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Article

The Lobed-Leaf Phenotype in Brassica juncea Is Associated with the BjLMI1 Locus as Evidenced Using GradedPool-Seq

by
Wen-Yuan Fu
1,2,
Jiu-Cui Teng
1,
Bing Tang
1,
Qing-Qing Wang
1,
Wei Yang
1,
Lian Tao
1,
Zheng-Jie Wan
2,
Kang-Yun Wu
3,
Guo-Fei Tan
1,* and
Ying Deng
1,*
1
Institute of Horticulture, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
2
Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
3
Institute of Sericulture (Pepper), Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(11), 2696; https://doi.org/10.3390/agronomy12112696
Submission received: 1 September 2022 / Revised: 20 October 2022 / Accepted: 27 October 2022 / Published: 30 October 2022
(This article belongs to the Special Issue Recognition and Utilization of Natural Genetic Resources for Advances in Plant Biology through Genomics and Biotechnology)
Browse Figures
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Abstract

:
The shape of the leaf is the primary phenotype which determines the commercial value of leaf mustard (Brassica juncea). However, there arefew reports on the lobed-leaf gene of B. juncea, and the molecular regulatory mechanisms underlying leaf margin formation are unknown. In this study, an F2 population derived from ‘MN001’ and ‘MU056’ was constructed. Genetic analysis revealed that the lobed-leaf trait is controlled by a major gene, and lobed leavesare dominant compared to round leaves. The GradedPool-Seq analysis combined with the re-sequencing results of parents identified a major interval on chromosome 10 of B. juncea’s genome A. The BjLMI1 gene (BjuA040054) was confirmed to be a candidate gene by gene ontology (GO) analysis, and it is homologous with LMI1 and encodes HD-Zip protein ATHB-51. A base substitution was observed in the conserved domain, and a 63 bp fragment deletion was found in the exon region between the two parents in the CDs region. The expression of BjLMI1 was significantly higher in the lobed-leaf parent than in the round-leaf parent. These findings provide insights into the molecular mechanism underlying leaf margin formation and will be valuable in the development of an ideal leaf shape in B. juncea.

1. Introduction

Leaf mustard (Brassica juncea) is a vegetable crop belonging to Brassica in Cruciferae. It is a naturally doubled allotetraploid after distant hybridization between B. campestris and B. nigra [1]. The leaves are the most important commercial organ of leaf mustard and play an important role in photosynthesis, respiration and photo-perception [2]. Leaf mustard is classified into round leaf, semi-lobed leaf and lobed leaf on the basis of leaf shape. The lobed character is easy to identify and observe, and is inherited stably, which is often used as an ideal morphological marker in leaf mustard breeding. The lobed-leaf character of B. juncea has strong adaptation on chilling stress and water stress, and it can resist high temperature and increase ventilation [3,4,5]. Furthermore, leaf mustard is used as a pickling material, and the petiole accounts for a higher proportion of the whole leaf in lobed leaves, which are beneficial for post-harvest processing. Thus, a better understanding of the molecular regulatory mechanism of lobed leaves in mustard will contribute to the leaf shape and its utilization in production.
Previous studies were mainly focused on the inheritance and gene mapping of lobed leaves. Several studies showed that the inherited character of lobed leaves is controlled by one pair of incomplete dominant genes, and it is dominant over the round leaf [6,7,8,9]. Some lobed leaf-related genes had been finely mapped in the chromosomes of plants. The homeodomain-leucine zipper (HD-Zip) class I transcription factor LATE MERISTEM IDENTITY1 (LMI1, also called ATHB-51) [10] can promote the formation of lobed leaves in A. thaliana [11]. LMI1 expresses in the distalleaf margin, where serrations fail to form in lmi1 recessivemutant leaves [10,11,12,13]. The smooth distal margin of lmi1 leaves is consistent with LMI1 acting as a growth repressor [11]. HD-Zip genes are a subset of homeobox genes, unique to plants, that contain homeodomains with tightly linked leucine zipper motifs [14]. Many HD-Zip proteins are involved in organ development and responses to biotic/abiotic stresses in plants [15,16]. The homologous to LMI1 genes have similar functions in other crops; for example, the LMI1 gene of legume barrel clover can also lead to lobed leaves [13]; Ni et al. (2017) mapped the lobed trait to a 32.1 kb section of B. napus A10, and two AtLMI1-like genes, BnaA10g26320D and BnaA10g26330D, were predicted. Subsequently, by transferring these two candidate genes into A. thaliana, the result showed that BnaA10g26320D is the dominant gene controlling lobed leaves [17]. Bol010029 and Bol010030, two genes homologous to ArabidopsisLMI1, were also identified as the candidate genes for the lobed trait of Brassica oleracea L. var. acephala [18,19]. It is also mapped to a candidate gene, Bra009510, for the lobed trait on B. napus A10, which is highly homologous to AtLMI1 of A. thaliana [8,9]. BnA10LMI1 null mutations in the lobed-leaf background were sufficient to produce unlobed leaves, cis-regulating the development of leaf lobes in B. napus [9]. At present, there are few reports on genes related to the lobed leaves of B. juncea. Heng et al. (2020) cloned a BjRCO gene from chromosome A10 via BSR-seq, which was associated with leaf morphological variation [20].
With the rapid development of plant genome research, the genome of allotetraploid mustard has been sequenced [21], providing a theoretical basis for further study on the formation mechanism of the lobed leaves of allotetraploid crops. In this paper, we present experimental data showing that lobed leavesare controlled by one pair of dominant genes. A GradedPool-seq analysis identified the LMI1-like gene, BjLMI1; the results obtained using qRT-PCR expression allowed us to postulate that BjLMI1 positively regulates HD-Zip protein expression at blade edge, resulting in lobed leaves. Finally, the result of the cloned alleles revealed that a base substitutionin the conserved domain or a fragment deletionin the exon region might lead to the loss of BjLMI1 function.

2. Materials and Methods

2.1. Materials and Phenotypic Data Collection

The materials used in the experiment were the female parent ‘MN001’ and the male parent ‘MU056’. ‘MU056’ is a high-generation inbred line obtained from local resources and purified by multiple-generation inbreeding. ‘MN001’ is a double haploid material obtained through microspore culture, which can be inherited stably after multiple generations of inbreeding [22]. The first filial generation (F1) was obtained by parental hybridization. F1 was selfed to produce an F2 population including 2000 individual plants. A total of 87 populations of the F4 inbred line were produced by the F2 population for two generations. These materials were used for phenotypic data collection and genetic analysis. The parents and F1 plants were used in qRT-PCR analysis, whereas the F2 plants were used in GradedPool-Seq analysis. A total of 102 plants were selected from 87 populations of F4 to verify the lobed-leaf gene. All materials were grown in the field at the Institute of Horticulture, Guizhou Academy of Agricultural Sciences, GuiZhou, China.

2.2. DNA Extraction and Construction of Mixing Pool

The GradedPool-Seq method [23] was used to map the lobed-leaf gene. Genomic DNA was extracted from young healthy leaves of parents and F2 individuals following the cetyltrimethylammonium bromide (CATB) method as described previously [24]. DNA was quantified using a Qubit2.0 fluorometer (Invitrogen Ltd., Paisley, UK) and 1% (v/v) agarose gel electrophoresis with a standard lambda DNA and an ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA). Equal amounts of DNA from 10 parent individuals were bulked for re-sequencing. Equal amounts of DNA from 100 round-leaf individuals were bulked to generate the round-leaf pool, 100 lobed-leaf individuals were bulked to generate the lobed-leaf pool, and 100 semi-lobed-leaf individuals were bulked to generate the semi-lobed-leaf pool for GradedPool-Seq.

2.3. Sequencing Library Construction and High-Throughput Sequencing

The DNA of each sample was fragmented into about 350 bp DNA fragments using Covaris S2/E210. Sheared DNA was end-repaired, and a single-nucleotide (A) overhang was added subsequently to the repaired fragments using Klenow fragment and dATP at 37 °C. Then, barcodes and Illumina sequencing adapters were ligated to the A-tailed fragments using T4 DNA ligase (TaKaRa, Dalian, China). The sequence depth of two parental lines was about 10×, whereas that of three F2 pools was about 30×. PCR was performed using diluted shearing-ligation DNA samples, dNTP, Q5 High-Fidelity DNA Polymerase (New England Biolabs (Beijing) Ltd., Beijing, China) and PCR primers. PCR products were then purified using Agencourt AMPure XP beads and pooled. The pooled samples were separated by 2% (v/v) agarose gel electrophoresis. About 500 bp fragments were excised and purified using the QIA Quick Gel Extraction Kit (Qiagen Inc., Santa Clara, CA, USA). Gel-purified products were then diluted for pair-end sequencing on an Illumina NovaSeq system using standard protocols.

2.4. SNP Identification and Genotyping

Low-quality reads (quality score < 20e) were filtered out, and then raw reads were sorted to each progeny according to barcode sequences. After the barcodes were trimmed from each high-quality read, clean reads from the same sample were mapped onto the B. juncea genome sequence using Burrows-Wheeler Aligner software (BWA, https://bio-bwa.sourceforge.net/, accessed on 20 March 2020) [25]. The latest version of the B. juncea genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_001687265.1, accessed on 20 March 2020) was used for aligning. Samtools software (https://samtools.sourceforge.net/, accessed on 20 March 2020) was used to mark duplicates, and then GATK (https://wiki.rc.usf.edu/index.php/Genome_Analysis_ToolKit_(GATK), accessed on 20 March 2020) was used for local realignment and base recalibration [26]. SNP calling analysis was performed by GATK with default parameters [27]. SNPs identified between the parents were regarded as polymorphic. Only biallelic SNPs were kept in the dataset. The genotypes of the three F2 pools were then recognized.

2.5. GradedPool-Seq Analysis

Association analysis was conducted according to the method of QTL-seq [28]. Briefly, SNP_index was calculated between the aa and ab pools. In the aa pool, SNP_index(aa) = Maa/(Faa + Maa), in which Maa and Faa are the depth of ‘MU056’ and ‘MN001’ origin SNPs, respectively. In the ab pool, SNP_index(ab) = Mab/(Fab + Mab), in which Mab and Fab are the depth of the ‘MU056’ and ‘MN001’ origin SNPs, respectively. Δ(SNP_index) = SNP_index(aa) − SNP_index(ab). The loss fitting of Δ(SNP_index) value was calculated, and the smooth value was obtained [29]. Based on the smooth value of the SLAF (specific-locus amplified fragment) marker from the same chromosome, the loess fitting curve was mapped. The smooth value of 99.8 percentile was taken as the threshold. SNP markers with a smooth value of Δ(SNP_index) greater than the threshold were thought to be associated with the target trait significantly.

2.6. Bioinformatics Analysis of Protein Encoded by Candidate Gene

The physical and chemical properties of the protein encoded by the candidate gene BjLMI1 were analyzed by the online tool ProtParam (http://web.expasy.org/protparam, accessed on 20 March 2020). The protein homology was searched by Blast in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 March 2020). The protein sequences of the leaf mustard BjLMI1 (BjuA040054) gene were compared with the homologous sequences of other plants by MEGA-X software, and the homologous protein evolutionary tree was constructed by neighbor-joining (NJ).

2.7. RNA Extraction and Reverse Transcription

Tissue samples were ground in liquid nitrogen, total RNA was extracted by TRIzol reagent (Invitrogen), and DNase I (Ferments) digestion was performed for 30 min at 25 °C to remove DNA according to manufacturer’s instructions. cDNA was synthesized from 2 mg of total RNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). cDNA was used as a template for cloning target genes and quantitative PCR analysis.

2.8. Gene Cloning

Primer pairs were designed to amplify the full length of the target gene from parents (Table S1). The PCR reactions were performed in a total volume of 26 μL according to PrimeSTAR polymerase (TaKaRa, Dalian, China). The products were electrophoresed through 1% agarose gels and extracted using a gel extraction kit (Qiagen, Valencia, CA, USA). The purified PCR products were then used for “+A base” reactions with rTaq. The 10 μL reaction volume included 0.5 μL of rTaq, 1 μL of 1:3 dNTP, 1 μL of 10× buffer, and the purified PCR products to a final volume of 10 μL. The mixture was heated to 72 °C for 10 min, and the PCR products were subsequently used for TA cloning [30]. Single bacterial colonies were validated by PCR, and triplicate positive clones of each leaf mustard line were sequenced by GeneCreate using the Sanger platform.

2.9. qRT-PCR Analysis of Candidate Gene

qRT-PCR was performed to elucidate the tissue-specific expression of the candidate gene at different times. After the material grew the first true leaf, the leaves until the sixth true leaf were taken. After bolting, the bolting leaves were taken. We chose fresh young leaves from parents and F1 in three biological replicates and froze them in liquid nitrogen.
Primer pairs (BjuA040054-F:5′-TGCTACACGATGAGGTGATGA-3′, BjuA040054-R:5′-GGGTAAGGCTGTTGCGAT-3′) and reference gene (BjuActin-F:5′-TCCATCCATCGTCCACAG-3′, BjuActin-R:5′-GCATCATCACAAGCATCCTT-3′) were subject toqRT-PCR analysis. It was performed with an SYBR Premix (TaKaRa, Dalian, China) in the Bio-Rad iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The values from triplicate reactions were averaged. The threshold cycle value of each gene was investigated and normalized to the Ct value of Cs-Actin to determine relative expression fold differences for each gene during different treatments [31].

2.10. SSR Primer Development and Validation of Candidate Gene

SSR primer sequences were derived from the re-sequencing information of ‘MU056’ and ‘MN001’. Samtools software (https://samtools.sourceforge.net/, accessed on 20 March 2020) was used to detect small repeat sequences with length less than 50 bp, and 15 pairs of SSR primers were designed with Primer Premier 5.0 software. The primers were used for genotype identification of F4 individuals. The DNA of parents, F1 and F4 was extracted using the CTAB method as previously described. The total PCR reaction system was 20 μL. In theband statistical method: the marker consistent with the female parent ‘MN001’ band pattern is a, the marker consistent with the male parent ‘MU056’ band pattern is b, and the heterozygous band pattern is h.

3. Results

3.1. Phenotypic and Genetic Analysis of the Lobed Leaf

According to the investigation and statistics of parents and F1 and F2 plants (Figure 1), all F1 plants had the lobed-leaf phenotype; 478 out of 2000 plants of the F2 population had a round-leaf phenotype, 501 plants were lobed-leaf, and 859 plants were semi-lobed-leaf. The separation ratio of lobed leaves (including semi-lobed leaves) versus round leaves in the F2 generation was 3:1 (χ2 = 0.886, p = 0.346), suggesting that the characteristics of lobed leavesare controlled by a major gene and the lobed leaf is dominant compared to the round leaf.

3.2. Bulked-Segregant-Analysis-Sequencing (BSA-Seq) and GradedPool-Seq Analysis

We constructed three mixed pools for BSA-seq. A total of 92.78 G, 84.71 G and 103.88 G (http://www.ncbi.nlm.nih.gov/bioproject/892164, accessed on 20 March 2020) of valid bases were generated from lobed-leaf, round-leaf and semi-lobed-leaf pools, with corresponding minimum Q30 values of 91.42%, 89.50% and 87.97%, respectively. After eliminating low-quality sequencing data through filtering and quality control, the subsequent analysis was fully guaranteed (Table S2). Two paternal lines, ‘MU056’ and ‘MN001’, were re-sequenced to generate short reads and aligned with the reference genome to obtain two consensus sequences. We used BWA to align mixed-pool reads with the consensus sequences to identify SNPs. Then, the correlation between SNP and lobed leaves was tested by Ridit. The correlation between SNP distribution and lobed leaves was reflected by the p value, and the average p value within a 0.4 Mb window size was computed using a 100 Kb step increment (Figure 2). A single genomic region, harbouring a cluster of SNPs with a high p value, was identified in the 18.4 Mb~19.0 Mb interval on chromosome 10 of B. juncea’s genome A.

3.3. Fine Mapping of the Lobed Leaf Genes

A total of 111 genes were obtained from the 0.6 Mb location interval of the lobed-leaf trait, and 108 genes were annotated by gene ontology (GO) according to the B. juncea genome database. Based on the screening of GO function, one candidate gene, BjuA040054 (https://db.cngb.org/brassica/#/GeneSequence/, accessed on 20 March 2020) ,was finally screened, which contains GO:0009965 (leaf morphogenesis); GO:0010434 (bract formation); GO:0010582 (floral meristem determinacy) and other important functions, and participates in the biological process of GO:0048510 (regulation of timing of transition from vegetation to reproductive phase). BjuA040054 is homologous with the At5G03790 (LATE MERISTEM-IDENTITY1, LMI1) gene of A. thaliana, the Bra009510 gene of B. campestris and the BnaA10g26320D gene of B. napus. Amino acid sequence analysis indicated that BjuA040054 encodes a box leucine zipper protein ATHB-51 containing a homeodomain superfamily domain at amino acid position 78–132, which is related to leaf morphological development. Therefore, BjLMI1 (BjuA040054) may be a candidate gene for the lobed leaf of B. juncea.

3.4. Phylogenetic Analysis of BjLMI1 Protein with Various Plant Species

Through analysis using the online tool ProtParam, the ORFs of BjuA040054 is 660 bp encoding the protein length 219 aa, the theoretical isoelectric point is 7.68, and the protein molecular weight is 2.55 kDa. We downloaded other plants’ amino acid sequences of LMI1 (or ATHB-51) from NCBI. A total of 15 homologous protein sequences from other plant species were downloaded, which contained BnA10.LMI1 (B. napus, GenBank: AWW43724.1) [9], Bra009510 (B. campestris) [8], AtLMI1(A. thaliana, GenBank: OAO91844.1) [10], ChLMI1 (Cardamine hirsuta, GenBank: AHW98968.1) [11], NdLMI1 (Nigella damascena, GenBank: QMT62769.1), CaATHB-51 (Cucurbita argyrosperma, GenBank: KAG7017408.1), CsATHB-51 (Camelina sativa, GenBank: XP_010452275.1), CrATHB-51 (Capsella rubella, GenBank: XP_006289048.1), VvATHB-51 (Vitis vinifera, GenBank: RVX11919.1), Cl ATHB-51 (Camellia lanceoleosa, GenBank: KAI7998737.1), PaATHB-51 (Potentilla anserina, GenBank: XP_050380442.1), SsATHB-51 (Solanum stenotomum, GenBank: XP_049407758.1), and SvATHB-51 (Solanum verrucosum, GenBank: XP_049360093.1). Alignment of these sequences revealed a high degree of conservation. A phylogenetic tree was constructed using the NJ algorithm (Figure 3). B. napus ATHB protein showed the highest percentage of identity in alignment, followed by B. oleracea and B. cretica, and they were clustered together.

3.5. Isolation of BjLMI1Gene

The sequence length of the BjLMI1 gene in the B. juncea genome is 1764 bp, which contains three exons and two introns. The CDs between from the start to stop codon is 660 bp, and the length of protein is 219aa. To detect sequence differences between parents, we designed primer pairs to clone the full length from ‘MU056’ and ‘MN001’. Sequencing indicated that the length of ‘MU056’ and ‘MN001’ was 1753 and 4155 bp, respectively. More bases were inserted into the intron of ‘MN001’ (Table S3). We compared the differences of CDs between parents, 659 and 591 bp in each line, that were 99.85% and 93.63 % similar to the reference sequence; only a single base was detected in ‘MU056’, anda total of 31 single-nucleotide substitutions and 3 base deletions were detected in ‘MN001’, with the longest deletion of 63 bases (Figure 4). We predicted a typical domain through Pfam (http://pfam.xfam.org/search/sequence, accessed on 20 March 2020). A homeobox domain was found between residues 78–132, and a single-base transition was detected in the homeobox domain for MN001, which caused serine to be substituted with leucin. Homeobox was a development-regulating gene belonging to the Hox gene family. The amino acid mutation occurred in this conserved module, leading to changes in the protein structure.

3.6. Tissue-Specific Expression

To further investigate the function of the BjLMI1 gene at different growth stages of leaf mustard, primer pairs were designed based on cDNA sequences to perform qRT-PCR analysis. The expression of BjLMI1 increased first and then decreased from the one-leaf stage to the bolting stage in the parents and F1 leaves (Figure 5A). The gene expression was not high in the one-leaf stage, two-leaf stage and bolting stage, and no significant difference was observed between the materials in the one-leaf stage and two-leaf stage. The gene expression was relatively high at the four- and five-leaf stages, and there was a very significant difference among materials during this period. The male parent ‘MU056’ had the highest gene expression at the five-leaf stage, and the female parent ‘MN001’ and F1 had the highest gene expression at the four-leaf stage. In seven different periods, the expression of genes in lobed leaves was higher than that in round leaves: MU056 > F1> MN001.
Then, the expression of BjLMI1 in the four- and five-leaf stages at the edge of leaves and inside leaves was analyzed using qRT-PCR (Figure 5B). The results showed that the gene expression in the edge of leaves was higher than that inside leaves at the four-leaf and five-leaf stages, and the gene expression at the five-leaf stage was higher than that in the four-leaf stage as a whole. At the same time, the expression of BjLMI1 in the edge of leaves and inside leaves at each stage was MU056 > F1 > MN001. The male parent ‘MU056’ had the highest gene expression at the five-leaf stage, and the gene expression at the edge of the leaf was significantly higher than that inside leaves at the four-leaf and five-leaf stages. The gene expression of the female parent ‘MN001’ was the highest at the five-leaf stage, and no significant difference was observed between each other. However, the gene expression of the leaf edge was significantly higher than that inside leaves at the four-leaf stage. The gene expression of F1 was the highest at the five-leaf stage, and the gene expression at the leaf edge was significantly higher than that inside leaves at the five-leaf stage. No significant difference was observed between each other at the four-leaf stage.

3.7. SSR Primer Sequences

Two parents and F1 were used to screen the polymorphisms of SSR primers. Only the SSR primer No. A10-5 showed obvious polymorphism (indicated by the red arrow in the figure), and F1 had complementary bands of two parents (Figure 6A). The other 14 pairs did not show obvious polymorphism, so the SSR primer numbered A10-5 was selected for subsequent validation. A total of 102 plants were selected from the F4 inbred line and screened by SSR molecular markers (Figure 6B,C). The bands showed that there were 30 plants with lobed leaves, 20 plants with semi-lobed leaves, and 52 plants with round leaves, which were consistent with the phenotypic investigation in the field.

4. Discussion

Lobed leaves in cruciferous vegetables are widely investigated. Understanding the genetic mechanism of lobed leaves plays an important role in changing plant morphology and improving crop yield. Lobed leaves can quickly respond and compete with limited light sources and improve photosynthetic efficiency [32,33]. In this study, the F1 population presented with semi-lobed leaves, and the round leaves were isolated in the F2 generation. The Chi-square test showed that the separation ratio of lobed leaf and round leaf in this population accorded with Mendelian genetic separation of 3:1, in which lobed-leaf contained semi-lobed-leaf plants. Therefore, we speculated that the lobed lead was controlled by a major gene. The inheritance law of this trait was the same in B. napus [7,8,9].
By constructing a large-scale F2 population and adding a middle-mixedpool based on the BSA method, GradedPool-Seq could quickly carry out QTL fine mapping of quantitative traits, which was applied to the study of genes related to the heterosis of rice grain yield for the first time [23]. Combined with GradedPool-Seq analysis, we selected F2, segregating the population to construct three mixed pools, and finally obtained the location interval of the lobed-leaf trait of leaf mustard within a physical distance of 0.6 Mb on chromosome 10 of genome A. Combined with gene annotation screening, a candidate gene, BjLMI1, related to leaf morphogenesis was finally obtained in the target interval. The gene was homologous with LMI1 and encoded HD-Zip protein (homeobox leucine zipper protein) ATHB-51. At the same time, BjLMI was homologous with the Bra009510 gene of B. campestris and the BnaA10g26320D gene of B. napus. We speculated that BjLMI1 was a candidate gene for controlling lobed leaves in leaf mustard.
Leaf growth typically involves an early stage of cell proliferation, followed by cell expansion associated withendoreduplication [34]. Cell proliferation provides new cells for organs; cells are constantly in mitosis;the increase in cell number promotes the further growth of leaf cells; and later, cell expansion controls final leaf size and shape [2,35]. Thetransition from cell proliferation to cell expansion in leaf cells is oftenassociated with increased levels of endoreduplication, analternative cell cycle in which the nuclear genome is replicatedin the absence of cell division [36]. Endoreduplication is considerably more widespread in plants, and contributes to the control of differentiation andmorphogenesis of various cell types. LMI1 and its putative homologous genes were considered to be the evolutionary key genes of leaf development. Previous studies had shown that the homologous gene RCO also plays a key role in leaf shape diversity in Arabidopsis, B. napus and C. hirsuta [11,17,37], but the loss of RCO in Arabidopsis leads to leaf simplification, while LMI1 positively regulates leaf margin formation. LMI1 encodes a homeobox-leucine zipper protein ATHB-51, which belongs to HD-Zip I protein, mainly expressed in thedistalleaf margin [10,11,12,16]. HD-Zips have four distinct subfamilies (I–IV), many of which have been found to be essential for plant development [14,15]. Some HD-Zips III proteins, such as ATHB14, ATHB9, REVOLUTA, and MIR165/166 are key regulators of adaxial domain formation in lateral organs such as leaves [38,39,40]. Vuolo et al. (2018) demonstrated that LMI1-activated endoreduplication regulated lobed leaf form by locally restricting cell proliferation. In proliferative tissues, endoreduplication reduced cell division and, ultimately, initial cell number, and caused cells to change from proliferative to expansive growth such that cell size increased but tissue growth was reduced [41]. Another gene, ATHB12 in the HD-Zip I subfamily, had a similar function, which promoted leaf growth by positive regulators of endoreduplication and cell expansion [42]. Quantitative expression analysis confirmed that the sequence and expression of this gene were different between the parents. BjLMI1 was highly expressed in lobed leaves and less expressed in round leaves, whereas the LMI1 gene was mainly expressed in the leaf margin. We speculated that during the initial stage of leaf development, BjLMI1 activated endoreduplication, resulting in the inhibition of cell proliferation in the leaf margin;endoreduplication reduces cell number, and this loss exceeds what can be compensated for by the subsequent expansive growth in cell size so the leaves show as lobed.By cloning the full length of the BjLMI1 gene, there was a base substitution in the conserved domain and a 63 bp fragment deletion in the exon region of MN001. Compared to MU056, substitution induced changes in the conserved domains of amino acid or fragment deletion, leading to the loss of BjLMI1 function, the leaves showed a round phenotype. Additionally, in other crops, for example, a tandem duplication of the LMI1 promoter resulted in increased expression and deep lobed leaf margin in cotton [43]. In rapeseed, a large insertion in the promoter of LMI1 also caused lobed leaves [9]. These findings suggest that BjLMI1 plays an active role in lobed-leaf formation.In the present study, we provide evidence that BjLMI1 positively regulates leaf-lobe formation in B. juncea, providing new insights into the molecular mechanism of leaf-lobe formation in Brassica crops. Subsequently, we will carry out transgenic verification on this gene and clone the newly discovered gene into round leaves in order to confirm the association between phenotype and genotype.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12112696/s1, Table S1. Primer sequences for gene cloning and SSR primer sequences; Table S2. Summary of BSA sequencing data for each sample; Table S3. Cloning of full length sequences of candidate genes.

Author Contributions

W.-Y.F., G.-F.T. and Y.D. conceived and designed the experiments; J.-C.T., B.T., Q.-Q.W. and W.Y. performed the experiments, prepared figures and/or tables; W.-Y.F. analyzed the data and wrote the manuscript; Z.-J.W., K.-Y.W., L.T., G.-F.T. and Y.D. proposed amendments to the first draft. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 3196180192), Guizhou Science and Technology Support Plan (No. 2022-086), Guizhou Science and Technology Support Plan (No. 2020-1Y090), Subsidy Fund of National Natural Science Foundation of Guizhou Academy of Agricultural Sciences (No. 2021-046), Guizhou Construction of Genetics and Breeding Laboratory of Vegetable Industry System (No. 2022-0102), Guiyang Science and Technology Cooperation Plan (No. 2021-5-1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phenotypic analysis of the lobed leaf. Representative photographs of lobed leaf in leaf mustard. (A) Parents and F1 generation; (B) F2 generation segregation phenotype.
Figure 1. Phenotypic analysis of the lobed leaf. Representative photographs of lobed leaf in leaf mustard. (A) Parents and F1 generation; (B) F2 generation segregation phenotype.
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Figure 2. BSA-seq and GradedPool-seq analyses identified the lobed-leaf locus. Note: the abscissa is the chromosome position coordinate, and the ordinate is the ratio of the points whose p value is lower than the threshold value to the total number of points in the window. The higher the position of the point, the closer the correlation with the trait. The threshold of p value is 10−10.
Figure 2. BSA-seq and GradedPool-seq analyses identified the lobed-leaf locus. Note: the abscissa is the chromosome position coordinate, and the ordinate is the ratio of the points whose p value is lower than the threshold value to the total number of points in the window. The higher the position of the point, the closer the correlation with the trait. The threshold of p value is 10−10.
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Figure 3. Phylogenetic relationships among ATHB proteins from different plant species. Phylogenetic tree inferred using the NJ method. Numbers indicate the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The tree is drawn to scale, and branch lengths indicate evolutionary distance. Evolutionary analyses were conducted in MEGA-X.
Figure 3. Phylogenetic relationships among ATHB proteins from different plant species. Phylogenetic tree inferred using the NJ method. Numbers indicate the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The tree is drawn to scale, and branch lengths indicate evolutionary distance. Evolutionary analyses were conducted in MEGA-X.
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Figure 4. Comparison of candidate gene sequences between parents. Base substitutions and deletions were indicated in gray and white. The red line indicated a conserved domain.
Figure 4. Comparison of candidate gene sequences between parents. Base substitutions and deletions were indicated in gray and white. The red line indicated a conserved domain.
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Figure 5. Tissue-specific expression of BjLMI1 gene. (A) Relative expression of BjLMI1 in different stages of parents and F1 leaves measured by qRT-PCR. (B) Relative expression of BjLMI1 in four-leaf stage and five-leaf stage at the edge of leaves and inside leaves. Values are mean ± SD (n = 3 biological and 3 technical replicates). Letters indicate p < 0.01 (Student’s t-test).
Figure 5. Tissue-specific expression of BjLMI1 gene. (A) Relative expression of BjLMI1 in different stages of parents and F1 leaves measured by qRT-PCR. (B) Relative expression of BjLMI1 in four-leaf stage and five-leaf stage at the edge of leaves and inside leaves. Values are mean ± SD (n = 3 biological and 3 technical replicates). Letters indicate p < 0.01 (Student’s t-test).
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Figure 6. SSR primer screening and candidate gene verification. (A) Amplification of the SSR primers in parents and F1; (B,C) SSR primer sequences validation of BjLMI1 gene. M: 2000 bp DNA ladder; P1: MN001; P2: MU056. The plant numbers of the F4 inbred line were 1~3 to 100~102.
Figure 6. SSR primer screening and candidate gene verification. (A) Amplification of the SSR primers in parents and F1; (B,C) SSR primer sequences validation of BjLMI1 gene. M: 2000 bp DNA ladder; P1: MN001; P2: MU056. The plant numbers of the F4 inbred line were 1~3 to 100~102.
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Fu, W.-Y.; Teng, J.-C.; Tang, B.; Wang, Q.-Q.; Yang, W.; Tao, L.; Wan, Z.-J.; Wu, K.-Y.; Tan, G.-F.; Deng, Y. The Lobed-Leaf Phenotype in Brassica juncea Is Associated with the BjLMI1 Locus as Evidenced Using GradedPool-Seq. Agronomy 2022, 12, 2696. https://doi.org/10.3390/agronomy12112696

AMA Style

Fu W-Y, Teng J-C, Tang B, Wang Q-Q, Yang W, Tao L, Wan Z-J, Wu K-Y, Tan G-F, Deng Y. The Lobed-Leaf Phenotype in Brassica juncea Is Associated with the BjLMI1 Locus as Evidenced Using GradedPool-Seq. Agronomy. 2022; 12(11):2696. https://doi.org/10.3390/agronomy12112696

Chicago/Turabian Style

Fu, Wen-Yuan, Jiu-Cui Teng, Bing Tang, Qing-Qing Wang, Wei Yang, Lian Tao, Zheng-Jie Wan, Kang-Yun Wu, Guo-Fei Tan, and Ying Deng. 2022. "The Lobed-Leaf Phenotype in Brassica juncea Is Associated with the BjLMI1 Locus as Evidenced Using GradedPool-Seq" Agronomy 12, no. 11: 2696. https://doi.org/10.3390/agronomy12112696

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