A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus
BMC Plant Biology volume 21, Article number: 568 (2021)
The determinate growth habits is beneficial for plant architecture modification and the development of crops cultivars suited to mechanized production systems. Which play an important role in the genetic improvement of crops. In Brassica napus, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross, but there are few reports on it. We fine mapped a determinate inflorescence locus, and evaluated the effect of the determinate growth habit on agronomic traits.
In this study, we assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity, enhancing lodging resistance, increasing plant branches and maintaining productivity. Genetic analysis in the determinate (4769) and indeterminate (2982) genotypes revealed that two independently inherited recessive genes (Bnsdt1, Bnsdt2) are responsible for this determinate growth trait. Bnsdt2 was subsequently mapped in BC2 and BC3 populations derived from the combination 2982 × 4769. Bnsdt2 could be delimited to an approximately 122.9 kb region between 68,586.2 kb and 68,709.1 kb on C09. BLAST analysis of these candidate intervals showed that chrC09g006434 (BnaC09.TFL1) is homologous to TFL1 of A. thaliana. Sequence analysis of two alleles identified two non-synonymous SNPs (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, qRT-PCR revealed that BnaC09.TFL1 expression in shoot apexes was significantly higher in NIL-4769 than in 4769, suggesting its essential role in sustaining the indeterminate growth habit.
In this study, the novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) that may be responsible for inflorescence traits were found.
In higher plants, inflorescences can be divided into indeterminate inflorescences and determinate inflorescences. To date, it has been found inflorescences with determinate growth habits have an important effect on agronomic traits in many plants. The determinate inflorescence mutant tfl1 was screened from wild-type Arabidopsis thaliana using ethylmethane sulphonate (EMS) treatment. The tfl1 mutant shoot apex produced a terminal flower, preventing further shoot apical meristem (SAM) differentiation, and exhibited reduced plant height, early flowering and a clear large-seed phenotype [1,2,3]. In leguminous crops, plants with determinate inflorescence, controlled by Vrdet1 (TFL1 homologue), showed earlier ripening of pods than those with indeterminate inflorescence . In sesame, studies of the determinate inflorescence mutant DS899 showed that compared with plants with indeterminate inflorescence, this plant showed reduced plant height (by approximately 26.4%), increased 1000-grain weight, and shortened growth and flowering periods, which played an important role in the yield improvement of sesame [5, 6]. Kaur and Banga assessed the phenotypic variation in 125 newly identified determinate B. juncea strains. The results showed that among the 125 determinate B. juncea strains, many determinate strains were superior to the indeterminate commercial varieties in some traits (earliness, plant height, pod number, yield, and oil content) . In general, determinate inflorescences have the characteristics of early flowering, reduced plant height and advanced maturity, which play an important role in the genetic improvement of crops.
The TFL1 gene and its homologue play an important role in determinate inflorescence. The TFL1 gene affects stem apex expansion and structure through interaction with the LFY, AP1 and FT genes, which are expressed in the apical meristem of A. thaliana. TFL1 can maintain the attributes of the inflorescence meristem (IM), while LFY and AP1 act as recognition genes in the floral meristem. In the IM, TFL1 mRNA is restricted to the inner cells, and the TFL1 protein is a mobile signal that is uniformly distributed throughout the meristem. In the floral meristem, the signal feedback of LFY and AP1 stimulates the movement of the TFL1 protein and maintains normal expression of the genetic attributes of the floral meristem, thus forming an indeterminate inflorescence that can continuously produce flowers . In addition, the durations of the early vegetative and reproductive stages of the TFL1 mutant were greatly shortened due to the overexpression of FT . Although FT and TFL1 have highly similar amino acid residues, these genes have opposite functions [10,11,12,13]. In cucumber, CsTFL1 interacts with CsNOT2a to inhibit determinate inflorescences and terminal flower formation, but low expression of CsTFL1 causes non-interaction with CsNOT2a to produce determinate growth . In Phaseolus vulgaris, PvTFL1y restored normal indeterminate growth, similar to that of wild-type A. thaliana, when it was introduced into the tfl1 mutant, which has determinate inflorescences . Therefore, the tfl1 gene is essential for the maintenance and control of determinate plant growth.
Rapeseed is one of the most important oil crops in China, among which B. napus is the most important cultivated species. Du et al.  found a determinate inflorescence mutant in microspore culture of B. napus. A genetic analysis showed that the determinate trait is controlled by one recessive gene, Bnsdt1. The Bnsdt1 gene was located on chromosome A10 of B. napus, and BnA10.tfl1 was identified as the gene controlling the trait of determinate inflorescence [16, 17]. However, we found that this trait is not controlled by one recessive gene in breeding. Therefore, a new study on the determinate inflorescence mutant 4769 was carried out. In the present study, the determinate inflorescence mutant 4769 of B. napus was analysed. We found a novel locus (Bnsdt2) of determinate inflorescence and performed fine mapping and cloning. The results will contribute to a better understanding of determinacy.
Effects of determinate growth habit on agronomic traits
To understand the effect of determinate growth habit on agronomic traits, we investigated some agronomic traits of the near-isogenic lines of determinate/indeterminate (4769 and NIL-4769) and eight hybrid combinations over 2 years (2019 and 2020) under standard agricultural conditions in Xining, China. First, these traits (initial flowering, final flowering and maturation) were surveyed and analysed for each plot. For the near-isogenic lines of the determinate/indeterminate plants (4769 and NIL-4769) and eight hybrid combinations, the number of days to initial flowering was not different between determinate and indeterminate lines in the 2 years. However, the final flowering and maturation times were significantly different between the determinate and indeterminate varieties in the 2 years (the determinate preceded the indeterminate varieties by 2–5 days) (Fig. 1A, Table S1). These results indicated that the determinate growth habit could shorten the time to the final flowering period and the mature period. Table S1 presents information about the three traits (initial flowering period, final flowering period, and mature period).
Subsequently, some yield-related traits were surveyed and analysed. The results showed that the determinate inflorescences had no effect on five yield-related traits (number of siliques per plant, seeds per silique, thousand-seed weight, seed yield per plant, yield per plot) in the near-isogenic lines of determinate/indeterminate plants (4769 and NIL-4769) and eight hybrid combinations during 2019 and 2020 (Fig. 1E, F; Table S2). Indeed, these traits did not significantly differ between the determinate and indeterminate groups. However, three other yield-related traits (plant height, number of branches per plant and lodging resistance) were significantly different between the determinate and indeterminate plants (Fig. 1B, C, D; Table S2).
The analysis above showed that compared with the indeterminate plant, determinate B. napus exhibited advancement of final flowering and maturity, reduced plant height, enhanced lodging resistance and increased number of branches per plant. However, it exhibited no penalty effect on the siliques per plant, thousand-seed weight, seeds per silique, seed yield per plant or yield per plot.
The segregation pattern of the growth habit using F2 and BC1 populations was investigated and analysed. Between the determinate and indeterminate genotypes, all F1 individuals displayed a complete indeterminate phenotype, indicating the dominance of the indeterminate growth habit over the determinate growth habit. In 2982 × 4769 F2 plants, 499 and 30 plants had indeterminate and determinate growth, respectively, which was consistent with the 15:1 segregation ratio. The BC1 plants were segregated in an approximate 3:1 ratio (BC1: indeterminate:determinate = 213:62) (Table 1). The results indicated that the determinate growth habit was controlled by two independently inherited recessive genes. Subsequently, we scanned the BC1 plants of the combination 2982 × 4769 with some markers closely linked to the Bnsdt1 gene, and the results showed that it contained the BnSDT1/Bnsdt1 locus. Therefore, we hypothesize that there was a new recessive locus, and the new determinate inflorescence gene was tentatively designated the Bnsdt2 gene.
Primary mapping of the Bnsdt2 gene
Five hundred - seventeen individuals of BC2 population were established by the ((2982 × 4769) × 4769) × 4769 cross to detect molecular markers linked to the Bnsdt2 gene. Six polymorphic markers linked to the Bnsdt2 gene were identified from 512 P + 3/M + 3 (256 P01–16/MC01–16 + 256 P01–16/MG01–16) and 512 EA + 3/M + 3 (256 EA01–16/MC01–16 + 256 EA01–16/MG01–16) AFLP primer combinations and named W01-W06 (Table 2). Next, a genetic linkage map was constructed and the results showed that W01-W05 and W06 were located on both sides of the Bnsdt2 gene (Fig. 2). Among these flanking markers of the Bnsdt2 gene, W01 and W06 were the most closely linked and were 6.1 and 19.2 cM away from the Bnsdt2 gene, respectively. Subsequently, six specific AFLP fragments were successfully cloned and sequenced. By BLAST analysis against the BRAD database (http://www.brassicadb.org/brad/), five AFLP markers (W02-W06) presented sequence homology to chrCnn_random of B. napus (the Darmor-bzh reference genome (Bna41: Brassica_napus V4.1)) (Table 3). The W01 was located on C06 of B. napus. Therefore, it can be inferred that the Bnsdt2 gene is located in the C genome of B. napus (chrCnn_random was not assembled on the C chromosome), presenting great difficulty in mapping.
Fine mapping of the Bnsdt2 gene
According to the physical locations of these markers on chrCnn_random, the Bnsdt2 gene is likely located in the 2.0 Mb–12.0 Mb and 49.0 Mb–71.0 Mb regions of chrCnn_random of B. napus (Table 3). Subsequently, we downloaded the region sequence from BRAD and developed 98 SSR markers. Finally, three SSR markers were found and named BnW07 to BnW09 (Table 4).
B. napus is an allotetraploid species (AACC) formed by Brassica campestris (AA) and Brassica oleracea (CC), and the Bnsdt2 gene is locked on the C chromosome. Therefore, we sequenced the specific fragments of these three SSR markers (BnW07 to BnW09) and compared them with the B. oleracea genome (CC). We found that these three markers were located on C09 (39 M–40 M) of the B. oleracea genome (BOL11: Brassica oleracea V1.1) (Table 4). In this interval, we developed 65 SSR markers and three SSR markers (BoW10 to BoW12) had polymorphism. Therefore, we hypothesize that the Bnsdt2 gene was located on C09. Genomic sequence information for Ningyou 7 (NY7) (http://ibi.zju.edu.cn/bnpedigome/) was obtained by third-generation genome sequencing technology in 2019. We subjected these six SSR markers to BLAST analysis, and the results showed that the Bnsdt2 gene is located on C09 of B. napus and delimited to the region around 68 Mb. Subsequently, five polymorphic SSR markers (from 71 SSR loci) were developed in this interval (BnW13-BnW17). Therefore, the Bnsdt2 gene was finally mapped to C09 of B. napus.
All individuals of the BC2 population were identified using these polymorphic markers on C09 of B. napus. The results showed that four markers (BnW07-BnW09 and BoW10) were located on one side of the Bnsdt2 gene, four markers (BnW13-BnW16) were located on the other side of the Bnsdt2 gene and three markers (BoW11, BoW12 and BnW17) co-segregated with the Bnsdt2 gene (Fig. 3). Among the markers flanking the Bnsdt2 gene, BnW09 and BnW16 were the most closely linked and were 0.3 cM and 0.2 cM from the Bnsdt2 gene, respectively (Table 4). The closely linked markers mentioned above were used for BLAST analysis against BRAD (http://www.brassicadb.org/brad/). The results showed that all of the markers were located on chrCnn_random of the Darmor-bzh reference genome (Bna41: Brassica_napus V4.1) (Table 4). More interestingly, four markers were located near 2 Mb, and another seven markers were located near 9 Mb and turned upside down (Fig. 3). Then, these markers were used for BLAST analysis against the reference genome of B. oleracea (BOL11: Brassica oleracea V1.1), and the results showed that these markers were located on C09 of B. oleracea (near 39 Mb). All the markers corresponding to these markers on our map appeared in the same order as those on C09 of B. napus (Fig. 3). Based on this order, the genomic region containing the Bnsdt2 gene was delimited to an interval of approximately 199 kb between 68,518.8 kb and 68,717.8 kb on C09.
To narrow the target region of Bnsdt2, we constructed 1426 individuals of BC3 population. Subsequently, six SSR markers (BnW18-BnW23) were developed and identified from the interval of 68,518.8 kb - 68,717.8 kb on C09 (Table 4). These six SSR markers and some previously used markers (BnW09, BoW11-BoW12, BnW16-BnW17) were employed to screen 1426 BC3 individuals. The results showed that the Bnsdt2 gene was positioned between BoW11 and BnW21. By BLAST analysis against BnPedigome, the Bnsdt2 gene was delimited to an interval of approximately 122.9 kb between 68,586.2 kb and 68,709.1 kb on C09 of B. napus (Fig. 4). In addition, we also found two co-dominant markers (BnW09 and BnW18) from the above markers, which could be used in molecular marker-assisted breeding of rapeseed.
Dissection of the Bnsdt2 target region
The candidate sequences were submitted to BnPedigome (http://ibi.zju.edu.cn/bnpedigome/) and TAIR database (http://www.arabidopsis.org/) for BLAST analysis. The results showed that the candidate region included 15 predicted genes and were homologous with 14 Arabidopsis genes (Table 5). According to the TAIR database of gene annotation of these genes, the chrC09g006434 (BnaC09.TFL1) gene is homologous to the AT5G03840 gene (Table 5). The AT5G03840 is the TERMINAL FLOWER 1 (TFL1) gene of A. thaliana, which encodes a phosphatidylethanolamine binding protein (PEBP). In Arabidopsis TFL1 mutants, the determinate inflorescence can be formed at the top of the inflorescence. Therefore, we inferred that chrC09g006434 (BnaC09.TFL1) gene was the most important candidate gene of the Bnsdt2, and select this gene for further study.
Sequence analysis of BnaC09.TFL1
The sequences of gDNA and CDS of the BnaC09.TFL1 gene were successfully obtained from parents and near-isogenic lines. Sequence alignment analysis showed that there were no sequence differences between the parents and the corresponding near-isogenic lines with the same inflorescence traits. Subsequently, the gDNA sequences of determinate and indeterminate inflorescences were analyzed and the results showed that there were many differences between gDNA of determinate and indeterminate inflorescences. It contains 22 single-base mutations,1 insertion (2 bases) and 2 deletions (6 bases and 2 bases) (Fig. 5). Amino acid sequence prediction and analysis of the BnaC09.TFL1 gene showed that two non-synonymous SNP mutations (T136C, G141C) were found in the first exon of the BnaC09.TFL1 gene. It leads to two amino acid substitutions (Phe replaces Leu and Leu replaces Phe) (Fig. 6), while the other amino acids were conserved (98.88%).
Expression pattern analysis of BnaC09.TFL1
We studied the expression pattern of BnaC09.TFL1 in near-isogenic lines (4769 and NIL-4769) using qRT-PCR. In order to avoid the effect of homologous copies, the qRT-PCR primers was designed at specific locations of the CDS sequences of determinate and indeterminate (Table S3). The results of expression analysis showed that the BnaC09.TFL1 expression had little difference in leaf and root organs, whereas the difference in the shoot apex was notable. The total expression level of BnaC09.TFL1 in the shoot apex was significantly higher than that in other organs, indicating that BnaC09.TFL1 was specifically expressed in the shoot apex. In addition, the expression of BnaC09.TFL1 in NIL-4769 was significantly higher than that in 4769, especially in P2 and P3, which represent the early stage before the formation of inflorescence traits (Fig. 7). Therefore, it is reasonable to hypothesize that BnaC09.TFL1 is a potential candidate gene for inflorescence traits.
Our study showed that the determinacy have no effect on initial flowering, but could advance final flowering and maturation (2–5 days) and shorten the growth period in both near-isogenic lines and hybrid combinations. At present, the phenomenon of shorten growth period had also been found in the determinacy of other plants. Such as A. thaliana [18, 19], Antirrhinum majus , Solanum lycopersicum  and Sesamum indicum [5, 6]. But the determinacy of these plants shorten the growth period by flowering early, while the determinacy of B.napus did not bloom early. The reason of short growth period of the determinacy in B.napus was analyzed. It was believed that the determinacy broke the advantage of inflorescence apex, accelerated the growth and development of each inflorescence, increased the number of flowering per unit time, shortened the flowering time of the plant. Compared with the indeterminacy, the determinacy could terminate flowering earlier and shorten the whole growth period. In addition, the determinacy of B.napus could reduce plant height and enhance lodging resistance. This was consistent with the research results in Sesamum indicum , Glycine max Merr , Vicia faba , B. juncea . The determinacy of B.napus could enhance the cause of the lodging resistance were analyzed. We hold the opinion that due to the degradation of the top of the determinacy, the length of each inflorescence became shorter, the plant height decreased, and each inflorescence was basically at the same level height, so that the whole plant was in a balanced state without tilting to a certain direction. Then, we studied the effect of the determinacy on yield, and the results showed that the determinacy had no negative effect on yield. This was consistent with the research results of other plants with determinate growth habit. We analyzed the causes of its formation and found that the determinacy could increase number of branches per plant, but had no effect on the siliques per plant, thousand-seed weight, seeds per silique, seed yield per plant or yield per plot. This indicated that the determinacy have the same number of siliques per plant as indeterminacy by increasing the number of branches, when the number of silique per branch becomes smaller. And it could ensure that the yield does not decrease.
With the discovery of determinate inflorescence mutants in A. thaliana , Antirrhinum majus , Sesamum indicum , Vigna radiata , Glycine max , Cucumis sativus  and B. juncea , among others, the genetics of determinate inflorescences have been investigated in these plants, and determinacy is controlled by one recessive gene. The determinate inflorescence strain (4769) of B. napus has been discovered among DH lines obtained from a spring B. napus × winter B. napus cross. Genetic analysis revealed that the determinate inflorescence resulting from the two combinations of 2014 × 4769 and 2092 × 4769 was controlled by one recessive gene, Bnsdt1. However, we found that it is not controlled by one recessive gene in breeding. Therefore, the DH lines 2982 and 4769 of B. napus were used as materials in the present study. The results showed that the determinate inflorescence (4769) resulting from the combination of 2982 × 4769 cells was controlled by two independently inherited recessive genes, namely, Bnsdt1 and Bnsdt2. The Bnsdt2 gene is a newly recessive locus. Zhang et al.  studied a natural mutant strain FM8 of B. napus with a determinate inflorescence, and genetic analysis showed that the inheritance of the determinate inflorescence was controlled by the interaction of two recessive genes and one recessive epistasis suppressor gene. The above findings indicate that the genetic pattern of determinate inflorescence mutants is different in B. napus. This may be caused by the following two reasons: on the one hand, the mutant type of determinate inflorescence is different; on the other hand, the parent materials of indeterminate inflorescence are closely related to determinate inflorescence when the genetic pattern of determinate inflorescence traits is analysed, which results in loss of the genetic locus.
In previous studies, most of the rapeseed genes that control quality traits were isolated by map-based cloning approaches [7, 27, 28]. We used this method to map the determinate trait of B. napus. In the early stages of the experiment, we could preliminarily inferred that the Bnsdt2 gene was located in the C genome of B. napus. But we didn’t know exactly where the Bnsdt2 gene was located on which chromosome of the C genome. Then we took the genomic sequence information of B. oleracea as reference and mapped the Bnsdt2 gene to the C09 chromosome. With the released of Ningyou 7 (NY7) genome sequence information in 2019. Finally, the Bnsdt2 gene was delimited to an interval of approximately 122.9 kb between 68,586.2 kb and 68,709.1 kb on C09 of B. napus. The above findings indicated that a suitable reference genome for gene mapping is very important. In the case of allopolyploid plants, the reference genome of the original ancestors could be referred to in the absence of a suitable reference genome. In addition, according to gene annotation of these genes in the intervals, we found that the gene chrC09g006434 (BnaC09.TFL1) was highly similar to the TFL1 gene in A. thaliana. Sequence analysis of BnaC09.TFL1 showed that the gDNA sequences had many differences between indeterminate and determinate sequences. The sequence contains 2 deletions (of 6 bases and 2 bases), 1 insert (of 2 bases) and 22 single base mutations (Fig. 7) and identifies two non-synonymous SNP mutations (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, we performed an analysis of the expression pattern of BnaC09.TFL1. The results showed that BnaC09.TFL1 was specifically expressed in the shoot apex. Therefore, it is reasonable to postulate that BnaC09.TFL1 is a potential candidate gene for the inflorescence trait.
In summary, these studies are important because we are not familiar with determinate inflorescences in B. napus. They also provide a basis for future breeding and gene cloning.
In this study, we identified a novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) was identified as suitable candidate genes for Bnsdt2 according to sequence analysis and qRT-PCR analysis. In addition, the determinate growth habit has positive effects on agronomic traits. It provides a new insight for plant architecture modification and the development of rapeseed cultivars suited to mechanized production systems.
Plant material and population construction
The DH lines 2982 and 4769 of B. napus were used as materials in the present study. The inflorescence of 2982 is indeterminate, and the inflorescence of 4769 is determinate (from the progeny of microspore culture [spring B. napus × winter B. napus] F1). Inheritance of the inflorescence trait was studied with F2 and BC1 populations. The inflorescence traits were investigated during the flowering period. Figure 8 shows two IM development phenotypes in near-isogenic lines.
The Bnsdt2 locus was separated by using the BC1 population derived from a 2982 × 4769 cross. In the BC1 population, we selected 10 indeterminate individuals (These 10 indeterminate individual were screened by some markers closely linked to Bnsdt1.Theoretically, the genotype of these 10 indeterminate individual were Bnsdt1Bnsdt1BnSDT2Bnsdt2.) and backcrosses with 4769 individuals to establish a BC2 isolate line (Fig. S1), which was sown in the Xining Experimental Base (Qinhai Province, China). The inflorescence traits of each line were investigated. Due to the existence of recombinant plants and the number plants too little in some isolated lines. The results showed that the separation ratio of indeterminate to determinate was 1:1 in 3 out of 10 lines. Therefore, we hypothesized that these three strains contain a dominant locus (BnSDT1 or BnSDT2). To verify that the three strains contained the BnSDT1 or BnSDT2 locus, 72 indeterminate individuals (each strain containing 24 individuals) were selected for scanning by the co-dominant markers closely linked to the Bnsdt1 gene. The results showed that the co-dominant markers did not show different bands in 72 indeterminate plants and showed the same banding pattern as the determinate. This indicates that the three strains are controlled by another locus, so their genotypes are speculated to be aaBb (Bnsdt1Bnsdt1BnSDT2Bnsdt2) (Fig. S1). Therefore, three strains containing the BnSDT2 locus were isolated from the BC2 population and used for mapping the Bnsdt2 gene. To conduct fine mapping of the Bnsdt2 gene, in the three BC2 isolate strains, we selected 15 indeterminate individuals (five indeterminate individuals each strains) and backcrosses with 4769 individuals to establish 1426 individuals of the BC3 population.. In order to assess the agronomic performance of the determinacy, we selected five indeterminate individuals (The background recovery rate was 100% by scanning of 38 SSR markers) self-cross twice in the BC3 population, and constructed near-isogenic lines 4769 (determinate) and NIL-4769 (indeterminate).
Field trial and trait evaluation
Eight hybrid combinations were operated between the near-isogenic lines 4769 (determinate)/NIL-4769 (indeterminate) and determinate lines 1583, 1589, 1593, and 1595. The near-isogenic lines and eight hybrid combinations were grown in Xining (Qinhai Province, China) for 2 years (2019 and 2020). A randomized complete block design was conducted with three replications. Each plot was planted with five rows, with 12 plants in each row and a distance of 15 cm between plants within each row and 30 cm between rows. During the entire reproductive stage, the initial flowering, final flowering and maturation were recorded for each plot. At the mature stage, 8–10 open-pollinated plants from each plot were selected to test agronomic traits, and the seeds were threshed by hand. For agronomic traits, the main tests included plant height (PH), number of primary branches (NPB), number of secondary branches (NSB), number of branches per plant (NBP), number of siliques per plant (NSP), seeds per silique (SS), thousand seed weight (TSW), seed yield per plant (SYP) and yield per plot (YP). Lodging was investigated before maturation of the plant, and the lodging index was calculated following the methods described by Shi . The data of these traits were analysed by using SPSS statistical software .
DNA extraction and amplified fragment length polymorphism (AFLP) analysis
DNA was extracted individually from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method . Two determinate and indeterminate bulks were constructed using equivalent amounts of DNA from 12 determinate and 12 indeterminate individuals in BC2 populations, respectively. Subsequently, two determinate and indeterminate bulks were used for bulked segregant analysis (BSA)  in combination with the AFLP technique. Genomic DNA was cleaved with the restriction enzymes PstI, EcorI and MseI. Pre-amplification was performed using the AFLP primers P0/MC, P0/MG, EA/MC, and EA/MG. The preamplified product was diluted (1:30) and used for selective amplification. AFLP amplification was performed as described by Vos et al. . The product of selective amplification was separated and silver stained as described for AFLP markers.
AFLP and SSR marker sequencing and identification of B. napus synteny
The expected AFLP and SSR bands were collected and purified following the methods described by Yi et al. . The purified products were ligated into the pMD18-T Easy vector (TaKaRa). Subsequently, M13-specific primers were used to detect the transformed clones. For each fragment, three positive clones were randomly selected and sequenced by Shanghai Sangon Biotechnology Corporation (Shanghai, China). After genetic mapping of The BC2 and BC3 populations, the sequences of these markers (six AFLP and 17 SSR markers) were used to identify putative homologous sequences within the B. napus genome. BLAST analysis was performed using the Brassica Database (BRAD) (http://www.brassicadb.org/brad/) and BnPedigome Database (http://ibi.zju.edu.cn/bnpedigome/).
Development of SSR markers
Because the chromosome sequence of B. napus has been published, the DNA sequence of the segment was downloaded from BRAD and the BnPedigome database according to the marker-locked region. SSR loci were detected using SSRHunter 1.3 software , and the SSR primers were designed using Primer 3 software . SSR amplification was performed as described by Lowe et al. . The amplified products were separated on a 6% denaturing polyacrylamide gel.
The BC2 populations (547 individuals) and BC3 populations (1426 individuals) were used for mapping the Bnsdt2 gene. The AFLP data, SSR markers and individual phenotypes were analysed with the JoinMap 4/MapDraw program [38, 39], and a partial linkage map of the region on the chromosome spanning the Bnsdt2 gene was constructed. The genetic distance (in cM) was calculated using the Kosambi function.
Cloning and expression analysis of the candidate gene
The BnaC09.TFL1 gene was amplified from the genomic DNA of 2982 and 4769 using the primers described in Table S3. The gDNA sequences were analysed using DNAMAN 8.0, and amino acid sequence prediction and analysis of the BnaC09.TFL1 gene were performed using Premier 5. For real-time qRT-PCR analysis, the shoot apex of near-isogenic lines (4769 and NIL-4769) were sampled at different developmental stages (P1: 2-leaf seedlings, P2: 4-leaf seedlings, P3: budding period, P4: initial flowering), frozen quickly in liquid nitrogen and stored at − 80 °C for RNA extraction. Total RNA was isolated by the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China). RNA was also extracted from the roots and leaves of 2-leaf seedlings. The first-strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. To measure the mRNA levels of genes, qRT-PCR was conducted using a LightCycler® 480 Instrument II (Roche, Basel, Switzerland) with SYBR Green Mix (TaKaRa, Dalian, China). The actin gene was selected as the reference gene for relative quantification of the candidate gene. The PCR conditions were as follows: 95 °C for 2 min followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s. A melting curve analysis was also performed to confirm the specificity of the primers. The data were processed using the 2−ΔΔCT method .
In this study, The methods were carried out in accordance to the current laws of China and international guidelines and legislation. The plant materials (or cultivars) were collected and preserved, with the appropriate permissions, by the Academy of Agricultural and Forestry Sciences of Qinghai University in China. All necessary permissions for planting and investigating these plant materials (or cultivars) were obtained from Academy of Agricultural and Forestry Sciences of Qinghai University in China, and the collection and research of these plant materials (or cultivars) have complied with the Convention on the Trade in Endangered Species of Wild Fauna and Flora.
Availability of data and materials
All data used during the study are included in this published article and its additional files.
Quantitative real-time PCR
Amplified fragment length polymorphism
Simple sequence repeat
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This research was financially supported by the National Natural Science Foundation of China (31960434), the Qinghai Provincial Natural Science Foundation of China (2019-ZJ-972Q), the Key Laboratory of Spring Rape Genetic Improvement of Qinghai Province (2020-ZJ-Y10) and the Earmarked Fund for China Agriculture Research System (CARS-12).
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Population construction. Table S1. Flowering analysis of near-isogenic lines ear-isogenic lines (NIL-4769 and 4769) and eight hybrid combinations. Table S2. Analysis of agronomic traits of near-isogenic lines (NIL-4769 and 4769) and eight hybrid combinations. Table S3. The primer sequences of gDNA, cDNA and qRT-PCR.
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Li, K., Xu, L., Jia, Y. et al. A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus. BMC Plant Biol 21, 568 (2021). https://doi.org/10.1186/s12870-021-03348-0