Identification and characterization of pleiotropic and epistatic QDRL conferring partial resistance to Pythium irregulare and P. sylvaticum in soybean

Pleiotropic and epistatic quantitative disease resistance loci (QDRL) were identified for soybean partial resistance to different isolates of Pythium irregulare and Pythium sylvaticum. Pythium root rot is an important seedling disease of soybean [Glycine max (L.) Merr.], a crop grown worldwide for protein and oil content. Pythium irregulare and P. sylvaticum are two of the most prevalent and aggressive Pythium species in soybean producing regions in the North Central U.S. Few studies have been conducted to identify soybean resistance for management against these two pathogens. In this study, a mapping population (derived from E13390 x E13901) with 228 F4:5 recombinant inbred lines were screened against P. irregulare isolate MISO 11–6 and P. sylvaticum isolate C-MISO2-2–30 for QDRL mapping. Correlation analysis indicated significant positive correlations between soybean responses to the two pathogens, and a pleiotropic QDRL (qPirr16.1) was identified. Further investigation found that the qPirr16.1 imparts dominant resistance against P. irregulare, but recessive resistance against P. sylvaticum. In addition, two QDRL, qPsyl15.1, and qPsyl18.1 were identified for partial resistance to P. sylvaticum. Further analysis revealed epistatic interactions between qPirr16.1 and qPsyl15.1 for RRW and DRX, whereas qPsyl18.1 contributed resistance to RSE. Marker-assisted resistance spectrum analysis using F6:7 progeny lines verified the resistance of qPirr16.1 against four additional P. irregulare isolates. Intriguingly, although the epistatic interaction of qPirr16.1 and qPsyl15.1 can be confirmed using two additional isolates of P. sylvaticum, the interaction appears to be suppressed for the other two P. sylvaticum isolates. An ‘epistatic gene-for-gene’ model was proposed to explain the isolate-specific epistatic interactions. The integration of the QDRL into elite soybean lines containing all the desirable alleles has been initiated.


Introduction
Soybean is a major crop around the world for its protein and oil content. In 2020, total world soybean production reached a historical record of 353.5 million tons with a production area of approximately 127.0 million hectares (FAOSTAT 2020). The U.S. soybean yield production in 2020 was 112.5 million tons, a 16% increase from the 96.7 million tons in 2019. Despite the continual increase in soybean production in the U.S. and the world, the impact of soybean diseases on yield cannot be ignored (Lin et al. 2022). For example, the soybean annual yield losses caused by diseases in the U.S. and Ontario, Canada ranged from 10.1 to 13.9 million tons from 2010 to 2014, accounting for 11.7-14.2% of total soybean production of the year (Allen et al. 2017). AllenMore than 35 soybean diseases have been considered economically important (Hartman et al. 2015) and seedling diseases are some of the most destructive diseases of soybean, causing 0.68 -1.76 million tons (25.1 -64.5 million bushels) of annual yield loss in the U.S. and Ontario, Canada (Allen et al. 2017).
Pythium spp., an oomycete pathogen, are some of the major causal agents of soybean seedling diseases (Griffin 1990;Arafa et al. 2020;Clevinger et al. 2021). Currently more than thirty Pythium species have been confirmed as soybean pathogens, causing seed rot, seedling dampingoff and root rot (Zhang et al. 1998;Huzar-Novakowiski and Dorrance 2018;Dorrance et al. 2004;Zitnick-Anderson and Nelson 2015;Broders et al. 2007;Radmer et al. 2017;Rojas et al. 2017;Li et al. 2019;Navarro and Krystel 2019;Clevinger et al. 2021). In a two-year survey of oomycete pathogens, more than 3,400 oomycete cultures were isolated from 11 major soybean producing states of the U.S and Ontario, Canada. Further analysis revealed that more than 86% of the recovered isolates were Pythium species, with P. sylvaticum and P. irregulare as two of the most prevalent and aggressive Pythium species (Rojas et al. 2017). Internal transcribed spacer (ITS) sequencing analysis indicated that P. irregulare and P. sylvaticum are phylogenetically close to each other and both belong to Clade F (Hyde et al. 2014;Rojas et al. 2017).
E13390 and E13901 are improved soybean lines from the Michigan State University soybean breeding program. Two QDRL with epistatic interactions for partial resistance to Phytophthora sansomeana were identified from a F4:5 mapping population (150029) derived from E13901 x E13390 in our previous study (Lin et al. 2021). Further investigation indicated that E13901 also imparts significantly higher level of partial resistance to P. irregulare and P. sylvaticum than E13390. Therefore, the objectives of this study aim to 1) identify and develop molecular markers for the QDRL imparting partial resistance to P. irregulare and P. sylvaticum from the F4:5 mapping population (150029), and 2) characterize the epistatic interaction and resistance spectrum of the QDRL to additional isolates of P. irregulare and P. sylvaticum using marker-assisted resistance spectrum analysis.

Plant materials and preparation of Pythium inoculum
The mapping population (150,029) consisted of 228 F4:5 recombinant inbred lines which were used for linkage map and QDRL mapping as described in Lin et al. 2021. Briefly, the MSU improved soybean line E13901 was crossed with E13390 in 2015 for F1, which was self-pollinated in the field in 2016. The F2 population was subjected to self-pollination using the single seed decent method for two generations in the greenhouse to obtain F4 seeds, which were subsequently planted next year in the field for F4:5 families. These F4:5 families were used for QDRL mapping. The F4:5 families were then advanced two generations using single seed decent to obtain F6:7 families, which were used for marker-assisted resistance spectrum (MARS) analysis.
P. irregulare isolate MISO 11-6 and P. sylvaticum isolate C-MISO2-2-30, from symptomatic soybeans in Michigan, were used for QDRL mapping (Rojas et al. 2017). There additional isolates of P. irregulare (ILSO 3-48C,  and four isolates of P. sylvaticum  were used in MARS analyses. The inoculum of P. irregulare isolate MISO 11-6 was prepared by placing 1200 ml of white millet and 500 ml of water into a 0.5 micron ventilated 3 mil polypropylene 20.3 cm L × 12.7 cm W × 50.8 cm D spawn bag (FungiPerfecti, Shelton, WA), rolled with the vent-side facing upward and placed into autoclavable pans and autoclaved for 275 min. The sterilized white millet was allowed to cool to room temperature for 24 h. Isolate MISO_11-6 was grown on CMA-PARP medium for 3-7 days, after which five colonized plates and five non-colonized CMA-PARP plates were aseptically placed in a sterile stainless-steel blender (Waring, VWR International) and 500 ml of sterile water added to the carafe. The contents were blended until a thick slurry developed. Approximately 100 ml of the P. irregulare slurry was poured in each spawn bag. After addition of the slurry, spawn bags were sealed three times with a heat sealer, then incubated for 14 days at 20-22℃ (room temperature). To enhance uniform millet colonization, the spawn bags were mixed every other day.
The inoculum of all other isolates was prepared by transferring a 5-mm agar plug from an actively growing isolate to 60 mm × 15 mm petri dish plates containing corn meal agar (CMA). The plates were then incubated at room temperature for 10-14 days until the pathogen fully colonized the plates. The inoculum was chopped into 4 mm × 4 mm pieces before use.

Evaluation of soybean resistance to Pythium pathogens
A modified layer test assay (Dorrance et al. 2008;Lin et al. 2018;Lin et al. 2020Lin et al. , 2021 was used for disease evaluation at the Michigan State University greenhouse facilities, with environmental conditions controlled at 24 °C-27 °C and 12-h photoperiod. The inoculation starts by filling seed starting trays (3.81 cm L × 2.54 cm W × 5.72 cm D/cell, T.O. Plastics, Inc.) with medium size vermiculite and soaked in tap water until the vermiculite was fully saturated. Then two 2 cm-deep, 1 cm-wide holes were made in each cell and 2 g of MISO11-6 inoculum, or one 4 mm × 4 mm piece of all other isolates was placed at the bottom of each hole. Two soybean seeds were then placed on top of the inoculum and softly pressed to ensure each seed was adhered to the inoculum.
Twelve seeds were planted as a single replicate for each line, with a total of three replicates for the inoculated group and three replicates for the non-inoculated treatment. After planting, all the trays were transferred to greenhouse benches covered with waterproof plastic for water retention. The benches were watered until the water reached over the level of the inoculum. After that, the benches were watered every other day to maintain a consistent water level until the day before data collection. Fourteen days after planting, the number of germinated seeds was counted, and the fresh root weight was measured using an electronic balance (Scout Pro, SP 4001; Ohaus Corp, Pine Brook, NJ). The responses of each soybean line challenged with P. irregulare and P. sylvaticum isolates were evaluated using the ratio of seedling emergence (RSE), the ratio of fresh root weight (RRW), and the disease resistance index (DRX) (Lin et al. 2021), where.
where RWI = total fresh root weight of an inoculated replicate/N, N represents the number of vigorous seedlings of each inoculated replicate and is estimated using the mean of germinated seeds across all the non-inoculated replicates. To ensure high quality of seeds, a cutoff of N ≥ 10 in each replicate was applied, and. RWC = total fresh root weight of a non-inoculated replicate / number of germinated seeds of the replicate, DRX ranges from 0 to 100, with 0 for complete susceptible and 100 for complete immunity.

DNA extraction and linkage map
For each F4:5 family, leaf samples from 12 F5 seedlings were bulk collected and used for DNA extraction as described in Lin et al. 2021. Briefly, the leaf samples were collected, and then placed at -80 °C freezer for storage and subsequently lyophilized before DNA extraction. DNA samples were extracted using a standard Cetyl Trimethyl Ammonium Bromide (CTAB) method and the resulting DNA pellet was dissolved in 200 μl 10 mM Tris-HCl buffer. DNA samples were quantified using an ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) for chip analysis. The genotyping of the population RSE = number of germinated seeds of an inoculated replicate∕N, was carried out using Illumina Infinium BARCSoySNP6K iSelect BeadChip genotyping array (Illumina, San Diego, USA) (Song et al. 2013), which yielded 978 polymorphic markers for linkage map construction using Joinmap software (v4.0, Ooijen 2006) with an independence LOD = 4.0 and a max recombination frequency of 0.5 (Lin et al. 2021). A total of 23 linkage groups were obtained, corresponding to the 20 chromosomes of soybean (Lin et al. 2021).

Statistics and QDRL mapping
SPSS software (IBMSPSS Statistics, IBMCorporation, Chicago, IL) was used in this study for statistical analysis. The software QTL Cartographer V2.5 (Wang et al. 2012) was used for composite interval mapping (CIM) with window size of 5 cM and the walking speed of 1 cM. The threshold of LOD score for statistical significance of QDRL effects was determined by 1000 permutations, and the LOD value corresponding to an experiment-wise Type I error rate of 5% (α = 0.05) was considered the threshold of significance (Churchill and Doerge 1994). The position of each QDRL was estimated as the point of maximum LOD score in the region under consideration.

Response of soybean lines to P. irregulare and P. sylvaticum
As expected, E13901 conferred significantly higher level of partial resistance to P. irregulare and P. sylvaticum than those of E13390. The RSE of E13901 against P. irregulare ranged from 0.67 to 0.92, with the mean of 0.81 ± 0.07, which was significantly higher than that of E13390, which ranged from 0.17 to 0.42, with the mean of 0.31 ± 0.07; The RRW of E13901 ranged from 0.39 to 0.52 with the mean of 0.44 ± 0.04, which was significantly higher than that of the other parent, which ranged from 0.04 to 0.19 with the mean of 0.12 ± 0.04. The DRX of the resistant parent ranged from 51.14 to 68.94 with the mean of 59.47 ± 5.17 and was significantly higher than the susceptible parent which ranged from 8.19 to 28.40, with the mean of 19.01 ± 5.86 (Table 1). The same pattern was observed for the partial resistance to P. sylvaticum. The mean of RSE, RRW, and DRX of E13901 were 0.97 ± 0.03, 0.71 ± 0.04, and 82.81 ± 3.45, respectively, which were significantly than those of E13390, which were 0.89 ± 0.07, 0.42 ± 0.08, and 61.25 ± 8.29, respectively (Table 2). Both lines conferred higher level of partial resistance against P. sylvaticum than P. irregulare. The partial resistance of F4:5 lines in population 150,029 to P. irregulare mostly ranged between the parental lines, with the means of RSE, RRW, and DRX at 0.59 ± 0.01, 0.25 ± 0.05, and 37.89 ± 0.64, respectively. The histogram analysis indicated that the distribution of partial resistance of 150,029 to P. irregulare appeared normal distribution (Fig. 1A, 1B, and 1C for RSE, RRW, and DRX, respectively). For resistance to P. sylvaticum, the means of RSE, RRW, and DRX of F4:5 lines in 150,029 were 0.97 ± 0.03, 0.69 ± 0.01, and 81.64 ± 0.41, respectively. The histogram of RRW and DRX appeared normal distribution, while that of RSE was left-skewed. Transgressive segregation was obviously observed as nearly half of the lines exhibited a higher level of partial resistance than the resistant parent ( Fig. 1D, 1E, and 1F).
Considering the close relationship of P. irregulare and P. sylvaticum, we were interested to explore if the partial resistance against the two pathogens were correlated. Pearson's correlation test was therefore applied as shown in Table 3 and Fig. 2. The correlation coefficients of RSE, RRW, and DRX between the partial resistance to P. irregulare and P. sylvaticum were 0.252, 0.172, and 0.163, respectively, all significantly correlated at p < 0.05 or p < 0.01 level.
QDRL mapping for partial resistance to P. irregulare and P. sylvaticum Using the 228 F4:5 lines, one QDRL (dubbed qPirr16.1) was detected for both RRW and DRX for resistance to P. irregulare (isolate MISO 11-6) using CIM method. qPirr16.1 was located at 1.91 cM on soybean chromosome 16, flanked by SNP markers Gm16_7743486_G_T and Gm16_7851145_G_A. The LOD scores of qPirr16.1 were 3.64 and 3.52 for RRW and DRX, respectively, which were higher than the LOD threshold of 3.30 and 3.20,  Table 4). Using CIM method, two QDRL were detected for soybean partial resistance to P. sylvaticum (dubbed qPsyl15.1 and qPsyl18.1, respectively). qPsy15.1 was located at 2.81 cM on soybean Chr. 15, between SNP markers Gm15_4315169_T_C and Gm15_4264903_C_T. The QDRL was detected using both RRW and DRX, with LOD scores of 4.02 and 4.07, respectively. The QDRL explained the RRW and DRX variations of 6.66% and 6.49%, respectively, and the additive effects were -0.024 and -1.742, respectively, indicating that the desirableresistant allele was from E13901 (Fig. 3B, Table 4). qPsyl18.1 was identified through RSE alone, with a LOD score of 3.68. qPsyl18.1 was located at 0.001 cM on Chr. 18, flanked by SNP markers Gm18_61528293_C_T and Gm18_61065114_G_A. The QDRL explained 6.51% of RSE variations with an additive effect of 0.011, which  indicated that the desirable allele of resistance was from E13390 (Fig. 3C, Table 4).

qPirr16.1 is a pleiotropic QDRL conferring resistance to both P. irregulare and P. sylvaticum in different manners
Because of the significant correlations of phenotypic responses to P. irregulare and P. sylvaticum, we hypothesized that a pleiotropic QDRL could be detected contributing resistance to both pathogens. To test this hypothesis, a joint phenotype was calculated by averaging the value of each index of each line for both pathogens. CIM mapping using the joint phenotypes identified a pleiotropic QDRL for DRX at 1.91 cM on chromosome 16, flanked by Gm16_7743486_G_T and Gm16_7851145_G_A, which overlaps with the qPirr16.1 locus (Fig. 3D, Table 4). The pleiotropic QDRL explained 6.85% of the variation of the joint DRX with LOD of 4.04 and its additive effect was -1.597, indicating that E13901 is the resistance donor ( Fig. 3D, Table 4). Interestingly, although qPirr16.1 was identified for resistance to P. irregulare, it was not detected in QDRL mapping for P. sylvaticum. Therefore, to verify the resistance of qPirr16.1 to P. sylvaticum, different genotypic groups (R, S, and H) were identified in the F4:5 mapping population using the flanking markers Gm16_7743486_G_T and Gm16_7851145_G_A. 71 lines with E13901 genotype were marked as R group, 97 lines with E13390 genotype were identified as S group, and 48 heterozygous genotypes on both marker loci were included in H group (Table S1).
Multiple comparisons between different genotypic groups against the P. irregulare isolate MISO 11-6 indicated that the R group was significantly higher than the S group in all RSE, RRW and DRX, confirming the partial resistance of qPirr16.1 to P. irregulare. The H group also showed a similar level of resistance as the R group and was significantly higher than the S group in all three traits, indicating that the resistance of qPirr16.1 is dominant to P. irregulare (Fig. 4A, 4B, and 4C). For P. sylvaticum, the RSE of the R group was not significant compared with S group, however, R group was significantly higher than the S group in RRW and DRX, confirming the partial resistance of qPirr16.1 to P. sylvaticum. Interestingly, the H group was as susceptible as the S group in both RRW and DRX, and was significantly lower than the R group, suggesting that the partial resistance from qPirr16.1 was recessive against P. sylvaticum. Therefore, qPirr16.1 is a pleiotropic QDRL which contributes dominant and recessive resistance to P. irregulare and P. sylvaticum, respectively.
Multiple comparisons using Least Square Difference (LSD) post hoc test indicated that neither qPirr16.1 nor qPsy15.1, or the combination of both QDRL contributed to the RSE for partial resistance to P. sylvaticum isolate C-MISO2-2-30 (Fig. 5A), which was consistent with the QDRL mapping results (Table 4, Fig. 4). For RRW, the RR group was significantly higher than the other three groups, whereas RS and SR group did not show significant differences than the SS group (Fig. 5B). An identical pattern was also observed for DRX, where the RR group was significantly higher than the other three groups and each QDRL alone did not contribute to a significantly higher level of resistance than the SS group (Fig. 5C). These results indicated that both qPirr16.1 and qPsy15.1 are required for partial resistance to P. sylvaticum isolate C-MISO2-2-30.

qPirr16. 1 confers resistance to three additional P. irregulare isolates
Marker-assisted resistance spectrum (MARS) test was performed for qPirr16.1 against three additional isolates of P. irregulare (ILSO 3-48C, . The flanking markers of qPirr16.1, Gm16_7743486_G_T and Gm16_7851145_G_A, were used to select the genotypes of F6:7 progeny lines which yielded 51 lines with homozygous-resistant genotypes (E13901 genotype, dubbed R group), and 67 lines with homozygous susceptible genotypes (E13390 genotype, dubbed S group). The 118 selected lines together with their parental lines were tested against the three isolates (Table S3), and the results are shown in Figure S1. For isolate ILSO 3-48C ( Figure S1 A, B, and C) and AR-127.S.2.3.A ( Figure S1 D, E, and F), the R group was significantly higher (p < 0.05) than the S group for RSE, RRW, and DRX. For isolate C-MISO2_5-14, the R group was significantly higher than the S group for RSE and DRX, but not significant for RRW. These results indicated that qPirr16.1 confers effective partial resistance against ILSO 3-48C,

qPirr16. 1 and qPsyl15.1 varies in resistance to four additional P. sylvaticum isolates
To test the resistance of qPirr16.1 and qPsy15.1 to other isolates of P. sylvaticum, the F6:7 progeny seeds of the four genotypic groups (RR, RS, SR, SS) were used (Table S3). Due to the limit of seeds, 18, 18, 18, and 30 F6:7 lines were used for each genotypic group, respectively. The 84 lines together with the parental lines were tested against four additional P. sylvaticum isolates (O_14-16, INSO 1-10c, NESO 2-13, and MISO_6-2) and results are shown in Figure S2.
The response of the two QDRL against O_14-6 was identical to that against C-MISO2-2-30 with an epistatic pattern, where the combination of qPirr16.1 and qPsyl15.1 conferred significantly higher resistance for RRW and DRX, but not for RSE, whereas each QDRL alone did not contribute resistance ( Figure S2 A1-A3).
The response of qPirr16.1 and qPsyl15.1 against INSO 1-10c was similar to C-MISO2-2-30 and O_14-6 in that each individual QDRL did not contribute resistance to the isolate alone, but the combination of them (RR group) was higher than the other groups for RRW and DRX. For RRW, the difference between the RR group and the RS group were not significant at p < 0.05 but was significant at p < 0.1 (p = 0.055). For DRX, the differences between the RR and RS or SS groups were not significant at p < 0.05, but the RR group was significantly higher than the SS group at p < 0.1 (p = 0.077) ( Figure S2 B1-B3).
However, the two QDRL behaves differently for isolates NESO 2-13 and MISO_6-2. For NESO 2-13, the SR group was significantly higher than the SS group for RSE, RRW,  Table 4. The RR group included 22 lines which were both E13901 alleles on both loci; The RS group included 28 lines with E13901 allele on qPsyl15.1 locus and E13390 allele on qPirr16.1 locus; The SR group included 29 lines with E13390 allele on qPsyl15.1 locus and E13901 allele on qPirr16.1 locus; The SS group included 39 lines with E13390 alleles on both loci. The multiple comparisons among groups were carried out using least square difference (LSD) with significance level at p < 0.05 and DRX, indicating that qPsyl15.1 confer partial resistance to the isolate. However, the resistance of both the RR and the RS groups were significantly lower than the SR and SS group, suggesting that qPirr16.1 enhanced soybean susceptibility to the isolate, and may inhibited the resistance of qPsyl15.1 ( Figure S2 C1-C3). The RR, RS, and SR groups did not show significant differences compared to the SS group against MISO_6-2, suggesting that neither qPirr16.1 nor qPsyl15.1, nor the combination of the two QDRL contribute resistance to the isolate. Instead, qPirr16.1 may play an inhibitor role against qPsy15.1 since the RSE and DRX of the RR group were significantly lower than the SR group against MISO_6-2.

Discussion
In this study, a novel QDRL, qPirr16.1, was identified conferring partial resistance to P. irregulare, which was further confirmed with three additional isolates of P. irregulare  using a MARS test. Further analysis found that qPirr16.1 is a pleiotropic QDRL and works together with another novel QDRL, qPsyl15.1, for partial resistance to three isolates of P. sylvaticum . Interestingly, qPirr16.1 confers dominant resistance against P. irregulare but recessive resistance against P. sylvaticum. Pleiotropic QDRL are not uncommon for partial resistance to Pythium diseases, suggesting there is likely some similarly of resistance mechanisms against different species of Pythium pathogens. For example, two QDRL were identified on chromosomes 13 and 17 that both confer partial resistance to P. irregulare and P. ultimum var. ultimum (Scott et al. 2019); A major QDRL was identified on Chr. 8 conferring resistance to P. irregulare, P. sylvaticum, and P. torulosum (Clevinger et al. 2021). Another large effect QDRL on Chr. 6 was also identified imparting partial resistance to P. sylvaticum and P. irregulare (Clevinger et al. 2021). All these pleiotropic QDRL as well as the novel ones identified in this study will be of particular importance for breeding partial resistance against multiple species of Pythium pathogens.
Partial resistance or quantitative resistance has been widely considered race non-specific, while isolate-specific QDRL have also been identified and confirmed (Marcel et al. 2008;Poland et al. 2009;St. Clair 2010;Lee et al. 2014;Mundt 2014;Stasko et al. 2016;Nelson et al. 2018;Karhoff et al. 2019). In the current study, qPirr16.1 conferred significant partial resistance to all four isolates of P. irregulare and therefore appears isolate non-specific against P. irregulare. However, the combination of qPirr16.1 and qPsyl15.1 showed clearly isolate-specific epistatic interactions with different isolates of P. sylvaticum. More interestingly, for some isolates of P. sylvaticum (C-MISO2-2-30, O_14-6, and INSO 1-10c), qPirr16.1 works together with qPsyl15.1 to enhance the resistance, whereas for other isolates, qPirr16.1 appears to suppress the resistance of the other QDRL. Isolate-specific QDRL have also been identified in soybean partial resistance to other diseases such as Ph. sojae and Ph. sansomeana. For instance, a QDRL on soybean Chr. 3 was identified conferring significant resistance to isolate 1.S.1.1 of Ph. sojae, but not to isolate OH30. In the same study, isolate-specific QDRL were identified on soybean chromosomes 6, 9, 13 and 18 that conferred resistance to only one of the two isolates of Ph. sojae (Lee et al. 2014). Lin et al. (2021) identified two isolate-specific QDRL (qPsan5.1 and qPsan16.1) conferring different patterns of resistance against eight isolates of Ph. sansomeana using the same population in this study. Interestingly, qPsan5.1 and qPsan16.1 also showed epistatic interactions to some of the isolates of Ph. sansomeana.
To explain the isolate specificity of minor QDRL, Parlevliet and Zadoks (1977) proposed a 'minor-gene-for-minorgene' (MGFMG) model which suggested that the genefor-gene interactions for major resistance genes also work for minor QDRL in a similar pattern. While the MGFMG model could explain the interactions of a single gene with the pathogens which could be confirmed in several studies (Marcel et al. 2008), it did not provide an explanation to the epistatic interactions of the QDRL as discovered in the current study and Ph. sansomeana (Lin et al. 2021). As such, we are proposing a 'epistatic gene-for-gene' model, where the host genes (either minor or major) with epistatic interactions contributed to the resistance and work together for the interaction with the pathogen in a gene-for-gene manner. This model can explain our observations of the epistatic isolate-specific partial resistance against P. sylvaticum and Ph. sansomeana and may be more universal in host-pathogen interactions than was previously known.
The f lanking markers of qPirr16.1 delimited a genomic region of 107 kb based on Williams82 reference genome (Gmax2.0). A total of seven genes were predicted in this region (www. soyba se. org) (Table S4) 1 3 including Glyma.16g077700 which encoded an mRNA splicing factor, which can regulate the pre-mRNA splicing (Mayeda and Krainer 1992) and reduces lytic virus progeny formation (Molin and Akusjärvi 2000). The flanking markers of qPsyl15.1 defined a 50.3 kb genomic region on soybean Chr. 15, including seven predicted genes (Table S4). Interestingly, one of the predicted genes, Glyma.15g055200, encoded a Fox domain protein.
Glyma.15g055200 may be considered as a candidate gene for resistance as the soybean F-box protein gene GmCOI1 has been shown to mediate jasmonate regulated plant defense response in Arabidopsis thaliana (Wang et al. 2005). The corresponding genomic region of qPsyl18.1 spanned a 463 kb interval and contained 72 predicted genes, including Glyma.18g294400 and Glyma.18g294800 which encoded leucine-rich repeat receptor-like protein kinase (LRR-RLK) and six genes (Glyma.18g287700, Glyma.18g287800, Glyma.18g287900, Glyma.18g289100, and Glyma.18g289600) encoding heat shock protein 70 (Table S4). LRR-RLK proteins has been shown to play a central role in defense pathways against the infection of plant pathogens (Afzal et al. 2008;Liu et al. 2011;Yeh et al. 2016;and Liu et al. 2017), and heat shock protein play important roles in plant disease resistance and virus infection (Lu et al. 2003;Gorovits et al. 2013;Jiang et al. 2014), and therefore may be considered candidate genes.
E13390 and E13901 are both recently improved soybean lines for high yielding and other desirable agronomic traits adaptive to the environments of central and southern Michigan, U.S. To initiate the integration of the desirable QDRL into new soybean varieties, the flanking markers of the three QDRL (qPirr16.1, qPsyl15.1, and qPsyl18.1) identified in this study, as well as the two QDRL (qPsan5.1 and qPsan16.1) for partial resistance to Ph. sansomeana (Lin et al. 2021) were used to identify superior progeny lines containing all the desirable alleles of the five QDRL. A total of three F6:7 lines were identified including 150020-167, 150029-192, and 150029-217 and the latter two, 150029-192 and 150029-217 were used as breeding parents (21P059 and 21P060, respectively) in 2021. Marker-assisted selection will be performed to select superior progenies containing the QDRL. Seed increase was also performed for the two lines which will be used for advanced yield trials next year.