Brassica campestris is an important oil crop of the genus Brassica in Brassicaceae. Flowering time is an important agronomic trait in the production of Brassica rapa. The early or late flowering time has an important impact on its yield and quality. There are many genes regulating the flowering time of Brassica campestris, and the gene regulatory network is complex. Moreover, environmental factors have important effects on flowering time and gene expression. A large number of flowering genes have been found in Brassica rapa, and the functions of some flowering genes have been verified (Liu et al., 2019; Su et al., 2020). Xiao (2012) identified 144 candidate gene markers related to flowering time in a double haploid population of Chinese cabbage. 10 QTLs related to flowering time were detected under different growth conditions, and candidate genes such as BrFLC2, SOC1 and FT were screened. Gong (2016) detected 2 QTLs controlling flowering of Chinese cabbage by genome-wide association analysis, which were in linkage groups A02 and A07, respectively. The closest gene to SNP-A026709555 on chromosome A02 is Bra035633, and the closest gene to SNP-A0711143455 on chromosome A07 is Bra012139. Both genes are not in the attenuation range, so other candidate genes related to flowering time located in the 2 QTLs attenuation regions need to be further found. Gao et al. (2017) re-sequenced 116 natural populations of Brassica rapa. And correlation analysis was conducted between resequencing results and flowering time of Brassica rapa under different conditions. 14 candidate genes related to flowering time of Brassica rapa, including FUL, PHYB, FPF1 and FT1, were finally obtained through gene collinearity and gene function annotation between Brassica rapa and Arabidopsis thaliana. Wu et al. (2012) analyzed the association between resequencing results and flowering time traits of 159 Brassica rapa and found that an InDel mutation in BrFLC2 gene was closely related to flowering time. Li et al. (2019) analyzed the molecular mechanism, evolution, expression and function of the members of the important gene family MAF/FLC that controls flowering time in Brassica rapa. The results showed that the AtMAFs gene in Arabidopsis thaliana had high sequence similarity with BrMAFs gene in Brassica rapa, but their gene functions were different between the two species. In addition, the analysis of gene expression level confirmed the triplication event of Brassica rapa gene.

With the continuous development of biological sequencing technology, it is more and more common to explore the molecular mechanism of plant morphology and traits by resequencing technology, especially in major crops and vegetables (Cheng et al., 2016). Genome-wide association analysis (GWAS) using resequencing technology plays an important role in the analysis of complex quantitative traits in plants. At present, GWAS has been successfully applied in the association analysis of plants and traits. Association analysis is widely used in model plants of various families. Xuan et al. (2019) screened new candidate genes BnaA.GL1.a, BnaC.SWEET4.a, BnaC.WAT1.a, and BnaC.WAT1.b related to leaf trichome formation in Brassica napus by GWAS indicating that sugar and auxin signaling are involved in leaf trichome initiation. 31 soybean flowering time traits related candidate genes Glyma10g36600 and Glyma19g37890 were obtained by resequencing and genome-wide association analysis (Song, 2018).

In addition, GWAS plays an important role in screening candidate genes related to flowering time traits in maize (Li et al., 2020), candidate genes related to seminal root length of maize seedlings under drought stress (Guo et al., 2019), candidate genes related to seed density within per silique and its related traits in Brassica napus (Ren et al., 2018), and candidate genes controlling continuous storage root formation and bulking in potato (Bararyenya et al., 2020). The study on flowering time is of great value for the genetic breeding of yield and quality of Brassica napus. In this study, 110 Brassica campestris collected nationwide were used as the research objects. The high-quality SNP polymorphism markers of 110 materials were obtained by resequencing. Combined with the flowering schedule type and its significantly associated SNP, the flowering time related candidate genes were determined according to the linkage disequilibrium intensity and gene annotation, which provided effective molecular markers and theoretical basis for the genetic breeding of early or late flowering materials.

1 Results and Analysis

1.1 Obtain of resequencing data

After strict filtering of the sequencing data, high-quality sequencing data were obtained. The total amount of sequencing data was 300.297 G, and the amount of high-quality data was 299.751 G. The genome size was 351 063 200 bp (excluding N), and the GC content was 36.83 %. The average alignment rate of population samples was 97.25%. The average sequencing depth of genome was 6.55 (only considering the sequence with alignment quality greater than 0), and the average coverage of samples was 83.85%. A total of 6 619 760 SNP loci were detected by SAMTOOLS software. After further strict filtration, a total of 140 164 high-quality SNP loci were finally obtained for subsequent analysis.

1.2 Flowering schedule type of Brassica campestris

The flowering time of 110 Brassica campestris was observed and recorded. The results showed that the flowering time of Brassica campestris from different sources was quite different (Table 1), and the variation range of flowering time was 122~156 d. Some materials from Sichuan, Yunnan, Tibet and Fujian flowered earlier, the flowering time was about 125 d, while some materials from Jiangsu and Qinghai flowered later, and the flowering time was more than 150 d. There were 24 materials with flowering time range of 122~125 d, 36 materials with flowering time range of 126~132 d, 25 materials with flowering time of 133~143 d, 16 materials with flowering time of 144~151 d, and 6 materials with the latest flowering time of 152-156 d. The flowering time of 110 materials showed partial normal distribution (Figure 1).

Table 1 Information and flowering time of Brassica campestris

Figure 1 The frequency distribution of flowering time of 110 Brassica campestris accessions

1.3 Analysis of population structure and genome-wide linkage disequilibrium

140 164 high-quality SNP loci obtained by resequencing were used to analyze the population structure of 110 materials. The results showed that △K had the maximum change when K=2 (Figure 2A). At the same time, the population phylogenetic tree analysis of 110 materials showed that the 110 Brassica campestris populations could be divided into two subgroups. The small subgroup included 28 materials, mainly from spring and part of semi-winter rapeseed in Tibet, Qinghai and Jiangsu, and the large subgroup included 82 materials, mainly from semi-winter rapeseed lines in China (Figure 2B). The population evolutionary tree analysis of 110 Brassica campestris materials was performed, and it was found that the internal distribution of each subgroup was relatively concentrated, and the distribution between the two subgroups was also closely related to the geographical origin of the material. In addition, the population genetic structure and principal component analysis (PCA) were carried out, and the results verified the phylogenetic tree results, that was appropriate to divide 110 population materials into two subgroups, and the materials with similar geographical locations had close genetic relationship. And clustered together in the figure, the farther the geographical distance was, the greater the genetic background difference was (Figure 2C; Figure 2D; Table 2).

Figure 2 Population structure of 110 Brassica campestris materials Note: A: Estimation of the number of sub-populations of 110 Brassica campestris L. materials; B: Neighbor-joining tree of the 110 B. campestris L. materials; C: Population structure of 110 B. campestris L. materials based on SNP markers; D: PCA of 110 B. campestris L. materials; E: The genome-wide LD decay in B. campestris L.

Table 2 Color information of 110 Brassica campestris samples in the figures

Genome-wide linkage disequilibrium analysis (Figure 2E) was performed using 140 164 high-quality SNP loci combined with flowering time of 110 B. campestris materials. The results showed that when R2 decreased from 0.167 to 0.08, the corresponding physical length was 19 kb. Therefore, the average genome-wide LD of B. campestris was 19 kb.

The GEMMA software was used to perform genome-wide association analysis on the flowering time of the population using the mixed linear model, and the linkage strength analysis was performed on the obtained signal points. When -log10(P) > 5, 4 signal sites significantly associated with flowering schedule type were obtained, among which 3 were located on chromosome A05, mutated from base C to T, 1 was located on chromosome A10, mutated from base G to A (Figure 3; Table 3).

Figure 3 Manhattan plot and qq plot of genome-wide association study for flowering time in B. campestris

Table 3 The strong association signals and functional annotation

1.4 Candidate gene prediction

The flowering time candidate sites were determined according to the significantly associated SNP sites and linkage disequilibrium interval, and the candidate genes related to flowering time in the site were further predicted. 9 candidate transcripts were screened from 4 significant association sites. Further analysis of the 9 transcripts showed that the 9 transcripts corresponded to 4 candidate genes, namely Bra010171, Bra036954, Bra030175, and Bra030176, which corresponded to At1g32540 (LOL1), At2g34960 (CAT5), At5g05580 (FAD8), and At1g74530 (unknown) genes in Arabidopsis thaliana. Whether these 9 transcripts and 4 candidate genes are related to flowering time will be further verified (Table 4).

Table 4 Candidate genes for flowering time of Brassica campestris

2 Discussion

Flowering time is an important agronomic trait affecting the yield and quality of Brassica rapa. There are many factors affecting the flowering time of Brassica rapa, including the complex gene regulatory network in plants and the external environmental factors. Previous studies have shown that the differential expression of BrFLCs (BrFLC1, BrFLC2, BrFLC3, BrFLC4, and BrFLC5) gene leads to the difference of flowering time in Brassica crops. Multi-copy BrFLCs genes regulate flowering time by the interaction of additive effects. The higher the level of FLC transcripts, the longer the flowering time of plants. The results showed that 5 natural antisense transcripts of BrFLC2 were induced to express and the expression of 4 BrFLC genes decreased gradually during vernalization of Chinese cabbage Bre. In addition, the results showed that transgenic plants overexpressing natural antisense transcripts of BrFLC2 did not require vernalization at low temperature, which confirmed that natural antisense transcripts of BrFLC2 could change the growth cycle of Chinese cabbage (Li et al., 2016). Li (2019) found that 2 insertions/deletions (76-bp) in BnFLC-C2 promoter region of Brassica napus were closely related to flowering time. Zhang (2018) found that the point mutation of BcFLC1 gene in WuCai (Brassica campestris L.) could affect its interaction with SVP gene, further affecting the expression of downstream FT and SOC1 genes, resulting in the late bolting phenotype of WuCai (Brassica campestris L.). Cheng (2017) et al. summarized 6 pathways regulating flowering time in Arabidopsis thaliana, and pointed out that FCA, FLD, FLK, FPA, FVE, FY and LD were involved in the autonomous flowering pathway, in which the flowering suppressor gene FLC played an important role in the autonomous flowering pathway. In recent years, there are more and more studies on agronomic traits of crops using genome-wide association analysis. Gao et al. (2017) used 116 natural populations of Brassica rapa as research materials. Resequencing technology and GWAS analysis were used to locate candidate genes related to flowering time of Brassica rapa, and 33 significant correlation signals related to flowering time were identified. Finally, 14 candidate genes related to flowering time of Brassica rapa were preliminarily identified, including Bra022475, Bra029347, Bra030284, Bra022192, and Bra024337, which were homologous genes of Arabidopsis FT1, FUL, GRP7, PHYB, and HYS1. In this study, 110 natural populations of Brassica campestris were used as the research objects to investigate their flowering time and resequencing the natural population. The obtained SNP markers were used for GWAS analysis, and 4 signal sites significantly associated with flowering schedule type were identified. A total of 9 candidate transcripts were selected from these 4 significant association sites. 4 candidate genes related to flowering time of Brassica campestris were obtained through gene collinearity and gene function annotation between B. Brassica campestris and Arabidopsis thaliana, which were homologous genes of Arabidopsis thaliana LOL1, CAT5, and FAD8. It has been reported that LOL1 gene may be involved in the response of Fagopyrum tataricum to abiotic stresses such as high concentration of salicylic acid, UV-B irradiation, and cold stress (Gao et al., 2015). Epple et al. (2003) identified two LSD1-like genes AtLOL1 and AtLOL2 in Arabidopsis thaliana. The results showed that AtLSD1 and AtLOL1 regulated the accumulation of Cu-ZnSOD by antagonism to determine the fate of cells. Yeh et al. (2011) found similar results by functional study of LSD1. The results showed that these LSD1 zinc finger proteins had regulatory functions on allergic cell death, plant spectral disease resistance, and abiotic environmental stresses such as low temperature and long sunshine. The candidate gene Bra036954 identified in this study is a homologous gene of CAT5 in Arabidopsis thaliana. CAT5 is a basic amino acid transporter with high affinity on the cell membrane, and its function may be to reabsorb the leaked amino acids at the leaf edge (Su et al., 2004; Zhao et al., 2012). The homologous gene corresponding to the candidate gene Bra030175 is the fatty acid desaturase gene FAD8. It has been reported that the FAD gene family is not only an important gene regulating plant fatty acid composition, but also involved in the regulation of plant cold stress, heat stress and drought stress response (Tovuu et al., 2016; Zhang et al., 2018). The functions of the 4 candidate genes identified by us in flowering time have not been reported. Therefore, whether the 4 candidate genes related to flowering time of Brassica campestris obtained in this study are involved in flowering regulation remains to be further identified.

3 Materials and Methods

3.1 Materials and reagents

A total of 110 test materials Brassica campestris were widely collected from various regions in China and abroad. The 110 materials were natural populations, whose names, field numbers, geographical sources and flowering time were recorded in Table 1. And all materials were planted in the open field at the base of Zhuanghang Comprehensive Experiment Station of Shanghai Academy of Agricultural Sciences (Altitude 3.3 m, 121°38′ E, 30°88′ N).

3.2 Investigation on flowering time

The flowering time from sowing to 1/4 number of plants is considered to be the first flowering stage, and the time required for each material from sowing to first flowering is recorded as its day to flowering (DTF). The flowering time of 110 Brassica campestris materials was observed and recorded at flowering stage.

3.3 Genome-wide resequencing

The young leaves of 110 Brassica campestris materials at seedling stage were selected, and 3 typical plants of each material were selected as 3 biological replicates. The DNA of each sample was extracted by CTAB method. Illumina PE150 sequencing method (Illumina Inc., USA) was used to perform double-terminal resequencing of 110 sample DNA, resulting in a 150 bp double-terminal sequence (Reads) with a total sequencing data of 300.297 G. The raw data obtained by sequencing are strictly filtered to obtain high-quality sequencing data. The effective high-quality sequencing data were compared to the reference genome of Chinese cabbage by BWA software (Li and Durbin, 2009), and the repeat sequences were removed by SAMTOOLS software (Li et al., 2009), and the SNP polymorphism of the population was detected. Then, the Bayesian model was used to detect the polymorphic loci in the population, and high-quality SNPs were obtained through the 3 strict filtering conditions of Q20 quality control, SNP site spacing requirements and SNP coverage depth requirements. Finally, the strong annotation function of ANNOVAR (Wang et al., 2010) was used to annotate the SNPs detection results.

3.4 Population genetic diversity analysis

The high-quality SNPs data obtained by resequencing were used for population evolutionary tree analysis, principal component analysis, and population genetic structure analysis of 110 Brassica campestris materials. Neighbor method was used to construct population evolutionary tree, and the leading value is calculated 1000 times. The guiding value was obtained after 1000 calculations. Population principal component analysis was used to draw the PCA distribution map by GCTA software and R software. Population genetic structure analysis was carried out by PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/), and the results were presented in the form of population genetic structure map.

3.5 Genome-wide linkage disequilibrium analysis

Haploview software was used for genome-wide linkage disequilibrium analysis of filtered high-quality SNP set sites.

3.6 Genome-wide association analysis

GEMMA software (http://www.xzlab.org/software.html) and mixed linear model were used to perform association analysis of flowering time traits in 110 Brassica campestris natural populations. According to the significance of association, potential candidate SNPs related to flowering time traits were screened out. Transcripts or genes in a certain area of upstream and downstream of the chromosome position of candidate SNPs were screened and functional analysis was conducted to predict candidate genes that may be related to flowering time of Brassica campestris.

Authors’ contributions

JJX conceived of this review, wrote the first draft, and carried out sequencing analysis and florescence observation and statistics. LYL, JMY, and ZJF were responsible for consulting and sorting out relevant documents, as well as sequencing data analysis. ZXR, WWR, and SC revised the manuscript. ZJY, and YLY were corresponding authors, who were responsible for this review. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the Growth Program for Young Talents in Shanghai Municipal Agricultural System [Hunongqing(2018) No.1-5].

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