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.

Simultaneous changes in anthocyanin, chlorophyll, and carotenoid contents produce green variegation in pink–leaved ornamental kale

Background Anthocyanin, chlorophyll, and carotenoid pigments are widely distributed in plants, producing various colors. Ornamental kale (Brassica oleracea var. acephala DC) which has colorful inner leaves is an ideal plant to explore how these three pigments contribute to leaf color. The molecular mechanisms of the coloration in ornamental kale could provide reference for exploring the mechanisms of pigmentation in other plants. Results In this study, we sequenced the transcriptome and determined the pigment contents of an unusual cultivar of ornamental kale with three different types of leaf coloration: pink (C3), light pink (C2), and variegated pink–green (C1). A total of 23,965 differentially expressed genes were detected in pairwise comparisons among the three types of leaves. The results indicate that Bo9g058630 coding dihydroflavonol 4–reductase (DFR) and Bo3g019080 coding shikimate O–hydroxycinnamoyltransferase (HCT) acted in anthocyanin biosynthesis in pink leaves. Bo1g053420 coding pheophorbidase (PPD) and Bo3g012430 coding 15–cis–phytoene synthase (crtB) were identified as candidate genes for chlorophyll metabolism and carotenoid biosynthesis, respectively. The transcription factors TT8, MYBL2, GATA21, GLK2, and RR1 might participate in triggering the leaf color change in ornamental kale. Anthocyanin content was highest in C3 and lowest in C1. Chlorophyll and carotenoid contents were lowest in C2 and highest in C1. Conclusions Based on these findings, we suspected that the decrease in anthocyanin biosynthesis and the increase in chlorophyll and carotenoid biosynthesis might be the reason for the leaf changing from pink to variegate pink–green in this unusual cultivar. Our research provides insight into the molecular mechanisms of leaf coloration in ornamental kale, contributing to a theoretical foundation for breeding new varieties.

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.

Calcium nitrate on growth and ornamental traits at salt-stressed condition in ornamental kale (Brassica oleracea L. var. Acephala)

Ornamental plants are used to improve the aestheticquality of urban and rural landscapes, recreational areas, interiorscapes and commercial sites. Any negative effects of salts on ornamental plant growth have to be taken into consideration mainly for their negative influences on aesthetic value of ornamental plants. The research was conducted to determine the effects of different concentrations of calcium nitrate on some stress parameters (i.e. leaf relative water content, chlorophyll, stomatal conductance, ion leakage in the leaf tissues) and some plant growth parameters (total leaf number, number of dried leaf, main stem diameter, plant crown diameter, plant height, fresh and dry weight of root) of ornamental kale (Brassica oleracea L. var. acephala) plant grown under saline conditions. Four doses of NaCl (NaCl1:30, NaCl2:60, NaCl3:90, NaCl4:120 mM), three doses of Ca(NO3)2 (CaN1:5, CaN2:10, CaN3:15 mM), groups combined together in all doses of NaCl and Ca (NO3)2 and control (NaCl0:0 + CaN0:0) were applied into the pots with three replications. Ca (NO3)2 was applied to the leaf surface of the plants in the pots to be applied as Ca (NO3)2 spray. When the plants reached a certain maturity (two weeks after transfer to the pot), salt stress applications were started. Ca (NO3)2 treatments have applied after one week from salt stress applications. Calcium nitrate treatements applied under salt stress conditions had positive effects and increasing in effect on all parameters examined except for chlorophyll and ion leakage in leaf tissues of the plant. With increasing in calcium nitrate concentration, morphological and physiological characteristics of plants were observed increases in general. It was concluded that the increase of the number of leaves by NaCl2+CaN3 application can be minimized with CaN3 (with 15 mM Ca (NO3)2) stress caused by 60 mM salt concentration According to the results of the research, calcium nitrate applications can be recommended for ornamental kale plants grown under salt stress conditions especially limiting the negative effects of stress and to minimize such damage.

Fine mapping of a candidate gene for cool-temperature-induced albinism in ornamental kale

Background The symptoms of cool-temperature-induced chlorosis (CTIC) are widely existed in higher plants. Although many studies have shown that the genetic mechanism of CTIC is generally controlled by recessive genes in model plants, the dominant inheritance of albinism has not been reported thus far. Here, two CTIC mutants, Red Kamome and White Kamome, were utilized to analyse the inheritance of the albino trait in ornamental kale. The objective of this investigation is to fine-map the target locus and identify the most likely candidate genes for albinism. Results Genetic analysis revealed that the albinism in the inner leaves of ornamental kale followed semi-dominant inheritance and was controlled by a single locus in two segregating populations. BSR-seq in combination with linkage analysis was employed to fine-map the causal gene, named AK (Albino Kale), to an approximate 60 kb interval on chromosome C03. Transcriptome data from two extreme pools indicated that the differentially expressed gene of Bol015404, which encodes a cytochrome P450 protein, was the candidate gene. The Bol015404 gene was demonstrated to be upregulated in the albino leaves of ornamental kale by qPCR. Additionally, the critical temperature for the albinism was determined between 10 °C and 16 °C by gradient test. Conclusions Using two independent segregating populations, the albino mutants were shown to be controlled by one semi-dominant gene, AK, in ornamental kale. The Bol015404 gene was co-segregated with albinism phenotypes, suggesting this unknown function P450 gene as the most likely candidate gene. The albino trait appeared caused by the low temperatures rather than photoperiod. Our results lay a solid foundation on the genetic control of albinism in ornamental kale.