Three cultivars Shixiya1, Hai7124, and TM-1, which belong to Gossypium arboretum (G. arboretum), Gossypium barbadense (G. barbadense) and Gossypium hirsutum (G. hirsutum), respectively, were used for gas chromatography combined with time-of-flight mass spectrometry (GC-TOF/MS) analysis. The cotton was planted in Chinese Academy of Agricultural Sciences, Anyang, China, and were cultivated under the same conditions. The seeds of the three cotton cultivars were harvested in the same period. Then, the samples were freeze-dried and stored at -80℃ until GC-TOF/MS analysis.
To illustrate the content distribution of catechin in different species, 47 accessions of G. arboretum, 37 accessions of G. barbadense, 144 accessions of G. hirsutum were chose from core collections of Gossypium used for measure catechin content by HPLC analysis respectively. Moreover, to explore the correlation between catechin and fatty acids in G. hirsutum, 144 accessions were measured for fatty acids by GC analysis.
Sample extract preparation and GC-TOF/MS analysis
Briefly, cottonseed sample powder (50 mg) was extracted with 0.48 mL 75% methanol containing 10 μL adonitol (0.5 mg/mL stock in dH2O) as internal standard. The resulting mixture was ultrasound treated for 5min, and then centrifuged (12000 rpm) at 4°C for 15 min. Transferred the supernatant (0.4 mL) to a new 2 mL GC/MS glass bottle and dried it completely in a vacuum concentrator. Then the extracts were oximated using 80 μL methoxyamine hydrochloride (20 mg/mL in pyridine) at 80℃ for 25 min. Subsequently, the samples were added 100 μL BSTFA regent (1% TMCS, v/v) and incubated at 70℃ for 1.5 h. The GC-TOF/MS was performed as described by Deng et al (Deng et al, 2020)
Data preprocessing and annotation
The Chroma TOF 4.3X software (LECO Corporation) and LECO-Fiehn Rtx5 database were used to preprocess and annotate the data of GC-TOF/MS analysis. In addition, the mass spectrum match and retention index match were also noted in metabolites identification (Kind et al. 2009).
Catechin extraction and analysis by High Performance Liquid Chromatography (HPLC)
Briefly, about 100 mg cottonseed powder was extracted using 3 mL of 80% acidified methanol (1% hydrochloric acid). After 30 min sonication, the homogenate was incubated for 12 h at 25℃. Then samples were centrifuged at 10000 rpm for 15 min. The supernatant was used by an Agilent 1100 series HPLC system (Agilent Technologies, USA) to determine the content of catechin (Peng et al. 2018).
Fatty acids extracted and analyzed by Gas chromatography (GC)
Extraction of fatty acids from mature cottonseeds was performed as described previously . Briefly, about 50 mg of cottonseed powder was added 1 mL of 0.3 M potassium hydroxide methanol solution, 1 mL of n-hexane containing 500 μg/mL C11:0 as an internal standard. After shaking for 30 seconds and holding at 25℃ for 1.5 h, the homogenate was added 1.5 mL of 0.9% (w/v) sodium chloride solution, followed by centrifugation (10000 rpm) at 25℃ for 5 min. The supernatant was separated by gas chromatography (Agilent7890-FID) according to previously described procedures (Dowd et al. 2010).
Multivariate analysis and statistics
To better visualization and subsequent analysis, the data of GC-TOF/MS was used to perform PCA and OPLS-DA analysis by the SIMCA software (V14.1, MKS Data Analytics Solutions, Umea, Sweden). The Student’s t-test and one-way ANOVA were used to compare differences between two groups and multiple groups, respectively. Pearson’s correlation coefficient analysis was performed by OriginPro 2021 (https://www.originlab.com/) to assess the correlation between catechin and fatty acids content. P-values < 0.05 or < 0.01 or < 0.001 were considered statistically significant.