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Article

Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

by
Yonghui Zhao
1,
Chuanhong Liu
1,
Bing Fang
2,
Shengnan Huang
1,
Nan Wang
1,
Chong Tan
1,
Jie Ren
1 and
Hui Feng
1,*
1
Department of Horticulture, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang 110866, China
2
Foreign Language Teaching Department, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(11), 1086; https://doi.org/10.3390/horticulturae8111086
Submission received: 17 October 2022 / Revised: 15 November 2022 / Accepted: 15 November 2022 / Published: 17 November 2022
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Versions Notes

Abstract

:
Leafy head formation is one of the most important characteristics of Chinese cabbage, and the process is regulated by a series of genes and environmental factors. In this study, a non-heading short leaf mutant slm was identified from an ethyl methane sulfonate mutagenesis (EMS) population of the heading Chinese cabbage line FT. The most significant phenotypic characteristics of slm was shortening leaves and increasing leaf numbers, which led to failure to form a leafy head. Genetic analysis showed that a single recessive gene Brslm was responsible for the mutant phenotype. Mutmap analysis suggested that Brslm was located on chromosome A07, and four candidate genes were predicted. KASP analysis demonstrated that BraA07g039390.3C was the target gene of the candidates. BraA07g039390.3C is a homologous to Arabidopsis CLV1 encoding receptor kinase with an extracellular leucine-rich domain. Sequencing analysis revealed that a single SNP from G to A occurred in 904th nucleotide of Brclv1, which resulted in the change of the 302nd amino acid from Asp to Asn. The SNP was co-segregated with the mutant phenotype in F2 individuals and located on the conserved domains. These results indicated that BrCLV1 was the mutant gene for slm which led to shortening leaves and increasing leaf numbers, disrupting the leafy heading formation in FT. These findings contribute to revealing the BrCLV1 function in leafy head formation in Chinese cabbage.

1. Introduction

Leafy head is the major product of Chinese cabbage (Brassica rapa L. ssp. pekinensis), while its formation and development are crucial characteristics in variety improvement. The four stages were summarized as seedling, rosette, folding and heading during the leafy head development, while the leaf shape, size and physiological function were different between these stages [1,2]. At the seedling stage, the primary leaves were flat and round with a longer petiole, mainly for photosynthesis [3]. At the rosette stage, the leaves expanded larger and upward growth, the petioles became shorter, and leaf wings began to develop [4]. At the folding stage, the outer leaves grew rapidly and started to bend inward, while the inner leaves differentiated and germinated from the shoot apical meristem (SAM). At the heading stage, leaves were longer, wrinkled and folded, and partly overlapped, accompanied by the gradual development of leaf wings, but the outer leaves bent inwards, which directly formed the outline of the leafy heads. The leaf primordium continued to split into new leaves and fulfilled the leafy head. The leafy head of Chinese cabbage is composed of leaves, and the specific shape and size of the leaves including length, width and angle are essential for leafy head formation [5,6,7].
Since leafy head is the primary agronomic characteristic of Chinese cabbage, its formation has been explored for decades. Some studies found that the formation of leaf head was closely related to leaf adaxial-abaxial (ab-ad) polarity development [1,8,9]. The differential growth rate of the abaxial and adaxial surfaces of the leaves contributes to leaf curvature and influences the leaf heads formation [2,6,10]. Some ad-ab polarity pathway genes has been reported which is involved in leafy head formation in Brassica rapa like BcpLH, as well as transcription factors BrpTCP4-1 and BrpSPL9-2 [2,6,9]. Studies have shown that auxin regulated speed and the direction of plant growth with uneven distribution in the abaxial and adaxial surfaces of leaves leads to the upward bending growth of leaves [11,12,13]. The hormone level in plants was crucial for leafy heads formation [8,14,15,16]. Li et al. [17] identified a non-heading mutant (fg-1) in heading Chinese cabbage. Their RNA-sequencing analysis showed that several auxin transports (BrAUX1, BrLAXs, and BrPINs), and responsive genes of auxin and ABA were significant differential expressions. Gao et al. [18] showed that a mutation in ent-kaurene synthase (KS), which is involved in gibberellin biosynthesis, resulted in a non-heading phenotype in Chinese cabbage. In addition, carbohydrate accumulation and sugar levels also play key roles in leafy heads formation [14,19].
SAM, acting as a stem cell reservoir, is the basis of plant shoot development and generates various organs through cell division and differentiation [20]. SAM can differentiate and form leaf primordia and flower primordia [21,22,23,24,25]. At Arabidopsis, the WUSCHEL (WUS)- CLAVATA (CLV) signaling pathway was a considerable SAM maintenance pathway. CLV1, a receptor kinase with an extracellular leucine-rich domain acting as extracellular signals, interacted with CLV3 along as a ligand-receptor pair in a signal transduction pathway by influencing the expression pattern of WUS to coordinate growth among adjacent meristematic regions and control the balance between meristem cell proliferation and differentiation. With respect to Arabidopsis, CLV1 affected the leaf shape and numbers at the rosette stage [26]. The double homozygous mutant of BnCLV3 exhibited more leaves than wild-type in Brassica napa [27]. However, there was less information on the leaf development function of CLV1 in Brassica rapa.
We identified a non-heading mutant slm with shortening leaves and increasing leaf numbers from an EMS mutagenesis population of Chinese cabbage double haploid (DH) line FT [28]. The mutant phenotype was controlled by a single recessive gene Brslm. Mutmap and KASP analysis proved that the target gene of Brslm was BraA07g039390.3C, which was a homologous to Arabidopsis CLV1 encoding the receptor kinase with an extracellular leucine-rich domain. Those results provide novel insights into understanding leafy head formation and highlighting the new functions of CLV1 in Chinese cabbage.

2. Materials and Methods

2.1. Plant Materials

FT, a DH heading Chinese cabbage line generated via microspore cultures, was used as wild-type for EMS mutagenesis. After immersing the germinated FT seeds in 0.8% EMS for 12 h, the seeds were washed in running water and planted. M0 plant was self-pollinated to generate M1. slm (short leaf mutant) was isolated from the M1 generation population and kept a stable mutant phenotype in multi-generation inbred lines. All plants were grown in a greenhouse at Shenyang Agriculture University (25 ± 3 °C day, 15 ± 3 °C night).

2.2. Genetic Analysis of slm

FT and slm were the two parents used to construct a population for genetic analysis. FT (P1) and slm (P2) were crossed and generated F1 (FT × slm) and rF1 (slm × FT). The F1 plant self-pollinated and produced F2 population. F1 backcrossed with FT and slm, respectively, and produced BC11 (F1 × FT) and BC12 (F1 × slm). The phenotype and segregation ratios of each population was recorded and analyzed based upon the chisquare (χ2) test.

2.3. Mutmap Analysis

The modified Mutmap method was used to map the mutant gene of mutant slm [29]. Fifty mutant phenotype individuals of the F2 population were selected to construct a DNA mixed pool. The DNA of two parents and F2 DNA mixed pool were extracted by DNA Secure Plant Kits (Tiangen, Beijing, China) to construct a DNA library with an insertion fragment of 400bp. These DNAs libraries were sequenced by next-generation sequencing (NGS) based on IlluminaHiSeq. After quality control analysis of the data, the raw data was filtered to generate clean data. The standard of data filtering mainly comprised joint pollution removal, quality filtering and length filtering. The clean data was then mapped to the reference genome (brassicadb.org/brad/datasets/pub/Genomes/Brassica_rapa/V3.0/) with a Burrows–Wheeler Aligner (BWA) [30]. SNP and INDEL were detected using GATK software [31] and annotated by ANNOVAR [32]. Before calculating the SNP index, the population SNP was further filtered. The filtering steps were as follows: (1) The SNP sites were retained, which were homozygous and different from the parents slm and FT; (2) The SNP sites conforming to the base mutation type (G-A, C-T) induced by EMS were retained; (3) Finally, 1106 SNP loci were obtained on 10 chromosomes of Brassica rapa, and then the SNP index was calculated based on the parental FT genotype. The distribution map of SNP index on chromosome was drawn based on a sliding-window analysis, which took 5 SNPs as the window size and 1 SNP as each step, calculated SNP index in each window, and took the position of reference genome as abscissa.

2.4. Kompetitive Allele Specific PCR (KASP)

KASP was employed to detect the SNP genotype of candidate SNP. Based on the result of the Mutmap analysis, allele-specific primers for four candidates SNP were designed and listed in Table S1. Two parents, F1 plant, F2 individuals of 99 mutant phenotype individuals and 90 wild-type phenotype individuals were used as templates to perform amplification. The PCR system and the procedure were described in Wang et al. [33].

2.5. Clone Sequencing

The promoter and full-length sequence of BrCLV1 in slm and FT was sequenced. The primers were designed using Primer Premier 5.0 software (Premier Inc., Charlotte, NC, USA), as shown in Table S2. The amplified PCR product was purified using a Gel Extraction Kit (CWBIO, Beijing, China) and introduced to pGEM-T Easy Vector (Promega, Madison, WI, USA). After transforming the vector into Top 10 competent cells (CWBIO, Beijing, China), the vector sequence sequenced by GENEWIZ (Tianjin, China). DNASTAR (DNASTAR, Inc., Madison, WI, USA) was used for sequence analysis.

2.6. Bioinformatic Analysis

Gene sequence and gene information of BrCLV1 were obtained from the Brassica database (http://brassicadb.cn/#/ (accessed on 29 January 2022)). The conserved domain of BrCLV1 was analyzed by SMART (http://smart.embl-heidelberg.de/ (accessed on 18 May 2022)). The transmembrane helices of BrCLV1 were predicted by TMHMM-2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0/ (accessed on 18 May 2022)). The presence of signal peptides and the location of their cleavage sites in proteins were predicted by SignalP—6.0 (https://services.healthtech.dtu.dk/service.php?SignalP/ (accessed on 18 May 2022)). In NCBI, the sequence of CLV1 homologs was downloaded from the NCBI database. The species name and accession numbers of the 25 CLV1 homologs were as follows: Brassica rapa (XP_009106236.1); Brassica napus (XP_013717912.1); Brassica oleracea var. Oleracea (XP_013591703.1); Raphanus sativus (XP_018445747.1); Eutrema salsugineum (XP_006390268.1); Camelina sativa (XP_010428620.1); Capsella rubella (XP_006300675.1); Arabidopsis lyrata subsp. Lyrata (XP_020891600.1); Arabidopsis thaliana (NP_177710.1); Tarenaya hassleriana (XP_010536408.1); Populus alba (TKS03250.1); Theobroma cacao (EOY15070.1); Gossypium arboreum (XP_017623398.1); Gossypium australe (KAA3484481.1); Gossypium hirsutum (XP_016741841.1); Gossypium raimondii (XP_012478138.1); Durio zibethinus (XP_022772630.1); Hibiscus syriacus (KAE8659851.1); Hevea brasiliensis (XP_021692733.1); Jatropha curcas (XP_012073772.1); Citrus sinensis (XP_006473681.1); Citrus clementina (XP_006435205.1); Populus trichocarpa (XP_002307734.1); Pistacia vera (XP_031283197.1); Populus euphratica (XP_011045060.1). The protein sequence of CLV1 homologs was analyzed using DNAMAN V6 software (Lynnon BioSoft, San Ramon, CA, USA). In order to further clarify its evolutionary relationship, a phylogenetic tree was constructed in 25 species using MEGA-X with Clustal W and the neighbor-joining method based on 1000 bootstrap replications [34].

2.7. Gene Expression Analysis

The gene expression level of BrCLV1 was studied in quantitative real-time (qRT)-PCR. Total RNA was extracted from slm and FT by employing a plant total RNA extraction kit (Tiangen, Beijing, China). First-strand cDNA was acquired by applying FastQuant RT Super Mix (Tiangen, Beijing, China). The gene-specific primers were designed by Primer Premier 5.0 and the Actin gene was used as an internal standard. The primers’ sequences are listed in Table S3. The qRT-PCR amplification procedures and reaction systems are described in Zhao et al. [35].

3. Results

3.1. Phenotypes of slm and Inheritance of the Mutant Character

Compared with wild-type FT, the leaf shape of slm was shorter and smaller during the whole growth period, whereas the leaf number increased (Figure 1 and Figure S1). At the heading stage, slm had longer phyllopodium and underdeveloped leaf wings with smallish leaves, and was unable to form a leafy head (Figure 1b,c).
The phenotypic analysis of the segregation population is shown in Table 1. It was observed that FT (P1) and slm (P2) could stably retain a wild-type phenotype and mutant-type phenotype, while F1 (P1 × P2) and rF1 (P2 × P1) returned to wild-type phenotype. This observation revealed that the mutant-type phenotype was controlled by the nuclear gene. For the F2 population, 317 individuals and 98 individuals had wild-type and mutant-type phenotypes, respectively, and the separation ratio was 3.23:1 (χ2 = 0.42), which was consistent with the Mendelian law of segregation. Meanwhile, the separation ratio of BC12 (F1 × P2) was 1.09:1 (χ2 = 0.083) and BC11 (F1 × P1) was the wild-type phenotype. According to the phenotype analysis of segregation population, it was shown that the mutant-type phenotype of slm was regulated by a single gene. In this article, we named the mutant gene as Brslm.

3.2. Mapping of Brslm via Mutmap

In accordance with the origin and inheritance of slm, we identified the target gene using a modified Mutmap method. Sequencing data statistics found that the quality of raw data was good (Table S4), and slm and mutant-phenotype DNA mixed pool (MP-pool) obtained 51,983,342 reads (98.88% of raw data) and 139,638,760 (98.86% of raw data) high-quality data (Table S5), respectively. The resequencing data of wild-type FT had been reported by Gao et al. [18]. More than 98.00% of the reads were mapped to the reference genome (Table S6). The sequencing data was then further filtered and the filtering steps were as follows: the SNP was retained which was homozygous both in parental slm and FT and was polymorphic between the two parents, the SNP was reserved which conformed to the base mutation type (G-A, C-T) induced by EMS. The SNP index of each polymorphic locus was calculated and the plot was mapped where there was an obvious peak on chromosome A07. Take the SNP index 95th percentile line as threshold line exceeded the and considered the region above the 95th percentile threshold line as the candidate region related to mutant traits (Figure 2). The final candidate region was a total of 5.20 Mb (A07: 23,513,543–28,718,531), containing 19 SNPs. By annotating the SNP loci in this region, six SNPs were located in exons, all of which could cause nonsynonymous mutations, the other thirteen were located in the non-coding region. Based on the mixed pool constructed by F2 mutant phenotype individuals, the SNP index of target SNP should be 1. Among the six SNPs, four SNPs (A07: 26,905,702; A07: 27,571,059; A07: 27,921,459; A07: 28278890) whose SNP index was equal to 1 were considered to be candidate SNP, and the genes they located were accordingly considered to be candidate genes (Table 2).

3.3. Identification of the Causal Gene of Brslm

Based on Mutmap analysis, the four SNPs location (A07: 26,905,702; A07: 27,571,059; A07: 27,921,459; A07: 28,278,890) further detected the genotype of F2 segregation population individuals by KASP. Parent FT, parent slm, F1 generation plant, F2 mutant phenotype individuals and F2 wild-type phenotype individuals were used to analyze SNP genotype and co-segregated analysis. At the location of A07: 26905702, the genotype of F2 mutant phenotype individuals were both T:T consistent with that of slm, while the genotype of F2 wild-type phenotype individuals were C:C or C:T, the genotype of FT was C:C and the genotype of F1 generation plant was C:T (Figure 3a and Table S7), which indicated that the genotype of SNP A07: 26,905,702 was co-segregated with the phenotype. At SNP (A07: 27571059), SNP (A07: 27921459) and SNP (A07: 28278890), the genotypes of FT were C:C, the genotypes of slm were T:T and F1 generation plant were T:C. However, 3, 6 and 10 recombinant events (C:C or C:T genotype) occurred in F2 mutant phenotype individuals at SNP (A07: 27571059), SNP (A07: 27921459) and SNP (A07: 28278890), respectively (Figure 3b–d and Table S7). Simultaneously, 1, 2 and 3 T:T event were generated in wild-type individuals of F2 individuals at SNP (A07: 27571059), SNP (A07: 27921459) and SNP (A07: 28278890), separately (Table S7). These results meant that the genotype of the three SNPs (A07: 27571059, A07: 27921459, A07: 28278890) were not completely co-segregated with phenotype, and ruled out these three SNPs as candidate SNPs. Thus, BraA07g039310.3C was the only candidate gene of Brslm and co-segregated between the genotype and phenotype in the F2 population. With reference to the annotation information in Brassica database, BraA07g039310.3C (BrCLV1) encoding a receptor kinase with an extracellular leucine-rich domain was homologous to CLV1 (At1g75820).

3.4. Candidate Gene Cloning Analysis

Clonal sequencing confirmed that the SNP (A07: 26905702) located on the 904th nucleotide of BrCLV1 was substituted from G to A in slm, which resulted in a change of the 302nd amino acid from D (Asp) to N (Asn) (Figure 4a). BrCLV1 was 3044 bp in the genomic sequence and consisted of 3 exons and 2 introns (Figure 4b), which was 2586-bp in the full-length coding sequence, and encoded a protein of 861 amino acids. The prediction of transmembrane helices and signal peptides detected that BrCLV1 was a transmembrane protein with a signal peptide (Figures S2 and S3, Tables S8 and S9). The analysis of the conserved domain showed that BrCLV1 had several leucine-rich repeats (LRR) domains, and a catalytic domain—Tyrosine kinase—in the C-terminal (Figure 4c). Sequence analysis showed that the SNP (A07: 26905702) occurred on the first exon and located on the LRR domain. The three-dimensional structure of BrCLV1 and Brclv1 found that the amino acid substitution from D to N at the 302nd of BrCLV1 caused changes in protein structure (Figure 4d,e). In NCBI, CLV1 homologs and other species were blasted, then the protein sequences in 25 species were compared (Figure 4f). It was the fact that the 302nd amino acid of BrCLV1 was highly conserved in 25 species. To explore the evolutionary relationship of these CLV1 homologs, a phylogenetic evolutionary tree was constructed (Figure S4). We found that B.rapa and AT belonged to the same branch, and the sequence similarity was 85.74%. In Arabidopsis, WUS-CLV signaling pathway regulated the differentiation of SAM and FM (floral meristem). slm had different leaf characteristics compared with wild-type FT, while leaf development began from SAM. Hence, we confirmed that BrCLV1 was the candidate Brslm gene.

3.5. Expression Analysis of BrCLV1

Quantitative real-time PCR (qRT-PCR) was performed on leaves of different stages to understand the expression of the BrCLV1 between slm and FT. The results showed that BrCLV1 expression patterns in leaves of slm at seedling stage, rosette stage, folding stage, heading stage were similar to those of FT (Figure 5). However, BrCLV1 expression level between FT and slm in these four stages was slightly different and the differences were not significant based on Student’s t-test (p < 0.05)

4. Discussion

Leafy head is the main product of Chinese cabbage, while the obstacle of the leafy head formation has a severe impact on its production. In this study, we identified a short leaf mutant slm, which cannot form a leafy head at the heading stage (Figure 1). Genetic analysis demonstrated that the mutant phenotype of slm was controlled by a single recessive gene Brslm (Table 1). Mutmap and KASP analysis indicated that BraA07g039310.3C (BrCLV1) was the target gene for Brslm (Figure 2, Table 2, Figure 3, Table S7). Sequencing results confirmed the SNP at 26,905,702 on A07 in BrCLV1 between FT and slm (Figure 4a). The SNP was located on LRR domain and highly conserved among 25 species (Figure 4). These findings indicated that the mutation of BrCLV1 disrupted the leafy head formation in the mutant due to shortening leaves and increasing leaf numbers. Limited by the imperfect transgenic system, a functional complementation experiment has not been carried out, but the results of Mutmap and KASP proved that BrCLV1 was the only candidate gene of Brslm.
Previous studies have manifested that CLV1 encoded receptor kinase with an extracellular leucine-rich domain has acted as a signaling molecule to participate in the WUS-CLV pathway, regulating the differentiation of SAM [36,37]. At Arabidopsis, clv1 with a non-synonymous SNP located in CLV1 showed enlarged shoot apical meristem, increased numbers of floral organs, expanded floral meristems and enhanced width of siliques [38]. In the present study, we identified the abnormal phenotype of floral organs and pods in slm (Figure S1). Furthermore, the changes of leaf shape and number in slm was observed. slm possessed more shortening leaves and increased leaf numbers than FT, yet it failed to develop into a leafy head during the heading stage. The expressive level of Brclv1 in slm showed no significant change, whereas the mutant site of Brclv1 was located on a highly conserved LRR domain. Diévart et al. [38] and Brody et al. [26] found that the LRR domain mutations of clv1 were invariably more severe phenotypically changed than those in the kinase domain. Mirzaei et al. [39] identified an EMS mutagenesis mutant of GmCLV1A in soybean, which displayed an increasing number of leaves and branches in comparison to the wild-type TILLING. Hirakawa et al. [40] proved that the CLAVATA peptide can induce multiple branches in Marchantia polymorpha. Brody et al. [26] identified two CLV1 parallel receptors, BAM1 and BAM2, which were BAM parallel receptors, and the triple mutant bam1-bam2-clv1 showed smaller leaves and an increased number of rosette leaves. Although the origin of Brassica rapa genome formed a whole-genome triplication (WGT) event of Arabidopsis, some characteristics of the two species were dissimilar, showing that Chinese cabbage formed leafy heads whereas Arabidopsis failed. Due to the unique Chinese cabbage characteristic of a leafy head, we considered that leafy heading formation may be associated with a divergent function of BrCLV1 compared with CLV1.
CLV1 bound directly with CLV3 to regulate the expression of WUS and participated in the growth and development of SAM [41,42]. At Brassica napa (AACC, 2n = 38), Yang et al. [27] obtained stable transgenic plant of double homozygous mutation of BnCLV3 (BnA4.CLV3 and BnC4.CLV3) through adopting the CRISPR/Cas9 system. Nonetheless, the double mutants of BnCLV3 produced more leaves, a fact which was only observed in clv3 of Arabidopsis, B.juncea, B.rapa [41,43]. In B.juncea, the mutant of CLV1 also showed no change of leaf phenotype [44], which was identical with Arabidopsis [21,45]. In contrast, slm possessed more leaves than wild-type, whose mutant candidate gene was BrCLV1. In addition, Xu et al. [46] predicted CsCLAVATA1 was a putative candidate gene for the dwarf phenotype of Csdw, in which internode length and plant height were significantly reduced. In the vegetative growth period, the stem of Chinese cabbage was compressed and no obvious distinction between nodes and internodes was detected. Nevertheless, the short internodes and dwarf could be observed after bolting during the reproductive growth period.
Leafy head formation of Chinese cabbage is a complex process and its molecular mechanism has not been fully explored [47,48,49]. Our results demonstrated that BrCLV1 was the candidate gene for the mutant phenotype of mutant slm It was the first report suggesting that CLV-WUS pathways were involved in the leafy head formation in Chinese cabbage. In higher plants, SAM is responsible for above-ground organ initiation and development and the regulatory role of SAM for leaf produce and development is further supported by wuschel (wus) mutants [50,51,52]. From the perspective of morphology, the leafy head formation of Chinese cabbage requires larger and sufficient leaves and their included angles [7,9,15,17,53]. We presumed that the reason why the mutant slm failed to form a leafy head was that the shortening leaves and undeveloped leaf wings made them leak light interactively at the leaf opex, and these shortening leaves gathered into clusters rather than forming a leafy head. In addition, there are more leaves in slm, and the shortening leaves may make it more difficult to hold the leafy head. These results provided some novel knowledge about leafy head formation in Chinese cabbage.

5. Conclusions

A single recessive gene Brslm was responsible for the disruption of leafy head formation through shortening leaves in the mutant slm. Mutmap and KASP analyses demonstrated that BrCLV1 was the target gene for Brslm. An SNP from G to A which occurred in the 904th nucleotide of Brclv1 resulted in the change of the 302nd amino acid from Asp to Asn. The SNP was located on the conserved domains and co-segregated with the mutant phenotype of F2 individuals. These findings are beneficial for the comprehension of BrCLV1 function in the leafy head formation of Chinese cabbage.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8111086/s1, Figure S1: Phenotype characterize of FT and slm; Figure S2: Prediction of transmembrane helices by TMHMM-2.0; Figure S3: Prediction of signal peptides by SignalP-6.0; Figure S4: The phylogenetic evolutionary tree of CLV1; Table S1: Primer sequences of Kompetitive allele specific PCR; Table S2: Primer sequences used for clone sequencing; Table S3: Primer sequences for qRT-PCR; Table S4: Quality statistics of raw data; Table S5: Statistics of high-quality (HQ) data; Table S6: Statistics of sequence alignment reference genome; Table S7: Genotype results of four candidate SNPs (SNP A07: 26905702, SNP A07:27571059, SNP A07: 27921459, SNP A07:28278890); Table S8: Prediction of transmembrane helices by TMHMM-2.0; Table S9: Prediction of signal peptides and the location of their cleavage sites in proteins by SignalP-6.0.

Author Contributions

Conceptualization, Y.Z. and H.F.; Data curation, Y.Z. and C.L.; Formal analysis, C.T. and J.R.; Funding acquisition, H.F.; Investigation, N.W.; Project administration, H.F.; Supervision, S.H.; Writing—original draft, Y.Z.; Writing—review & editing, B.F. All authors have read and agreed to the published version of the manuscript.

Funding

The research was supported by National Natural Science Foundation of China (Grant No. 31972405) and Graduate Student Innovation Cultivation Project of Shenyang Agricultural University (2021YCXB15).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw sequence data of the Mutmap has been uploaded to the Sequence Read Archive at NCBI under the accession number SRR22245392.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The phenotype characteristics of FT and slm. (a) The phenotype characteristics of FT and slm at seedling stage. (b) Longitudinal anatomical drawing in leafy head of FT and slm. (c) Split diagram of leafy head in slm and FT.
Figure 1. The phenotype characteristics of FT and slm. (a) The phenotype characteristics of FT and slm at seedling stage. (b) Longitudinal anatomical drawing in leafy head of FT and slm. (c) Split diagram of leafy head in slm and FT.
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Figure 2. The distribution of SNP index on chromosomes. SNP index on 10 chromosomes based on Mutmap analysis. The x-axis represents the chromosome position. The y-axis represents the SNP index value. The black line is the mean value of the SNP index under the window. The pink dashed line is the threshold line of SNP index (0.92).
Figure 2. The distribution of SNP index on chromosomes. SNP index on 10 chromosomes based on Mutmap analysis. The x-axis represents the chromosome position. The y-axis represents the SNP index value. The black line is the mean value of the SNP index under the window. The pink dashed line is the threshold line of SNP index (0.92).
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Figure 3. Verification of the candidate genes by KASP in F2 individuals. (a) The genotype result at the SNP location of A07: 26905702. (b) The genotype result at the SNP location of A07: 27571059. (c) The genotype result at the SNP location of A07: 27921459. (d) The genotype result at the SNP location of A07: 28278890.
Figure 3. Verification of the candidate genes by KASP in F2 individuals. (a) The genotype result at the SNP location of A07: 26905702. (b) The genotype result at the SNP location of A07: 27571059. (c) The genotype result at the SNP location of A07: 27921459. (d) The genotype result at the SNP location of A07: 28278890.
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Figure 4. Sequence analysis of BrCLV. (a) Alignments of the nucleotide and amino acid sequences of BrCLV1 between FT and slm. The red box indicates the mutation site of slm. (b) Gene structure of BrCLV1 and the mutation site of slm. (c) Conserved domain of BrCLV1 and the mutation site of slm. (d) The three-dimensional structure of BrCLV1. The highlighted amino acid structure represents the 302nd amino acid D of BrCLV1. (e) The three-dimensional structure of Brclv1. The highlighted amino acid structure presents the 302nd amino acid N of Brclv1. (f) Sequence alignment of CLV1 homologs in 25 species and the mutation site of slm.
Figure 4. Sequence analysis of BrCLV. (a) Alignments of the nucleotide and amino acid sequences of BrCLV1 between FT and slm. The red box indicates the mutation site of slm. (b) Gene structure of BrCLV1 and the mutation site of slm. (c) Conserved domain of BrCLV1 and the mutation site of slm. (d) The three-dimensional structure of BrCLV1. The highlighted amino acid structure represents the 302nd amino acid D of BrCLV1. (e) The three-dimensional structure of Brclv1. The highlighted amino acid structure presents the 302nd amino acid N of Brclv1. (f) Sequence alignment of CLV1 homologs in 25 species and the mutation site of slm.
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Figure 5. Expression of BrCLV1 in leaves during diversified stages. Expression of BrCLV1 in seedling leaf, rosette leaf, folding stage, heading leaf of FT and slm was measured by qRT-PCR.
Figure 5. Expression of BrCLV1 in leaves during diversified stages. Expression of BrCLV1 in seedling leaf, rosette leaf, folding stage, heading leaf of FT and slm was measured by qRT-PCR.
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Table
Table 1. Genetic analysis of slm in Chinese Cabbage.
Table 1. Genetic analysis of slm in Chinese Cabbage.
GenerationWild-Type PhenotypeMutant-Type PhenotypeTotalSegregation Ratioχ2 Test
P1 (FT)100 100
P2 (slm) 100100
F1 (P1 × P2)23 23
rF1 (P2 × P1)21 21
BC11 (F1 × P1)47 47
BC12 (F1 × P2)2523481.09:10.083
F2317984153.23:10.42
Table
Table 2. Candidate SNP information in the candidate region by Mutant analysis.
Table 2. Candidate SNP information in the candidate region by Mutant analysis.
NumberChromosome: PositionWT- GenotypeMut- GenotypeSNP IndexGene IDAnnotations (Blastx to AT)
1A07: 26905702CT1BraA07g039310.3CPutative receptor kinase with an extracellular leucine-rich domain
2A07: 27571059CT1BraA07g040690.3CAMP-dependent synthetase and ligase family protein
3A07: 27921459CT1BraA07g041310.3CE3 ubiquitin-protein ligase
4A07: 28278890CT1BraA07g042160.3CBinding protein
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Zhao, Y.; Liu, C.; Fang, B.; Huang, S.; Wang, N.; Tan, C.; Ren, J.; Feng, H. Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Horticulturae 2022, 8, 1086. https://doi.org/10.3390/horticulturae8111086

AMA Style

Zhao Y, Liu C, Fang B, Huang S, Wang N, Tan C, Ren J, Feng H. Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Horticulturae. 2022; 8(11):1086. https://doi.org/10.3390/horticulturae8111086

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Zhao, Yonghui, Chuanhong Liu, Bing Fang, Shengnan Huang, Nan Wang, Chong Tan, Jie Ren, and Hui Feng. 2022. "Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)" Horticulturae 8, no. 11: 1086. https://doi.org/10.3390/horticulturae8111086

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