Promoter variations in a homeobox gene, BrLMI1, contribute to leaf lobe formation in Brassica rapa ssp. chinensis Makino

doi:10.21203/rs.3.rs-2851677/v1

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Promoter variations in a homeobox gene, BrLMI1, contribute to leaf lobe formation in Brassica rapa ssp. chinensis Makino

https://doi.org/10.21203/rs.3.rs-2851677/v1

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published 14 Aug, 2023

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Leaves are the main consumed organ in leafy non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis Makino), and the shape of the leaves is an important economic trait. However, the molecular regulatory mechanism underlying lobed-leaf trait in non-heading Chinese cabbage remains unclear. Here, we identified a stable incompletely dominant major locus qLLA10 for lobed leaves formation in non-heading Chinese cabbage. Based on map-based cloning strategies, BrLMI1, a LATE MERISTEM IDENTITY1 (LMI1)-like gene, was predicted as the candidate gene for qLLA10. Genotyping analysis showed that promoter variations of BrLMI1 in two parents are responsible for elevating the expression in the lobed-leaf parent and ultimately causing the alternation in leaf shape between the two parents, and the promoter activity of BrLMI1 was significantly affected by the promoter variations. BrLMI1 was exclusively localized in the nucleus and expressed mainly at the tips of each lobe. Leaf lobe development was perturbed in BrLMI1-silenced plants produced by virus-induced gene silencing assays, and ectopic overexpression of BrLMI1 in Arabidopsis led to deeply lobed leaves never seen in the wild type, which indicates that BrLMI1 is required for leaf lobe formation in non-heading Chinese cabbage. These findings suggested that BrLMI1 is a positive regulatory factor of leaf lobe formation in non-heading Chinese cabbage and that cis-regulatory variations lead to the phenotype of lobed or entire leaf margins, thus providing the theoretical basis for unraveling the molecular mechanism underlying lobed leaves phenotype in Brassica crops.

Leaf lobe formation

HD-Zip transcription factor

cis-regulatory variations

non-heading Chinese cabbage

BrLMI1 is a positive regulatory factor of leaf lobe formation in non-heading Chinese cabbage, and cis-regulatory variations lead to the phenotype of lobed or entire leaf margins

Non-heading Chinese cabbage, originating in China, is an important economic vegetable crop worldwide and includes several groups of varieties. As a leafy vegetable, the leaves are the main organ of consumption and nutrition in non-heading Chinese cabbage (Nugrahedi et al. 2015). The leaf shape of this crop is a major factor affecting leaf function and plant architecture and ultimately the yield and quality of the crops (Karamat et al. 2021). Leaf shape variations mainly involve in the shape of the leaf margins, including entire, serrated or deeply lobed. Compared with entire- or serrated-leaf plants, deeply lobed-leaf plants seem to be better adapted to environmental stress due to the optimized canopy structure (Peppe et al. 2011). In practice, as an ideal indicator trait, leaf lobes can be used in crops breeding, especially in hybrid production and purity identification (Tu et al. 2013). Thus, mining novel genes controlling leaf morphogenesis and revealing the molecular mechanism regulating leaf shape development will contribute to leaf shape improvement and utilization in non-heading Chinese cabbage breeding.

Leaf shape development and regulation involve complex crosstalk, including various environmental signals, plant hormones and complex genetic networks (Nikovics et al. 2006). Through forward and reverse genetic studies, a few genes involved in lobed leaf formation have been characterized, such as the class I KNOTTED-LIKE HOMEOBOX (KNOX1) family, encoding homeodomain transcription factors, and upregulation of KNOX1 genes in leaves contributes to the lobes or leaflets formation (Bhatia et al. 2021). LATE MERISTEM IDENTITY1 (LMI1), encoding a homeodomain leucine zipper (HD-Zip) transcription factor, is necessary for serrated-leaf formation in Arabidopsis, and the lobed leaves phenotype in cotton, watermelon, rapeseed, and legume barrel clover is also regulated by polymorphic LMI1-like genes (Wang et al. 2021). The REDUCED COMPLEXITY (RCO) gene, another HD-Zip I transcription factor, is required for leaflet formation in Cardamine hirsute (C. hirsuta) (Vlad et al. 2014). NAM-ATAF1/2-CUC2 (NAC) transcription factors are important in controlling leaf margin boundary formation (Hibara et al. 2006). Overexpression of CUP-SHAPED COTYLEDON2 (CUC2) and CUC1 and CUC3, two other members of the NAC gene family, leads to the lobed leaves development (Takada et al. 2001). Teosinte branched1/Cycloidea/Proliferating cell factor (TCP) transcription factors regulate leaf shape and leaf size by directly regulating leaf development-related specific genes, miRNAs and plant hormones (Yu et al. 2021).

Brassica species have extensive leaf morphology diversity, and the genetic patterns of leaf lobe phenotype in several Brassica crops have been reported. In rapeseed, a major QTL BnLLA10 affecting leaf lobe formation was detected on chromosome A10, BnA10.RCO and BnA10.LMI1 were proven to determine the formation of lobed leaves (Hu et al. 2018; 2020). In ornamental kale, the feathered-leaf phenotype is regulated by candidate gene BoALG10, which encodes an α-1,2 glycosyltransferase (Feng et al. 2020; 2022). In Brassica rapa, a quantitative trait locus (QTL) at chromosome A10 was considered to be the major locus controlling lobed leaves formation (Wang et al. 2015). The altered expression of BrTCP15 resulted in a change in leaf shape (Kawakatsu et al. 2021). Although some QTLs related to lobed leaves were identified on chromosomes A02, A03, A06, A08, and A10 (Kubo et al. 2010), no additional genes responsible for lobed leaf formation have been identified, and the molecular regulatory mechanisms involved in leaf morphological diversity have yet to be elucidated in B. rapa.

In this study, a stable major QTL qLLA10 for lobed leaf formation was identified by traditional QTL mapping based on different environmental conditions and genetic backgrounds. After that, we confirmed the function of BrLMI1 in regulating leaf shape variation using turnip yellow mosaic virus (TYMV)-induced gene silencing (VIGS) technique in the lobed-leaf parent and ectopic overexpression in Arabidopsis, and identified that the BrLMI1 promoter variations are associated with different leaf shapes between the two parents. The co-segregating BrLMI1-specific insertion and deletion (InDel) marker can be used in molecular marker-assisted selection (MAS) breeding. Overall, these results provide a theoretical foundation for leaf shape improvement in non-heading Chinese cabbage.

Plant materials and mapping populations

Three non-heading Chinese cabbage inbred lines, Jingshuicai (JSC), Heiyebaicai (HYBC) and Wutacai (WTC), were used to develop segregating populations for lobed leaves. The female parent JSC shows deeply lobed leaves, and the two male parents HYBC and Wutacai WTC have entire leaves. JSC was crossed with HYBC and WTC to generate two F₁ hybrids that were self-crossed to obtain the F₂ populations for phenotype and genetic analysis, respectively.

An F₂ segregating population developed from the JSC × HYBC cross (239 individuals, designated 2015JH) was used for traditional QTL mapping for the lobed-leaf trait and was planted in the Tongzhou experimental farm in the autumn of 2015. Subsequently, another F₂ population from lobed-leaf JSC and entire-leaf WTC (506 individuals, designated 2017JW) and the larger JSC×HYBC F₂ population (890 individuals, designated 2018JH) were constructed for QTL verification and fine mapping of lobed leaves, which were cultivated at the central farm in spring 2017 and winter 2018, respectively.

The lobed leaf phenotype was investigated on mature leaves at the rosette stage using a 1–9 scale (1, 3, 5, 7, 9): 1 = entire leaf, 5 = semilobed leaf, and 9 = lobed leaf, which were largely similar to HYBC/WTC, F₁ and JSC. Scale 3 is an intermediate phenotype between scale 1 and scale 5, and scale 7 is an intermediate phenotype between scale 5 and scale 9.

QTL mapping of lobed-leaf trait

Molecular markers, including small InDel and single nucleotide polymorphism (SNP) markers, were developed based on the comparison of resequencing data of three parents, and were used to analyze individuals of the F₂ population 2015JH (Li et al. 2020). Genetic linkage analysis was performed by JoinMap 4.0 based on the genotype data of 2015JH population (Van et al. 2006). The map distances were calculated using Kosambi mapping function (Kosambi, 1943). The presence of QTLs was detected by MapQTL 5.0 via a composite interval mapping (CIM) procedure at a threshold of LOD = 3.0 (Van et al. 2004).

To narrow down the preliminary QTL region, extreme recombinants form 2018JH F₂ population were screened by two flanking markers. To fine map the qLLA10, additional SNP markers (Table S1) in the target interval of qLLA10 were designed and used for genotyping the recombinants as described by Li et al. (2020).

Expression level analysis of BrLMI1

The BrLMI1 expression patterns were examined. Various segments of the fifth leaves from two parents at the rosette stage were sampled and total RNA was extracted using TRIzol reagent. The cDNA synthesis was performed using SuperScript Reverse Transcriptase (Invitrogen). A Roche thermocycler (LightCycler480, Roche, Switzerland) was used to perform quantitative real-time PCR. The GAPDH gene in Chinese cabbage and ACTIN2 gene (At3g18780) in Arabidopsis were selected as the internal controls. Three independent biological and technical replicates were designed for each qRT-PCR reaction.

Phylogenetic analysis of BrLMI1

The homologs of BrLMI1 proteins were searched against the NCBI, Brassica database (BRAD) and TAIR database. Multiple sequence alignments were carried out by DNAMAN, and neighbor-joining phylogenetic tree was constructed using MEGA 6.0 software with 1000 bootstrap replicates.

Promoter activity analysis of BrLMI1

The 2736-bp and 2000-bp sequences of BrLMI1 promoter from JSC and HYBC were PCR-amplified and recombined with EcoRI linearized luciferase (LUC) vector CP461. Both recombination vectors were transferred into Agrobacterium GV3101 and then injected into Nicotiana benthamiana leaves as described by Liang et al (2022). After 48–96 h, the LUC activity was analyzed by a Dual-Luciferase Assay System (Promega, VPE1910).

Subcellular localization of BrLMI1

The coding DNA sequence (CDS) was amplified from JSC and cloned into the binary vector pSAT6-EYFP-N1 digested by EcoRI and SalI. The 35S::BrLMI1-GFP and 35S::GFP vectors were transferred into mesophyll protoplasts of Chinese cabbage as described by Li et al (2018). The green fluorescent protein (GFP) signals were subsequently observed under a laser scanning confocal microscope (Zeiss LSM 700).

GUS staining

The BrLMI1 promoter (2736-bp) was PCR-amplified from JSC and subcloned into the pMDC162 vector. The proBrLMI1::GUS plasmid was transformed into wild-type Arabidopsis plants, and homozygous T₃proBrLMI1::GUS positive lines were used as subsequent experimental materials. The samples from proBrLMI1::GUS positive lines was immersed in GUS staining buffer (Beijing, Huayueyang), incubated at 37°C overnight in darkness and then washed with 75% (v/v) ethanol until the chlorophyll was completely removed.

Functional analysis of BrLMI1

For overexpression vector construction, the CDS of BrLMI1 was amplified from JSC and recombined with KpnI / SpeI linearized binary vector pCAMBIA1301 driven by the CaMV35S promoter. The recombinant 35S::BrLMI1 plasmid was then transformed into wild-type Arabidopsis via Agrobacterium tumefaciens strain GV3101as previously described (Li et al. 2018)

For knockout assays, the VIGS method was adopted to silence BrLMI1 in lobed-leaf JSC, as described previously (Zhang et al. 2020), with appropriate modifications. A 40-nt sequence specific to BrLMI1 was selected, and the corresponding 80-nt palindromic sequence with SnaBI restriction site was synthesized by Sangon Biotech and ligated into pTY-S vector. Two recombinant pTY-BrLMI1 plasmids were generated and transformed into competent Super Stbl3 for large quantities using Plasmid Giga Kits (OMEGA, United States). Two or three-week-old JSC seedlings were used for inoculation. Firstly, silicon carbide powder was sprinkled onto the fully expanded leaves surface, and then tiny wounds were made through gentle friction using index finger. Secondly, 10 µl solution of purified plasmids with working concentration (300–500 µg/µl) was added to each leaf and spread evenly using fingers. After 2 minutes, the treated leaves were washed with water. About three fully expanded leaves per JSC seedling were inoculated. Infiltrated plants were incubated in darkness for 24 h and then placed in a greenhouse with a temperature regimen of 25℃ day/18℃ night. The seedlings treated with pTY-S plasmid and ddH₂O were set as positive and negative controls, respectively. The second virus infiltration was carried out a week later. To obtain silencing effect, three or four infiltrations were needed. The phenotypes were observed 10 days after the last infection.

Phenotypic characterization and inheritance of lobed-leaf trait

To investigate the genetic patterns underlying the lobed-leaf trait in non-heading Chinese cabbage, two crosses between a parent line (JSC) with deeply lobed-leaf and two entire-leaf parent lines (HYBC and WTC) were conducted. All F₁ plants exhibited semilobed leaves that were similar to those of the lobed-leaf parent JSC, indicating that the lobed leaves phenotype is an incompletely dominant character (Fig. 1a, b; Fig. S1). The lobe complexities in the three F₂ populations (2015JH, 2017JW, 2018JH) showed continuous variation (Fig. 1c, d, e), implying that the lobed leaves phenotype in JSC is quantitatively inherited.

Classical QTL mapping identifies BrLMI1 as a candidate lobed-leaf gene

To map the potential loci controlling lobed leaves in non-heading Chinese cabbage, we performed classical QTL mapping for lobed leaves. A major QTL associated with lobed leaves, qLLA10 (lobed leaf A10), was identified at an LOD threshold of 3.0 using composite interval mapping (CIM). qLLA10 was located in the 18.07–20.29 Mb region flanked by SNP markers A10_18069317 and A10_20293321 on chromosome A10 (Fig. 2a, Table S2), and explained the maximum phenotypic variation (47.8%). To assess qLLA10 in a different genetic background, we examined 2017JW F₂ population derived from the JSC ×WTC cross and found that qLLA10 was in the same chromosome interval as in 2015JH and explained 40% of phenotypic variation (Table S2), indicating that qLLA10 is a major QTL for lobed-leaf trait, which was consistently detected in multiple genetic and environmental backgrounds.

To narrow down the qLLA10 locus, individuals from the larger JSC×HYBC F₂ population (2018JH) were screened using flanking markers, and 27 extreme recombinants were identified (Table S3). Using six newly developed SNP makers between A10_18069317 and A10_20293321 (Table S1), we genotyped these extreme recombinants, which were divided into 11 types according to their genotypes. As a result, qLLA10 was narrowed down to a 149-kb interval flanked by two SNP markers A10_19974351 and A10_20135947 on chromosome A10 (Fig. 2b).

Within the 149-kb interval of qLLA10, 24 genes were annotated based on the Brassica database. According to the comparative genomic annotation in Arabidopsis thaliana (TAIR database) (Table S4), only two tandemly duplicated genes, BraA10g032440.3C (hereafter BrRCO) and BraA10g032450.3C (hereafter BrLMI1), are predicted to be related to leaf shape diversity, and they both encode HD-Zip I transcription factors (Fig. S3). BraA10g032440.3C is the homolog of RCO, which is required for leaflet formation in C. hirsuta (Vlad et al. 2014). BraA10g032450.3C is a homolog of LMI1 in Arabidopsis, which participates in the formation of leaf serration (Saddic et al. 2006).

We further compared the genomic DNA sequences between JSC and HYBC, including the ~ 3-kb promoter sequence, the gene body, and the 2-kb 3′ flanking region. Multiple sequence alignment revealed that their coding sequences are precisely the same, while a 736-bp long segment insertion and a 4-bp oligonucleotide deletion exist at the − 1301 bp and − 1398 bp promoter region of BrLMI1 in JSC compared with HYBC, respectively. (Fig. 2c, Fig. S4). Nonetheless, no variation was detected in the coding or promoter sequences of BrRCO. Therefore, BrLMI1 was considered to be the candidate gene involved in lobed leaf formation in non-heading Chinese cabbage.

BrLMI1 expression pattern and subcellular localization

Next, we investigated the detailed expression patterns of BrLMI1 in JSC and HYBC. In JSC, leaf samples were collected from three segments, including leaf tips of each lobe (#1) and two small regions at the base of terminal (#2) and lateral (#3) leaflets of the fifth rosette leaf. In HYBC, the corresponding leaf base (#1) and leaf margin (#2) (Fig. 3a) were also sampled. qRT-PCR results indicated that BrLMI1 expression was very low in all leaf segments in HYBC, whereas BrLMI1 was expressed in nearly all leaf segments in JSC, with the highest expression at the tips of each lobe (Fig. 3b). These findings revealed that the differential expression of BrLMI1 may be associated with the formation of leaf lobes.

We transformed the proBrLMI1::GUS vector into Arabidopsis plants (Col-0), and homozygous T₃ generation transgenic positive lines were used for further staining. BrLMI1 expression was readily observed in all tissues in the seedling stage (Fig. 3c). In slightly older and still expanding leaves, BrLMI1 expression was more pronounced in the leaf margins and was very strong in the serrations (Fig. 3d, e). In the bolting and flowering stage, the margins and serrations in the bracts were stained (Fig. 3f, g). In blooming flowers, BrLMI1 was expressed in the margins of sepals, petals, and carpels (Fig. 3h).

For subcellular localization observation, the recombinant p35S::BrLMI1-GFP plasmid was constructed and transiently expressed in Chinese cabbage protoplasts. The fluorescent signals of p35S::BrLMI1 were exclusively localized in the nucleus as shown in Fig. 3i.

Verification of the function of BrLMI1 in regulating leaf lobe formation

To further verify the BrLMI1 function, a 35S::BrLMI1 vector overexpressing the CDS of BrLMI1 driven by the CaMV35S promoter was constructed and transformed into Arabidopsis Col-0 plants. Approximately 13 independent hygromycin-resistant T₂ plants were obtained. BrLMI1 expression levels in three representative lines were detected by qRT-PCR, which showed that BrLMI1 expression in overexpression lines was significantly upregulated compared with the wild type. All the transgenic lines produced highly lobed leaves that have never been seen in the wild type (Fig. 4a, b, c, d). Additionally, BrLMI1 was involved in other organs development of transgenic lines. Some 35S::BrLMI1 overexpression lines exhibited delayed bolting and significantly increased rosette leaves. In the reproductive stage, the numbers of bracts and secondary inflorescences in some lines also significantly increased (Fig. S5). These results indicated that BrLMI1 showed pleiotropic effects on the development of plant organs.

We further confirmed the functions of BrLMI1 in regulating lobed leaf formation in non-heading Chinese cabbage by using VIGS system in lobed-leaf JSC. To construct the recombinant pTY-BrLMI1 vectors, target sequences of BrLMI1 were inserted into the pTY-S vector and the high-concentration purified recombinant pTY-BrLMI1 plasmids were used to infect seedlings. In BrLMI1-silenced seedlings, the expression level of BrLMI1 was nearly five times downregulated. Three representative plants with lower BrLMI1 expression levels showed a visibly reduced lobed-leaf phenotype compared with controls (Fig. 4e, f, g, h). Taken together, these findings showed that BrLMI1 positively regulates the formation of lobed leaves in non-heading Chinese cabbage.

Verification of BrLMI1 -specific markers

To explore whether the sequence variations in BrLMI1 promoter are responsible for leaf shape variability, the promoter activity of BrLMI1 in JSC and HYBC was tested by transient transcription activity assays. As shown in Fig. 5, the promoter activity of BrLMI1^JSC was significantly higher than that of BrLMI1^HYBC. Eleven lobed leaf lines and 82 non-lobed leaf lines in the natural population were randomly selected for promoter variation identification of BrLMI1 to further verify the promoter variation correlated with the phenotypes. The results indicated that the 736-bp insert was present only in the lines with lobed leaves (Fig. S6). These results strongly demonstrated that the cis-regulatory variations in BrLMI1 played a vital role in determining leaf margin morphology in non-heading Chinese cabbage.

Leaf shape is both a complex and commercially important developmental trait in leafy vegetables that can be affected by genetic and environmental factors. In this study, we revealed a dominant gene BrLMI1 that regulates leaf lobe formation in non-heading Chinese cabbage, and the cis-regulatory diversification of BrLMI1 was the most conspicuous variation between the two parents. The significantly upregulated expression levels and higher promoter activity of BrLMI1 in JSC suggested that the promoter variations of BrLMI1 are directly related to leaf lobe information in B. rapa. These findings further strengthen the evidence that LMI1 and its putative paralogs are evolutionary hotspots for leaf shape diversification in plants. For instance, in upland cotton, there is a tandem duplication in GhLMI1-D1b promoter, which led to the upregulated expression and occurrence of lobe leaf margin (Andres et al. 2017). The variations in promoters of BnA10.LMI1 are responsible for lobed leaves formation in rapeseed (Hu et al. 2018). In ornamental kale, the promoter variations of BoLMI1a lead to different expression patterns in two parents (Zhang et al. 2021). In Cucurbita pepo, CpDll expression was affected by promoter sequence variations, which is positively correlated with lobed leaves development (Bo et al. 2022).

LMI1 is a direct target of LEAFY, and activates CAULIFLOWER (CAL) expression together with LEAFY. Besides, LMI1 also functions in bracts and serrations formation (Vuolo et al. 2018). Compared with the wild type, the lmi1 mutants produce entire leaves without serrations, significantly increased numbers of bracts and secondary inflorescences, whereas no significant alteration was founded in rosette leaves number or flowering time (Saddic et al. 2006). In Medicago truncatula, MtLMI1s regulate leaf shape formation by activating the expression of SMOOTH LEAF MARGIN1 (SLM1) (Wang et al. 2021). As a transcription factor, the BrLMI1 protein was localized in the nucleus in our study, which indicated that BrLMI1 may be involved in leaf shape diversity by regulating downstream target genes. However, we have not clarified which promoter variation is responsible for expression alterations of BrLMI1 in the two parents. Further exploration of upstream regulatory factors will facilitate the identification of key promoter variations that affect BrLMI1 transcription. In addition, mining the downstream target genes and interacting proteins will be necessary for unraveling the regulatory network of BrLMI1 regulating leaf development. Moreover, the delayed bolting and significantly increased numbers of rosette leaves and bracts in BrLMI1 overexpression lines were observed, which were similar to those of lmi mutants in Arabidopsis (Fig. S5). These different phenotypes probably reflect that the regulatory network of BrLMI1 maybe different from that of LMI1 in leaf lobe development.

Class I HD-Zip transcription factors have been proven to participate in the development of many plant organs, especially in leaf development. For instance, ATHB1 was found to affect true leaf development and hypocotyl elongation when overexpressed in tobacco (Aoyama et al. 1995). The overexpression plants of HAHB4, an HD-Zip transcription factor in sunflower, produced rounder leaves compared with the wild type (Dezar et al. 2005). Overexpression of ATHB13 resulted in altered development of cotyledons and leaves (Hanson et al. 2001). ATHB16 negatively regulates leaf cell expansion, and transgenic plants with increased ATHB16 expression exhibit smaller leaves and reduced flowering time (Wang et al. 2003). These results indicated that BrLMI1 and other HD-Zip I transcription factor genes are highly pleiotropic in regulating the development of plant organs. In addition, all these results also suggested that HD-Zip I transcription factor genes may have a conservative function in regulating leaf lobe information going through dicotyledon. Further investigation of additional HD-Zip I transcription factor members will help uncover the complex network forming lobed leaf.

Previous reports indicated that RCO, another HD-Zip I transcription factor, is required in shaping leaf diversity in C. hirsuta, Arabidopsis lyrata, B. juncea and B. napus (Sicard et al. 2014; Vlad et al. 2014; Heng et al. 2020; Hu et al. 2020). In our study, BrRCO and BrLMI1 were both in the target region of qLLA10, and the evolutionary relationship of the two genes is close, whereas there was no variation in the coding or promoter regions of BrRCO between the two parents. Therefore, whether BrRCO plays a vital role in modulating leaf lobe formation in non-heading Chinese cabbage remains an intriguing question.

The lobed-leaf phenotype can only be observed when the true leaf is fully unfolded, which makes traditional breeding challenging, and high-throughput genotyping methods are needed. InDel markers have been extensively used in the identification of germplasm resources in B. oleracea (Liu et al. 2017; Han et al. 2018)d rapa (Zhang et al. 2018). However, to make the InDel marker more effective in actual crop breeding, the InDel sequences should be functional; otherwise, a high proportion of false breeding materials would be selected. In B. oleracea, a BoLMI1a-specific marker designed according to promoter variations was developed and used for marker-assisted selection in leaf shape improvement (Zhang et al. 2021). Here, the InDel in the BrLMI1 promoter strongly affected the transcription levels and promoter activity of BrLMI1 in parent lines. These results indicated that the insert is functional and the BrLMI1-specific InDel markers responding to it can be used for marker-assisted selection in non-heading Chinese cabbage.

In conclusion, we revealed that BrLMI1 was a positive regulator of leaf lobe formation in non-heading Chinese cabbage and that the regulatory network of BrLMI1 may be different from that of LMI1 in leaf lobe development. In addition, promoter modifications to BrLMI1 were responsible for the functional variation between two parents. Overall, our findings provide theoretical foundation for unraveling the molecular mechanism of lobed leaves phenotype in Brassica crops.

Author contribution statement

SY, ZW, PL, and FZ designed the experiments. PL, LW, and YW performed the experiments. PL and ZW analyzed the data. PL, HL and ZW wrote the manuscript. TS and SY constructed the mapping populations.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (No. 32272714), the Young Talent Award of Beijing Agricultural and forestry Science and Science Innovation Program (KYCX202001-07), the Youth Foundation of Beijing Academy of Agriculture and Forestry Sciences (QNJJ202239), the Innovation and Capacity-Building Project of BAAFS (KJCX20230123 KJCX20230126, KJCX20230403) and the China Postdoctoral Science Foundation (2019T120066). We are grateful to Dr. Jinghua Yang (Zhejiang University) for his help in VIGS assays.

Data availability

The datasets and plant materials generated during this study are available in the article and supplementary materials.

Conflict of interest The authors declare that there are no conflicts of interest.

Ethics approval The authors note that these experiments complied with the ethical standards for scientific conduct.

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Supplementarytables.xlsx
Table S1 Primers used for fine mapping, gene amplification, qRT-PCR, vector construction and inDel markers. Table S2 Quantitative trait loci (QTL) for leaf lobe detected in different F₂ populations. Table S3 Genotypes and phenotypes of the extreme recombinants used for fine mapping. Table S4 Annotated genes in the 149-kb region between A10_19974351 and A10_20135947.
SupplementaryFigures.pdf
Fig. S1 Characteristics of leaf lobes in JSC, WTC and their F₁ hybrids. Fig. S2 QTL likelihood curves of the LOD score of lobed leaves phenotype in F₂populations. Fig. S3 Phylogenetic tree of BrRCO (BraA10g032440.3C) and BrLMI1(BraA10g032450.3C) with their orthologs and paralogs from Arabidopsis. Fig. S4 (a) Genomic sequence, (b) amino acid sequence and (c) promoter sequence alignment of BrLMI1 in the JSC and HYBC genomes. Fig. S5 BrLMI1 showed pleiotropic effects on the development of plant organs. LMI1 overexpression transgenic lines exhibited highly lobed leaves and delayed bolting time (a) and an increased number of bracts (b), rosette leaves (c), and secondary inflorescences (d). Fig. S6 InDel marker designed based on the deletion in the promoter of BrLMI1 showing co-segregation with the lobed-leaf trait in parents and 93 different inbred lines. The red line indicates inbred lines with lobed leaves.

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Promoter variations in a homeobox gene, BrLMI1, contribute to leaf lobe formation in Brassica rapa ssp. chinensis Makino

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Abstract

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Key message

Introduction

Materials and methods

Plant materials and mapping populations

QTL mapping of lobed-leaf trait

Subcellular localization of BrLMI1

GUS staining

Results

Phenotypic characterization and inheritance of lobed-leaf trait

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

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