The 510 soybean materials provided by the Biotechnology Center of Jilin Agricultural University and Guangzhou University of Chinese Medicine, the soybean materials were planted in 2017–2018 at Changchun, China (123.53° E, 23.84° N), in 2018–2019 at Qingdao, China (120.41° E, 36.39° N), in 2019–2020 at Guangdong, China (113.41° E, 23.31° N). A randomized complete block design was used. The field was divided into three blocks and those were subdivided into eight sections. After that we did natural drying, then seeds were threshed for linoleic acid determination. Based on the collection, the soybean of two groups were identified with obvious differences in linoleic acid content, and were named Group 1 and Group 2. The names of the soybean lines and the fatty acid content are shown in Table. S2.
Determination of fatty acids in soybean Seeds
The content of linoleic acid and other four fatty acids (stearic acid, palmitic acid, oleic acid and linolenic acid) in soybean seeds were determined by NIRSTM DS 2500 (FOSS, Hillerod, Denmark). SPSS version 22.0 software (SPSS Inc, Chicago, IL, USA) was used to calculate the correlation coefficient of fatty acid in soybean seeds.
Genotyping of soybean germplasms
Total genomic DNA was extracted from leaves of each soybean line using a CTAB method according to Murray & Thompson (Murray and Thompson, 1980). The 510 soybean materials were genotyped by Specific-Locus Amplified Fragment Sequencing (SLAF-seq) and SNP molecular markers were developed. The sequencing service is supported by Beijing Biomarker Biotechnology company. DNA extraction is the first step in sequencing. SNP molecular markers are used for GWAS analysis and genetic evolutionary correlation analysis. The restriction endonuclease combination was RsaI-HaeIII.
Genome-wide association analysis (GWAS)
Based on the SNP markers obtained by SLAF-Seq technology, the correlation values between SNP markers and oleic acid content were obtained. TASSEL software can calculate the Q matrix of sample population structure according to the K matrix, and finally get a correlation value of each SNP maker. The results of each model of each trait were annotated based on the10-6 level of significance. In this experiment, Manhattan map were constructed by using Haploview software (BROAD Inc, Chicago, USA). The Manhattan map was used to represent the correlation between genotype data and phenotypic data. In this study, the candidate genes were predicted by using Swiss-Prot and NR databases.
Quantitative reverse transcription-PCR
The total RNA was extracted using Eastep® Super total RNA extraction Kit (TaKaRa, USA), then cDNA synthesis was performed using a reverse transcription kit (Omega. USA). The qRT-PCR analysis was performed using a Bio-Rad CFX system (Amersham Biosciences, Little Chalfont, Buckinghamshire,UK). Gene-specific primer pairs P3: (5’-TTGCCTGTCTAGATCCACAGCTGGTACCGAT-3’) and P4(5’-TTGTGACCTCGACCTATTGGCGTTACCAATT-3’) were used to amplify GmWRI14. The lectin gene (GenBank: A5547-127) was used as the reference gene. The reference gene was amplified with primer pairs P5: (5’-GCACTTAAGATACTCTAGGTAC-3’) and P6: (5’-CCACCTCCCTACTATCCATT-3’). The amplification reaction conditions were pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 10 s, annealing at 53 °C for 20 s, extension at 72 °C for 15 s, and the amplification reaction conditions of the gene GmWRI14 are pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 35 s, 35 cycles, and extension at 72 °C for 10 min. Three biological replicates were used for each gene.
Vector construction and plant transformation
The 1,815-bp cDNA of the GmWRI14 gene from the Bacillus thuringiensis strain HBF-18was ligated into the BamHI-SacI site of pCAMBIA3300 to place the coding region under the regulatory control of the 35S promoter and nos terminator (Fig. S1). The recombinant plasmid was named pCAMBIA3300- GmWRI14, and then introduced into Agrobacterium tumefaciens strain LBA4404 using the freezing-thaw method (Holsters et al., 1978). We introduced GmWRI14 into the soybean cultivar JN38 (Approval number 2012010), the seed of JN38 possesses good agronomic characteristics with high linoleic acid content. The transformation process was divided into five sequential steps: bacterial inoculation, cocultivation, resting, selection and plant regeneration.
RNA-seq library preparation and sequencing
Plants samples were processed for total RNA extraction using Eastep® Super total RNA extraction Kit (TaKaRa, USA), RNA quality was checked using a Nanodrop 2000c (Thermo Scientific, Hudson, NH, US). RNA-seq library preparation and sequencing were performed using the protocols described previously (Kumar et al., 2012; Sultan et al., 2012; Zhong et al., 2011). Fold change for gene expression was calculated by normalizing Ct values at each developmental stage against endogenous control (Gmβ-actin:Gm15g05570) using the 2-ΔΔCt method (Livak and Schmittgen, 2001).
The phenotypic data was measured and recorded using Microsoft Excel 2020 software. each data with three replicates. Differential saliency analysis, analysis of variance, correlation analysis and descriptiveness were performed by using SPSS 19.0 (IBM Corp, Armonk, NY, USA) software (Livak and Schmittgen, 2001). The statistical significance at P ≤ 0.01 was calculated. The positive and negative maps and histograms were constructed by using Graphpad Prism software (Graphpad Company, San Diego, CA).