The Effect of Resistant Soybean on Male and Female Development and Adult Sex Ratios of Heterodera glycines

To determine whether currently used sources of resistance (soybean Plant Introductions [PI] 548402, 88788, 90763, 437654, 209332, 89772, and 548316) influence sex ratios in H. glycines, four inbred lines of the nematode characterized by zero or high numbers of females on resistant soybean were used to observe the number of adult males produced. Nematodes were allowed to infect soybean roots for 5 days in pasteurized sand. Infected plants were washed and transferred to hydroponic culture tubes. Males were collected every 2 to 3 days up to 30 days after infestation (DAI), and females were collected at 30 DAI. Resistance that suppressed adult females also altered adult male numbers. On PI 548402, 90763, and 437654, male numbers were low and close to zero, whereas on PI 88788, male numbers were higher (α = 0.05). In a separate experiment, the same PIs were infected by an inbred line that tested as an HG Type 0 (i.e., the numbers of females that developed on each PI were less than 10% of the number that developed on the standard susceptible soybean cultivar Lee). In this experiment, male numbers were similar to female numbers on PI 548402, 90763, 437654, and 89772, whereas male numbers on PI 88788, 209332, and 548316 were higher than those of females (α = 0.05). In all experiments, the total number of adults that developed to maturity relative to the number of second-stage juveniles that initially penetrated the root was less on resistant than on susceptible soybean (P ≤ 0.05), indicating that resistance influenced H. glycines survival and not sexual development.     

Characterization of the Virulence Phenotypes of Heterodera glycines in Minnesota

Knowledge of the virulence phenotypes of soybean cyst nematode, Heterodera glycines populations is important in choosing appropriate sources for breeding resistant cultivars and managing the nematode. We investigated races of 59 H. glycines populations collected from 1997 to 1998 and races and HG Types of 94 populations collected in 2002 from soybean fields across southern and central Minnesota. In the 1997 to 1998 samples, race 3 was predominant and represented 78% of the populations. The remaining populations were 11.9% race 1, 1.7% race 4, 6.8% race 6, and 1.7% race 14. In the 2002 samples, the populations were classified as 15.3% race 1, 77.6% race 3, 2.4% race 5, 3.5% race 6 and 1.2% race 9. Percentage of 1997 to 1998 populations with female indices (FI) higher than 10 were 10.2% on Pickett 71, 3.4% on Peking, 13.6% on PI 88788, 3.4% on PI 90763, 1.7% on PI 209332, and 1.7% on PI 437654. Percentage of 2002 populations with FI >10 was 1.1% on Peking, 17.0% on PI88788, 14.9% on PI 209332, 33.0% on PI 548316, 11.7% on Pickett 71, and 0% on the other three indicators, PI 90763, PI 437654, and PI 89772. The line PI 548316 was relatively susceptible to the Minnesota H. glycines populations and may not be recommended for breeding resistant cultivars in the state. There was no noticeable change of frequencies of virulence phenotypes in response to the use of resistant cultivars during 1997 to 2002 in Minnesota except that FI increased on the PI 209332.     

Changes in the Reproduction of Heterodera glycines on Different Lines of Glycine max

Selection for ability of soybean cyst nematode (SCN), Heterodera glycines, to reproduce on soybeans with different sources of resistance divides some SCN race 4 field populations into two distinct subpopulations. These subpopulations reproduce well on ''Bedford'' and plant introduction (PI) 88788 or PI 89772 and PI 90763 but not on both pairs of soybean lines. The ability of these subpopulations to reproduce on the four soybean lines was reversed by changing the soybean line used as a host during a second cycle of selection. When SCN populations previously selected for reproduction on Bedford and PI 88788 were selected for their ability to reproduce on D72-8927 and J74-88, the ability of these populations to reproduce on Bedford and PI 88788 decreased significantly and their ability to reproduce on PI 89772 and PI 90763 increased significantly. Conversely, when SCN populations, previously selected for reproduction on P189772 and P190763, were selected for their ability to reproduce on Bedford, the reproduction of these populations on Bedford increased significantly and reproduction on PI 89772 and PI 90763 decreased significantly. Selection for ability of a SCN race 4 field population to reproduce on soybean lines derived from SCN race 4 resistant PIs resulted in the same division of the field population into two distinct subpopulations. These data substantiate earlier proposals to rotate cultivars with different genes for SCN resistance as a means of managing SCN populations.     

Rhg1 alleles from soybean PI 437654 and PI 88788 respond differentially to isolates of Heterodera glycines in the greenhouse

The production of resistant soybean [Glycine max (L.) Merr.] cultivars is the most effective means for controlling losses from soybean cyst nematode (SCN) (Heterodera glycines Ichinohe). The major resistance gene in most SCN resistance sources is rhg1, which has been mapped as a quantitative trait locus onto linkage group G. Our objective was to determine whether the SCN resistance sources PI 437654 and PI 88788 have different functional alleles at rhg1 based on resistance phenotypes. Populations segregating for resistance alleles at rhg1 from both PI 88788 and PI 437654 and at Rhg4, a second SCN resistance gene from PI 437654, were developed. These populations were screened for resistance to the H. glycines inbred isolates PA3 (HG type 7) and TN14 (HG type 1.2.5.7) in the greenhouse and evaluated with molecular markers linked to both rhg1 and Rhg4. Each isolate test was repeated, and the evaluations were done on a single-plant and a line-mean basis in Test 1, and solely on a single-plant basis in Test 2. Across two tests with the TN14 isolate, plants with the PI 437654 allele for a marker linked to rhg1 had significantly (P<0.0001) less SCN reproduction than plants carrying the PI 88788 allele. A marker linked to Rhg4, however, was not significantly associated with resistance to TN14. Across two tests with the PA3 isolate, alleles of rhg1 from both sources gave a resistant reaction, although plants homozygous for the PI 88788 allele had significantly (P<0.05) greater resistance than plants with the PI 437654 allele. The marker allele from PI 437654 linked to Rhg4 was significantly (P<0.0005) associated with greater resistance than the PI 88788 allele in both PA3 tests, and resistance was dominant. There was a significant interaction between alleles at rhg1 and Rhg4 in both PA3 tests. These results suggest that PI 437654 and PI 88788 each have a different functional SCN resistance allele at or close to rhg1. These allelic differences have implications that breeders should consider before incorporation into cultivars.     

Changes in the Heterodera glycines Female Index as Affected by Ten-year Cropping Sequences

The objective of this experiment was to measure the change in female index (FI) of Heterodera glycines from bioassays on Bedford, Peking, PI 89772, and PI 90763 soybean (Glycine max) for 12 cropping sequence treatments over a 10-year period. Cropping sequences included continuous plantings of Forrest, Peking, and D72-8927 soybean (all resistant to race 3); Bedford, Nathan, and D75-10710 soybean (all resistant to races 3 and 14); a Bedford-corn (Zea mays) rotation; a rotation of Bedford, Essex (susceptible), and Forrest; and a 70:30 blend of Bedford and Forrest. The FI from bioassays with PI 89772 and PI 90763 decreased over time from 24.3 to 1.6 with treatments involving continuous Bedford, Nathan, and D75-10710 and the Bedford-corn rotation. The FI increased in bioassays using Bedford with treatments involving Bedford, Nathan, D75-10710, the Bedford-Forrest blend, and the two rotations. Results of this field experiment confirm greenhouse experiments in which reciprocal changes occur in FI on PI 89772 and PI 90673 compared with FI on Bedford.     

QTL, additive and epistatic effects for SCN resistance in PI 437654

PI 437654 is a unique accession because of its resistance to nearly all HG types (races) of soybean cyst nematode (Heterodera glycines Ichinohe; SCN). Objectives of this study were to confirm and refine the locations and gene action associated with SCN resistance previously discovered in PI 437654, and to identify new QTLs that may have been missed because of low coverage with genetic markers used in previous studies. Using 205 F_7:9 RILs and 276 SSR and AFLP molecular markers covering 2,406.5 cM of 20 linkage groups (LGs), we confirmed and refined the locations of major SCN resistance QTLs on LG-A2, -B1, and -G previously identified in PI 437654 or other resistant sources. We found that these major QTLs have epistatic effects among them or with other loci for SCN resistance. We also detected some new QTLs with additive or epistatic effects for SCN resistance to different HG types (races) on all LGs except LGs-B2 and -D1b. The QTL on LG-G was associated with resistance to HG types 2.5.7, 1.2.5.7, 0, and 2.7 (races 1, 2, 3, and 5), and it contributed a large proportion of the additive effects. The QTL on LG-A2 was associated with resistance to HG types 2.5.7 and 0 (races 1 and 3). The QTL on LG-B1, associated with resistance to HG types 2.5.7, 0, 2.7 (races 1, 3, and 5), was the similar QTL found in PI 90763 and PI 404198B. In addition to QTL on LGs-A2, -B1 and -G, a novel additive QTL associated with SCN resistance to HG types 0, 2.7, and 1.3.5.6.7 (race 3, 5, and 14) was identified on LG-I flanked by Sat_299 and Sat_189. Several minor QTLs on LGs-C1, D1a, H, and K were also found to be associated with SCN resistance. Confirmation of the new resistance QTL is underway by evaluating another RIL population with a different genetic background.     

Selection Against Heterodera glycines Males by Soybean Lines with Genes for Resistance

Soybeans with genes for resistance select against Heterodera glycines with the corresponding genes for avirulence. There may be a differential effect of sex with some specific gene interactions, which would influence the magnitude of gene frequency changes. No effect on H. glycines males was detected with one selected nematode population and the resistant soybean line PI88788. The selective effect of PI89772 against male nematodes was greater with two inbred nematode populations than with one selected (on PI88788) population, presumably due to differences in H. glycines gene frequencies. ''Peking'' also had few males with the one inbred nematode population, whereas Forrest and ''Pickett 71'' had intermediate numbers. Apparently Forrest and Pickett 71 did not get all the Peking genes for resistance that affect male as well as female nematode development. Other H. glycines-soybean genes stop only females, since there were few or no cysts, except on the susceptible soybean Williams. The number of males'' phenotype will help identify specific genes in both organisms.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Selection and Inbreeding of Heterodera glycines on Glycine max

Few soybean cyst nematodes (SCN), Heterodera glycines, of a diverse gene pool developed into females on soybeans PI 89772 or PI 209332. Nematodes surviving the selection pressure were then inbred for nine generations by single cyst transfers on the same selecting soybean line. These nematodes appeared to tolerate concurrent selection and inbreeding. Effects of selection-inbreeding, selection only, and secondary selection were evaluated by relative ability to produce cysts on 11 soybean lines. The genetic differences of PI 89772 (also Peking and Pickett 71) and PI 209332 were reaffirmed. The random effects of inbreeding indicated that Ilsoy and Williams may have genes for resistance different from those in PI 89772 or PI 209332. Egg inoculum obtained from soil resulted in very few cysts in some tests. Fresh egg inoculum (from cysts on 27-30-day-old plants) generally resulted in more cysts and more consistent results. Concurrent with the change in inoculum, there was a large increase in relative numbers of cysts on several soybean lines but no change on other lines; the true cause of this large interaction is unknown. Secondary selection of two inbreds was effective and suppressed cyst numbers on the line on which one inbred was selected initially. These results are consistent with the allelism linkage of some SCN genes reported previously.     

Genetic Analysis of Parasitism in the Soybean Cyst Nematode Heterodera Glycines

A genetic analysis of parasitic ability in the soybean cyst nematode Heterodera glycines was performed. To identify and characterize genes involved in parasitism, we developed three highly inbred H. glycines lines, OP20, OP25 and OP50, for use as parents for controlled crosses. Through these crosses, we have identified genes in the inbred parents that control reproduction of the nematode on hosts that carry resistance genes. These genes, designated as ror-* for reproduction on a resistant host, segregate in a normal Mendelian fashion as independent loci. Host range tests of F(1) generation progeny indicated that at least one parasitism gene in both the OP20 and OP50 lines for host PI 88788 was dominant. Parasitism genes in OP50 for hosts ``Peking' and PI 90763 are recessive. Two types of single female descent populations, a single backcrossed BC(1)F(2)-derived and a double backcrossed BC(2)F(1)-derived, were established on the susceptible soybean cultivar ``Lee 68.' Host range tests for parasitism in these lines demonstrated the presence of two independent genes in OP50, one for host PI 88788 designated ror-1 and one for host PI 90763 designated ror-2. OP20 carries two independent genes for parasitism on PI 88788, designated as alleles kr3 and kr4.     

Identification of QTLs associated with resistance to soybean cyst nematode races 2, 3 and 5 in soybean PI 90763

Soybean cyst nematode (SCN) is a major soybean pest throughout the soybean growing regions in the world, including the USA. Soybean PI 90763 is an important SCN resistance source. It is resistant to several SCN populations including races 2, 3 and 5. But its genetics of resistance is not well known. The objectives of this study were to: (1) confirm quantitative trait loci (QTLs) for resistance to SCN race 3 in PI 90763 and (2) identify QTLs for resistance to SCN races 2 and 5. QTLs were searched in Hamilton × PI 90763 F_2:3populations using 193 polymorphic simple sequence repeats (SSRs) covering 20 linkage groups (LGs). QTLs for resistance to SCN were identified on LGs A2, B1, E, G, J and L. The same QTL was suggested for resistance to different SCN races where their 1-LOD support intervals of QTL positions highly overlapped. The QTL on LG G was associated with resistance to races 2, 3 and 5. The QTL on LG B1 was associated with resistance to races 2 and 5. The QTL on LG J was associated with resistance to races 2 and 3. The QTLs on LGs A2 and L were associated with resistance to race 3. The QTL on LG E was associated with resistance to race 5. We conclude that LGs A2 and B1 may represent an important distinction between resistance to SCN race 3 and resistance to SCN races 2 and 5 in soybean.     

Attempt to Select a Cyst Nematode Population on Soybean Plant Introduction 437654

A method of selecting soybean cyst nematode (Heterodera glycines Ichinohe) on segregating soybean progeny was evaluated for developing a population capable of reproducing on PI 437654. Direct selection on PI 437654 was not possible, since no cysts developed on it. Cysts were selected for 12 nematode generations on F₃ and F₄ plants of Forrest x PI 437654. No cysts of the selected population were produced on PI 437654, but more males were produced on it by the selected population than by the base population. The number of cysts on Forrest and other soybean lines considered to have some of the same genes for resistance increased with selection as expected. The increase in number of males on these other lines with some of the same genes for resistance as Forrest was greater than anticipated, indicating that these lines may have some of the same genes as PI 437654.     

Cytological expression of early response to infection by Heterodera glycines Ichinohe in resistant PI 437654 soybean.

The soybean PI 437654 is resistant to all known races of the soybean cyst nematode (SCN) in the U.S.A. and became a new source of resistance genes in cultivar development. Race 3, a wide-ranging nematode pathotype, was used to examine root cells of PI 437654 and susceptible 'Essex', 2, 3, and 5 days after inoculation (DAI). In initial response to SCN, both genotypes formed syncytia by cell wall dissolutions. Hypertrophy of syncytium component cells and hyperplasia of cells near syncytia were observed. At 2 DAI, incompatible response of PI 437654 to SCN was exhibited: limited cell hypertrophy, inhibition of syncytium growth, initiation of necrosis, and wall appositions. At 3 DAI, cellular events appeared to be a sum of the operative mechanisms for SCN resistance: irregular wall thickening, pronounced wall appositions, necrosis, and nuclear breakdown followed by cytoplasmic collapse. The cells surrounding the syncytia showed necrosis, wall apposition, and accumulation of electron-dense bodies. By 5 DAI, syncytia and neighboring cells were totally devoid of ground plasma and the degeneration process was completed. The normal route for early syncytium development in 'Essex' (increased number of organelles, intense vacuolization, accumulation of dense deposits in vacuoles, and wall ingrowths) suggests the involvement of portions of the developmental pathway of differentiating tissues in organogenesis. Early onset of SCN resistance 2 DAI in PI 437654 suggests rapid activation of genes in a cascade reaction leading to cell death. Key words : soybean, nematode, syncytium, cell death.     

Effect of Soil Temperature and pH on Resistance of Soybean to Heterodera glycines

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a major pest of soybean, Glycine max L. Merr. Soybean cultivars resistant to SCN are commonly grown in nematode-infested fields. The objective of this study was to examine the stability of SCN resistance in soybean genotypes at different soil temperatures and pH levels. Reactions of five SCN-resistant genotypes, Peking, Plant Introduction (PI) 88788, Custer, Bedford, and Forrest, to SCN races 3, 5, and 14 were studied at 20, 26, and 32 C, and at soil pH''s 5.5, 6.5, and 7.5. Soybean cultivar Essex was included as a susceptible check. Temperature, SCN race, soybean genotype, and their interactions significantly affected SCN reproduction. The effect of temperature on reproduction was quadratic with the three races producing significantly greater numbers of cysts at 26 C; however, reproduction on resistant genotypes remained at a low level. Higher numbers of females matured at the soil pH levels of 6.5 and 7.5 than at pH 5.5. Across the ranges of temperature and soil pH studied, resistance to SCN in the soybean genotypes remained stable.     

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance

Background

Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance.

Results

Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F₅–derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000.

Conclusions

Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1531-3) contains supplementary material, which is available to authorized users.     

Molecular characterization of resistance to Heterodera glycines in soybean PI 438489B

Soybean (Glycine max L. Merr.) plant introduction (PI) 438489B is a newly found germplasm source that has resistance to multiple soybean cyst nematode (Heterodera glycines Ichinohe, SCN) races. We studied the inheritance of resistance to SCN races 1, 2, 3, 5 and 14 in PI 438489B using F₂ and F_2:3 families, which were generated by crossing to the susceptible cultivar ’Hamilton.’ The objectives of this study were to investigate the inheritance for resistance to SCN races in PI 438489B, to find molecular markers associated with resistances, and to study the allelic relationships among resistance loci for different SCN races. The results showed that the responses to SCN races were approximately normally distributed with large environmental effects, and were also highly correlated, which implied that genes giving resistance to different races were similar. The narrow-sense heritabilities of resistance to all five SCN races ranged from 0.55 to 0.88. Fifty one restriction fragment length polymorphism (RFLP) markers and 64 simple sequence repeat (SSR) markers were found to be polymorphic in the F₂ population. Quantitative trait loci (QTLs) associated with resistance to SCN races were anchored on soybean linkage groups (LGs) A1, A2, B1, B2, C1, C2, D1a, E and G. These QTLs explained 47.3%, 45.8%, 51.5%, 34.5% and 37.2% of the total phenotypic variances, respectively, for each race we investigated. Some QTLs for different races encompassed the same region of flanking markers; therefore, QTLs for multiple races may be linked or pleiotropic effects may be involved. Some loci provided resistance in a race-specific manner. Resistance to SCN race 14 had a different pattern compared to other races. Our results indicated that resistance to race 14 did not include loci on LGs A2 and G. These flanking markers associated with QTLs could be used to select for resistance to multiple SCN races in soybean breeding programs. Received: 25 March 2000 / Accepted: 4 August 2000