BnIR, Brassica napus multi-omics database (information resource)

BnIR Brassica napus multi-omics information resource ————A multi-omics database with various tools for Brassica napus research and breeding

e.g. FLC or AT1G02970

About phenotype

In Phenotype portal, we collected 118 traits of 2,512 accessions, including 50 traits of 525 inbred lines^[1], 36 glucosinolate related traits of 288 accessions^[2], five traits of 991 accessions^[3-5], three flowering time traits of 210 accessions^[6], and 27 traits of 505 accessions^[7-9]. These phenotypic data are grouped into five modules based on their data sources, and users can browse these phenotypic datasets in the corresponding modules.

Tutorials for each sub-module

Phenotype

References

// 525 inbred lines (Bus et al. 2011)

This module comprises of two phenotypic datasets from 525 lines. The population was constructed by Bus, A., et al (2011). Of them, 509 inbreds were grown in six replicates, for 30 days in an α-lattice design with 24 blocks of 24 pots in a green-house experiment. The 20 seedling development traits were assessed by Körber, N. et al (2015) to cover a wide range of aspects as well as developmental stages during seedling growth which could be measured with high throughput methods. And 30 agronomic and seed quality traits of 410 inbreds was collected by Körber, N. et al (2016).

[1] Bus, A., Körber, N., Snowdon, R.J. & Stich, B. Patterns of molecular variation in a species-wide germplasm set of Brassica napus. Theor Appl Genet 123, 1413-1423 (2011).
[2] Körber, N. et al. Seedling development traits in Brassica napus examined by gene expression analysis and association mapping. BMC Plant Biology 15, 136 (2015).
[3] Körber, N. et al. Agronomic and Seed Quality Traits Dissected by Genome-Wide Association Mapping in Brassica napus. Front Plant Sci 7, 386 (2016).

// Glucosinolate proﬁle (Kittipol, V. et al. 2019)

This phenotypic data comprises of leaf and root glucosinolate (GSL) proﬁles across a diversity panel of 288 B. napus lines and were collected by Kittipol, V. et al (2019).

Detailed methods about growth of plant material for glucosinolate content A subset of 288 B. napus accessions was grown in long day (16/8 h, 20 ℃/14 ℃) under controlled glasshouse conditions (University of York, UK). Within this panel, there are 56 Modern Winter OSR, 65 Winter OSR, 6 Winter Fodder, 121 Spring OSR, 26 Swede and 14 Exotic varieties. Four biological replicates of each accession were grown in root trainers with Terra-Green for ease of root harvesting, supplemented weekly with a half concentration of Murashige and Skoog growth medium adjusted to pH6.5 with KOH. The experiment was arranged as randomized four-block design with one plant per lines in each block. Four weeks after sowing, the third true leaf and the whole root system were harvested from each plant. At harvest, leaves were cut at the base, wrapped in a labelled aluminum foil and immediately frozen in liquid nitrogen. Plants were removed from the tray, had the roots washed, dried with paper towel and cut. All samples were wrapped in labelled aluminum foils and immediately frozen in liquid nitrogen and stored at -80 ℃.

[1] Kittipol, V. et al. Data in support of genetic architecture of glucosinolate variations in Brassica napus. Data Brief 25, 104402 (2019).

// 991 accessions (Wu D, et al. 2019)

The 991 rapeseed (B. napus) germplasm accessions include 658 winter, 145 semi-winter, and 188 spring types, which correspond roughly to their geographical distributions in Europe, Asia, and the Eurasian continent/North America, respectively, and were collected by Wu, D. et al(2019). Flowering time of 926 accessions were surveyed by Wu, D. et al(2019). The 290 accessions were grown in the experimental field of Changxing Agricultural Experiment Station of Zhejiang University (30°02′N and 119°93′E) in 2017 and the Experimental Farm of Jinhua Academy of Agricultural Sciences (29°05′N and 119°38′E) in 2018, respectively and their seed oil contents were measured by Wang, H. et al(2021) and leaf trichome traits were surveyed by Xuan, L. et al(2020).

[1] Wu, D. et al. Whole-genome resequencing of a worldwide collection of rapeseed accessions reveals the genetic basis of ecotype divergence. Mol. Plant 12, 30-43 (2019).
[2] Wang, H. et al. Genome-wide association study reveals a patatin-like lipase relating to the reduction of seed oil content in Brassica napus. BMC Plant Biol 21, 6 (2021).
[3] Xuan, L. et al. Genome-wide association study reveals new genes involved in leaf trichome formation in polyploid oilseed rape (Brassica napus L.). Plant Cell Environ 43, 675-691 (2020).

// 210 accessions (Song JM, et al. 2020)

A natural population including 210 rapeseed accessions was collected from the world major rapeseed-growing countries to represent the genetic diversity of rapeseed by Wang, B. et al. This natural population was planted in the experimental field in two SOR cultivation areas (Xining, Qinghai, 36° 35′ N, 101° 47′ E and Lanzhou, Gansu, 36° 02′ N, 103° 50′ E) in Northwest China in the 2013 and 2014 growing seasons and one SWOR cultivation area (Wuhan, 30° 36′ N, 104° 18′ E, China) in the 2014-2015 growing season. Flowering time was investigated and recorded by Song, J.M. et al.

[1] Wang, B. et al. Dissection of the genetic architecture of three seed-quality traits and consequences for breeding in Brassica napus. Plant Biotechnol. J. 16, 1336-1348 (2018).
[2] Song, J.M. et al. Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Brassica napus. Nat. Plants 6, 34-45 (2020).

// 505 accessions (Tang S, et al. 2021)

1. Salt stress

The 505 natural B. napus accessions was used to investigate growth in normal and salt stress conditions at seed germination and seedling stages. The related traits at the germination stage under 0 mM, 150 mM (T1), and 215 mM NaCl (T2), and at the seedling stage under 0 mM and 215 mM NaCl (T2) were analyzed by Zhang, G. et al. The germination potential (FYS_CK, FYS_T1, and FYS_T2), germination rate (FYL_CK, FYL_T1, and FYL_T2), shoot length (GSL_CK and GSL_T1), and root length (GRL_CK and GRL_T1) at the germination stage were measured. At the seedling stage, the traits under the normal and salt stress conditions were observed, such as plant height (PH_CK and PH_T2), root length (RL_CK and RL_T2), root dry weight (RDW_CK and RDW_T2), aboveground dry weight (ADW_CK and ADW_T2), total dry weight (TDW_CK and TDW_T2), leaf area (LA_CK and LA_T2), malondialdehyde content (MDA_CK and MDA_T2), proline content (Proline_CK and Proline_T2), chlorophyll content under (SPAD_CK and SPAD_T2), and relative electrical conductivity (REC_CK and REC_T2). In order to better reﬂect salt stress responses, the salt tolerance coeﬃcient (STC) was calculated as the ratio of trait value under salt stress condition to that under normal condition, which was represented by the suﬃx type “trait_R1” under low salt stress and “trait_R2” under high salt stress.

[1] Zhang, G. et al. Genome-wide association studies of salt tolerance at seed germination and seedling stages in Brassica napus. Front. Plant. Sci. 12 (2022).

2. Salt-alkali stress

These materials consisting of 505 natural accessions of B. napus were used as research objects under control, low salt-alkali, and high salt-alkali stress conditions at seedling and mature stages. Treatment Conditions and Experimental Design Wuyuan County (105°12′ -109°53′ E, 40°13′ -42°28′ N) of Bayan Nur City is located in the western part of the Inner Mongolia Autonomous Region, which belongs to the hinterland of the Hetao Plain. The ingredients of saline-alkali land are dominated by sulfate, chloride, chloride-sulfate or sulfate-chloride in the Hetao Plain. The 3 treatment groups were designed, including control (Salt:0.1-0.25%, pH: 7.5-8.), low salt-alkali (Salt:0.35-0.53%, pH: 8.-8.5), and high salt-alkali (Salt:0.64-1.05%, pH: 8.3-9.) conditions. Each germplasm of 505 B. napus accessions was sown in 4 rows, and the space between each row was 20 cm. Each row contained 8 individual plants, and the space between each individual plant was 20 cm. At the same time, the second technical repetition was set up in the adjacent plots in diﬀerent salt-alkali lands. The sowing time is May 7, 2017. Before sowing, 225 kg/hm2 of complex fertilizer (N:P2O5:K2O, 25:10:16; total nutrients ≥ 51%) was applied as base fertilizer to maintain normal growth and development of plants on May 7, 2017. In addition, 150 kg/hm2 of complex fertilizer as upplemental fertilizer was also applied on July 1, 2017. The irrigation was conducted on May 7, 2017, May 17, 2017, and July 1, 2017, respectively. Its salt content and pH value in control, low salt-alkali, and high salt-alkali stress conditions were measured at seedling and mature stages. Then, some agronomic or growth indicators were determined at the seedling (July 9, 2017) and harvest stages (September 9, 2017), respectively. Shihezi City (84°58′ -86°24′ E, 43°26′ -45°20′ N) is located in the northern part of Xinjiang Uygur Autonomous Region, which belongs to the Southern margin of Junggar Basin. The ingredients of saline-alkali land are dominated by soda, chloride, sulfate-chloride, chloride-sulfate, or sulfate in Shihezi City. In order to further verify and screen extreme materials under salt-alkali condition, 2 treatment groups were designed, including control soil (Salt:0.05-0.15%, pH: 6.8-7.3), and high salt-alkali soil (Salt:0.80-1.15%, pH: 8.5-10). Each germplasm of 505 B. napus accessions was sown in 4 rows, and the space between each row was 20 cm. Eight individual plants were grown in each row for one replicate, and the space between each individual plant was 20 cm. At the same time, another technical repetition was conducted in the adjacent plots. The sowing date is July 10, 2018. Before sowing, 225 kg/hm2 of complex fertilizer (N: P2O5:K2O, 25:10:16; total nutrients ≥ 51%) was applied as base fertilizer to maintain normal growth and development of plants on July 10, 2017. In addition, 150 kg/hm2 of complex fertilizer as supplemental fertilizer was also applied on August 4, 2017. The irrigation was conducted on July 10, 2017, August 4, 2017, and August 20, 2017, respectively. Then, the response of the extreme materials at the seedling stage were observed on September 19, 2018.

[1] Zhang, G., Peng, Y., Zhou, J., Tan, Z., Jin, C., Fang, S., Zhong, S., Jin, C., Wang, R., Wen, X., et al. (2022). Genome-Wide Association Studies of Salt-Alkali Tolerance at Seedling and Mature Stages in Brassica napus. Front. Plant Sci. 13

3. Cadmium accumulation

The 419 rapeseed accessions were cultivated in three environments (E1: greenhouse, 2013; E2: greenhouse, 2014; E3: natural environment, 2014) at the seedling stage. Shoot and root Cd concentrations and Cd translocation were compared after 3-week exposure to 5 mg L−1 Cd2+ in the hydroponic solutions. The h2 values for Cd concentration in shoot, Cd concentration in root, and Cd translocation were 85.3, 78.1, and 69.8%, respectively (Table 1). The results indicated that these Cd-related traits for a given genotype in the three environments are almost identical. The fact that the shoot Cd concentration was higher than the root Cd concentration implies that the former is less inﬂuenced by the environment than the latter.

Trait: RCC, root Cd concentration; SCC, shoot Cd concentration; RDW, roots dry weight; SDW, shoots dry weight; CT, Cd translocation coeﬃcient.

[1] Chen, L. et al. Genome-Wide Association Study of Cadmium Accumulation at the Seedling Stage in Rapeseed (Brassica napus L.). Front Plant Sci 9, 375 (2018).

4. Branch angle and plant height of Sun, C. et al.

The 520 diverse rapeseed accessions were grown using a randomized complete block design with three replicates on the experimental farms at Changsha (N 28.22◦, E 113.00◦), Chongqing (N 29.82◦, E 106.43◦), and Nanjing (N 32.05◦, E 118.78◦), China, in the 2012/2013, and 2013/2014 growing seasons. Each line was grown in plots with two rows and 12-15 plants in each row. Five to eight representative plants in the middle of each plot were selected to measure plant height at the BBCH 89 growth stage (fully ripe). Plant height was measured as the height from the base of the stem to the tip of the main inﬂorescence.

As for the study of branch angle, 530 diverse rapeseed accessions were grown in the 2012/2013 and 2013/2014 growing season using a randomized complete block design with three replications on experimental farms at Changsha (N 28.22°, E 113.00°), Wuhan (N 30.52°, E 114.32°) and Nanjing (N 32.05°, E 118.78°) China. Each line was grown in a plot with two rows and 12-15 plants in each row. The phenotypic investigation started approximately three weeks after the final flowering stage. In 2012/2013, we measured the five branches from the top of each plant, and four plants for each accession from two replicates were selected. In 2013/2014, we extended the sample size to 12.

[1] Sun, C. et al. Genome-Wide Association Study Dissecting the Genetic Architecture Underlying the Branch Angle Trait in Rapeseed (Brassica napus L.). Sci Rep 6, 33673 (2016).
[2] Sun, C. et al. Genome-Wide Association Study Provides Insight into the Genetic Control of Plant Height in Rapeseed (Brassica napus L.). Front Plant Sci 7, 1102 (2016).

5. Seed phytate

The 505 B. napus diverse accessions were collected worldwide, including 420 semi-winter, 59 spring, 16 winter, and 10 unknown types, collected from major breeding centres across China. A total of 443 lines originated in China, 28 from Europe, 10 from Japan, 5 from Canada, 6 from Australia, 3 from Korea, and 10 unknown.

The association panel was used to conduct two years of ﬁeld trails at the experimental site of Huazhong Agricultural University in Wuhan (114.32°E, 30.52°N) from October 2013 to May 2014, and from October 2015 to May 2016. Phytate was analysed using a modiﬁcation of an existing method¹.

[1] Haug, W. and Lantzsch, H.J. Sensitive method for the rapid determination of phytate in cereals and cereal products, J Sci Food Agric., 34, 1423-6 (1983).
[2] Liu, H., Li, X., et al. Integrating a genome-wide association study with transcriptomic data to predict candidate genes and favourable haplotypes influencing Brassica napus seed phytate. DNA Research 28 (2021).

6. Plant height and branch number in response to low-phosphorus stress

The association panel of Brassica napus comprises 403 cultivars and inbred lines, including 350 semi-winter, 44 spring, 8 winter and 1 unknown type, collected from major B. napus breeding centres across China. Of them, 361 lines originated in China, 21 from Europe, 8 from Japan, 5 from Canada, 4 from Australia, 3 from Korea and 1 unknown. The panel was grown in the field at LP supply (P, 0 kg ha-1) and SP supply (P, 40 kg ha-1) with three replications at Meichuan Town, Wuxue city, Hubei province, China (115·55°E, 29·85°N) from 2018 to 2019 (Trial 1) and from 2019 to 2020 (Trial 2). The soil was sandy loam soil. The topsoil (0-30 cm) was collected before sowing (before fertilization) for determination of the available nutrient concentrations. We used 0·5 M NaHCO3 (pH 8·5) to measure available soil P. All the plots received basal fertilizer, and the application rate was as follows (per hectare), 108 kg of N (supplied as urea), 0 or 40 kg of P (supplied as calcium superphosphate), 87 kg of K (supplied as potassium chloride) and 6 kg of B (supplied as borax). These fertilizers were thoroughly mixed and applied in bands near the crop rows. The remaining N (72 kg ha-1) was top-dressed as urea in equal amounts at the four- to five-leaf stage. Each accession had four rows and each plot had eight plants in each row. At the mature stage, six plants in each plot were selected to measure PH and BN. the PH was the length of the plant from the base of the stem to the tip of the main inflorescence, and BN was calculated as the number of primary branches arising from the main shoot. PHr and BNr were defined as the ratio of PH_LP to PH_SP and that of BN_LP to BN_SP, respectively.

[1] Liu, H., Wang, J., et al. Genome-wide association study dissects the genetic control of plant height and branch number in response to low-phosphorus stress in Brassica napus. Annals of Botany 128:919-930 (2021).