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Article

Fine Mapping and Functional Verification of the Brdt1 Gene Controlling Determinate Inflorescence in Brassica rapa L.

by
Cuiping Chen
1,†,
Xuebing Zhu
1,2,†,
Zhi Zhao
1,2,
Dezhi Du
1,2 and
Kaixiang Li
1,2,*
1
Academy of Agricultural and Forestry Sciences of Qinghai University, Xining 810016, China
2
Key Laboratory of Spring Rape Genetic Improvement of Qinghai Province, Rapeseed Research and Development Center of Qinghai Province, Xining 810016, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(2), 281; https://doi.org/10.3390/agronomy14020281
Submission received: 25 December 2023 / Revised: 21 January 2024 / Accepted: 23 January 2024 / Published: 27 January 2024
(This article belongs to the Special Issue Advances in Crop Molecular Breeding and Genetics)
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Abstract

:
Brassica rapa, a major oilseed crop in high-altitude areas, is well known for its indeterminate inflorescences. However, this experiment revealed an intriguing anomaly within the plot: a variant displaying a determinate growth habit (520). Determinate inflorescences have been recognized for their role in the genetic enhancement of crops. In this study, a genetic analysis in a determinate genotype (520) and an indeterminate genotype (515) revealed that two independently inherited recessive genes (Brdt1 and Brdt2) are responsible for the determinate trait. BSA-seq and SSR markers were employed to successfully locate the Brdt1 gene, which is localized within an approximate region 72.7 kb between 15,712.9 kb and 15,785.6 kb on A10. A BLAST analysis of these candidate intervals revealed that Bra009508 (BraA10.TFL1) shares homology with the A. thaliana TFL1 gene. Then, BraA10.TFL1 (gene from the indeterminate phenotype) and BraA10.tfl1 (gene from the determinate phenotype) were cloned and sequenced, and the results indicated that the open reading frame of the alleles comprises 537 bp. Using qRT-PCR, it was determined that BraA10.TFL1 expression levels in shoot apexes were significantly higher in NIL-520 compared to 520. To verify the function of BraA10.TFL1, the gene was introduced into the determinate A. thaliana tfl1 mutant, resulting in the restoration of indeterminate traits. These findings demonstrate that BraA10.tfl1 is a gene that controls the determinate inflorescence trait. Overall, the results of this study provide a theoretical foundation for the further investigation of determinate inflorescence.

1. Introduction

Oil crops belonging to the genus Brassica hold significant importance in global agriculture. This genus comprises a diverse range of plant species, including the species Brassica rapa L., Brassica napus L., and Brassica juncea L. [1,2]. Brassica rapa is a diploid species (2n = 20, AA) and has a worldwide distribution [3]. It includes vegetable crops and oil seed crops [4]. Oil seed crops have excellent characteristics, such as barren, drought, and cold resistance [5,6]. Currently, the rapeseed cultivation area in China is approximately seven million hectares, with B. rapa accounting for about 15% of this area [7]. Brassica rapa plays an irreplaceable role in the provinces of the Yangtze River basin and the Northwest Plateau due to its short growth period, particularly in the rotation of rice and rapeseed. All Brassica crops possess an indeterminate growth habit, which is influenced by competition for resources within the plant canopy, both within and between plants [8]. This competition often leads to incomplete seed filling, immature pods, and sterility at the tip of the plant at maturity [9]. Additionally, certain varieties with indeterminate inflorescence growth habits have drawbacks that negatively impact yield, such as taller plants, an increased vulnerability to lodging, longer growth periods, and inconsistent ripening periods [10]. Transforming plants from indeterminate to determinate inflorescence offers a new approach for breeders. Thus, it is necessary to research the molecular mechanisms underlying the determinate inflorescence traits in B. rapa to enhance rapeseed genetics.
There are many research reports on determinate inflorescence [8,11,12,13]. Kaur and Banga [8] identified the determinate gene Sdt1, which was responsible for regulating determinate inflorescence on the B5 chromosome of Brassica juncea. Li et al. [9,14] discovered a double haploid (DH) line 4769 that exhibited a determinate inflorescence trait. Further investigation revealed that this trait was controlled by two independently inherited recessive genes (Bnsdt1 and Bnsdt2). Wan et al. [15] employed BSA-Seq technology to identify two QTL loci associated with determinate inflorescence in a mutant of B. napus. These loci were found on the C02 and C06 chromosomes, respectively. Chen et al. [10] discovered that the determinate inflorescence natural mutant 6138 material of B. napus is controlled by a single recessive gene, BnDM1. Furthermore, they successfully identified the location of the gene on the C02 chromosome.
The TERMINAL FLOWER 1 (TFL1) gene and its homolog play an important role in determinate inflorescence. The genetic mechanism of the TFL1 gene in A. thaliana has been extensively studied [16,17,18]. The TFL1 gene is predominantly expressed in the central region of the apical meristem of Arabidopsis [19]. Its interaction with genes, such as LEAFY (LFY), APETALA1 (AP1), and FLOWERING LOCUS T (FT), influences the development of the stem tip of A. thaliana, leading to the formation of distinct inflorescences [20,21]. TFL1, being the main inhibitor of flower development, interferes with the development of flowers by binding to the transcription factor FLOWERING LOCUS D (FD) and inhibiting FT. This inhibition is crucial in regulating the expression of downstream flowering integrators AP1 and LFY [22].
Additionally, there are many reports about TFL1 in other plants. In rice, the TFL1 homologous gene RCN initiates its regulatory pathway by binding to 14-3-3 proteins, followed by its interaction with FD to regulate flower recognition genes [23]. However, CsTFL1 in cucumbers does not directly interact with CsFD, CsFDP, or Cs14-3-3. Instead, it interacts with CsNOT2a, binding CsFD or CsFDP and affecting the transcription of CsAP1 and CsLFY [24]. Therefore, it can be concluded that the regulatory pathway of TFL1 in tall plants is a relatively complex process.
Although there has been extensive research on determinate inflorescence in other plants [25,26,27], the genetic patterns and molecular basis of determinate inflorescence remain unclear in B. rapa. In order to investigate the genetic inheritance patterns controlling determinate inflorescence in B. rapa, this study focused on the determinate inflorescence mutant 520 of B. rapa. Two genes (Brdt1 and Brdt2) associated with determinate inflorescence were identified and further subjected to the fine mapping and functional verification of Brdt1. These findings enhance our theoretical understanding of determinate inflorescence and lay the groundwork for future investigations.

2. Materials and Methods

2.1. Plant Material and Population Construction

Lines 520 and 515 of B. rapa were used as materials in the present study. The inflorescence of 520 was determinate (a natural mutant) (Figure 1a), while the inflorescence of 515 was indeterminate (Figure 1b). At maturity, they will exhibit different morphological structures (Figure 1c,d). Initially, F1 generation was obtained through a hybrid cross between lines 515 and 520, completed in 2018. Subsequently, they were selfed to produce the F2 generation, and the F1 generation was backcrossed with the recessive parent line 520 to generate the BC1F1 generation (2019). The F2 and BC1F1 populations were used for the genetic analysis of inflorescence traits. The inflorescence traits were investigated during the flowering period in 2020.
The Brdt1 gene was identified using the BC1F1 population. From this population, 12 indeterminate individuals were selected for backcrossing with line 520. The resulting BC2F1 isolate line was then planted at the Xining Experimental Base in Qinghai Province, China, in 2020. Subsequently, the inflorescence traits of each line were investigated and it was found that the indeterminate to determinate separation ratio was 1:1 in 9 out of 12 lines. The dominant BrDT1 gene was identified in an isolated line, with an indeterminate to determinate ratio of 1:1. To establish the BC3F1 population, 10 indeterminate individuals were selected for backcrossing with individuals from line 520, resulting in a population of 847 individuals (2021). From this BC3F1 population, 18 indeterminate individuals were chosen and backcrossed with individuals from line 520 to create the BC4F1 population, which consisted of 1267 individuals (2022). This BC4F1 population was used for the fine mapping of the Brdt1 gene (Figure S1). Additionally, near-isogenic lines were constructed for gene expression analysis, including line 520 (determinate) and NIL-520 (indeterminate).

2.2. Morphological Observation and Paraffin Sectioning

The inflorescence variations were observed under the fluorescent microscope (4× magnification), based on stem apex meristem development in accordance to the method described by Kobayashi et al. [28]. In this study, a stereoscopic fluorescence microscope (Nikon SMZ25, Tokyo, Japan) was used to observe the inflorescence SAM in homozygous plants at different stages, including the 2-, 4-, 6-, and 8-leaf stages. The aim was to investigate the differences in apical SAM. The methods for paraffin sectioning plant SAMs were followed as described in previous studies [29]. The samples were treated with 70% formalin-acetic acid-alcohol (FAA), followed by Safranin O staining, rinsing, dehydration, clearing, infiltration, embedding, slicing, sealing, and examination using a Nikon microscope (Nikon, Tokyo, Japan).

2.3. DNA Extraction and BSA-Seq

DNA was extracted individually from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method [30]. To map the Brdt1 gene, 20 plants with indeterminate inflorescence and 20 plants with determinate inflorescence were selected from the BC3F1 population. Separate indeterminate and determinate bulks were created. The two parents and bulks were subjected to bulked segregant analysis (BSA) at Novogene Biological Company in Beijing, China. The Illumina HiSeq TM PE150 sequencing method was employed. Subsequently, the Burrows–Wheeler alignment (BWA) tool aligned the whole-genome sequencing (WGS) reads to the reference genome of ‘chiifu’ v1.5 (BRAD (http://brassicadb.org/brad/)). Single-nucleotide polymorphisms (SNPs) were detected using the Haplotype Caller of Genome Analysis Toolkit (GATK, version 3.7). The candidate region was determined based on the SNP index.

2.4. Development of SSR Marker

The initial localization of the determinate inflorescence gene Brdt1 and the location of its homologous gene in B. napus chromosomes helped determine the approximate range of Brdt1 on the A10 chromosome. The sequence segment was downloaded from the BRAD (http://brassicadb.org/brad/) database, and SSR loci were detected using SSRHunter 1.3. Using the Primer 3 software (Premier Biosoft International, Palo Alto, CA, USA) to design the SSR primers. SSR amplification was performed following the method described by Lowe [31]. The co-dominance of SSR markers was detected using 6% polypropylene gel electrophoresis.

2.5. Mapping

The Brdt1 gene was mapped using the BC3F1 populations (847 individuals) and BC4F1 populations (1267 individuals). The SSR markers and individual phenotypes were analyzed using the JoinMap 4/MapDraw program, resulting in the construction of a partial linkage map for the chromosome region containing the Brdt1 gene.

2.6. Cloning and Sequence Analysis of the Candidate Gene

The Brdt1 gene was amplified from the gDNA of parents 515 and 520. Primer 3.0 software was used to design the specific primers (Bra.TFL1-orf-F/Bra.TFL1-orf-R) of the gene (Table S1). The amplification process was as follows: pre-denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, extension at 72.0 °C for 60 s, and terminal extension at 72 °C for 10 min. The amplified sequences were cloned using the PMD19-T vector and E. coli DH5α methods. The positive clones were verified using M13-specific primers (Tsingke Biotech, Beijing, China). The gDNA sequences were analyzed using DNAMAN8.0.

2.7. Plasmid Construction and Plant Transformation

To assess the functionality of the candidate gene, a primer was designed based on the Bra009508 (TFL1 homologous) sequences obtained from NCBI (National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/)). The primers were modified to include the EcoRI and PstI enzyme restriction sites, as well as 15 bp sequences from both ends of the pCAMIBA2300 vector. The amplification of the gDNA fragment, which consisted of the upstream, full-length gene, and downstream sequences, was performed using Phusion Hot Start High Fidelity DNA Polymerase (NEB, Ipswich, MA, USA) and the recombinant primers (CEBra.TFL1-F/CEBra.TFL1-R, Table S1). The indeterminate inflorescence line 515 served as the source of DNA for this amplification. To create the complementation plasmid pBraA10. TFL1: BraA10. TFL1, a genomic fragment was sequenced and digested with EcoRI and PstI. The digested fragment was then ligated into the pCAMIBA2300 vector using a One Step Cloning Kit (Vazyme, Nanjing, China). Finally, the confirmed recombinant plasmid was introduced into GV3101 (Agrobacterium tumefaciens).
The inflorescence impregnation method was implemented to transform Arabidopsis thaliana (L.) Heynh. Specifically, the fused construct was introduced into the A. thaliana tfl1-2 mutant. PCR was conducted to verify and identify the positive transgenic plants (PT-Bra.TFL1-F/PT-Bra.TFL1-R, Table S1).

2.8. RNA Extraction and qRT-PCR

Total RNA was extracted from 520 and NIL-520 materials at different developmental stages, including the 2-leaf seedlings, budding stage, bolting stage, and root, stem, and leaf tissues during the bolting stage. The extraction was performed using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China) with three biological replicates. The extracted RNA samples were frozen in liquid nitrogen and stored at −80 °C. First-strand cDNA was synthesized using the PrimeScriptTM RT Reagent Kit (TaKaRa, Dalian, China) following the manufacturer’s protocol. The primers (qRT-braA10-1F/qRT-braA10-1R, Table S1) for qRT-PCR were designed using Primer-BLAST in NCBI (Primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 22 January 2024)). The actin gene was selected as the reference gene for the relative quantification of the candidate gene (Actin-F/Actin-R, Table S1). qRT-PCR was conducted using a CFX Opus 96 instrument (Bio-Rad, Hercules, CA, USA). The reaction system consisted of 25 μL, including 2 μL of gene-specific primer (10 ng μL−1) (Table S1), 2 μL of cDNA (50 ng μL−1), 12.5 μL of TB Green Premix Ex Taq II (TliRNaseH Plus) (TaKaRa, Dalian, China), and 8.5 μL of sterile water. The PCR conditions were 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. The data were processed using the 2−ΔΔCt method.

3. Results

3.1. Observations of the SAM Apex in B. rapa

The formation of the SAM was observed using a stereoscopic fluorescence microscope and paraffin section (Figure 2). The aim was to determine the point at which the apices of indeterminate inflorescence and determinate inflorescence exhibit morphological differences during the growth process. This observation showed that B. rapa exhibits normal inflorescence differentiation at the SAM of indeterminate and determinate inflorescences during the two-to-six-leaf stage (Figure 2a–c,e–g). However, at the eight-leaf stage, indeterminate inflorescences continued to undergo normal inflorescence differentiation (Figure 2d), while the SAM of determinate inflorescences exhibited variation and could not differentiate inflorescence tissue normally (Figure 2h). The results of paraffin sections also indicated that during the two-to-six-leaf stage, the indeterminate and determinate shoot apex meristem displayed the same shapes (Figure 2i–k,m–o). However, in the eight-leaf stage, the indeterminate normal process continued (Figure 2i), and the determinate apices had already exhibited variation and could not differentiate into normal inflorescence meristems (Figure 2p). These findings provide morphological evidence for the formation period of determinate inflorescences and offer insights for observing determinate inflorescences in B. rapa.

3.2. Genetic Analysis

This study investigated and analyzed the growth habits of the F2 and BC1F1 populations. It was observed that all F1 individuals exhibited a complete indeterminate phenotype, indicating the dominance of indeterminate growth over determinate growth. In 515 × 520 F2 plants, 297 plants displayed indeterminate growth while 27 plants showed determinate growth, consistent with a segregation ratio of 15:1. The BC1 plants exhibited a segregation ratio of approximately 3:1 (indeterminate-to-determinate = 168:50) (Table 1). These findings suggest that determinate growth habits are controlled by two independently inherited recessive genes, and determinate inflorescence genes were tentatively designated Brdt1 and Brdt2.

3.3. Primary Mapping of the Determinate Gene Brdt1 Using BSA-Seq

The BC3F1 population obtained from a cross between 515 and 520 was used for BSA resequencing [32] to determine the position of the Brdt1 gene. After sequencing the two bulks and their respective parents, a total of 54.429 Gb clean reads were obtained after quality control filtering. Specifically, the 515 parent, the 520 parent, the indeterminate bulk, and the determinate bulk accounted for 10.454 Gb, 10.731 Gb, 17.035 Gb, and 16.209 Gb, respectively. The sequencing data showed that more than 90.15% of the bases in both pools and parents had a quality score of more than 30 (Q30), and more than 96.13% had a quality score of more than 20 (Q20). Additionally, the average GC content was between 38.03% and 39.31%. The sequencing data were analyzed, and a total of 659,356 SNPs were identified between 515 and 520 by aligning with the ‘chiifu’ v1.5 reference genome. Out of these SNPs, 604,936 homozygous SNPs were found between the two parents, and these were used to calculate the SNP index for the two descendants. A graph of the ΔSNP index was plotted against the genomic regions (Figure 3a), revealing a significant peak in the 1.55 Mb region from 14.76 Mb to 16.31 Mb on chromosome A10 (Figure 3b). This finding suggests that the Brdt1 gene, located in this specific region, could be a potential candidate locus.

3.4. Fine Mapping of the Brdt1 Gene

Based on the findings derived from the BSA-Seq analysis, it is likely that the Brdt1 gene is located in the 14.76–16.31 Mb regions of A10 of B. rapa. Subsequently, the region sequence was downloaded from BRAD (http://brassicadb.org/brad/) and 60 SSR markers were developed. Among these, nine markers with polymorphism were identified and named BrSSR1 to BrSS9 (Table 2). A total of 847 BC3F1 individuals were screened using polymorphic SSR markers to assess the genetic distance between the Brdt1 gene and the SSR markers and determine the order of these markers. Subsequently, the recombinants for each marker were recorded. The genetic distance was computed, and the genetic linkage map was constructed using JoinMap 4.0/MapDraw software V2.1. The results indicate that BrSSR1–BrSSR3 were located on one side of the Brdt1 gene, BrSSR5–BrSSR9 were located on the other side of the Brdt1 gene, and BrSSR4 co-segregated with the Brdt1 gene (Figure 4a). Among the markers flanking the Brdt1 gene, BrSSR3 and BrSSR5 displayed the closest linkage, with distances of 0.3 cM and 0.2 cM from the Brdt1 gene, respectively (Table 2). These closely linked markers were subjected to BLAST analysis against BRAD (http://brassicadb.org/brad/). All markers were mapped to A10 of the ‘chiifu’ v1.5 reference genome (Table 2). Furthermore, the order of these markers on the map perfectly aligned with their counterparts on A10 of B. rapa. Based on this established order, the genomic region harboring the Brdt1 gene was precisely delimited within an approximate interval of 232.3 kb, from 15,580.0 to 15,812.3 kb on A10 (Figure 4b).
To narrow down the target region of Brdt1, a total of 1267 individuals from the BC4F1 population were used. Three SSR markers (BrSSR10–BrSSR12) were identified within the interval of 155,800 to 158,123 kb on A10 (Table 2). We utilized SSR markers including the previously used markers (BrSSR3–BrSSR5) and these SSR markers to conduct a screening of BC4F1 individuals. Our findings revealed that the Brdt1 gene was positioned between BrSSR4 and BrSSR11 (Figure 5a). Using BLAST analysis against BRAD (http://brassicadb.org/brad/), the Brdt1 gene was further narrowed down to an interval of approximately 72.7 kb, specifically between 15,712.9 kb and 15,785.6 kb on A10 of B. rapa (Figure 5b).

3.5. Dissection of the Brdt1 Target Region

The candidate intervals were then submitted to the BRAD (brassicadb.cn) and TAIR (https://www.arabidopsis.org/) databases for BLAST analysis. The analysis revealed that the candidate region included 18 predicted genes (Figure 5c) from the B. rapa reference genome and showed homology to 17 genes on the A. thaliana chromosome (Table 3). Notably, according to the TAIR database of the gene annotation of these genes, the Bra009508 (BraA10.TFL1) gene is homologous to the AT5G03840 gene, which is known as the TERMINAL FLOWER 1 (TFL1) gene. The AT5G03840 gene encodes a phosphatidylethanolamine-binding protein (PEBP). In Arabidopsis TFL1 mutants, determinate inflorescence can be formed at the SAM of the inflorescence. Based on this information, it was inferred that the Bra009508 (BraA10.TFL1) gene was the most promising candidate gene for Brdt1 and selected for further study.

3.6. Expression of BraA10.TFL1 in Different Tissues of B. rapa

A qRT-PCR analysis was conducted to investigate the expression levels of the BraA10.TFL1 gene in different stages and tissues of B. rapa growth. Specifically, the gene was quantified in the shoot apex during the two-leaf stage, budding stage, and bolting stage of both indeterminate and determinate inflorescences. Additionally, gene expression was measured in the root, stem, and leaf tissues during the bolting stage. The findings revealed that the expression level of the BraA10.TFL1 gene remained relatively consistent in the root, stem, and leaf tissues during the bolting stage. However, significant differences were observed in the expression of the BraA10.TFL1 gene in the shoot apex between the 520 and NIL-520 lines, encompassing the two-leaf stage, budding stage, and bolting stage. Furthermore, the expression of BraA10.TFL1 in the NIL-520 line was notably higher than in the 520 line, particularly during the P2 and P3 phases. These results emphasize the crucial role of the BraA10.TFL1 gene in the development of distinct inflorescences (Figure 6). Therefore, it is reasonable to postulate that BraA10.TFL1 is a potential candidate gene for the inflorescence trait.

3.7. Cloning and Sequencing Analysis of the BraA10.TFL1/BraA10.tfl1 Gene

The sequences of gDNA and CDS of BraA10.TFL1/BraA10.tfl1 were amplified from the indeterminate inflorescence line 515 and the determinate inflorescence line 520. DNAMAN 8.0 software was used to analyze the gDNA and CDS sequences of the determinate and indeterminate inflorescences. The results revealed the presence of a gDNA sequence measuring 1066 bp and a 537 bp cDNA sequence in inflorescence lines 515 and 520, respectively. These sequences contained four exons and three introns. The sequence analysis of BraA10.TFL1 (BraA10.TFL1_DNA) and BraA10.tfl1 (BraA10.tfl1_DNA) in B. rapa revealed two SNP mutations (G 434 T and C 569 T) in the intron region (Figure 7a). Amino acid sequence prediction and the analysis of the BraA10.TFL1/BraA10.tfl1 genes were performed using Premier 5. The results showed no differences in amino acid sequences (Figure 7b).
Previous studies have reported that the gene BnA10.tfl1, responsible for determinate inflorescence, is located on the A10 chromosome of B. napus. A comparison of the sequences of the BnA10.tfl1 and the BnA10.TFL1 gene, which controls indeterminate inflorescence, revealed 22 differences [33]. Among these differences, two SNPs resulted in a change in two amino acids (Phe to Leu and Leu to Phe), potentially leading to a transition from indeterminate to determinate inflorescence. To further investigate this, the gene and amino acid sequences of BraA10.TFL1 and BraA10.tfl1 from B. rapa were compared with the BnA10.TFL1 and BnA10.tfl genes from B. napus. The results indicated that BraA10.tfl1 and BnA10.TFL1 had the same size, consisting of 1066 bp, and showed high homology with only two differences out of 1066 bases. However, there were 20 sequence differences between BraA10.tfl1 and BnA10.tfl1, resulting in changes in two amino acids (Phe to Leu and Leu to Phe). These unexpected findings highlight the importance of conducting functional validation to confirm the accuracy of the predicted genes.

3.8. BraA10.TFL1 Rescues the tfl1-2 Mutant Phenotype in A. thaliana

The complete genomic DNA sequence of BraA10.TFL1 from the 515 line was amplified and utilized to construct pBraA10.TFL1: BraA10.TFL1 in the PCAMBIA2300 vector. This vector contained a 3437 bp genomic fragment that included the BraA10.TFL1 gene, with 1842 bp upstream, 1066 bp coding region, and 529 bp downstream (Figure S2). After the transformation process, we introduced this construct into the A. thaliana tfl1-2 mutant (determinate inflorescence) (Figure 8b). Both the transgenic A. thaliana lines and tfl1-2 mutants exhibited the expected outcomes (Figure 8b–d). The transgenic lines showed indeterminate inflorescence stem growth, similar to wild-type A. thaliana (Figure 8a). A total of 12 positive transgenic plants were obtained. The terminal flowers of tfl1-2 mutants suppressed the differentiation of the SAM, resulting in the development of determinate inflorescence. It is important to note that the T1 A. thaliana plants displayed multiple buds, indicating an indeterminate phenotype (Figure 8c,d). In conclusion, these findings provide evidence supporting the functional similarities between BraA10.TFL1 and TFL1.

4. Discussion

Inflorescences significantly impact the yield of B. rapa [34,35,36]. In previous studies, the formation of determinate inflorescences changed plant height and the flowering and maturity stages. This not only improved the plant type, but also facilitated mechanized harvesting. Among the determinate mutant plants currently studied, A. thaliana [37], B. juncea [8], and Vigna radiata [38], it has been observed that the determinant inflorescence is regulated by a recessive gene. Zhang et al. [39] examined a natural mutant strain, FM8, of B. napus with a determinant inflorescence, and genetic analysis revealed that the inheritance of this inflorescence type is controlled by the interaction of two recessive genes and one recessive epistasis suppressor gene. Li et al. [14] found that the determinate inflorescence strain of B. napus was controlled by two independently inherited recessive genes (Bnsdt1 and Bnsdt2). However, the determinate inflorescence line 520 of B. rapa was used as material in this study, and it was found that it is controlled by two independent inherited recessive genes, Brdt1 and Brdt2. This result may be due to the different mutant types of determinate inflorescence. Therefore, further in-depth research is necessary to understand the genetic mechanisms governing determinate inflorescence genes in rapeseeds.
This study utilized stereoscopic fluorescence microscopy and paraffin sectioning to observe the growth stages of the shoot apical meristem (SAM) in both determinate and indeterminate inflorescences. The results showed that the SAMs of determinate inflorescences (Figure 2e–g,m–o) were structurally similar to those of indeterminate inflorescences (Figure 2a–c,i–k) at the two-, four-, and six-leaf stages. However, the SAMs of determinate inflorescences (Figure 2h,p) showed differences compared to indeterminate inflorescences (Figure 2d,l) in the eight-leaf stage. The apex of the determinate SAM exhibited variation similar to the shape of a floral organ, which may cause determinate growth. Other studies have reported the observation of determinate SAMs forming terminal flowers, either singularly or in multiples [40,41]. While the phenotypes exhibited variation among different plants with determinate inflorescences, they had one fundamental characteristic in common: the shoot apical meristem (SAM) maintained its differentiation, producing fresh floral tissue, thereby causing a transition from an indeterminate to a determinate growth pattern.
In previous studies, the genes responsible for controlling quality traits were identified using bulk segregant sequencing (BSA-Seq) and map-based cloning approaches [42,43,44,45,46]. This study employed a method to map the determinate trait of B. rapa. Initially, the genomic sequence information of ‘chiifu’ v1.5 (B. rapa) was used as a reference to successfully map the Brdt1 gene to the A10 chromosome. The Brdt1 gene was delimited to an interval of approximately 72.7 kb, ranging from 15,712.9 kb to 15,785.6 kb on A10 of B. rapa. Furthermore, a gene annotation analysis within this interval identified a highly similar gene, Bra009508 (BraA10.TFL1), which is homologous with the TFL1 gene in A. thaliana. A sequence analysis of BraA10.TFL1 revealed two SNP differences (G 434 T; C 569 T) in the intron region between indeterminate and determinate sequences. Subsequently, an expression pattern analysis of BraA10.TFL1 was conducted, which demonstrated its specific expression in the shoot apex. Genetic transformation experiments in A. thaliana further confirmed the functionality of the BraA10.TFL1 gene. When introduced into the A. thaliana tfl1-2 mutant, the T1 A. thaliana plants reverted to an indeterminate state. Jia et al. [33] conducted a study in which they transferred the TFL1 homologous gene BnA10.TFL1 from B. napus into a determinate Arabidopsis mutant. The researchers observed that this transfer restored determinate expression in Arabidopsis, resulting in indeterminate inflorescence. These findings provide evidence that BraA10.TFL1 shares a similar function to TFL1.
In this study, it is noteworthy that the sequence alignment results indicated no difference in the amino acid sequences encoded by the determinate inflorescence and indeterminate inflorescence sequences. However, the genetic transformation results demonstrated that the BraA10.TFL1 gene from indeterminate inflorescence was successfully introduced into the A. thaliana tfl1-2 mutant (regenerating plants from B. rapa through genetic transformation is challenging). As a result, the mutant’s phenotype could be restored to indeterminate inflorescence. Based on these findings, it is hypothesized that the promoter region of the gene may contain a functional region that contributes to determinate inflorescence formation. However, additional evidence is necessary to substantiate this hypothesis.

5. Conclusions

As a novel combination of materials was used in this study, genetic analysis revealed that the determinate inflorescence is controlled by two independent recessive genes (Brdt1 and Brdt2). Morphological observations indicated that the 520 strain with determinate inflorescence exhibited the characteristic at the eight-leaf stage. One of the genes, referred to as Brdt1, was mapped using BSA sequencing and SSR marker development. The gene was fine-mapped to the 15,712.9–15,785.6 kb interval on chromosome A10. Within this interval, it was found that the gene Bra009508 (BraA10.TFL1) is homologous to the AT5G03840 gene in Arabidopsis, which is annotated as the TFL1 gene. Moreover, when the BraA10.TFL1 gene from an indeterminate inflorescence plant was transferred to the A. thaliana tfl1-2 mutant, the determinate traits became indeterminate. These findings suggest that BraA10.TFL1 may play a role in controlling the determinate inflorescence trait. Overall, this research provides novel insights into the molecular mechanism of oilseed breeding for determinate inflorescences.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14020281/s1, Figure S1: Population construction; Figure S2: The 3437 bp genomic fragment incorporating the BraA10.TLF1 gene. Table S1: The primer sequences used in this study.

Author Contributions

C.C. and X.Z. performed the research and wrote the manuscript; Z.Z. conducted the data analysis. K.L. and D.D. designed the research and revised the article. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Qinghai Provincial Natural Science Foundation of China (2022-ZJ-975Q).

Data Availability Statement

No new data were created.

Acknowledgments

We are grateful to Yongping Jia, Xutao Zhao, Lingxiong Zan, Liren Xie, and the Oil Crop Research Institute of the Chinese Academy of Agricultural Sciences for their help in purchasing A. thaliana mutants.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Two IM development phenotypes in B. rapa. (a) The determinate phenotype (520). (b) The indeterminate phenotype (515). (c) The line 520 phenotype at maturity. (d) The line 515 phenotype at maturity.
Figure 1. Two IM development phenotypes in B. rapa. (a) The determinate phenotype (520). (b) The indeterminate phenotype (515). (c) The line 520 phenotype at maturity. (d) The line 515 phenotype at maturity.
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Figure 2. Microscope observation and paraffin section of the shoot apical meristem (SAM) of the indeterminate (a–d,i–l) and determinate inflorescences (e–h,m–p). (a,e,i,m) SAMs of indeterminate and determinate inflorescences at the two-leaf stage. (b,f,j,n) SAMs of indeterminate and determinate inflorescences at the four-leaf stage. (c,g,k,o) SAMs of indeterminate and determinate inflorescences at the six-leaf stage. (d,h,i,p) SAMs of indeterminate and determinate inflorescences at the eight-leaf stage. Red arrows indicate growth points. In the eight-leaf stage, the determinate inflorescence was beginning to appear.
Figure 2. Microscope observation and paraffin section of the shoot apical meristem (SAM) of the indeterminate (a–d,i–l) and determinate inflorescences (e–h,m–p). (a,e,i,m) SAMs of indeterminate and determinate inflorescences at the two-leaf stage. (b,f,j,n) SAMs of indeterminate and determinate inflorescences at the four-leaf stage. (c,g,k,o) SAMs of indeterminate and determinate inflorescences at the six-leaf stage. (d,h,i,p) SAMs of indeterminate and determinate inflorescences at the eight-leaf stage. Red arrows indicate growth points. In the eight-leaf stage, the determinate inflorescence was beginning to appear.
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Figure 3. ΔSNP index Manhattan plot graphs. (a) The blue line indicates the 95% threshold value, and the purple line indicates the 99% threshold value. Different colors represent different chromosomes. (b) SNP index of delta chromosome A10. The red line is the SNP-index mean line, the green line is the 95% threshold line, and the purple line is the 99% threshold line.
Figure 3. ΔSNP index Manhattan plot graphs. (a) The blue line indicates the 95% threshold value, and the purple line indicates the 99% threshold value. Different colors represent different chromosomes. (b) SNP index of delta chromosome A10. The red line is the SNP-index mean line, the green line is the 95% threshold line, and the purple line is the 99% threshold line.
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Figure 4. Mapping of Brdt1 gene. (a) A partial genetic linkage map around the Brdt1 gene. (b) A partial physical map of linkage markers around the Brdt1 gene. The red region indicates candidate intervals, corresponding to 15,580.0–15,812.3 kb, identified through bulk segregant sequencing and SSR markers.
Figure 4. Mapping of Brdt1 gene. (a) A partial genetic linkage map around the Brdt1 gene. (b) A partial physical map of linkage markers around the Brdt1 gene. The red region indicates candidate intervals, corresponding to 15,580.0–15,812.3 kb, identified through bulk segregant sequencing and SSR markers.
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Figure 5. Fine mapping of Brdt1 gene. (a) A partial genetic linkage map around the Brdt1 gene. (b) A partial physical map of linkage markers around the Brdt1 gene. The red region indicates candidate intervals corresponding to 15,712.9–15,785.6 kb. (c) Results of a BLAST analysis using sequences from candidate intervals against the ‘chiifu’ v1.5 genome.
Figure 5. Fine mapping of Brdt1 gene. (a) A partial genetic linkage map around the Brdt1 gene. (b) A partial physical map of linkage markers around the Brdt1 gene. The red region indicates candidate intervals corresponding to 15,712.9–15,785.6 kb. (c) Results of a BLAST analysis using sequences from candidate intervals against the ‘chiifu’ v1.5 genome.
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Figure 6. Expression analysis of the BraA10.TFL1 gene among different stages and tissues of the NIL-520 and 520 lines in B. rapa. P1, P2, and P3 are two-leaf stage, budding stage, and bolting stage, respectively. The expression levels in the root, stem, and leaf tissues were consistent during the bolting stage. ** indicates significant differences at p < 0.01.
Figure 6. Expression analysis of the BraA10.TFL1 gene among different stages and tissues of the NIL-520 and 520 lines in B. rapa. P1, P2, and P3 are two-leaf stage, budding stage, and bolting stage, respectively. The expression levels in the root, stem, and leaf tissues were consistent during the bolting stage. ** indicates significant differences at p < 0.01.
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Figure 7. Sequence analysis of BraA10.TFL1. (a) The gDNA sequences of BraA10.TFL1 (indeterminate) from the 515 line and BraA10.tfl1 (determinate) from the 520 line were aligned with BnA10.TFL1 (indeterminate) and BnA10.tfl1 (indeterminate) from B. napus. The green box indicates the position of SNP differences between indeterminate 515 and determinate 520. Blue indicates the bases that are different in the sequence. (b) Amino acid sequence alignment between BraA10.TFL1 (indeterminate) and BraA10.tfl1 (determinate) with BnA10.TFL1 (indeterminate) and BnA10.tfl1 (indeterminate) from B. napus. Blue indicates amino acids with differences in the sequence.
Figure 7. Sequence analysis of BraA10.TFL1. (a) The gDNA sequences of BraA10.TFL1 (indeterminate) from the 515 line and BraA10.tfl1 (determinate) from the 520 line were aligned with BnA10.TFL1 (indeterminate) and BnA10.tfl1 (indeterminate) from B. napus. The green box indicates the position of SNP differences between indeterminate 515 and determinate 520. Blue indicates the bases that are different in the sequence. (b) Amino acid sequence alignment between BraA10.TFL1 (indeterminate) and BraA10.tfl1 (determinate) with BnA10.TFL1 (indeterminate) and BnA10.tfl1 (indeterminate) from B. napus. Blue indicates amino acids with differences in the sequence.
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Figure 8. Architecture of terminal racemes in transgenic A. thaliana. (a) Wild-type A. thaliana (WT). (b) The A. thaliana tfl1-2 mutant. (c,d) T1 A. thaliana transgenic plants returned to an indeterminate phenotype.
Figure 8. Architecture of terminal racemes in transgenic A. thaliana. (a) Wild-type A. thaliana (WT). (b) The A. thaliana tfl1-2 mutant. (c,d) T1 A. thaliana transgenic plants returned to an indeterminate phenotype.
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Table 1. Segregation of inflorescence traits in the F2 and BC1 populations.
Table 1. Segregation of inflorescence traits in the F2 and BC1 populations.
CombinationF1/RF1PopulationNo. of
INDT. Plants		No. of
DT. Plants		Expected RatioX2 Value
515 × 520indeterminateF22972715:12.40
BC1F1168503:10.49
Table 2. Information about the markers that were closely linked to Brdt1.
Table 2. Information about the markers that were closely linked to Brdt1.
Type of MarkerNameSize of MarkerPhysical Position (kb)Chromosome of ‘chiifu’ v1.5
SSRBrSSR117415,476,191A10
SSRBrSSR215815,505,412A10
SSRBrSSR318515,580,043A10
SSRBrSSR416615,712,914A10
SSRBrSSR517515,812,313A10
SSRBrSSR621515,859,041A10
SSRBrSSR718215,872,725A10
SSRBrSSR823815,912,798A10
SSRBrSSR923415,918,222A10
SSRBrSSR1017415,731,259A10
SSRBrSSR1115115,785,574A10
SSRBrSSR1218215,793,126A10
Table 3. Results of BLASTN searches using the candidate interval gene.
Table 3. Results of BLASTN searches using the candidate interval gene.
Gene of B. rapaHomologous Gene in A. thalianaPutative Function
Bra009492AT5G04030unknown
Bra009493AT5G04020Calmodulin-binding
Bra009494AT5G04010F-box family protein
Bra009495AT5G03990FK506-binding-like protein
Bra009496AT5G03980SGNH hydrolase-type esterase superfamily protein
Bra009497AT5G00970F-box family protein
Bra009498AT5G03960IQ-domain 12
Bra009499AT5G03940Chloroplast signal recognition particle 54 KDa subunit protein
Bra009500AT5G03910ABC2 homolog 12
Bra009501AT5G03905Iron-sulfur cluster biosynthesis family protein
Bra009502AT5G03900Iron-sulfur cluster biosynthesis family protein
Bra009503AT5G03900Iron-sulfur cluster biosynthesis family protein
Bra009504AT5G03890unknown
Bra009505AT5G03880Thioredoxin family protein
Bra009506AT5G00893unknown
Bra009507AT5G03850Nucleic acid-binding, OB-fold-like protein s28
Bra009508AT5G03840TFL1 (TERMINAL FLOWER 1); PEBP (phosphatidylethanolamine binding protein) family protein
Bra009509AT5G03795Exostosin family protein
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Chen, C.; Zhu, X.; Zhao, Z.; Du, D.; Li, K. Fine Mapping and Functional Verification of the Brdt1 Gene Controlling Determinate Inflorescence in Brassica rapa L. Agronomy 2024, 14, 281. https://doi.org/10.3390/agronomy14020281

AMA Style

Chen C, Zhu X, Zhao Z, Du D, Li K. Fine Mapping and Functional Verification of the Brdt1 Gene Controlling Determinate Inflorescence in Brassica rapa L. Agronomy. 2024; 14(2):281. https://doi.org/10.3390/agronomy14020281

Chicago/Turabian Style

Chen, Cuiping, Xuebing Zhu, Zhi Zhao, Dezhi Du, and Kaixiang Li. 2024. "Fine Mapping and Functional Verification of the Brdt1 Gene Controlling Determinate Inflorescence in Brassica rapa L." Agronomy 14, no. 2: 281. https://doi.org/10.3390/agronomy14020281

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