Abstract
Soybean cyst nematode (SCN, Heterodera glycine Ichinohe) is a major threat to global soybean yield. Resistance genes at the rhg1 locus from PI 88788 are majorly utilized in 95% of the U.S. breeding programs. Continuous use of this resistance source leads to a shift in the virulence of SCN populations and overcomes host resistance. Therefore, it is necessary to identify alternative SCN resistance sources to combat this ever-changing pest. Previously, we identified an exotic soybean line, PI 567516C, which carries a novel qSCN10 (O) locus for SCN resistance demonstrating different resistance responses compared to the known rhg1 and Rhg4 loci. Here, we narrowed the qSCN10 QTL region to 142-kb (containing 20 genes). Based on gene expression, gene ontology, in-silico analysis, and QTL-based haplotyping, two genes were identified for functional characterization. Overexpression of the transcription factor TGA1-related and Shugoshin C-terminus in the SCN-susceptible Williams 82 reduced the cyst number by 6.4-fold (84.6%) and 5.3-fold (81.2%), respectively. GmTGA1-10 and GmSCT-10 Tilling mutants showed high cyst numbers. The two genes associated with the qSCN10 QTL have significant potential to reduce the SCN population. They also offer an alternative source of durable SCN resistance that is independent of rhg1 and Rhg4.
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Introduction
Soybean cyst nematode (SCN, Heterodera glycine Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and was first found in the U.S. in North Carolina in 19541, and since then, it has spread to all soybean production areas2. Syncytium, a physiological sink that takes energy from the host by competing for water and nutrient uptake, is formed when the nematode sends a signal to the initial feeding cell. The effector proteins secreted by nematodes are the key to unraveling the complete mechanism of parasitism in general, and the effectiveness of different strains with a wide range of virulence3. Effector proteins cause suppression or reprogramming of host defense in susceptible soybeans. The host resistance mechanism plays a crucial role during its interaction with the nematode’s effector proteins4,5. Identification and functional characterization of host resistance genes and their interactions with effector proteins and understanding their corresponding transcription factors that regulate effector-triggered immunity and hormonal signaling networks will advance our understanding of parasitism suppression in resistant soybeans6,7. Molecular mechanisms of host defense during SCN invasion are still poorly understood. Most of the identified effector proteins physically interact with and modulate the functions of host proteins involved in various defense mechanisms.
Nematode management strategies require a holistic approach that includes nematicide seed treatment, crop rotation, and the use of host plant resistance8,9. Because there are many nematode species in soil, identifying the correct species is essential for determining the most effective control measure. Genetic resistance plays a significant role in controlling SCN populations and offers a positive return on investment. Over the last decade, farmers planted SCN-resistant cultivars as the most efficient management strategy to control SCN. However, over 95% of resistant cultivars carry the same source of resistance derived from plant introduction (PI) 88788 that carries SCN resistance genes harbored at the rhg1-b locus and provides protection to SCN races 3 (HG type 0) and 14 (HG type 1.3.5.6.7)9. Although PI 88788-based cultivars are still more effective than susceptible lines in reducing the number of SCN eggs in plants10, planting varieties with a single source of resistance causes a tremendous risk of increasing nematode virulence associated with evolutionary changes and natural fitness between the pathogen and its host9,11. The continuous use of PI 88788- type resistance caused nematode population shifts, also referred to as “race shift”, by decreasing SCN races 3 and 14 and increasing the prevalence of other races that can reproduce on resistant cultivars and the subsequent loss of genetic effectiveness over the last ten years11,12,13. Therefore, SCN management strategies used in the past are no longer efficient due to limited genetic diversity in available soybean breeding programs. Many soybean farmers do not realize their fields offer a buffet for nematodes, despite the use of “resistant” soybean varieties. Non-effective varieties grown on fields with increasing SCN population densities could be compared to a ticking time bomb, and field management is the most challenging9. Finding new resistant sources seems to provide real solutions to cope with nematode virulence and pathotype specialization against host resistance genes.
Currently, hundreds of SCN-resistant soybean varieties are derived from either PI 88788 (95% of modern varieties) or Peking (3%), and the same sources of resistance are used in most soybean breeding programs14. This limited genetic diversity has created a bottleneck in the development of new soybean varieties carrying novel SCN resistance genes, essential for effectively countering this constantly evolving pest. It is more critical than ever to identify and characterize additional sources of SCN resistance for more effective and durable control of this ever-changing pathogen. Utilizing a global germplasm pool and advanced molecular breeding tools has a tremendous opportunity to develop new varieties with improved resistance targeting additional SCN races.
Soybean accession PI 567516C displays different reactions to multiple SCN races, which are different from known sources of resistance PI 88788 and Peking15. PI 88788 and Peking carry known genes at the rhg1 and Rhg4 loci, which are race-specific and cause complete resistance to nematode infection16,17,18,19,20. Recently, the molecular mechanisms of these genes involving the GmSNAP18 (rhg1-a), the GmSHMT08 (Rhg4), and a novel pathogenesis-related protein has been revealed21,22. Under SCN infection, GmSNAP18, GmSHMT08, and GmPR10-08 transcripts were induced in the SCN-resistant line (cv. Forrest), showing a physical interaction at the molecular level which triggers an extreme reaction in the host that prevents cyst formation and limiting cyst structure at a very minimum level21,22. Additionally, few other genes involved in SCN resistance have been discovered during the last decade, including the GmSNAP11, t-SNARE-syntaxins (chrs. 12 and 16), and GmSYP31A (chr. 02) in Peking type of resistance and the rhg1-b (AAT, α- SNAP, WI12), NSF, and γ-SNAP (chr. 07) in PI 88788 type of resistance23. Unlike other known sources of SCN resistance, PI 567516C shows broad-spectrum resistance to multiple SCN races15. This phenomenon suggests a different mode of action as a race-nonspecific type of resistance compared with the known Rhg genes. Few nematodes are capable of infecting and reproducing on PI 567516C roots24. The broad-spectrum resistance caused by novel genes could be explained by the production of proteins compatible with conserve nematode effector proteins common to many SCN virulent populations. Another explanation could involve restricting nematode in water and nutrients or activating the plant dead signaling pathway. Here, we describe the identification of two novel genes at the qSCN10 locus that contribute to SCN resistance in PI 567516C. A transcription factor TGA1-related was initially reported to regulate effector-triggered immunity and hormonal signaling networks25. The second gene, Shugoshin C-terminus, was previously described to be required with SGO2 for complete protection of centromeric cohesion during anaphase I mitosis, guiding chromosome cohesion during cell division, and also may function as a stress regulator by binding to and regulating RNA Polymerase (RNAP) II and regulating the transcription of heat shock protein genes26. However, to our knowledge, no previous study has shown their involvement in plant pathogen resistance nor for broad-spectrum resistance to SCN.
Results
Progeny test of additional recombinants and high-throughput SCN phenotyping
We identified about 18 individual recombinants as cross-recombinants in the target qSCN10 region by two SNP markers: O1_Gm10_42597820 and O15_Gm10_42898898. To further genotype these recombinants, four additional SNP markers (O7_Gm10_42756962, O9_Gm10_42780065, O11_Gm10_42817297, and O12_Gm10_42819414) were designed between the two SNP markers to dissect crossover points (Table S1). Six recombinants (W1300, W1500, W1101, W299, W661, and W1783) were selected for SCN phenotyping because the QTL region contains distinct crossover sites (Fig. 1). The progeny tests were carried out for these six recombinants against SCN HG type 1.3.5.6.7 type in the greenhouse, as previously reported24, in which HG Type 1.3.5.6.7 has been shown the most significant reduction in the FIs in NILs for the qSCN10 locus24.
The target region of qSCN10 was narrowed down to a 142-kb interval between O7_Gm10_42756962 and O15_Gm10_4298898 in the soybean genome using 6 key near-isogeneic lines (NIL) recombinants. Here, black bars indicate PI 567516C derived sequences, whereas white bars indicate Magellan derived sequences. The SCN phenotypes were demonstrated on the right as resistant (R) and susceptible (S) based on SCN phenotyping assays. The female index is shown as mean ±SE (n > 10), and the Tukey’s HSD test determined significant differences.
As expected, Magellan (recurrent parent) showed susceptibility with a FI count of 131, while PI 567516C (donor parent & source of the qSCN10 resistant locus) showed resistance with FI count of 14 against HG Type 1.3.5.6.7 (Table S2). Most of the recombinants containing genomic fragments between the markers O7_Gm10_42756962 and O15_Gm10_4298898 from PI567516C showed significant reductions in FIs according to Tukey’s HSD test (p < 0.001), ranging from 54% to 73%, in W1783, W299 and W1101 lines compared to Magellan. We did not find any significant differences in the other two recombinants: W1300 and W1500. These genotypic and phenotypic results narrowed the qSCN10 locus to a 142- kb interval between markers O7_Gm10_42756962 and O15_Gm10_4298898 (Fig. 1). We developed additional KASP markers (Table S3) from the fine-mapped region that can be used to genotype these recombinants; however, these markers are unable to further decrease the target interval.
Haplotype analysis of the qSCN10 QTL using the 106 diverse soybean panel
Previously, we conducted haplotype analysis to pinpoint the causal variation of several traits, including salt tolerance27 and SCN resistance28. Haplotyping analysis using 106 different soybean accessions provided specific features at the promoter region and coding sequence of the rhg1 and Rhg4 genes that correlate with resistance to SCN. Using the same approach, we revealed the lack of functional redundancy within the rest of the GmSHMT gene family members in resistance to soybean cyst nematode, including all four classes (cytosol, nucleus, mitochondrion, and chloroplast)20. While several haplotypes at the GmSHMT08c are resistant to SCN, haplotyping analysis of the 106 lines showed that none of the GmSHMT gene family members presented specific GmSHMT haplotypes that correlate with SCN resistance. The possible role of the GmPR08-Bet VI in resistance to SCN, newly cloned genes that confer SCN resistance, was also explored using the natural variations within the gene in different soybean germplasms. In fact, soybean lines belonging to the GmPR08-Bet VI-c haplotype showed resistance to moderate resistance reaction to SCN. However, soybean lines associated with the GmPR08-Bet VI-a and GmPR08-Bet VI-b haplotypes exhibited a susceptible to moderate susceptible resistance reaction against SCN. Thus, haplotype clustering analysis using the whole genome resequencing data from a collection of 106 diverse soybean germplams (15X) was crucial to identify allelic variants and haplotypes within several SCN-resistant candidate genes.
Similarly, in this study, we used the WGRS data from the 106 diverse soybean accessions comprising wild, landraces, and elite lines29 and conducted QTL-based haplotype analysis27 to identify allelic variation at the qSCN10 QTL (Fig. 2). Briefly, SNP haplotypes were analyzed by generating map and genotype data files, and clustering results for the qSCN10 QTL genomic regions were depicted using FLAPJACK30. SNPs from each line were grouped by using a neighbor-joining (NJ) tree and further assessed for synonymous or non-synonymous differences through amino acid sequence translation. SNP diversity metrics, including average pairwise variation within populations (θπ), Watterson's estimator (θw), and Fst, were calculated using methods outlined earlier29. We identified six major haplotypes (H1– H6) based on SNP variations. PI 567516C was represented by H1 which carried a unique set of SNPs as compared to known resistant lines with the rhg1 and Rhg4 loci.
SNPs were positioned relative to the genomic position in the genome version W82.a2. The SNPs in the black background are different from the reference genome (‘Williams 82’).
We performed in-depth haplotype analysis in all the genes in qSCN10 locus among all 106 soybean germplasms and found multiple SNPs that cause an altered amino acid sequence (non- synonymous) in several genes belonging to H1 haplotype group harboring PI 567516C (Fig. 3, Supplementary Data 1). For instance, GmTGA1 (Glyma.10G194800) gene has 5 non- synonymous SNPs (Y49S, V74F, S86T, A88T and I200F), GmNUP1 (Glyma.10G194700) has 10 non-synonymous SNPs (R126G, P173S, H538N, R587G, C911S, F927L, R977S, R1043S, M1063L, and S1143A) (Figure S1), GmLLR1-PK-10 (Glyma.10g195700) has 11 non- synonymous SNPs (R172S, Y196N, S267A, F303L, K334E, E350G, I388S, V144I, S431N G891D, and T675I), and the Shugoshin-1 (Glyma.10G196400) has 1 non-synonymous SNP in PI 567516C in H1 haplotype group at the qSCN10 locus that highly correlate with SCN resistance (Fig. 3, Supplementary Data 1). This analysis confirmed that PI 567516C has a unique SNP variation in qSCN10 locus, leading to an amino acid change. This analysis helped narrow down the candidate genes and focus on the H1 haplotype (Supplementary Data 1).
Schematic graphs depict the position of amino acid change (non-synonymous SNP/indel) for GmSCT1, GmLRR10, and GmTGA1 genes. The SNPs in orange (GmSCT1), blue (GmLRR10) and green (GmTGA1) background in H1 haplotype group containing PI 567516C are different from the reference genome (‘Williams 82’). In the gene model figure (top of the figure), the box denotes exons, the black bar represents introns, and the red lines represent SNP/ changes. SNPs/ haplotypes were located relative to the genomic position in the genome version W82.a4.
SNPs in promoter regions and UTRs can alter gene function and expression levels as shown earlier31,32. Several researchers have successfully shown that alteration in promoter sequences harboring SNPs leads to variation in phenotypes such as enhanced disease tolerance and increased yield33,34. Here, we performed haplotype analysis for promoters of three genes from the qSCN10 QTL namely GmTGA1-10 (Glyma.10g194800), GmSCT-10 (Glyma.10G196400) and GmLRR- PK-10 (Glyma.10g195700). These genes harbor unique SNPs and Indels in their promoter regions in resistant line PI 567516C (HN025) than susceptible WI82 (Supplementary Data 2). This analysis indicates that SNPs/Indels detected in GmTGA1-10, GmSCT-10, and GmLRR-PK- 10 promoters may be contributing to altered transcript abundance and thereby SCN resistance in PI 567516C.
Identification of potential candidate genes for resistance to SCN at the qSCN10 locus in PI 567516C
The 142-kb interval in the qSCN10 locus contains 20 candidate genes for SCN resistance (Table 1). After narrowing the QTL from 379-kb to a 142-kb region, we followed a three-step procedure to select promising candidate genes for further functional characterization.
The presence of non-synonymous SNPs was crucial to identify several genes for resistance to SCN, including the two major genes in Peking-type resistance; the rhg1-a (GmSNAP18) and the Rhg4-a (GmSHMT08). The GmSHMT08 showed the presence of two non-synonymous SNPs (R130P and Y358N)17 and the GmSNAP18 showed 8 non-synonymous SNPs (Q203K, D208E, E285Q, D286Y, D286H, D287E, −288A, −288V and L289I)16 between the SCN-resistant and SCN-susceptible soybean varieties28. Therefore, as the first selection criterion, we compared non-synonymous SNPs within these 20 candidate genes between Magellan and PI 567516C parents. Out of the 20 candidate genes, only seven genes contained non-synonymous SNPs. Nine non-synonymous SNPs were present in the GmNUP1-10, five non-synonymous SNPs in GmTGA1-10, and one non-synonymous SNPs in Shugoshin-1 (GmSCT-10), leucine-rich repeat receptor-like protein kinase (GmLLR1-PK), Myb family transcription factor (GmMYB-TF), K4BPD4 MATE efflux family protein, and the unknown protein (Glyma.10G195100) (Table 2, Fig. 4). While the rest of the genes (13 genes) did not show any non-synonymous SNPs (Fig. 4). Therefore, we selected these 7 candidate genes containing non-synonymous SNPs, and discarded the rest of the 13 candidate genes at the qSCN10 locus since they presented similar coding sequences between the SCN-susceptible line Magellan and the SCN-resistant PI 567516C.
No expression data were available for Glyma.10G195400, Glyma.10G195500, and Glyma.10G196000 genes. Non- synonymous SNPs (A) represents the number of Non-synonymous SNPs present at the 20 candidate genes at the qSCN10 locus between the 2 parents only (Magellan and PI567516C). Non- synonymous SNPs (B) represents the number of Non-synonymous SNPs present at the 20 candidate genes at the qSCN10 locus among all the 106 soybean germplasms analyzed.
Next, as a second criterium to narrow further the list of candidate genes that might play a role in SCN resistance, we compiled gene expression under SCN infections in PI 567516C of the selected seven candidate genes35. Soybean breeding to target and functionally characterize essential genes to improve important agronomic traits (i.e. oil, protein, yield, resistance to biotic and abiotic stresses) is challenging due to the presence of two duplication events that occurred in the soybean genome about 13 and 59 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies36. The use of gene expression analysis was very useful to select top candidate genes that are involved in several quantitative traits19,20,37,38,39,40,41,42. Previously, we performed expression analysis of soybean root tissue from SCN-infected and non-infected using the two soybean lines; Magellan and PI 567516C35. Gene expression analysis showed that out of the seven candidate genes at the qSCN10 locus showing non-synonymous SNPs, three genes showed higher transcript abundance under SCN infection in PI 567516C including the Shugoshin C-terminus (GmSCT-10; Glyma.10G196400), the leucine-rich repeat receptor-like protein kinase (GmLRR-PK-10; Glyma.10g195700), and the transcription factor TGA1-related (GmTGA1-10; Glyma10g194800). GmSCT-10 and GmTGA1-10 were induced at an early stage (2 days after SCN infection) in the resistant soybean line PI 567516C, while GmLRR-PK-10 transcripts were induced at the late stage of infection (10 days after SCN infection). GmSCT-10 transcripts were induced up to 3.5-fold under SCN infection, GmTGA1-10 transcripts were 3.6-fold induced, while GmLRR-PK-10 transcripts were 2-fold induced under SCN infections in roots (Fig. 4). However, the rest of the other four genes showed either moderate gene expression, barely detected, or non-detected transcripts. Thus, due to the low or absence of transcripts induction/expression under SCN infection in the resistant PI 567516C soybean roots (Fig. 4), these four genes were not selected for further functional characterization. Considering the previous haplotyping analysis (gene indicating the presence of non-synonymous SNPs) (Figs. 2, 3) and gene expression analysis (genes displaying increased gene expression in the root of PI 567516C in comparison to Magellan) (Fig. 4, S2), only three genes (GmSCT-10, GmTGA1-10, and GmLRR-PK-10) were chosen for further characterization and deemed as potential candidates for SCN resistance.
The third criterium consisted of the analysis of putative cis-elements in the promoter region (−2 Kb) upstream of the translation start codon of the three selected genes; GmSCT-10, GmTGA1- 10, and GmLRR-PK-10. The analysis showed several losses and gains in cis-elements between PI 567516C & Magellan (Supplementary Data 3–5). In the case of GmTGA1-10, while 86 matrix families were conserved between PI 567516C and Magellan, five matrix families were specifically present in PI 567516C and eight in Magellan promoter (Fig. 5A, Supplementary Data 6-A). For the GmSCT-10, while 80 matrix families were conserved between PI 567516C and Magellan, two matrix families were specifically present in PI 567516C and two were present in the Magellan promoter (Fig. 5B, Supplementary Data 6-B). For the GmLRR-PK-10 gene, no differences were observed between the resistant and susceptible lines since all 92 matrix families were conserved between PI 567516C and Magellan (Fig. 5C, Supplementary Data 6-C).
A–C Venn diagram showing common and non-common cis-elements within the three-candidate gene’s promoter (GmSCT-10, GmTGA1-10, and GmLRR/RLK-10) in PI 567516C and Magellan. D Among all the cis-element identified, five cis-elements (FORC, TCPF, HOCT, TRIH, and E2FF) are specifically present at the GmTGA1-10 promoter (Supplementary Data 3), and 2 cis- elements (SCAP and BBZF) are only present at the GmSCT-10 promoter (Supplementary Data 4) in the resistant PI 567516C. E, F Only cis-elements that were uniquely present either in PI 567516C or in Magellan were compared. The analysis showed the presence of a common element, NAC domain- containing protein 87, between GmSCT-10 and GmTGA1-10 genes (but not in GmLRR-10) in the Magellan promoter.
Among all the cis-elements identified, the following five cis-elements FORC, TCPF, HOCT, TRIH, and E2FF are specifically present at the GmTGA1-10 promoter (Supplementary Data 3), and the following two cis-elements SCAP and BBZF are only present at the GmSCT-10 promoter (Fig. 4, Supplementary Data 4) in the resistant PI 567516C. The exclusive five cis-elements in GmTGA1-10 are majorly involved in fungal and oomycete pathogen response (FORC), cell cycle regulators (E2FF), histone modifications (HOCT) indicating their crucial involvement in plant immune response, and cell cycle regulations (Supplementary Data 3). While the other two GmSCT-10 unique cis-elements play a key role in protein-protein interactions along with biotic stress (BBZF) and cell cycle regulation (SCAP) (Supplementary Data 4). suggesting the importance of this gene in conferring SCN resistance in PI 567516C.
Moreover, we investigated if the three GmSCT-10, GmTGA1-10 and GmLRR-PK10 genes have any common unique cis-elements in their promoters among the two parents. While none of the GmSCT-10, GmTGA1-10, and GmLRR-PK10 carried a common cis element at the PI 567516C promoter (Fig. 5E, Supplementary Data 6-K), the “NAC domain-containing protein 87” was found to be commonly present in the promoter of GmSCT-10 and GmTGA1-10 genes (while absent in GmLRR-PK10) in Magellan, but not in PI 567516C (Fig. 5F, Supplementary Data 6-L). The presence of the “NAC domain-containing protein 87” cis-elements in both GmSCT-10 and GmTGA1-10 genes in the SCN susceptible soybean Magellan, but not in the SCN-resistant PI 567516C indicated that this cis-element might play a role in SCN resistance.
Thus, promoter analysis demonstrated that both GmSCT-10 and GmTGA1-10 genes showed several important cis-elements including “NAC domain-containing protein” that was commonly present in the promoter regions of GmSCT-10 and GmTGA1-10 genes, while absent in GmLRR- PK10 promoter. Therefore, promoter analysis pointed to the possible involvement of GmSCT-10 and GmTGA1-10 genes in SCN resistance, but not GmLRR-PK10, and thus were selected for further functional characterization. Taken together, and considering all previous three-step procedures described above, both GmSCT-10 and GmTGA1-10 were selected for functional characterization including gene overexpression using hairy root composite plant transformation in SCN-susceptible Williams 82 soybean lines, in addition to mutational analysis using TILLING-by-sequencing+ technology.
Functional characterization of GmTGA1-10 and GmSCT-10 using hairy root composite plant transformation
To test the effect of the GmSCT-10 and GmTGA1-10 genes on SCN resistance, we overexpressed their corresponding coding sequences in susceptible genotype Williams 82. To conduct the GmSCT-10 and GmTGA1-10 overexpression analysis, the nucleotide coding sequence of the GmSCT-10 and GmTGA1-10 genes, in addition to the GmSHMT08 (Rhg4-a) (positive control) and WI82::EV (empty vector, negative control) were overexpressed under the control of a soybean ubiquitin promoter using a transgenic hairy root system. As expected, WI82-WT and pG2RNAi2::EV showed a susceptible reaction to SCN infection (HG type 0) with 102 and 96 cyst numbers (CN) on average, respectively (Fig. 6). Overexpression of the positive control pG2RNAi2::GmSHMT08 in transgenic hairy roots decreased the CN to 10% showing a resistant reaction to SCN (HG type 0) when compared to WI82-WT and pG2RNAi2::EV. Most importantly, overexpression of pG2RNAi2::GmSCT-10 and pG2RNAi2::GmTGA1-10 genes reduced the CN from 96 contained in pG2RNAi2::EV to 15 and 18, respectively (Fig. 6). Thus, overexpression of both GmSCT-10 and GmTGA1-10 genes in susceptible Williams 82 transgenic roots reduced cyst by 6.4-fold (84.6%) (P < 0.0001) and 5.3-fold (81.2%) (P < 0.0001) when compared to the control (pG2RNAi2::EV) (Fig. S3).
The data shown represent the averages from all three biological repeats (n = 53 for pG2RNAi2::GmSCT-10 and n = 42 for pG2RNAi2::GmTGA1-10). F-WT: Forrest wild-type (n = 19), PI 567516C-WT: PI 567516C wild- type (n = 10), and WI82-WT: Williams 82 wild-type (n = 9) (Supplementary Data 8). GmSHMT08 (Rhg4) was used as a positive control (n = 14). pG2RNAi2::EV (Empty Vector) was used as negative control (n = 10). Connecting letters indicate significant differences between the tested lines as determined by ANOVA (P <0.0001). The experiments were repeated three times, and similar results were obtained.
Investigating the role of GmTGA1-10 and GmSCT-10 in a closely related source of SCN resistance using TbyS+
Hairy root transformation brought evidence that the two GmTGA1-10 and GmSCT-10 genes play a role in SCN resistance in PI 567516C. While we could not isolate any EMS TILLING mutants in PI 567516C for the GmTGA1-10 and GmSCT-10 genes, we decided to investigate the role of both GmTGA1-10 and GmSCT-10 genes using other closely related sources of SCN resistance in soybeans such as PI 407729. PI 407729 is genetically and phenotypically similar to PI 567516C24,28. Both PI 407729 and PI 567516C carry the rhg4-b coding sequence susceptible haplotype (Williams 82-like). Additionally, PI 407729 and PI 567516C share the same Rhg4-a promoter (Peking-like)28. PI 407729 is known to have broad resistance to SCN HG types (2.5.7, 1.2.5.7, 0, 2.5.7, 1.3.5.6.7) [races 1, 2, 3, 5, 14], which is similar to PI 567516C, and does not require the typical SCN resistance Rhg4-a haplotype24,28. Altogether, SCN resistance offered by both PI 407729 and PI 567516C are independent of the resistant rhg1 and Rhg4 loci.
To investigate the role of the GmSCT-10 and GmTGA1-10 genes in closely related sources of SCN resistance, we screened our soybean EMS mutant collection using a reverse genetic approach. We developed earlier a high-throughput TILLING-by-Sequencing+ (TbyS+) technology, along with universal bioinformatics tools, to identify mutations across soybean populations37,39,42. Based on seed quality and availability, we selected three Shugoshin C- terminus and three transcription factor TGA1-related EMS TILLING mutants. At least ten plants of each of the TILLING mutants were screened against HG type 0 and HG type 2.5.7 SCN races. The identified GmTGA1-10 E213K and GmSCT-10M417I PI 407729 background mutants have been next mapped on their corresponding protein homology models. GmTGA1-10E213Kwas mapped at the DOG1 conserved (controlling seed dormancy) (Fig. 7A), while GmSCT-10M417I was mapped at the Shugoshin C-terminus (protects Rec8 at centromeres during meiosis) (Fig. 7B).
In cyan, DOG1 conserved domain (A) at the GmTGA1-10 protein (conserved residues 47-124; NCBI domain architecture ID 10624400) which controls seed dormancy and (B) Shugoshin C-terminus at the GmSCT-10 protein (conserved residues 395-419; NCBI domain architecture ID 10540851) which protects Rec8 at centromeres during meiosis. SCN HG type 0 (race 3) (C) and HG type 2.5.7 (race 5) (D) screening of the isolated EMS TILLING Gmtga1-10 and Gmsct-10 mutants. Connecting letters indicate significant differences between the tested lines as determined by ANOVA (***P <0.001, **P < 0.005, *P <0.05) (n = 5, 6) (Supplementary Data 9).
To elucidate the impact of the isolated TILLING mutations, both GmTGA1-10E213K and GmSCT-10M417I mutants were screened for their reaction under SCN infections. Under HG type 0 SCN infection, the PI 407729 wild-type presented a moderate resistant reaction to SCN (21 CN on average) (Fig. 7C). The transcription factor GmTGA1-10E213K EMS mutant showed increased CN to 53 in average (max of 61 CN) (P <0.0001) when compared to the wild-type (Fig. 7C). The Shugoshin C-terminus GmSCTM417I mutant showed also increased CN to 26 in average (max of 32 CN) when compared to the wild-type (Fig. 7C).
Under HG type 2.5.7 SCN, PI 407729 wild-type showed a moderate resistant reaction to SCN with 18 CN on average. The transcription factor TGA1-related GmTGA1-10E213K mutant showed increased CN to 33 in average (max of 43 CN) (P <0.05) when compared to the wild-type (Fig. 7D). The Shugoshin C-terminus GmShugoshin-C-terM417I mutant showed increased CN to 35 in average (max of 41 CN) (P <0.005), when compared to the wild-type (Fig. 7D). Therefore, the TILLING mutants validated the role of the Shugoshin C-terminus and transcription factor TGA1-related genes in SCN resistance (Fig. 7D).
Discussion
Novel genes for broad-spectrum SCN resistance
The present study discovered two novel genes in PI 567516C that are required to confer resistance to SCN. Unraveling the genes responsible for SCN resistance has historically presented a significant challenge to the soybean community. Two major SCN resistance types are known, the PI 88788-type that requires the resistant rhg1-b allele and the Peking-type requires both the Rhg4-a and rhg1-a resistant alleles. PI 88788 type resistance requires 8 copies of the rhg1-b allele, while the Peking-type resistance requires both the Rhg4-a (2 copies) and rhg1-a (3 copies) resistant alleles and this allele combination is absent in PI 567516C28. Unlike the Peking and PI 88788-type of resistance, PI 567516C is a unique source that shows broad-spectrum resistance to SCN at least against five HG types of SCN (2.5.7 (race 1), 1.2.5.7 (race 2), 0 (race 3), 2.5.7 (race 5), and 1.3.5.6.7 (race 14))15. Additionally, although PI 567516C carries the rhg1- a like Peking-type, it contains the Rhg4-b susceptible allele (unlike the Rhg4-a resistant allele). Moreover, the Rhg4 promoter haplotype at PI 567516C is also different than the resistant haplotype promoter in Peking and PI88788 type of resistance (Supplementary Data 7). Thus, our previous studies of haplotype clustering and copy number variation28 (Supplementary Data 7) and fine-mapping analysis24 demonstrated that PI 567516C shows different haplotype and copy number variations at the rhg1 and Rhg4 loci as compared to the resistant haplotypes contained in PI 88788 and Peking types of resistance. Mapping the qSCN10 locus on chr. 10 in PI 567516C facilitated the discovery of a new SCN resistance type (independent of resistant rhg1 and Rhg4-a loci), leading to the identification of novel SCN-resistant genes in soybeans24. In this study, the qSCN10 region was narrowed down to 142 kb (Fig. 1), in which 20 genes were identified and further investigated in detail based on the haplotype (Figs. 2, 3), gene expression analyses (Fig. 4), coupled with the presence of non-synonymous SNPs. The resequencing of diverse genetic populations represents a potent strategy for uncovering traits and has been applied across a range of organisms, encompassing humans43 and a number of other species44,45. The WGRS dataset enables the detection of functional variations and offers an extensive inventory of genome-wide polymorphisms in closely related accessions. Among all the genes, two genes namely GmSCT-10 and GmTGA1-10 were identified as potential candidates harboring non-synonymous SNPs underlying the qSCN10 locus regulating SCN resistance. The functional validation of candidate genes was investigated in two ways; overexpression of candidate genes and mutagenesis through the TbyS+ approach. Overexpression of GmSCT-10 and GmTGA1-10 into susceptible W82 roots demonstrated reduced cyst count (more than 80%). PI 567516C presents a resistant reaction to PA5 (FI=2), but a moderate reaction to PA1 (FI = 27), PA2 (FI = 18), PA3 (FI=15), and PA14 (FI=15)28. Overexpression of both genes in the SCN- susceptible variety Williams 82 via composite hairy root transformation significantly reduced the cyst numbers from 96.6 on average contained in WI82::EV (control) to 15.03 (84.6%) and 18.69 (81.2%), when compared to 28 cyst numbers on average contained in PI 567516C. The observed effect of both GmSCT-10 and GmTGA1-10 genes was even stronger than the previously identified GmPR08-Bet VI gene, whose overexpression in transgenic Williams 82 roots decreased SCN cysts by 65%21. Therefore, the obtained data revealed the effectiveness of those genes in conferring resistance out of the PI 567516C genetic background.
Additionally, GmSCT-10 and GmTGA1-10 mutants from PI 407729 showed increased CN compared to the wild-type (Fig. 7C-D) under HG type 0 and HG type 2.5.7 indicating the crucial involvement of Shugoshin C-terminus and transcription factor TGA1-related genes in SCN resistance. The obtained data show that both genes at the qSCN10 locus not only are effective in conferring resistance in PI 567516C, but also in the closely related sources of SCN such as PI 407729, as shown via mutational analysis. Although overexpression analysis showed that individual tested genes decrease cyst numbers by more than 80%, and since the overexpression analysis and mutagenized lines have shown the effect of these genes individually, it is not possible at this time to rule out the effect of both genes to provide resistance in PI567516C.
This is not the first time that several genes within the same locus have been shown to play a role in SCN resistance. Previous studies have also demonstrated the involvement of several genes within the rhg1-b locus in conferring resistance46,47. The three genes; an amino acid transporter, alpha-SNAP, and wound-inducible protein were reported to play a role in SCN resistance in PI 88788 source of resistance, although their mode of action and link among their functional role remains unclear.
Shugoshin/microtubule mediated novel regulation of SCN resistance
Shugoshin-1 (Sgo1) is a crucial protein involved in the maintenance of centromeric cohesion and pairing during mitosis, preventing premature separation of homologous chromosomes before recombination is complete48,49. In addition to regulating various aspects of chromosome segregation and stability during cell division. Shugoshin is involved in the regulation of kinetochore-microtubule attachments and cohesin, which are crucial for chromosome movement during mitosis and maintaining chromosomal stability during cell division respectively.
Microtubules are involved in organizing and guiding the movement of the stylet within the plant cells. Upon nematode invasion, soybean plants activate defense responses to restrict the nematode's movement and development. In this case, microtubules may play a role in the establishment and regulation of these defense responses. Microtubules are an essential component of the plant's cytoskeleton and have important functions in cell division, and cell shape maintenance and actively involved in the establishment and regulation of cellular and defense responses to various stresses, including nematodes50. Microtubules are vital components in the complex interaction between nematodes and plants50,51,52.
SCN induces the formation of feeding sites called syncytia within the plant roots. The syncytia are multinucleated cells formed by the fusion of neighboring plant cells, and they serve as feeding sites for the nematode. Microtubules are involved in the reorganization of the plant cytoskeleton during syncytium formation and involved in regulating gene expression in response to nematode (SCN) infection and therefore, participate in the control of gene transcription, translation, and the transport of RNA molecules, influencing the plant's ability to mount an effective defense response against the nematode52. It has been reported that a “giant cell mini cell plate” may form a physical barrier to separate the two daughter nuclei that are required for the multiple rounds of mitosis in developing giant cells, which result in a functional feeding site53. MAP65-3 plays a key role in microtubule arrays organization during mitosis and cytokinesis during plant cell division. A defect in giant cell mini cell plate formation in the absence of MAP65-3 would lead to the accumulation of mitosis defects (cell wall stubs and connected nuclei) during repeated mitoses53. These defects may prevent the development of functional feeding cells, resulting in the death of the nematode.
Altogether, these studies confirm critical role of microtubule in nematode resistance including soybean cyst nematode. SCN resistance involves a sophisticated interplay between the nematode and the plant's defense responses. Understanding the roles of microtubules in SCN resistance can provide valuable insights into the molecular mechanisms of defense and may contribute to the development of more effective strategies to combat this damaging nematode pest.
Transcription factor-mediated novel regulation of SCN resistance
Transcription factors (TFs) regulate the expression of a specific set of genes by binding to promoter regions. They play essential roles in various cellular processes, including plant's response to biotic and abiotic stresses54,55,56. While limited information is available regarding the role of TGA (TGACG-binding), TFs in SCN resistance, and/or their specific involvement in microtubule dynamics related to SCN resistance; it is worth mentioning that the field of plant- nematode interactions are complex and multifaceted. Transcription factors can be part of the signaling pathways that are activated upon nematode infection and may modulate the expression of genes involved in the plant's defense response57,58,59.
A transcription factor TGA1-related was previously reported to regulate effector-triggered immunity and hormonal signaling networks25. Nematode effectors are crucial components of the interaction between plant-parasitic nematodes and their host plants (SCN-soybeans). Effectors are molecules produced by nematodes and secreted into the plant to manipulate plant cellular processes, suppress the plant's defense mechanisms, and facilitate the nematode's successful infection and establishment60,61,62. In soybeans, HgSLP-1, an esophageal-gland protein that is secreted by the nematode during plant parasitism, was co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact7. Several pathogen effectors and their host targets involving transcriptional regulation during plant immunity were identified in several plant species, including Arabidopsis thaliana, chili pepper (Capsicum annuum), rice (Oryza sativa), and black cottonwood (Populus trichocarpa)63,64,65,66,67.
Mutations in TFs, including WRKYs, TGAs, NACs, CBP60s/SARD1, ERFs, bZIPs, bHLHs, MYBs, CAMTAs, and TCPs, altered plant disease resistance against different pathogens68,69,70,71,72,73. The GmTGA1-10 (Glyma.10G194800) has 30 paralogs including genes for TGA transcriptional factor, Basic leucine zipper (bZIP) transcription factor, and camp-response element binding protein. Out of the 30 GmTGA1-10 paralog genes, the GmTGA2-13 (Glyma.13G193700) member was previously shown to respond to SCN resistance21. GmTAG2- 13 transcripts were induced in both compatible and incompatible reactions under SCN infections, whereas GmTAG2-13 transcripts were significantly induced in the resistant line only after treatment with salicylic acid21. Overexpression of the At-TGA2 in transgenic soybean roots also showed a reduction of SCN cysts by less than 50%74. However, no other study reported the direct involvement of TGA in resistance to SCN. Thus, to the best of our knowledge, this is the first report showing the involvement of the GmTGA1-10 and GmSCT-10 in conferring resistance to SCN in PI 567516C type of resistance. Unlike the other type of SCN resistance requiring either the rhg1-b locus (case of PI 88788) or the rhg1-a and Rhg4-a loci (case of Peking-type) Rhg4, the PI 567516C type resistance requires GmTGA1-10 and GmSCT-10 for its resistance to SCN.
Furthermore, the presence of NAC domain cis-element in GmSCT-10 and GmTGA1-10 promoter in the SCN susceptible soybean suggested that this cis-element may play a role in SCN susceptibility. Previous studies have shown the implication of the NAC domain-containing proteins in root-knot nematode stressed soybean roots75, and also was found within a QTL region for resistance to the three SCN HG Types, 0, 2.5.7, and 1.2.3.5.6.7, in the common bean (Phaseolus vulgaris)76. In soybeans, NAC domain-containing proteins were shown to be induced under HG Type 2.5.7 SCN infection in roots77.
Although the present study revealed the roles of GmSCT-10 and GmTGA1-10 at the qSCN10 (O) locus in resistance to SCN, their involvement and mode of action in cell division, microtubule dynamics, and effector-triggered immunity still need to be further elucidated.
Material and methods
Genetic population and progeny test of additional recombinants
A novel QTL (qSCN10) was mapped for SCN resistance in a cross of Magellan and PI 567516C and subsequently confirmed in the near-isogenic background to confer resistance to multiple SCN HG types15. Our previous study developed a BC4F2 backcross population to introgress the qSCN10 region from the exotic resource to the elite parent, Magellan, using marker- assisted backcrossing (MABC) method24. The initial fine-mapping effort was able to narrow the qSCN10 region to 379-kb interval between the markers SNP2 (Gm10:42,430,713) and SNP-O8 (Gm10:42,809,800)24. In the current study, 18 additional recombinant progenies were identified and advanced to homogenize the recombinant chromosomal segments. Further, these recombinants were planted in a greenhouse of the SCN phenotyping facility at the University of Missouri.
SCN bioassays
For SCN bioassays, several soybean accessions, progenies of the Magellan × PI 567516C population, and SCN indicator lines were evaluated for resistance to SCN HG Type 1.3.5.6.7 (race 14), while transgenic hairy roots developed in this study were screened with HG type 0 (race 3) as it is known historically as the most abundant race in the United States field. Homogenous nematode population of HG Type 1.3.5.6.7 and HG Type 0 have been maintained for more than 30 generations. In each experiment, all 18 recombinants were tested with parental lines and indicator lines (Peking, PI 88788, PI 90763, PI 437654, PI 209332, PI 89772, PI 54831 along with Pickett) as well as susceptible lines ‘Hutcheson’ and ‘Lee 74’ for HG type test78. SCN bioassays were performed in a greenhouse at the University of Missouri following a well-established method24,79,80. Briefly, soybean seeds were germinated in paper pouches for 3–4 days and then transplanted into PVC tubes (100 cm3) (one plant per tube). The tubes were filled with steam-pasteurized sandy soil and packed into plastic containers prior to transplanting. Each container held 25 tubes and was suspended over water baths maintained at 27 °C. Five plants of each indicator line were arranged in a randomized complete block design (RCBD). Two days after transplanting, each plant was inoculated with approximately 2000 SCN eggs. Thirty days post-inoculation, nematode cysts were washed from the roots of each plant and counted using a fluorescence-based imaging system80. The female index (FI %) was estimated to evaluate the response of each plant to each HG type of SCN using the following formula: FI (%) = (average number of female cyst nematodes on a given individual/ average number of female nematodes on the susceptible check). Five replications were included for each tested line and organized in an RCBD per experiment. Two independent experiments were performed, and the final values are the averages of the female index of two independent experiments.
Development of new markers and Kompetitive allele-specific PCR (KASP) assay
New SNP markers for KASP assay (LGC Genomics, UK) were developed to dissect the chromosomal crossover points of the newly identified recombinants. Briefly, whole genome resequencing (WGRS) data for PI 567516C29 and Magellan81 were used to obtain the genome sequence information of the qSCN10 locus. The sequence data were further rechecked by investigating read alignments in the Integrative Genomics Viewer tool82. We selected a set of SNPs and designed two allele-specific forward primers (along with tail sequences) and one common reverse primer for KASP assays (Table S1). For the genotyping assay, genomic DNA was extracted from young trifoliate leaves of the parental lines, Magellan and PI 567516C, and individual recombinant progenies using a standard CTAB (cetyl trimethyl ammonium bromide) method83 with some minor modifications. KASP reactions were performed as follows; we added 5 µl of 2X premade KASP master mix, 5 µl of 10–25 ng/µl gDNA, and 0.14 µl of primers mix for 10 µl reaction volume. These reactions were kept in the PCR cycling conditions at 95°C 15 min, followed by ten touch-down cycles at 94°C of 20 s, at 65–57 °C of 20 s (drop 0.8 °C per cycle) and then 23 cycles at 94°C of 20 s and at 57°C of 1 min. All the reactions were performed in Roche Light Cycler 480 Instrument II (Roche Applied Sciences, Indianapolis, IN, USA) using the fluorescent end-point genotyping method.
Analysis of Putative Cis-Elements at the GmTGA1-10, GmSCT-10, and GmLRR-PK-10 Promoters
Putative cis-elements in the upstream region (-2Kb upstream) of GmTGA1-10, GmSCT-10, and GmLRR-PK-10 promoters were searched using the programs PLACE, Plant PAN 2.0, and MatInspector84,85,86. Additional filtering was carried out based on motif score and redundant repeated motifs. Next, significant motifs were searched manually using PLACE for the putative role in effector-triggered immunity, hormonal signaling networks, and biotic stress84.
Expression analysis
Soybean root tissue from SCN-infected and non-infected Magellan and PI 567516C that were used for RNAseq expressions analysis were compiled as shown earlier35. Briefly, gene expression profiles of PI 567516C and Magellan in response to the SCN inoculation at different time points (0, 2, 3, 5, 10 DPI) were performed. Three independent experiments were conducted using a randomized complete block design (RCBD). Ten seedlings per line were included for each replication. Total RNAs were extracted using RNeasy QIAGEN KIT and RNA-seq library preparation and sequencing were performed at Novogen INC using the Illumina Hiseq 2500 platform. Briefly, messenger RNA has been purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the 1st strand cDNA was synthesized using random hexamer primers followed by the second strand synthesis. The library was prepared using end repair, A- tailing, adapter ligation, size selection, amplification, and purification steps. These libraries were checked and quantified and then pooled for RNA sequencing on the Illumina platform. Raw RNA reads were processed through fastp software and clean reads were obtained by following Q30 content analysis. After quality check, clean reads were mapped to the Glycine max v2 reference genome using Hisat2 v2.0.587. To identify differentially regulated genes between two samples, the following criteria were selected: 2-fold, with a t-test p value <0.05, and the absolute signal intensity difference between the two samples >10. DESeq2 has been used to study differential expression analysis between samples. Functional classification of differentially regulated genes was analyzed using MapMan. The SRA data was submitted to NCBI with bioproject accession number: PRJNA 1163880.
Haplotype clustering analysis
The WGRS data of 106 diverse soybean lines sequenced at approximately 17X genome coverage29 have been utilized to identify allelic variants in the qSCN10 QTL. Haplotype analysis of qSCN10 QTL was conducted using a previously described pipeline27. Briefly, SNPs from qSCN10 QTL were extracted from the whole genome resequencing data using SNPviz (V2.0) tool (https://soykb.org/SNPViz2/). The imported data file converted into map (.map) and genotype (.geno) file to upload into Flapjack software to visualize the graphical genotypes and identify associated haplotypes as detailed manual30. This SNPs underlying qSCN10 were further analyzed by calculating the similarity matrix and principal components to generate dendrogram. The dendrogram and genotype matrix (light blue and black color) was then converted to image files, followed by assigning haplotype number (H1 – H6) based on the major clades in dendrogram. Furthermore, gene IDs were manually inserted in the exact position on the genotype matrix (Fig. 2). Next, the major genes with large effect SNP and higher expression upon SCN infection (Fig. 3) such as GmTGA1-10, GmSCT-10, and GmLRR-PK-10 were clustered as mentioned above followed by overlaying SCN phenotypic data in excel. Transcript sequence-based annotation (Wm82.a2.v1) was used to classify synonymous and non-synonymous SNPs by translating into amino acid sequences. The haplotype analysis and SCN cyst count phenotypic data were overlayed with SNP matrix (haplotypes) corresponding to individual lines and specific clusters.
Cloning and transgenic soybean composite hairy root transformation
The functional characterization of the selected two candidate genes, including the transcription factor TGA1-related (Glyma.10G194800) and Shugoshin C-terminus (Glyma.10G196400) genes, has been validated using the transgenic composite hairy root system21,88. cDNA sequence from both genes were amplified from soybean PI 567516C root cDNA by RT–PCR, in addition to the GmSHMT08 (Rhg4-a) that used as a positive control, were cloned into the pG2RNAi2 vector under the control of the soybean ubiquitin (GmUbi) promoter. Cloning was carried out between AscI and AvrII sites in the pG2RNAi2 vector to generate pG2RNAi2::GmTGA1-10 and pG2RNAi2::GmSCT-10. Williams 82 composite hairy roots transformed with pG2RNAi2::empty vector were used as a negative control. The pG2RNAi2 vector carries a GFP-selectable marker for planta selection. Transgenic Williams 82 composite hairy roots transformed with pG2RNAi2::GmTGA1-10 and pG2RNAi2::GmSCT-10 were produced by injecting Agrobacterium (K599) suspensions three times into the hypocotyl directly below soybean cotyledons using a 3-mL syringe (BD#309578). After injection, composite hairy roots from at least 50 independent soybean transgenic plants per construct were grown and propagated in vermiculite. Plants were covered with plastic domes and sprayed consistently with water to maintain humidity in a growth chamber for 1–2 weeks. Plants were fertilized once a week with NPK 20-20-20. Plants with GFP-positive composite hairy roots at ~2–3 inches long were transferred to sandy soil before SCN screening. Growth conditions and SCN screenings with HG type 0 was performed, as mentioned earlier. SCN HG type 0 is known historically as the most abundant race in the United States field. After 30 days, cysts were counted under a stereomicroscope. The experiment was independently conducted three times with a minimum of 15 to 20 independent composite hairy root lines per construct per experiment. The results were plotted and analyzed for statistical significance by using analysis of variance (ANOVA) using the JMP Pro V12 software88.
Development of an EMS-mutagenized Forrest population
The soybean lines PI 407729 and PI 567516C were used to develop two EMS mutagenized populations. The wild-type seeds from the two soybean genotypes were mutagenized with 0.6% EMS89, and planted to harvest families of M2 generation. It included 1,536 from PI 407729 and 1,440 from PI 567516C, which were subsequently advanced to the M3 generation at the Horticulture Research Center at Southern Illinois University, Carbondale, IL. In total, 2,976 M3 EMS mutants (from PI 407729 and PI 567516C) were used to pool EMS mutants within the two candidate genes (GmTGA1-10 and GmSCT-10) using TILLING-by-Sequencing+.
TILLING-by-Sequencing+
A two-dimensional pooling strategy was implemented to reduce the number of pools. Samples were vertically pooled into pools of 24 samples and horizontally into pools of 48 samples, as shown earlier37,39,42. Then, specific probes were designed to target regions of interest at the transcription factor TGA1-related gene (Glyma.10G194800) and shugoshin C-terminus gene (Glyma.10G196400). After library preparation and probe design, the developed TILLING-by- Sequencing+ workflow (based on capture-seq enrichment and target recovery technology) uses magnetic beads for targeting desired genes with higher efficiency and specificity before proceeding with next-generation sequencing of the pooled DNA. The result was saved as a FASTQ file, then raw reads were subject to quality control using FastQC90, trimming, and filtering of low-quality reads using Trimmomatic91. The clean reads were aligned to the Williams 82 reference genome (Wm82.a4) using BWA92. SAM tools were used to filter and sort the bam files to serve as input for variant calling using Freebayes93 and CRISP94. The VCF files resulting from variant calling were filtered by VCFtools and visualized in IGV for demultiplexing95. Using the VCF files, the SNPs corresponding to the induced mutations within the mutant population were found. After identifying the desired PI 407729 GmTGA1-10 and GmSCT-10 TILLING mutants, their seeds were phenotyped for their HG type 0 (race 3) and HG type 2.5.7 (race 5) SCN reaction, since PI 407729 wild-types show resistance to moderate resistance to both SCN races.
Homology modeling of Transcription Factor TGA1-related and Shugoshin C-Terminus protein and mutational analysis
Homology modeling of putative Transcription Factor TGA1-related and Shugoshin C- Terminus protein structures was conducted with Deepview96 and Swiss Model Workspace software97 using the protein sequence from “Williams 82” and an available crystal structure as a template; PDB accession I1NHU8.1.A for Transcription Factor TGA1-related and I1LCL1.1.A for Shugoshin C-Terminus. All residues were modeled against the two templates with a sequence identity of 100%. Mutation mapping and visualizations were performed using the UCSF Chimera package98.
Statistics and reproducibility
The data presented in Fig. 6 were performed with the analysis of variance by a T-student test means comparison, using JMP Pro V18 software. The data shown represents the averages from all three biological repeats. Connecting letters indicate significant differences between the tested lines as determined by ANOVA (P <0.0001). The experiments were repeated three times, and similar results were obtained. The data presented in table S2 were generated using the mean of 10 replications and statistical analysis was performed by using Tukey's HSD test which demonstrated the significant difference between the tested lines (P <0.0001).
Conclusion
The exotic soybean line, PI 567516C, harbors previously unknown SCN resistance genes that provide broad-spectrum resistance to SCN, distinct from the known Rhg4 and rhg1 genes. In this study, we further conducted fine-mapping the qSCN10 QTL region, which was detected in PI 567516C. Through rigorous genotyping, expression analysis, and QTL-based haplotyping, we successfully selected the two most promising candidate genes within a 62.4-kb interval. Among these candidates, GmTGA1-10 and GmSCT-10 exhibited distinctive allelic variations in both coding and promoter regions, providing the basis for further gene functional characterization. Overexpression of GmTGA1-10 and GmSCT-10 genes in susceptible genotype (Wm82) significantly reduced the FI by (84.6%) and (81.2%), respectively. These genes responsible for SCN resistance in PI 567516C hold significant potential in countering shifts in SCN populations and virulence. These genes can be introduced to high-yielding varieties by using newly developed molecular markers for MAS to enhance the durability of soybean resistance to SCN. Moreover, the obtained results provide a basis for novel molecular mechanisms involving cell division, microtubule dynamics, and effector-triggered immunity that warrants in-depth investigation. The current study’s findings provided valuable genetic resources for the sustainable management of SCN.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data supporting the findings of this study are available within the paper. Supplementary data associated with this study can be found in the online version. The SRA data was submitted to NCBI with bioproject accession number: PRJNA1163880. All other data provided in this manuscript are available from the corresponding author. Numerical source data for the graphs presented in Figs. 6, 7 can be found in Supplementary Data 1 & 2, respectively. Source data of the ANOVA analysis for the graph presented in Fig. 6 can be found in Fig. S3.
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Acknowledgements
Principal Investigator (PI) Henry Nguyen and Co-PI Khalid Meksem greatly acknowledge the funding support from the United States Department of Agriculture- National Institute of Food and Agriculture (USDA-NIFA) Award No. 2019-67013-29370) and the United Soybean Board (Grant No. 2220-172-0155). The authors thank Jinrong Wan for his advice on data analysis.
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H.T.N. and K.M. oversaw the project and conceived the research idea; N.L. and S.S.C. designed the project experiments and wrote the manuscript; N.L. conducted composite hairy root transformation analysis and gene over-expression, Tilling-by-seq+, EMS mutants development and identification, gene cloning, protein homology modeling, cis-element promoter analysis, expression analysis, and data interpretation; SSC conducted fine mapping, progeny testing, SCN phenotyping, KASP marker development, cloning, expression analysis, SCN testing of transgenic plants and data interpretation; V.D. and G.P. performed haplotype analysis and edited the manuscript; H.Y. and T.V. re-confirmed the earlier discovery of novel SCN resistance source from PI 567516C by developing the mapping population, recombinant progeny testing, and data analysis, D.K. and A.E.B. performed cis-element analysis. All authors discussed the results and contributed to the final manuscript
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Lakhssassi, N., Chhapekar, S.S., Devkar, V. et al. Discovery of two tightly linked soybean genes at the qSCN10 (O) locus conferring broad-spectrum resistance to soybean cyst nematode. Commun Biol 8, 259 (2025). https://doi.org/10.1038/s42003-025-07633-8
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DOI: https://doi.org/10.1038/s42003-025-07633-8
