Molecular mapping and candidate gene identification of two major quantitative trait loci associated with silique length in oilseed rape (Brassica napus L.).

Rapeseed is a significant global source of plant oil. Silique size, particularly silique length (SL), impacts rapeseed yield. SL is a typical quantitative trait controlled by multiple genes. In our previous study, we constructed a DH population of 178 families known as the 158A-SGDH population. In this study, through SL QTL mapping, we identified twenty-six QTL for SL across five replicates in two environments. A QTL meta-analysis revealed eight consensus QTL, including two major QTL: cqSL.A02-1 (11.32-16.44% of PVE for SL), and cqSL.C06-1 (10.90-11.95% of PVE for SL). Based on biparental resequencing data and microcollinearity analysis of target regions in Brassica napus and Arabidopsis, we identified 11 candidate genes at cqSL.A02-1 and 6 candidate genes at cqSL.C06-1, which are potentially associated with silique development. Furthermore, transcriptome analysis of silique valves from both parents on the 14th, 21st, and 28th days after pollination (DAP) combined with gene function annotation revealed three significantly differentially expressed genes at cqSL.A02-1, BnaA02G0058500ZS, BnaA02G0060100ZS, and BnaA02G0060900ZS. Only the gene BnaC06G0283800ZS showed significant differences in parental transcription at cqSL.C06-1. Two tightly linked insertion-deletion markers for the cqSL.A02-1 and cqSL.C06-1 loci were developed. Using these two QTL, we generated four combinations: A02SGDH284C06158A, A02SGDH284C06SGDH284, A02158AC06158A, and A02158AC06SGDH284. Subsequent analysis identified an ideal QTL combination, A02158AC06SGDH284, which exhibited the longest SL of this type, reaching 6.06 ± 0.10 cm, significantly surpassing the other three combinations. The results will provide the basis for the cloning of SL-related genes of rapeseed, along with the development of functional markers of target genes and the breeding of rapeseed varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01464-x.

CD93 regulates breast cancer growth and vasculogenic mimicry through the PI3K/AKT/SP2 signaling pathway activated by integrin ß1.

In women, breast cancer (BC) accounts for 7%-10% of all cancer cases and is one of the most common cancers. To identify a new method for treating BC, the role of CD93 and its underlying mechanism were explored. MDA-MB-231 cells were used in this study and transfected with si-CD93, si-MMRN2, oe-CD93, si-integrin ß1, or oe-SP2 lentivirus. After MDA-MB-231 cells were transfected with si-NC or si-CD93, they were injected into nude mice by subcutaneous injection at a dose of 5 × 106/mouse to construct a BC animal model. The expression of genes and proteins and cell migration, invasion and vasculogenic mimicry were detected by RT‒qPCR, western blot, immunohistochemistry, immunofluorescence, Transwell, and angiogenesis assays. In pathological samples and BC cell lines, CD93 was highly expressed. Functionally, CD93 promoted the proliferation, migration, and vasculogenic mimicry of MDA-MB-231 cells. Moreover, CD93 interacts with MMRN2 and integrin ß1. Knockdown of CD93 and MMRN2 can inhibit the activation of integrin ß1, thereby inhibiting the PI3K/AKT/SP2 signaling pathway and inhibiting BC growth and vasculogenic mimicry. In conclusion, the binding of CD93 to MMRN2 can activate integrin ß1, thereby activating the PI3K/AKT/SP2 signaling pathway and subsequently promoting BC growth and vasculogenic mimicry.

Sef1, rapid-cycling Brassica napus for large-scale functional genome research in a controlled environment.

KEY MESSAGE: We demonstrated a short-cycle B. napus line, Sef1, with a highly efficient and fast transformation system, which has great potential in large-scale functional gene analysis in a controlled environment. Rapeseed (Brassica napus L.) is an essential oil crop that accounts for a considerable share of global vegetable oil production. Nonetheless, studies on functional genes of B. napus are lagging behind due to the complicated genome and long growth cycle, this is largely due to the limited availability of gene analysis and modern genome editing-based molecular breeding. In this study, we demonstrated a short-cycle semi-winter-type Brassica napus 'Sef1' with very early-flowering and dwarf phenotype, which has great potential in large-scale indoor planting. Through the construction of an F2 population of Sef1 and Zhongshuang11, bulked segregant analysis (BSA) combined with the rape Bnapus50K SNP chip assay method was used to identify the early-flowering genes in Sef1, and a mutation in BnaFT.A02 was identified as a major locus significantly affecting the flowering time in Sef1. To further investigate the mechanism of early flowering in Sef1 and discover its potential in gene function analysis, an efficient Agrobacterium-mediated transformation system was established. The average transformation efficiency with explants of hypocotyls and cotyledons was 20.37% and 12.8%, respectively, and the entire transformation process took approximately 3 months from explant preparation to seed harvest of transformed plants. This study demonstrates the great potential of Sef1 for large-scale functional gene analysis.

Optimizing glyphosate tolerance in rapeseed by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing.

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.

Genome-wide identification of cold responsive transcription factors in Brassica napus L.

BACKGROUND: Cold stress is one of the primary environmental factors that affect plant growth and productivity, especially for crops like Brassica napus that live through cold seasons. Till recently, although a number of genes and pathways involved in B. napus cold response have been revealed by independent studies, a genome-wide identification of the key regulators and the regulatory networks is still lack. In this study, we investigated the transcriptomes of cold stressed semi-winter and winter type rapeseeds in short day condition, mainly with the purpose to systematically identify the functional conserved transcription factors (TFs) in cold response of B. napus. RESULTS: Global modulation of gene expression was observed in both the semi-winter type line (158A) and the winter type line (SGDH284) rapeseeds, in response to a seven-day chilling stress in short-day condition. Function analysis of differentially expressed genes (DEGs) revealed enhanced stresses response mechanisms and inhibited photosynthesis in both lines, as well as a more extensive inhibition of some primary biological processes in the semi-winter type line. Over 400 TFs were differentially expressed in response to cold stress, including 56 of them showed high similarity to the known cold response TFs and were consistently regulated in 158A and SGDH284, as well as 25 TFs which targets were over-represented in the total DEGs. A further investigation based on their interactions indicated the critical roles of several TFs in cold response of B. napus. CONCLUSION: In summary, our results revealed the alteration of gene expression in cold stressed semi-winter and winter ecotype B. napus lines and provided a valuable collection of candidate key regulators involved in B. napus response to cold stress, which could expand our understanding of plant stress response and benefit the future improvement of the breed of rapeseeds.

Mapping of Two Major QTLs Controlling Flowering Time in Brassica napus Using a High-Density Genetic Map.

Research on the flowering habit of rapeseed is important for the selection of varieties adapted to specific ecological environments. Here, quantitative trait loci (QTL) for the days-to-flowering trait were identified using a doubled haploid population of 178 lines derived from a cross between the winter type SGDH284 and the semi-winter type 158A. A linkage map encompassing 3268.01 cM was constructed using 2777 bin markers obtained from next-generation sequencing. The preliminary mapping results revealed 56 QTLs for the days to flowering in the six replicates in the three environments. Twelve consensus QTLs were identified by a QTL meta-analysis, two of which (cqDTF-C02 and cqDTF-C06) were designated as major QTLs. Based on the micro-collinearity of the target regions between B. napus and Arabidopsis, four genes possibly related to flowering time were identified in the cqDTF-C02 interval, and only one gene possibly related to flowering time was identified in the cqDTF-C06 interval. A tightly linked insertion-deletion marker for the cqFT-C02 locus was developed. These findings will aid the breeding of early maturing B. napus varieties.