Skip to main content

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

Abstract

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.

Peer Review reports

Background

Leaves are essential organs that play an important role in plants, including carbon assimilation, gas exchange, water transport and nutrient distribution [1]. Leaf shape can significantly affect both leaf function and plant architecture [2, 3]. A typical variation in leaf shape involves the leaf margin, which can be unlobed, serrated or lobed [4]. Lobed leaves can be easily visualized even in the primary leaf stage, which can be used as an indicator trait for hybrid production [5, 6]. Compared to unlobed- or serrated-leaf lines, plants with lobed leaves are better adapted to environmental stresses [7, 8]. With improved heat transfer and light energy absorption, lobed leaves are advantageous for high-density planting and mechanized production [9]. Additionally, lobed leaves are also a graceful decorative trait for ornamental plants such as kale [4].

Ornamental kale (Brassica oleracea L. var. acephala) is an attractive ornamental crop owing to its polymorphic, colorful leaves [10]. Lobed-leaf genes have been genetically analyzed and mapped in some Brassica species. For example, the lobed-leaf trait in B. rapa is controlled by major gene or polygenic effects [11,12,13,14]. In B. napus, the incomplete dominant lobed-leaf gene BnLL1 was mapped to the distal end of chromosome A10 [15]. In ornamental kale, some studies have shown that the lobed-leaf trait exhibits incomplete dominance over the unlobed-leaf trait [16]. Genetic analysis of an interspecific hybrid between B. napus and Rorippa indica (L.) Hiern revealed that the lobed-leaf trait is controlled by a dominant gene [9]. Moreover, Ren et al. mapped a quantitative trait locus (QTL) associated with lobed leaves to chromosome 9 of ornamental kale flanked by insertion-deletion (InDel) markers LYIn39 and LYIn40, with genetic distances of 0.17 cM and 0.11 cM, respectively [4].

With the development of high-throughput sequencing technology and the release of B. oleracea draft genomes [17, 18], a growing number of genes that govern important traits have been mapped in this species. Bulk segregant analysis (BSA) is a rapid and accurate gene mapping method that was first developed and performed in plants [19]. This method is characterized by bulk genotyping of a pool of segregants that share the same phenotype. InDel has been considered as an ideal source for marker design due to its high-density distribution and genotyping efficiency. Using InDel markers, many genes/QTLs have been mapped in B. oleracea, including the yellow-green leaf gene ygl-1 [20], the purple leaf gene BoPr [21], QTLs associated with heading traits [22], male sterility genes [23, 24] and the petal color gene BoCCD4 [25].

Lobed-leaf trait is a unique variation in kale that can be produced by infrequent genetic mechanisms. In the present study, we developed F1, F2 and BC1 populations descended from the ornamental kale inbred line 18Q2513 (with lobed leaves) and 18Q2515 (with unlobed leaves). A rare dominant inheritance pattern was identified for lobed-leaf trait using these populations. Furthermore, the lobed-leaf gene BoLl-1 was fine-mapped to a narrow interval using BSA-seq and linkage analysis. The findings provide new insight into the molecular mechanism underlying leaf shape formation in ornamental kale.

Results

Genetic analysis of leaf shape in ornamental kale

The leaf shape throughout all the F1 plants (comprising 16 individuals) generated by crossing 18Q2513 (lobed-leaf, Fig. 1a) with 18Q2515 (unlobed-leaf, Fig. 1b) was lobed; thus, the lobed-leaf trait is dominant over the unlobed-leaf trait in these two ornamental kale lines. The F2 population comprised 120 individuals, with 92 displaying lobed leaves and 28 unlobed leaves. According to a chi-square test, the segregation ratio is 3:1. The BC1P1 population contained 850 individuals, with 429 lobed-leaf individuals and 421 unlobed-leaf individuals, and the segregation ratio was confirmed to be 1:1 by a chi-square test. The 200 BC1P2 individuals all had lobed leaves (Table 1). These results indicate that the lobed-leaf trait is controlled by a single dominant gene, which was named BoLl-1.

Fig. 1
figure1

Leaf phenotypes of the parental lines. a 18Q2513 with lobed leaves. b 18Q2515 with unlobed leaves. Bar = 4 cm

Table 1 The Chi-square (χ2) goodness-of-fit test ratios of leaf shape segregation in BC and F2 populations

Fine mapping of the BoLl-1 gene by BSA-seq and linkage analyses

To identify markers associated with lobed leaves, the SNP index and Δ(SNP index) between the two bulks were calculated using high-quality SNPs. The average SNP-index and Δ(SNP-index) of the two bulks across a 1-Mb genomic interval were measured using a 10-kb sliding window and plotted against the genome position. The highest peak region, which was considered to be the candidate interval associated with BoLl-1, contains approximately 1.33 Mb (53.34–54.67 Mb) on chromosome 9 according to the ‘TO1000’ reference genome (Fig. 2a). For the candidate region of BoLl-1, 3280 SNPs between parental lines were identified, 410 of which are effective; 593 InDels were identified, 35 of which are effective (Table S1).

Fig. 2
figure2

Fine mapping of the BoLl-1 gene in ornamental kale. a Plot of the Δ(SNP-index) value obtained from the two bulks. The top line indicates the threshold line. The x-axis represents the position of nine chromosomes and the y-axis represents the Δ(SNP-index). b Linkage map of the BoLl-1. The left panel is a genetic map of BoLl-1 in the target region (units: cM). The right panel is the corresponding physical map of BoLl-1 (units: Mb)

To further delineate the location of BoLl-1, 16 InDel and seven SNP markers (by comparing resequencing data of the parents with the sequence of the TO1000 reference genome) within the 1.33-Mb candidate region and its flanking regions (600 kb on each side) were designed. Ultimately, five InDel and three SNP markers showed polymorphisms between the two parents. A total of 429 recessive individuals of the BC1P1 population were subsequently used for BoLl-1 fine mapping.

A linkage map consisting of five InDel and three SNP markers was constructed using MapDraw (Fig. 2b). The SNP markers SL4 and SL6 were found to be tightly linked to BoLl-1, with genetic distances of 0.6 cM and 0.6 cM, respectively. Based on the marker locations in the reference genome, BoLl-1 was ultimately delimited to a 127-kb region (53680797–53,808,289 bp) on chromosome C09.

Prediction and expression analysis of the candidate genes

Based on the ‘TO1000’ reference genome [18], 21 genes were identified within the 127-kb interval (Table 2). According to annotations from the Brassica oleracea genome and BLASTX (best hit) to A. thaliana, only two genes Bo9g181710 and Bo9g181720 are related to the formation of leaf shape. These two genes are homologues of the LATE MERISTEM IDENTITY 1 (LMI1) gene in Arabidopsis, which encode a homeodomain leucine zipper class I (HD-Zip I) meristem identity regulator that plays an important role in leaf morphogenesis and bract formation. Thus, we designated that Bo9g181710 and Bo9g181720 were candidate genes controlling lobed leaf shape in ornamental kale.

Table 2 The 21 putative gene models in the target mapping region

To analyze the expression patterns of Bo9g181710 and Bo9g181720, qRT-PCR was performed using young leaves from 28-day-old seedling of the parents. The expression level of Bo9g181710 in lobed-leaf 18Q2513 was significantly higher than that in unlobed-leaf 18Q2515, whereas no significant difference in Bo9g181720 expression between the parental lines was detected (Fig. 3).

Fig. 3
figure3

Expression patterns of Bo9g181710 and Bo9g181720 as determined by qRT-PCR between 18Q2513 and 18Q2515. BoActin served as the equal loading control. The error bars represent standard errors of three biological replicates. Asterisks represent significant differences (p < 0.01)

Sequence analysis of the candidate genes

To determine the causal relationship between the candidate genes and leaf shape formation, a comparative sequence analysis of the Bo9g181710 and Bo9g181720 genes body (DNA) and ~ 3-kb promoter region was performed using genomic DNA from 18Q2513 and 18Q2515. No sequence variations (between the parental lines) in the coding sequences of Bo9g181710 were detected, while a 1737-bp deletion (1466 bp upstream of the transcription start site), a 92-bp insertion (1466 bp upstream of the transcription start site) and an SNP (765 bp upstream of the transcription start site) were identified within the Bo9g181710 promoter region of 18Q2513 (Fig. 4a). Conversely, no variation was detected in either the promoter or coding regions of Bo9g181720 between the 18Q2513 and 18Q2515. Combined with expression analysis, we speculated that the Bo9g181710 may control the formation of leaf shape in ornamental kale, and renamed it BoLMI1a. In addition, we renamed the Bo9g181720 to BoLMI1b.

Fig. 4
figure4

Gene structure and protein alignment of BoLMI1a. a The BoLMI1a gene structure as well as promoter variations between 18Q2513 and 18Q2515 are shown; horizontal blue arrows represent specific primers for amplifying the promoter and genomic sequences and detecting the promoter variations of BoLMI1a. b Sequence alignment of the BoLMI1a and BoLMI1b proteins and its seven homologues from other cruciferous species. The homeobox domain as well as the leucine zipper domain are indicated

Sequence analysis further revealed that BoLMI1a, which consists of three exons and two introns, encodes a putative 219-amino acid protein containing a homeobox domain and a leucine zipper domain (Fig. 4). Sequence alignment of the BoLMI1a and BoLMI1b proteins and its seven homologues from other cruciferous species revealed that BoLMI1a shared a high degree of similarity with its homologues in B. napus (98.17%) and B. rapa (91.32%) but a relatively lower degree of similarity with BoLMI1b (59.41%) and Camelina sativa (54.92%) (Fig. 4b). Furthermore, a phylogenetic analysis of the BoLMI1a and BoLMI1b proteins and its close homologues was carried out to evaluate their evolutionary relatedness. The results showed that BoLMI1a is closely related to B. napus ATHB-51 and is located in the same clade as other cruciferous plants, indicating that they may be derived from the same ancestor gene (Fig. 5).

Fig. 5
figure5

Phylogenetic analysis of BoLMI1a and BoLMI1b proteins and its 23 homologues from other species. Numbers are bootstrap values

Verification of BoLMI1a-specific markers

Using the co-dominant marker CMLMI1 and the dCAPS marker DMLMI1, we determined whether the variations the BoLMI1a promoter are also present in 118 different cabbage inbred lines (with unlobed leaves) and another ornamental kale inbred line 18Q2523 (with lobed leaves). The results indicated that the insertion, deletion (detected by co-dominant marker CMLMI1) and the SNP (detected by dCAPS marker DMLMI1) were present only in the lobed-leaf ornamental kale inbred line 18Q2523 (Fig. 6; Fig. S1). These markers exhibited 100% accuracy which can be used for marker-assisted selection. Overall, the analyses strongly indicated that the variations in the promoter of BoLMI1a exist only in lobed-leaf ornamental kale inbred lines and may be responsible for the change in leaf shape from unlobed to lobed.

Fig. 6
figure6

Amplicons of the co-dominant marker CMLMI1 in parents and 118 different cabbage inbred lines and ornamental kale inbred lines 18Q2523. M represents the DNA ladder, P1 is lobed-leaf inbred line 18Q2513, and P2 is unlobed-leaf inbred line 18Q2515. Lane 1 is ornamental kale inbred line 18Q2523 with lobed leaves, Lanes 2–119 are 118 different cabbage inbred lines with unlobed leaves

Discussion

In previous studies, the lobed-leaf trait was reported to be controlled by an incomplete gene or a QTL in ornamental kale [4, 16, 26]. In the present study, we analyzed the inheritance of leaf shape using F2 and BC populations derived from a cross of lobed-leaf ornamental kale with unlobed-leaf ornamental kale, showing that the lobed-leaf trait is controlled by a single dominant nuclear-encoded gene.

Ren et al. mapped the lobed-leaf gene BoLl to chromosome 9 of ornamental kale flanked by InDel markers LYIn39 and LYIn40, with genetic distances of 0.17 cM and 0.11 cM, respectively [4]. Two candidate genes, Bol010029/Bo9g181710 and Bol010030/Bo9g1181720, were revealed, but no sequence variations were found in their promoter and coding regions according to the B. oleracea ‘02–12’ (cabbage) [17] and ‘TO1000’ (Chinese kale like) genomes [18]. Therefore, the authors did not conclude which gene controlled the formation of leaf shape in ornamental kale. In our study, based on the ‘TO1000’ genome, the BoLl-1 gene was finely mapped to a 127-kb (53680797–53,808,289 bp) interval on chromosome 9. SNP markers SL4 and SL6 were tightly linked to BoLl-1, flanking the gene at genetic distances of 0.6 cM and 0.6 cM, respectively. Sequence analysis of the parental alleles revealed no sequence variations in the coding sequence of Bo9g181710, whereas three variations were identified in the promoter region. In contrast, no sequence variations were detected in the promoter and coding regions of Bo9g181720. The expression level of Bo9g181710 in lobed-leaf 18Q2513 was significantly higher compared with unlobed-leaf 18Q2515, though the expression level of Bo9g181720 was similar between the parental lines. Thus, we further confirmed that the Bo9g181710 may control the formation of leaf shape in ornamental kale.

In B. napus, Hu et al. reported that a 2624-bp insertion (317 bp upstream of the transcription start site) and three SNPs were identified in the BnA10.LMI1 promoter sequence, along with 12 SNPs in the 3′ flanking sequence, which were considered to be the cause of the lobed-leaf formation [27]. In ornamental kale, the genes that determine leaf shape are not fully understood. Ren et al. mapped the BoLl gene and found no sequence variations in the promoter and coding regions of candidate [4]. In our study, three variations, including an SNP, a 1737-bp deletion, and a 92-bp insertion (765 bp, 1466 bp, and 1466 bp upstream of the transcription start site, respectively) were identified in the BoLMI1a promoter region compared with the ‘TO1000’ reference genome. Through verification analyses of BoLMI1a-specific markers corresponding to the promoter variations revealed that the variations existed only in lobed-leaf ornamental kale inbred lines. These variations may strongly enhance the transcription levels of BoLMI1, thus changing leaf shape from unlobed to lobed.

Leaf shape plays an important role in the reproduction and evolution of plants. Increasing evidence indicates that lobed leaves can improve photosynthesis efficiency and agronomic profitability [7, 28,29,30,31]. LMI1-like genes encoding an HD-Zip I transcription factor have been functionally identified in several plants, and they were reportedly involved in leaf shape formation [27, 32,33,34,35]. For example, Hu et al. [27] identified the BnA10.LMI1 gene, which was responsible for the lobed-leaf shape in Brassica napus. In addition, the BnA10.LMI1 knockout mutations in the HY (with lobed leaves) background were sufficient to produce unlobed leaves. In this study, we identified an LMI1-like gene, BoLMI1a, which was the strong candidate gene underlying the lobed-leaf trait in ornamental kale. Thus, our findings further strengthen the potential for revealing the molecular mechanism underlying leaf shape formation, and we showed that BoLMI1a-specific markers (CMLMI1 and DMLMI1) can be used for marker-assisted selection in ornamental kale breeding.

Conclusions

In this study, the lobed-leaf trait is shown to be controlled by a single dominant gene, BoLl-1, in ornamental kale. The BoLl-1 gene was fine-mapped to a 127-kb fragment. A homologue of Arabidopsis LMI1, BoLMI1a was identified as a strong candidate gene. Three variations were identified in the promoter region of BoLMI1a. The expression of BoLMI1a in lobed-leaf parent 18Q2513 was significantly up-regulated compared with unlobed-leaf parent 18Q2515. This study lays a foundation for cloning BoLMI1a and provides new insight into the formation of leaf shape in ornamental kale.

Methods

Plant materials

The 18Q2513 female parent (P1) is an ornamental kale inbred line with lobed leaves; the 18Q2515 male parent (P2) is an ornamental kale inbred line with unlobed leaves. 18Q2513 was crossed with 18Q2515 to generate an F1 population. An F2 population was generated from self-pollination of the F1 plants; BC1P1 and BC1P2 were then generated by BCs of F1 × 18Q2513, F1 × 18Q2515, respectively.

Additionally, 118 different cabbage inbred lines (with unlobed leaves) and another ornamental kale inbred line 18Q2523 (with lobed leaves), were screened for BoLl-1 promoter variations. All of the plant materials used in the present study were grown in a 25 °C ± 2 °C greenhouse (16 h light/8 h dark photoperiod) at the seedling stage and then transplanted to the field after 1 month. Daily watering and fertilization were performed regularly until the plants enter the flowering stage (about 3 months of vernalization from December to February of the next year). All the plant materials are from the Institute of Vegetables and Flowers, Chinese Academy of Agriculture Sciences (IVFCAAS, Beijing, China).

Genetic analysis and whole-genome resequencing

Leaf shape was investigated visually. Segregation ratios for the F2 and BC1 populations were analyzed by chi-square (χ2) tests using SAS software.

Fifty lobed-leaf BC1 and fifty unlobed-leaf BC1 individuals were selected to construct two bulks. Genomic DNAs were isolated from the individuals within the two bulks and two parental lines using the Plant Genomic DNA Kit (Tiangen, Beijing, China), following the manufacturer’s instructions. The quality of the DNAs was ensured using spectrophotometric analysis and agarose gel electrophoresis. Equally high-quality genomic DNAs from the two bulks and two parental lines were then used to construct paired-end sequencing libraries, which were subsequently sequenced with an Illumina Hi-Seq 2500 sequencer by the Beijing Genomics Institute (BGI) (Shenzhen, China). SNP-index and sliding-window analyses were performed as previously described [36].

Marker development and fine mapping of the BoLl-1 gene

InDel and SNP markers were designed based on candidate region resequencing data for the two parents. Markers were designed with amplicon lengths of 100–180 bp, GC contents of 40–50% and Tm values of 52–58 °C. The markers that were polymorphic between the parents were then used to analyze unlobed-leaf individuals in the BC1P1 populations.

Genomic DNA was extracted from 28-day-old seedling young leaves of the parents and BC1P1 individuals using a modified cetyltrimethylammonium bromide (CTAB) protocol [37]. The DNA concentration was subsequently determined using a spectrophotometer (BioDrop, UK) and adjusted to 40–50 ng/μL.

The 10-μL PCR reaction mixture consisted of 2 μL DNA template, 1 μL 10× PCR buffer (Mg2+ included), 0.8 μL dNTPs (2.5 mM each), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.2 μL Taq DNA polymerase (5 U/μL), and 5.2 μL ddH2O. The reactions were performed in accordance with the follows: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 45 s; and then 72 °C for 10 min. The amplicons were separated by 8% polyacrylamide gel electrophoresis (160 V for 1.2 h), and the gel was stained with silver nitrate.

For each marker, individuals consistent with the 18Q2513 (lobed-leaf) allele, the 18Q2515 (unlobed-leaf) allele, and the F1 allele were categorized as ‘a’, ‘b’, and ‘h’, respectively. Genetic distances between markers were calculated by the Kosambi map function [38], and a genetic map was constructed using MapDraw [39].

Candidate gene analysis

To identify the lobed-leaf gene BoLl-1, genes located within the candidate interval were analyzed based on annotations for the B. oleracea ‘TO1000’ reference genome (http://plants.ensembl.org/Brassica_oleracea/Info/Index) [18]. The expression patterns of candidate genes Bo9g181710 and Bo9g181720 were investigated using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from 28-day-old seedling young leaves of the parents using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s protocol, and PrimeScript™ RT Reagent Kit (Takara, Japan) was used to reverse transcribe cDNA from the total RNA extracted. qRT-PCR was carried out using a CFX96 Real-Time System (Bio-Rad) with SYBR Premix Ex TaqII Reagent Kit (Takara, Japan). Three biological and three technical replicates were included for each experiment. The relative expression level of each gene was calculated using the 2−ΔΔCt method [40]. The qRT-PCR primers used are listed in Table S2, and B. oleracea actin was employed as a control.

Gene-specific markers GL10 (primers GL10-F and GL10-R) and GL20 (primers GL20-F and GL20-R) (Table S2) were used to amplify the promoter and genomic sequences of Bo9g181710 and Bo9g181720, respectively. The resulting PCR products were analyzed by electrophoresis on 1% agarose gels, followed by sequencing and alignment. The co-dominant marker CMLMI1 (primers CMLMI1-F1, CMLMI1-F2 and CMLMI1-R) and the derived cleaved amplified polymorphic sequence (dCAPS) marker DMLMI1 (primers DMLMI1-F and DMLMI1-R) (Table S2) were used to detect variations in the promoter of BoLMI1a in 118 different cabbage and ornamental kale inbred lines.

BLASTP searches were conducted using the amino acid sequence of BoLMI1a to search for homologues within the protein database of the National Center for Biotechnology Information (NCBI) and the B. oleracea reference genome ‘TO1000’. Protein sequence alignment was performed with MAFFT (v7.037) [41]. FastTree (LG + JTT model) was used to construct phylogenetic trees [42].

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. The raw sequencing data used during this study are available in the NCBI SRA database (Accession number: PRJNA729727, https://www.ncbi.nlm.nih.gov/sra/PRJNA729727). The B. oleracea reference genome ‘TO1000’ used in this study can be found at the link: http://plants.ensembl.org/Brassica_oleracea/Info/Index. The A. thaliana genome can be found at the link: https://www.arabidopsis.org/. The protein database of National Center for Biotechnology Information (NCBI) can be found at the link: https://www.ncbi.nlm.nih.gov/. All these databases are open to public access.

Abbreviations

BC:

Backcross

SNP:

Single Nucleotide Polymorphism

LMI1 :

LATE MERISTEM IDENTITY 1

QTL:

Quantitative Trait Locus

InDel:

Insertion/Deletion

BSA:

Bulk Segregant Analysis

dCAPS:

Derived Cleaved Amplified Polymorphic Sequences

NCBI:

National Center for Biotechnology Information

References

  1. 1.

    Tsukaya H. Mechanism of leaf-shape development. Annu Rev Plant Biol. 2006;57:477–96.

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Tsukaya H. Leaf shape: genetic controls and environmental factors. Int J Dev Biol. 2005;49:547–55.

    PubMed  Article  Google Scholar 

  3. 3.

    Nikovics K, Blein T, Peaucelle A, Ishida T, Morin H, Aida M, et al. The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. Plant Cell. 2006;18:2929–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Ren J, Liu Z, Du J, Fu W, Hou A, Feng H. Fine-mapping of a gene for the lobed leaf, BoLl, in ornamental kale (Brassica oleracea L. var. acephala). Mol Breed. 2019;39:40.

    Article  CAS  Google Scholar 

  5. 5.

    Pu HM, Fu SZ, Qi CK, Zhang JF, Wu YM, Gao JQ, et al. Inheritance of divided leaf trait of rapeseed (Brassica napus) and application in hybrid breeding. Chin J Oil Crop Sci. 2001;23:60–2.

    Google Scholar 

  6. 6.

    Zhuang J, Zhou XR, Li SL, Gu LD. Breeding of divided leaf two-type line of recessive genic male sterility in Brassica napus L. Acta Agric Shanghai. 2003;19(2):17–9.

    Google Scholar 

  7. 7.

    Vogel S. Leaves in the lowest and highest winds: temperature, force and shape. New Phytol. 2009;183:13–26.

    PubMed  Article  Google Scholar 

  8. 8.

    Peppe DJ, Royer DL, Cariglino B, Oliver SY, Newman S, Leight E, et al. Sensitivity of leaf size and shape to climate: global patterns and paleoclimatic applications. New Phytol. 2011;190:724–39.

    PubMed  Article  Google Scholar 

  9. 9.

    Tu YQ, Sun J, Dai XL, Tang J, Tu WF, Shao HQ. Character and genetic analysis of lobed-leaf traits in Brassica napus. Chin J Oil Crop Sci. 2013;35:93–6.

    Google Scholar 

  10. 10.

    Schmidt S, Zietz M, Schreiner M, Rohn S, Kroh LW, Krumbein A. Genotypic and climatic influences on the concentration and composition of flavonoids in kale (Brassica oleracea var. sabellica). Food Chem. 2010;119:1293–9.

    CAS  Article  Google Scholar 

  11. 11.

    Song K, Slocum MK, Osborn TC. Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. Campestris). Theor Appl Genet. 1995;90:1–10.

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Kubo N, Saito M, Tsukazaki H, Kondo T, Matsumoto S, Hirai M. Detection of quantitative trait loci controlling morphological traits in Brassica rapa L. Breed Sci. 2010;60:164–71.

    CAS  Article  Google Scholar 

  13. 13.

    Wang Y, Liu X, Ji X, Zhang L, Liu Y, Lv X, et al. Identification and validation of a major QTL controlling the presence/ absence of leaf lobes in Brassica rapa L. Euphytica. 2015;205:761–71.

    CAS  Article  Google Scholar 

  14. 14.

    Kawakatsu Y, Sakamoto T, Nakayama H, Kaminoyama K, Igarashi K, Yasugi M, et al. Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan. Hortic Res. 2021;8:132.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Ni X, Huang J, Ali B, Zhou W, Zhao J. Genetic analysis and fine mapping of the LOBED-LEAF 1 (BnLL1) gene in rapeseed (Brassica napus L.). Euphytica. 2015;204:29–38.

    CAS  Article  Google Scholar 

  16. 16.

    Feng X, Li X, Yang X, Zhu P. Fine mapping and identification of the leaf shape gene BoFL in ornamental kale. Theor Appl Genet. 2020;133:1303–12.

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Liu S, Liu Y, Yang X, Tong C, Edwards D, Parkin IA, et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun. 2014;5:3930.

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Parkin IA, Koh C, Tang H, Robinson SJ, Kagale S, Clarke WE, et al. Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol. 2014;15(6):R77.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. 19.

    Michelmore RW, Paran I, Kesseli RV. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci U S A. 1991;88(21):9828–32.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Liu X, Yang C, Han F, Fang Z, Yang L, Zhuang M, et al. Genetics and fine mapping of a yellow-green leaf gene (ygl-1) in cabbage (Brassica oleracea var. capitata L.). Mol Breed. 2016;36:1–8.

    Article  CAS  Google Scholar 

  21. 21.

    Liu X, Gao B, Han F, Fang Z, Yang L, Zhuang M, et al. Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala). BMC Genomics. 2017;18:230.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Lv H, Wang Q, Zhang Y, Yang L, Fang Z, Wang X, et al. Linkage map construction using InDel and SSR markers and QTL analysis of heading traits in cabbage. Mol Breed. 2014;34:87–98.

    CAS  Article  Google Scholar 

  23. 23.

    Han F, Yuan K, Kong C, Zhang X, Yang L, Zhuang M, et al. Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S. Theor Appl Genet. 2018;131(12):2651–61.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Ji J, Yang L, Fang Z, Zhuang M, Zhang Y, Lv H, et al. Complementary transcriptome and proteome profiling in cabbage buds of a recessive male sterile mutant provides new insights into male reproductive development. J Proteome. 2018;179:80–91.

    CAS  Article  Google Scholar 

  25. 25.

    Zhang B, Han F, Cui H, Li X, Ren W, Fang Z, et al. Insertion of a CACTA-like transposable element disrupts the function of the BoCCD4 gene in yellow-petal Chinese kale. Mol Breed. 2019;39(9):130.

    Article  CAS  Google Scholar 

  26. 26.

    Zhu P, Feng X, Cheng M, Pan Z, University SA. Genetic analysis of feathered-leaved related traits in Brassica oleracea var. acephala. Acta Botan Boreali-Occident Sin. 2016;36(2):0288–95.

    Google Scholar 

  27. 27.

    Hu L, Zhang H, Yang Q, Meng Q, Han S, Nwafor CC, et al. Promoter variations in a homeobox gene, BnA10.LMI1, determine lobed leaves in rapeseed (Brassica napus L.). Theor Appl Genet. 2018;131(12):2699–708.

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Pettigrew WT, Heitholt JJ, Vaughn KC. Gas-exchange differences and comparative anatomy among cotton leaf-type isolines. Crop Sci. 1993;33:1295–9.

    Article  Google Scholar 

  29. 29.

    Chang L, Fang L, Zhu Y, Wu H, Zhang Z, Liu C, et al. Insights into interspecific hybridization events in allotetraploid cotton formation from characterization of a gene regulating leaf shape. Genetics. 2016;204:799–806.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Vuolo F, Mentink RA, Hajheidari M, Bailey CD, Filatov DA, Tsiantis M. Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity. Genes Dev. 2016;30:2370–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Zhu QH, Zhang J, Liu D, Stiller W, Liu D, Zhang Z, et al. Integrated mapping and characterization of the gene underlying the okra leaf trait in Gossypium hirsutum L. J Exp Bot. 2016;67:763–74.

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Andres RJ, Coneva V, Frank MH, Tuttle JR, Samayoa LF, Han SW, et al. Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of upland cotton (Gossypium hirsutum L.). Proc Natl Acad Sci U S A. 2017;114:E57–66.

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Saddic LA, Huvermann B, Bezhani S, Su Y, Winter CM, Kwon CS, et al. The LEAFY target LMI1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER. Development. 2006;133:1673–82.

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Sicard A, Thamm A, Marona C, Lee YW, Wahl V, Stinchcombe JR, et al. Repeated evolutionary changes of leaf morphology caused by mutations to a homeobox gene. Curr Biol. 2014;24:1880–6.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Vlad D, Kierzkowski D, Rast MI, Vuolo F, Ioio RD, Galinha C, et al. Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene. Science. 2014;343:780–3.

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Song J, Li Z, Liu Z, Guo Y, Qiu LJ. Next-generation sequencing from bulked-segregant analysis accelerates the simultaneous identification of two qualitative genes in soybean. Front Plant Sci. 2017;8:919.

    PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8:4321–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Kosambi D. The estimation of map distances from recombination values. Ann Eugenics. 1944;12:172–5.

    Article  Google Scholar 

  39. 39.

    Liu R, Meng J. Map draw: a Microsoft excel macro for drawing genetic linkage maps based on given genetic linkage data. Hereditas. 2003;25(3):317–21.

    PubMed  Google Scholar 

  40. 40.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2CT method. Methods. 2001;25:402–8.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009;26:1641–50.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgments

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31872948). The work was performed in the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China. All funders were not involved in the design, data analysis, and writing of the study.

Author information

Affiliations

Authors

Contributions

Y. Zhang conceived and designed the experiments; B. Zhang, W. Chen and X. Li performed the experiments and analyzed the data; B. Zhang and Y. Zhang wrote and revised the paper; and W. Ren, L. Chen, F. Han, Z. Fang, L. Yang, M. Zhuang, H. Lv, and Y. Wang coordinated and designed the study. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Yangyong Zhang.

Ethics declarations

Ethics approval and consent to participate

All the plant materials are from the Institute of Vegetables and Flowers, Chinese Academy of Agriculture Sciences (IVFCAAS, Beijing, China). The utilization of these plant materials in this study complies with the guidelines and legislation of China.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Details of SNP and InDel variations in the candidate region of BoLl-1.

Additional file 2: Table S2.

Primer sequences of the markers used in this study.

Additional file 3: Figure S1.

Amplicons of the dCAPS marker DMLMI1 in parents and 118 different cabbage inbred lines and ornamental kale inbred lines 18Q2523. M represents the DNA ladder, P1 is lobed-leaf inbred line 18Q2513, and P2 is unlobed-leaf inbred line 18Q2515. Lane 1 is the ornamental kale inbred line 18Q2523 with lobed leaves, Lanes 2–119 are 118 different cabbage inbred lines with unlobed leaves.

Additional file 4: Figure S2.

The original, full-length gel and blot images of Fig. 6. Figure S3. The original, full-length gel and blot images of Fig. S1.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, B., Chen, W., Li, X. et al. Map-based cloning and promoter variation analysis of the lobed leaf gene BoLMI1a in ornamental kale (Brassica oleracea L. var. acephala). BMC Plant Biol 21, 456 (2021). https://doi.org/10.1186/s12870-021-03223-y

Download citation

Keywords

  • Ornamental kale
  • Lobed leaf
  • Fine mapping
  • Promoter variation
  • Enhanced expression