Map-based cloning and promoter variation analysis of the lobed leaf gene BoLMI1a in ornamental kale (Brassica oleracea L. var. acephala)

Background Leaf shape is an important agronomic trait in ornamental kale (Brassica oleracea L. var. acephala). Although some leaf shape-related genes have been reported in ornamental kale, the detailed mechanism underlying leaf shape formation is still unclear. Here, we report a lobed-leaf trait in ornamental kale, aiming to analyze its inheritance and identify the strong candidate gene. Results Genetic analysis of F2 and BC1 populations demonstrate that the lobed-leaf trait in ornamental kale is controlled by a single dominant gene, termed BoLl-1 (Brassica oleracea lobed-leaf). By performing whole-genome resequencing and linkage analyses, the BoLl-1 gene was finely mapped to a 127-kb interval on chromosome C09 flanked by SNP markers SL4 and SL6, with genetic distances of 0.6 cM and 0.6 cM, respectively. Based on annotations of the genes within this interval, Bo9g181710, an orthologous gene of LATE MERISTEM IDENTITY 1 (LMI1) in Arabidopsis, was predicted as the candidate for BoLl-1, and was renamed BoLMI1a. The expression level of BoLMI1a in lobed-leaf parent 18Q2513 was significantly higher compared with unlobed-leaf parent 18Q2515. Sequence analysis of the parental alleles revealed no sequence variations in the coding sequence of BoLMI1a, whereas a 1737-bp deletion, a 92-bp insertion and an SNP were identified within the BoLMI1a promoter region of parent 18Q2513. Verification analyses with BoLMI1a-specific markers corresponding to the promoter variations revealed that the variations were present only in the lobed-leaf ornamental kale inbred lines. Conclusions This study identified a lobed-leaf gene BoLMI1a, which was fine-mapped to a 127-kb fragment. Three variations were identified in the promoter region of BoLMI1a. The transcription level of BoLMI1a between the two parents exhibited great difference, providing new insight into the molecular mechanism underlying leaf shape formation in ornamental kale.

Fine Mapping and Identification of the Lobed-Leaf Gene in Radish (Raphanus Sativus L.)

Leaf shape is one of the important factors affecting the yield and quality of radish (Raphanus sativus L.). In this study, an F2 population obtained from the cross of the lobed-leaf cultivar J4 with the serrated-leaf cultivar WA was used for genetic analysis and gene mapping of the lobed-leaf trait. The lobed-leaf trait is controlled by a single dominant nuclear gene, and dominant over the serrated-leaf trait. Through bulked segregant analysis (BSA) and genotyping by sequencing (GBS), the lobed-leaf gene was initially mapped to the genomic region from 0.66 Mb to 8.19 Mb on chromosome R7. By using nine insertion/deletion (INDEL) markers, the gene was further narrowed down to a 1.53 Mb region. The comparative analysis of the collinear region between Raphanus sativus and Brassica napus identified Rs390250, an HD-Zip I transcription factor as the most probable candidate gene. Phylogenetic analysis supported that Rs390250 is an RCO (REDUCED COMPLEXITY) orthologous gene, and the single nucleotide variation (C425T) of Rs390250 which caused an amino acid substitution from serine (S) to lysine (L) in conserved leucine zipper domain, may destroy its DNA-binding function and be supposed to response for the morphological variation.

Promoter Variations Strongly Enhance the Transcription Level of the BoLMI1 Gene, Causing Lobed Leaves in Ornamental Kale (Brassica Oleracea L. Var. Acephala)

Background: Leaf shape is an important agronomic trait in ornamental kale (Brassica oleracea L. var. acephala). Although some leaf shape-related genes have been reported in ornamental kale, the detailed mechanism underlying leaf shape formation is still unclear. Here, we report a lobed-leaf trait in ornamental kale, aiming to analyze its inheritance and identify the strong candidate gene.Results: Genetic analysis of F2 and BC1 populations demonstrate that the lobed-leaf trait in ornamental kale is controlled by a single dominant gene, termed BoLl-1. By performing whole-genome resequencing and linkage analyses, the BoLl-1 gene was finely mapped to a 127-kb interval on chromosome C09 flanked by SNP markers SL4 and SL6, with genetic distances of 0.6 cM and 0.6 cM, respectively. Based on annotations of the genes within this interval, Bo9g181710, an orthologous gene of LATE MERISTEM IDENTITY 1 (LMI1) in Arabidopsis, was predicted as the candidate for BoLl-1, and was renamed BoLMI1. The expression level of BoLMI1 in lobed-leaf parent 18Q2513 was significantly higher compared with unlobed-leaf parent 18Q2515. Sequence analysis of the parental alleles revealed no sequence variations in the coding sequence of BoLMI1, whereas a 1737-bp deletion, a 92-bp insertion and an SNP were identified within the BoLMI1 promoter region of parent 18Q2513. Verification analyses with BoLMI1-specific markers corresponding to the promoter variations revealed that the variations were present only in the lobed-leaf ornamental kale inbred lines.Conclusions: This study demonstrated that promoter variations strongly enhance the transcription level of BoLMI1 and cause the leaf shape change from unlobed to lobed, providing new insight into the molecular mechanism underlying leaf shape formation in ornamental kale.

An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector

Background: Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, represents an effective technology for the analysis gene functions in plants. However, no VIGS vectors are available for Brassica oleracea till now. The gene silencing vectors TRV, pTYs and CaLCuV were chose to improve the VIGS system in cabbage, using phytoene desaturase (PDS) gene as an efficient visual indicator for VIGS.Results: We successfully silenced the expression of PDS and observed the photobleaching phenomenon in cabbage by pTYs and CaLCuV, with the latter displaying higher efficiency and lower cost. Then, the factors potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including targeting fragment strategy, inoculation method and incubation temperature, were compared. The optimized CaLCuV-based VIGS system is as follows: approximately 500 bp insert sequence, Agrobacterium OD600=1.0, vacuum osmosis method at the bud stage, and the incubation temperature of 22 °C, through which we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected two other genes in cabbage including Mg-chelataseH subunit (ChlH) and lobed-leaf 1 (LL1) to knock down their expression, and we observed the expected yellow or lobed leaves. In addition, we successfully applied the CaLCuV-based VIGS system in two other representative Brassica crops including B. rapa and B. nigra and thus expanded its application scope.Conclusion: These results demonstrate that CaLCuV-based VIGS effectively work in cabbage and can be used for analyzing gene function. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crops.

Stephania polygona (Menispermaceae), a new species from Southern Vietnam

A new species, Stephania polygona N.H. Xia & V.T. Chinh, is described and illustrated from southern Vietnam. It is similar to S. subpeltata and S. elegans in general appearance, but differs by its polygonous lobed leaf blade, five to eight veins, and longer, red purple sepals.