After 6 to 7 years of continuous self-pollination under the single-seed descent (SSD) method, subsequent molecular marker selection, SCN-infestation testing, and further agronomic trait evaluations in 2023, six advanced breeding lines, namely, QingF6-67, QingF6-98, QingF6-99, HHF7-3-10, HHF7-6-6, and HHF7-6-10, were developed. The pedigree breeding scheme is shown in Figure 1 .

All selected advanced breeding lines in this study were erect types regarding growth and had yellow seeds. The key agronomic traits of QingF6-67, QingF6-98, and QingF6-99 were surveyed in northeast China, which had a height of 82.12 ± 3.11 cm, 77.41 ± 2.31 cm, and 106.66 ± 3.50 cm, respectively, and few branches on the mature plants. The 100-seed weights were 21.48 ± 0.75 g, 19.59 ± 0.85 g, and 21.1 ± 0.84 g, respectively, compared with 18.85 ± 0.64 g of the donor parent. They all had a yellow coat and hilum ( Figure 2 ). HHF7-3-10, HHF7-6-6, and HHF7-6-10 were surveyed in the Huang-Huai Valleys and had a height of 80.02 ± 2.74 cm, 91.22 ± 3.79 cm, and 86.00 ± 2.63 cm, respectively, with four branches on the mature plants on average. The 100-seed weights were 21.60 ± 0.73 g, 18.91 ± 0.73 g, and 19.12 ± 0.47 g compared with 18.3 ± 0.50 g of the donor parent. They all had a yellow coat and brown hilum except for HHF7-3-10, which had a black hilum ( Figure 2 ). Other agronomic traits, such as first pod height, node number, pod number per plant, and grain yield per plant, were also recorded ( Table 1 ). Moderate branching, upright growth, and concentrated fruit distribution are key to increasing soybean yield, except for resistant disease.

The contents of protein, fat, and isoflavones in the seeds of the six advanced breeding lines and three resistance donor parents were measured ( Table 2 ). Oil content ranged from 19.96% to 22.74% in QingF6-67, QingF6-98, and QingF6-99, compared with 19.61% in the donor parent Kangxian 12. The protein content ranged from 42.3% to 43.5% in HHF7-3-10, HHF7-6-6, and HHF7-6-10 compared with 41.7% in the donor parent Zhonghuang 57 and 41.3% in the donor parent Handou10. The six primary components of soybean isoflavones including daidzin, daidzein, and genistein, were also measured. Overall, the total soybean isoflavone content was 1,071.19 mg/kg, 856.72 mg/kg, and 700 mg/kg in QingF6-67, QingF6-98, and QingF6-99, respectively, compared with 692.72 mg/kg in the donor parent Kangxian 12. This shows that the isoflavone content increased in each advanced breeding line. The soybean isoflavone content was 603.31 mg/kg, 971.86 mg/kg, and 724.82 mg/kg in HHF7-3-10, HHF7-6-6, and HHF7-6-10, respectively, compared with 529.65 mg/kg in the donor parent Zhonghuang57 and 1,074.56 mg/kg in the donor parent Handou10.

KASP assays were performed for all the single-nucleotide polymorphisms (SNPs) in the advanced breeding lines at the rhg1 and Rhg4 loci using the soybean lines with known reactions to SCN as a control ( Table 3 ; Supplementary Table S1 ). GSM383 is an SNP functional marker to differentiate Peking-source with a G allele in the Rhg1 loci and PI88788-sources with a C allele in the Rhg1 loci for resistance. All six advanced lines and the control lines Peking and PI437654 have similar haplotypes. All are the Peking/PI437654-source marker haplotype for the rhg1 and Rhg4 resistance alleles (GSM381=G, GSM383=G, and GSM191=G).

SCN resistance phenotypes of the advanced breeding lines with a resistance-associated genotype, including 28 that originated from the Huang-Huai Valleys and 19 that originated from Northeast China, were evaluated in detail by inoculating races 1, 2, 3, and 5 in separate bioassays ( Table 4 ; Supplementary Table S2 ). Only the data of the six lines that were finally selected are shown here. All six lines were found to be resistant to races 1, 3, and 5. Three lines from Northeast China were resistant to race 1 with a female index (FI) that ranged from 0.5 to 2, and three lines from the Huang-Huai Valleys were moderately resistant to race 1 with an FI that ranged from 12 to 13.3. All six lines were resistant to races 3 and 5, while those that were sensitive or moderately sensitive to race 2 had varied FI values. Briefly, QingF6-67, QingF6-98, QingF6-99, HHF7-3-10, HHF7-6-6, and HHF7-6-10 were resistant to races 1, 3, and 5 (resistance types like Peking). The SNPs in rhg1 and Rhg4 are associated with this type of SCN resistance. All the sources used as resistant parents in this study exhibit Peking-type resistance, with the phenotypic and genotypic analysis results of Zhonghuang57 and Handou10 published by Lian et al. (2023), and of Kangxian12 published by Chen et al. (2023).

Five soybean cultivars that are members of maturity group I, namely, Kangxian12 (resistant parent), Hefeng55, Kenfeng18, Heinong48, and Zhongpin03-5366, and three soybean cultivars that are primarily members of maturity group III, namely, Zhonghuang57 (resistant parent), Handou10 (resistant parent), and Zheng9525, were used as parents in this study. Additionally, Lee, Lee 68, PI 437654, Peking, and PI 88788 were used as the controls in the bioassay evaluations for SCN resistance and KASP analyses. In particular, of the donor parents used in this study, Kangxian12 is resistant to races 1, 3, and 5 but has a low 100-seed weight and oil content. Zhonghuang57 exhibits resistance to races 1-5 but possesses less desirable characteristics, including improper plant height and excessive branching. Handou10 exhibits resistance to races 1, 3, and 5 but it is limited to spring planting in the central and northern parts of Hebei Province. Lee is susceptible to SCN and was used as the susceptible check. The HG Type 2.5.7 (race 1), HG Type 1.2.5.7 (race 2), HG Type 7 (race 3), and HG Type 2.5.7 (race 5) SCNs used in the study were isolated from single cysts.

QingF6-67, QingF6-98, and QingF6-99 have the same resistant parent, designated Kangxina12, and were developed in Northeast China at Heilongjiang Academy of Agricultural Sciences (Harbin, China). The F1 seeds were continuously self-fertilized until the sixth generation using SSD. HHF7-3-10, HHF7-6-6, and HHF7-6-10 were crossed between the F1 from Zheng9525 crossed with Zhonghuang57 and F1 from Zheng9525 crossed with Handou10 in the HeNan Academy of Agricultural Sciences (Zhengzhou, China). These F1 seeds from two-way hybrids obtained in 2016 were self-crossed continuously until the seventh generation using SSD. The F6/F7 plants with favorable agronomic traits were selected and planted into rows for larger seed harvests for marker, bioassay, and further evaluations of their agronomic traits in 2022. In total, 48 single plants were harvested from the F6 generation which originated from Northeast China, and 51 single plants from the F7 generation which originated from the Huang-Huai Valleys. A genotype analysis to evaluate the SCN resistance loci using F6:7 or F7:8 seeds was conducted, and a bioassay to subsequently evaluate the resistance to SCN resistance was conducted in a controlled climate room by inoculating 2,500 eggs for each plant tested. Only the lines that contained the SCN resistance loci were examined in the bioassay, as determined by the genotype results, which resulted in bioassay tests of 28 out of the 51 lines cultivated in the Huang-Huai Valleys and 19 out of the 48 lines cultivated in Northeast China. The key agronomic traits of selected advanced breeding lines were evaluated separately at two locations, i.e., the Qiqihar Soybean Breeding Experimental Center of Heilongjiang (19 lines tested, N47°14′08”, E123°40′33”) and the Huang-Huai National Regional Trial Experimental Center (28 lines tested, N35°21′1″, E113°43′1″). Each line was sown in a plot of size 6.4 m² with 4 meters in each row and four rows. Plant height (cm), first pod height (cm), node number, branch number, pod number per plant, grain yield per plant (g), 100-seed weight (g), seed coat color, and hilum color were recorded. All determined agronomic traits for each line were based on average values of ten randomly selected plants. The recommended crop production practices were followed throughout the experiment to reach the maximum yield potential of the crop. In 2023, abundant rainfall led to severe lodging of soybeans. Finally, six lines were selected by comprehensively considering both their resistance and agronomic traits.

For marker analyses, the DNA was isolated from the fresh leaves of 2-week-old plants using magnetic beads (MagAttract 96 DNA Plant Core Kit; Qiagen, Germany). Its concentration was determined by a NanoDrop 2000 ultra micro-UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and 5 ng of DNA was used to detect the integrity of DNA in 1% agarose gel electrophoresis and stored at -20 °C. The primers of GSM381 (Gm18: 645407), GSM383 (Gm18: 1643660), and GSM191 (Gm08: 8361148) were synthesized using the KASP marker sequence described by Shi et al. (2015). The cycling conditions were used as described by KBiosciences (Herts, UK), and the fluorescent end-point genotyping method was conducted using a Roche LightCycler instrument (Roche, Basel, Switzerland). The plants tested were genotyped with the functional SNP markers (two for rhg1 and one for the Rhg4 locus). GSM191 identified the resistant (G) and sensitive (C) loci in the Rhg4 allele, and GSM381 discriminates between resistant and susceptible Rhg1 alleles. In addition, GSM383 is a functional SNP that can differentiate Peking(G) and PI88788(C) loci in the Rhg1 allele.

The protein contents in soybean seeds were determined according to the modified Kjeldahl method (Bradstreet, 1954). Briefly, 200 mg heat-dried fine powder was weighed into a micro Kjeldahl digestion unit and digested with 15 mL H₂SO₄ and composite catalyst (CuSO₄ and K₂SO₄) overnight. The samples were then incubated under the following successive temperature conditions: 160°C, 15 min; 220°C, 30 min; 350°C, 30 min; 450°C, 120 min. Nitrogen content was determined using an automatic Kjeldahl apparatus (KjeltecTM 8400, FOSS, Denmark). Protein percentage was calculated by percentage N multiplied by the factor 6.25.

The fatty acid content in soybean seeds was determined using the method described by Fan et al. (2015) with minor modifications. Briefly, dried mature seeds were ground into a fine powder and then transferred into a 15 mL glass tube filled with 2 mL extraction buffer (chloroform: isopropanol, 2:1). Subsequently, they were treated with 2 mL 1% sulfate in methanol (v/v) at 80°C for 1 h and then extracted for FAME determination with 3 mL hexane and 1 mL 0.9% (w/v) NaCl using 1 mL hexane phase for FAME detection on a gas chromatography equipped with a DB-23 column (Agilent, CA, USA). The samples were then incubated under the following successive temperature conditions: 120°C for 5 min; increased by 4°C min^-1 to 190°C and held for 12 min; continued to increase to 210°C at 2.5°C min^-1 and maintained for 10 min at the final temperature. Furthermore, 1 µL of each sample was injected and detected by a gas chromatography-flame ionization detector (7890A, Agilent, USA) at 280°C. The absolute difference between two independent measurement results obtained under identical conditions should not exceed 10% of the arithmetic mean.

Soybean seed isoflavones were extracted and characterized following the protocol from Zhang et al. (2007). Briefly, seeds were ground into powder, which was then sifted through a 40-mesh screen. Degreasing was performed with petroleum ether at 65°C for 2 h, and then samples were dried at 37°C to constant weight. Furthermore, 250 mg of skimmed powder was dissolved in 10 mL of 80% methanol at room temperature for 2 h, and the mixture was subsequently distilled at 80°C for 12 h. After centrifugation at 12,000 rpm for 15 min, 1 mL of the supernatant solution was filtered into a separate HPLC vial for isoflavones determination. The standard solutions of six major isoflavone components (daidzin, glycitin, genistin, daidzein, glycitein, and genistein) with purity over 98% (Sigma-Aldrich, USA) were dissolved in 100 mg/L of methanol preparations to establish the calibration curves. Employing high-performance liquid chromatography (HPLC, Shimadzu, Kyoto Prefecture, Japan), isoflavone components in seeds were identified and quantified according to the retention times and peak areas of six standard isoflavone solutions. The sum of the contents of these six major components represented the total isoflavone contents. All samples were analyzed in triplicate.

Soybean cyst nematode populations of races 1, 2, 3, and 5 were propagated in a controlled climate room under non-sterile conditions on susceptible SCN plants to rear sufficient numbers of eggs. The fully developed brown cysts from the roots were harvested and prepared for inoculation. For environmentally controlled resistance bioassays, only the accessions that harbored the resistant alleles were evaluated for their resistance to SCN following a well-established greenhouse bioassay at the Henan Academy of Agricultural Sciences. Five plants were tested for each line, indicator lines of the Riggs model, including Pickett, Peking, PI 88788, and PI 90763 (Riggs and Schmitt, 1988), and the susceptible check (Lee 68) were arranged. Seedlings that had germinated after 3 days at 25°C were transplanted into disposable plastic cups and filled with sterile soil that was sterilized at 150°C for 1.5 h. Furthermore, 3 or 4 days after transplantation (one seedling/cup), the eggs of races 1, 2, 3, or 5 were inoculated on the roots. Approximately 2,500 fresh eggs were inoculated on each plant using a peristaltic pump. The experiments were maintained at 28°C/25°C (day/night) and watered daily. At 25–30 days post-inoculation, the cysts were collected by carefully washing the roots of each plant and imaged using a fluorescence-based imaging system developed by the Soybean Research Group of Henan Academy of Agricultural Sciences. The cysts were then counted, and the FI was calculated using the percentage of the mean number of cysts per plant of each line relative to the mean number of cysts per plant of the susceptible control line ‘Lee’ to evaluate the resistance level to SCN.