we identified a nonsynonymous mutation at the 58th nucleotide of exon1 in BrFLC1 (named as Pe1+58 (A/C)) by screening resequencing data of 199 B. rapa accessions and verified this mutation as being related to flowering time variation. Strong linkage inheritance was detected between this locus and a previously reported splicing site mutation at intron 6 of BrFLC1 (Pi6+1 (G/A)), showing as co-occurrence of BrFLC1Pe1+58(A) and BrFLC1Pi6+1(G), named as haplotype H1: AG, or co-occurrence of BrFLC1Pe1+58(C) and BrFLC1Pi6+1(A), named as haplotype H2: CA. The frequency distribution of BrFLC1 haplotypes skewed to the haplotype H1 in turnip, broccoletto, mizuna, komatsuna, and taicai, while it was skewed to the haplotype H2 in caixin, pak choi, zicaitai, and wutacai. The frequencies of the two haplotypes were comparable in Chinese cabbage. This indicated that BrFLC1 haplotypes were related to B. rapa intraspecific diversification. Further analysis of a Chinese cabbage collection revealed that accessions from the spring ecotype preferred to keep H1: AG and almost all accessions from the summer ecotype were H2: CA. The early flowering haplotype of BrFLC1 was purified in summer Chinese cabbage, indicating that BrFLC1 had been strongly selected during genetic improvement of summer Chinese cabbages. A significant difference in flowering time of F2 individuals with the homologous BrFLC1Pi6+1(G) allele but different BrFLC1Pe1+58 (A/C) alleles, indicated that this locus had independent genetic effects on flowering time. The newly identified allelic diversity of BrFLC1 can be used for breeding of resistance to premature bolting in B. rapa vegetables.

Establishment of Agrobacterium-mediated genetic transformation and application of CRISPR/Cas9 gene-editing system to Chinese cabbage (Brassica rapa L. ssp. pekinensis)

2020 ◽  
Author(s):  
Xiaonan Li ◽  
Haiyan Li ◽  
Yuzhu Zhao ◽  
Peixuan Zong ◽  
Zongxiang Zhan ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Transformation Method ◽  
Sequencing Analysis ◽  
Related Gene ◽  
Transformation System ◽  
Regeneration System ◽  
Regeneration Rate ◽  
Functional Studies

Abstract Background: Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.Results: In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without a pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5 to 4.0 months. Average transformation frequency is up to 10.83%. Furthermore, using this transformation system, the CRISPR/Cas9 technology was successfully applied in Chinese cabbage by knocking out a self-incompatibility-related gene SRK. Gene sequencing analysis in the positive transgenic lines revealed various mutations, including deletions, insertions, and substitutions. Conclusion: A simple, stable and efficient genetic transformation method was established for Chinese cabbage and successfully applied to the CRISPR/Cas9 system. The results of this study pave the way for further gene functional studies and genome editing in Chinese cabbage.

scholarly journals Transcriptome and small RNA combined sequencing analysis of cold tolerance response in non-heading Chinese cabbage

2020 ◽  
Author(s):  
Jin Wang ◽  
Qinxue Zhang ◽  
Xiong You ◽  
Xilin Hou
Keyword(s):  
Gene Expression ◽  
Cold Tolerance ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Small Rna ◽  
Target Genes ◽  
Differentially Expressed ◽  
Sequencing Analysis ◽  
Heading Chinese Cabbage ◽  
Gene Expression Levels

Abstract Background: Non-heading Chinese cabbage ( Brassica rapa ssp. chinensis ), as an important leaf vegetable grown worldwide. However, there is currently no enough transcriptome and small RNA combined sequencing analysis of cold tolerance, which hinders further functional genomics research.Results: In this study, 63.43 Gb of clean data was obtained from the transcriptome analysis. The clean data of each sample reached 6.99 Gb, and the basic percentage of Q30 was 93.68% and above. The clean reads of each sample were sequence aligned with the designated reference genome ( Brassica rapa, IVFCAASv1 ), and the efficiency of the alignment varied from 81.54% to 87.24%. According to the comparison results, 1,860 new genes were discovered, of which 1,613 were functionally annotated. Among them, 13 common differentially expressed genes were detected in all materials, including 7 up-regulated and 6 down-regulated. At the same time, we used quantitative real-time PCR to confirm the changes of these gene expression levels. In addition, we sequenced miRNA of the same material. Our findings revealed a total of 34,182,333 small RNA reads, 88,604,604 kinds of small RNA, among which the most common size was 24 nt. In all materials, the number of common differential miRNAs is 8. According to the corresponding relationship between miRNA and its target genes, we carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the set of target genes that each group of differentially expressed miRNAs. Through the analysis, it is found that the distribution of candidate target genes in different materials is different. We not only use transcriptome sequencing and small RNA sequencing, but also use experiment to prove that the expression level of differentially expressed genes are obtained by sequencing. Sequencing combined with experiments proved the mechanism of some differential gene expression levels after low temperature treatment.Conclusions: In all, this study provides a resource for genetic and genomic research under abiotic stress in Pak-choi.

scholarly journals Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs

2021 ◽  
Vol 22 (9) ◽  
pp. 4631
Author(s):  
Haemyeong Jung ◽  
Areum Lee ◽  
Seung Hee Jo ◽  
Hyun Ji Park ◽  
Won Yong Jung ◽  
...  
Keyword(s):  
Flowering Time ◽  
Brassica Rapa ◽  
Negative Feedback ◽  
Chinese Cabbage ◽  
Feedback Loop ◽  
Molecular Mechanisms ◽  
Transcriptome Profiling ◽  
Negative Feedback Loop ◽  
Transcription Analysis ◽  
Nitrogen Levels

Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, “4004” (early bolting) and “50” (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line “50” with or without vernalization. In agreement with the transcription analysis, the shoots in line “50” had substantially higher nitrogen levels than those in “4004”. Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line “50”, whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line “4004”. CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.

scholarly journals Characterization of Squamosa-Promoter Binding Protein-Box Family Genes Reveals the Critical Role of MsSPL20 in Alfalfa Flowering Time Regulation

2022 ◽  
Vol 12 ◽  
Author(s):  
Lin Ma ◽  
Xiqiang Liu ◽  
Wenhui Liu ◽  
Hongyu Wen ◽  
Yongchao Zhang ◽  
...  
Keyword(s):  
Flowering Time ◽  
Binding Protein ◽  
Expression Patterns ◽  
Crop Improvement ◽  
Critical Role ◽  
Functional Differentiation ◽  
Future Research ◽  
Systematic Analysis ◽  
Squamosa Promoter Binding Protein ◽  
Delayed Flowering

SQUAMOSA Promoter-binding protein-Like (SPL) genes affect a broad range of plant biological processes and show potential application in crop improvement by genetic modification. As the most widely planted forage crop in the world, biomass and abiotic stresses tolerance are important breeding targets for alfalfa (Medicago sativa L.). Nevertheless, the systematic analysis of SPL genes in alfalfa genome remains lacking. In the present study, we characterized 22 putative non-redundant SPL genes in alfalfa genome and uncovered the abundant structural variation among MsSPL genes. The phylogenetic analysis of plant SPL proteins separated them into 10 clades and clade J was an alfalfa-specific clade, suggesting SPL genes in alfalfa might have experienced gene duplication and functional differentiation within the genome. Meanwhile, 11 MsSPL genes with perfect matches to miRNA response elements (MREs) could be degraded by miR156, and the cleavage sites were gene specific. In addition, we investigated the temporal and spatial expression patterns of MsSPL genes and their expression patterns in response to multiple treatments, characterizing candidate SPL genes in alfalfa development and abiotic stress tolerant regulation. More importantly, overexpression of the alfalfa-specific SPL gene (MsSPL20) showed stable delayed flowering time, as well as increased biomass. Further studies indicated that MsSPL20 delayed flowering time by regulating the expression of genes involved in floret development, including HD3A, FTIP1, TEM1, and HST1. Together, our findings provide valuable information for future research and utilization of SPL genes in alfalfa and elucidate a possibly alfalfa-specific flowering time regulation, thereby supplying candidate genes for alfalfa molecular-assisted breeding.

scholarly journals The Floral Repressor GmFLC-like Is Involved in Regulating Flowering Time Mediated by Low Temperature in Soybean

2020 ◽  
Vol 21 (4) ◽  
pp. 1322 ◽  
Author(s):  
Jing Lyu ◽  
Zhandong Cai ◽  
Yonghong Li ◽  
Haicui Suo ◽  
Rong Yi ◽  
...  
Keyword(s):  
Low Temperature ◽  
Flowering Time ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Protective Mechanisms ◽  
Floral Repressor ◽  
Mobility Shift Assay ◽  
Delayed Flowering ◽  
Soybean Leaves

Soybean is an important crop that is grown worldwide. Flowering time is a critical agricultural trait determining successful reproduction and yields. For plants, light and temperature are important environmental factors that regulate flowering time. Soybean is a typical short-day (SD) plant, and many studies have elucidated the fine-scale mechanisms of how soybean responds to photoperiod. Low temperature can delay the flowering time of soybean, but little is known about the detailed mechanism of how temperature affects soybean flowering. In this study, we isolated GmFLC-like from soybean, which belongs to the FLOWERING LOCUS C clade of the MADS-box family and is intensely expressed in soybean leaves. Heterologous expression of GmFLC-like results in a delayed-flowering phenotype in Arabidopsis. Additional experiments revealed that GmFLC-like is involved in long-term low temperature-triggered late flowering by inhibiting FT gene expression. In addition, yeast one-hybrid, dual-luciferase reporter assay, and electrophoretic mobility shift assay revealed that the GmFLC-like protein could directly repress the expression of FT2a by physically interacting with its promoter region. Taken together, our results revealed that GmFLC-like functions as a floral repressor involved in flowering time during treatments with various low temperature durations. As the only the FLC gene in soybean, GmFLC-like was meaningfully retained in the soybean genome over the course of evolution, and this gene may play an important role in delaying flowering time and providing protective mechanisms against sporadic and extremely low temperatures.

scholarly journals Fine Mapping and Analysis of Candidate Genes for qFT7.1, a Major Quantitative Trait Locus Controlling Flowering Time in Brassica Rapa L

2021 ◽  
Author(s):  
Gaoyang Qu ◽  
Yue Gao ◽  
Xian Wang ◽  
Wei Fu ◽  
Yunxia Sun ◽  
...  
Keyword(s):  
Quantitative Trait Locus ◽  
Flowering Time ◽  
Brassica Rapa ◽  
Quantitative Trait ◽  
Recurrent Parent ◽  
Coding Region ◽  
Late Flowering ◽  
Synonymous Mutations ◽  
Trait Locus ◽  
Delayed Flowering

Abstract In Brassica rapa, flowering time (FT) is an important agronomic trait that affects the yield, quality, and adaption. FT a complicated trait that is regulated by many genes and is affected greatly by the environment. In this study, a chromosome segment substitution line (CSSL), CSSL16, was selected that showed later flowering than the recurrent parent, rapid-cycling inbred line of B. rapa (RcBr). Using Bulked Segregant RNA sequencing, we identified a late flowering quantitative trait locus (QTL), designated as qFT7.1, on chromosome A07 based on a secondary-F2 population derived from the cross between CSSL16 and RcBr. qFT7.1 was further validated by conventional QTL mapping. This QTL explained 39.9% (logarithm of odds = 32.2) of the phenotypic variations and was fine mapped to a 56.4-kb interval using recombinant analysis. Expression analysis suggests that BraA07g018240.3C, which is homologous with ATC (encoding Arabidopsis thaliana CENTRORADIALIS homologue), a gene for delayed flowering in Arabidopsis as the most promising candidate gene. Sequence analysis demonstrated that two synonymous mutations existed in the coding region and numerous bases replacements existed in promoter region between BraA07g018240.3C from CSSL16 and RcBr. The results will increase our knowledge related to the molecular mechanism of late flowering in B. rapa, and lay a solid foundation for the breeding of late bolting in B. rapa.

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