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Dep. of Crop Sciences, Univ. of Illinois, 1101 W. Peabody Dr., Urbana, IL 61801
* Corresponding author (bdiers{at}uiuc.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: HG, Heterodera glycines • J2, second-stage juvenile • LG, linkage group • MG, maturity group • NIL, near isogenic line • PCR, polymerase chain reaction • Pf, final population density • Pi, initial population density • PI, plant introduction • QTL, quantitative trait locus • Rf, reproductive factor • SCN, soybean cyst nematode • SSR, simple sequence repeat
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic mapping studies have shown that PI 88788 has a major SCN resistance quantitative trait locus (QTL) on LG G (Concibido et al., 1997; Diers et al., 1997) that maps to the same region where major SCN resistance QTL map in PI 437654 (Webb et al., 1995), PI 209332 (Concibido et al., 1996), ‘Peking’, PI 90763 (Concibido et al., 1997), and PI 89772 (Yue et al., 2001). This QTL has been designated rhg1 and has been shown to provide greater than 50% of the resistance to several Heterodera glycines (HG) Types of SCN (Concibido et al., 1996; 1997; Niblack et al., 2002).
Field studies throughout the midwestern USA have been performed on cultivars containing resistance derived from PI 88788 to test for the effects of this resistance source on SCN reproduction, population shifts (Colgrove et al., 2002), and other agronomic traits (MacGuidwin et al., 1995; Wheeler et al., 1997; Chen et al., 2001a; Wang et al., 2003a). MacGuidwin et al. (1995) studied the impact of PI 88788 resistance on yield in fields with varying levels of SCN. They found that the yield of ‘Bell’, which has SCN resistance from PI 88788, yielded more than the susceptible cultivars Corsoy 79 and BSR 101 by 43 and 30%, respectively. They also found that Bell significantly reduced SCN population densities, and that a higher correlation existed between final (Pf) egg counts and yield than did final cyst counts and yield.
Chen et al. (2001a) tested 47 SCN resistant cultivars over 3 yr, 34 of which had known PI 88788 resistance, for their SCN development and associated yield. They showed initial SCN population densities (Pi) had a large effect on yield. They also found that the average difference in yield between resistant and susceptible cultivars at a site increased with increasing Pi, but yield potential varied greatly among resistant cultivars. When the average Pi was high [>1000 SCN eggs (100 cm3 soil)–1], data were more consistent among plots in a field. In contrast, when the average Pi was low [<500 SCN eggs (100 cm3 soil)–1] no difference in yield was observed between resistant and susceptible cultivars.
Although tests have been performed to study SCN population development on cultivars with resistance from PI 88788, the effect of the rhg1 region alone on SCN reproduction has not been studied. Because of the importance of rhg1 in breeding SCN resistant cultivars, it is necessary to understand these effects. The effects of a single genetic region can be studied in NILs segregating for the gene of interest. Our objectives were to use two populations of NILs to (i) measure the effect of rhg1 on SCN reproduction in the greenhouse and field and (ii) measure the effect of the genetic region where rhg1 maps on agronomic traits in the field.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The SR-2 NIL population was developed from a single F6 plant from the S22-C3 by S42-M1 cross that was heterozygous for the region containing rhg1. F7 seeds from this heterozygous plant were grown and tested with the sequence characterized amplified region marker CTA (Meksem et al., 2001), which was mapped 1 cM from rhg1. The BR-2 NIL population was developed from a single F4 plant from the Bell by Colfax cross that was heterozygous for the rhg1 region. A F4–derived line was developed from this plant and the line was advanced to the F7 generation in bulk. F7 plants in the line were genotyped with the simple sequence repeat (SSR) marker Satt309, which is 0.4 cM from rhg1 (Cregan et al., 1999). For both the SR-2 and BR-2 populations, plants that were predicted to be homozygous resistant and susceptible were selected and individually threshed to form the two populations of F7–derived NILs.
Lines in SR-2 were classified as 15 resistant and 17 susceptible, whereas lines in BR-2 were classified as 22 resistant and 22 susceptible. In the fall of 2000, seed from the selected plants were sent to Hawaii and grown two generations to increase seed. The F7–derived lines from both populations were planted in a different study (Kopisch-Obuch et al., 2005) during the 2001 growing season to evaluate the effect of rhg1 on yield under low SCN pressure. F7:11 seed harvested from that study was planted in our 2002 study, and F7:12 seed harvested from the 2002 study was planted in the 2003 study. Because the SR-2 population was developed from a single F6 plant, 1/32 of the genome was expected to segregate in the population, while 1/8 of the genome was expected to segregate in BR-2, which was developed from a F4 plant. This limited background segregation enabled us to measure confidently the effect of the rhg1 genetic region.
Genetic Marker Analysis
The marker analysis was done on DNA extracted from young leaf tissue by the quick extraction method described by Bell-Johnson et al. (1998). Polymerase chain reaction (PCR) was performed according to Cregan and Quigley (1997). The PCR products were separated by electrophoresis on 6% (w/v) nondenaturing polyacrylamide gels (Wang et al., 2003b). Before electrophoresis, 50 µL of 10 mg mL–1 ethidium bromide solution was added to approximately 500 mL of 0.5x TBE buffer in the lower reservoir of the gel apparatus to yield a final concentration of 1 µg mL–1 ethidium bromide. The PCR products were viewed under UV light. All lines in the SR-2 population were tested with the marker CTA and all lines in the BR-2 population were tested with Satt309. In addition, a subset of six lines from each population were tested with all available SSR markers within 20 cM of rhg1 to estimate the size of the segregating region flanking the gene in each population.
Greenhouse Test
Near isogenic lines in each population were tested in a greenhouse for resistance to PA3, a HG Type 0 (Race 3 phenotype) isolate of SCN. This isolate was obtained from Dr. Prakash Arelli (USDA-ARS, Jackson, TN) and was maintained on the susceptible cultivar Hutcheson. The SCN resistance testing was done with a thermo-regulated water bath system according to Niblack et al. (2002). The water bath system maintains a constant soil temperature of 27 ± 1°C, providing optimal conditions for SCN growth and uniformity. Each NIL population was evaluated in a separate test arranged in a completely randomized design with at least three replications per entry. Entries in each test included NILs and parents of the population, seven HG Type indicators, and the susceptible check ‘Lee 74’. Each soybean entry was germinated on germination paper for 48 h at 24°C. On the day of transplanting, cysts were crushed to release the eggs to prepare the inoculum (Riggs and Schmitt, 1991). The eggs and second-stage juveniles (J2)s were counted under a microscope in five 25-µL samples to quantify the inoculum. Seedlings from each entry were selected on viability and uniformity and each was planted into a separate 25-mm-diam x 20-cm-long PVC tube filled with steam-sterilized sandy soil that had been infested with approximately 1500 eggs + J2s. The tubes were packed with soil into large plastic crocks before planting. The crocks were suspended in the water bath and the infected plants were grown with 16 h of light, fertilized biweekly, and given the appropriate amount of water for 30 d. Approximately 30 d after soil infestation, the cysts and females were washed from the roots of each plant, collected on a 250-µm-aperture wire mesh sieve and counted under a stereomicroscope. A female index (FI) was calculated for each entry with the following formula (Golden et al., 1970): (N1/N2) x 100, where N1 is the average number of females per entry and N2 is the average number of females on Lee 74.
Field Test
The NILs were tested in 2002 and 2003 in fields naturally infested with SCN to evaluate SCN reproduction and agronomic traits. Each population of NILs was planted both at Urbana and Newton, IL, in randomized complete blocks with three replications at each location. Both locations in 2002 were planted on 4 June, the Urbana location in 2003 was planted on 13 May, and the Newton location in 2003 was planted on 28 May. A different field was used each year at the two locations to create four test environments. Each plot measured 1.5 m wide by 3.2 m long with two rows spaced 0.76 m apart. From each plot, a soil sample consisting of 10 2.5-cm-diam cores 0.2 m deep was taken within 80 mm of the rows in the area between the two rows during the VC to V1 growth stages (Fehr et al., 1971). The samples were stored at 5°C until processed. The cores for each sample were mixed and a 100-cm3 sample of soil was processed with a semiautomatic elutriator (Univ. of Georgia Science Instrument Shop, Athens, GA) to extract the cysts from the soil (Byrd et al., 1976). The cysts were then ground with a rubber stopper to release the eggs and washed through nested 150-, 75-, and 25-µm-aperture wire mesh sieves. Eggs caught on the 25-µm sieve were stained with 3-mL egg staining solution (0.35 g acid fuchsin, 250 mL lactic acid, 750 mL water) (Southey, 1986) and microwaved on high until the sample had boiled for at least 30 s. The sample was diluted with water, bringing the volume up to 100 mL. A 5-mL sample was counted with 40x magnification to provide an estimate of the Pi in each plot. Before the plants matured, a second sample was taken from each plot with the same methods used for the initial samples to provide an estimate of the Pf in each plot. Characterization of the SCN field isolates was performed with an HG Type test from a composite soil sample from each of the four environments. The reproductive factor (Rf), or change in nematode population density was calculated as (Pf/Pi). The agronomic traits measured were as follows: plant height; maturity, measured as the number of days after 31 August when at least 95% of the pods had reached mature color; lodging, measured at maturity on the scale of 1 = all plants erect to 5 = all plants prostrate; and seed yield, measured on a 130 g kg–1 moisture basis.
Data Analysis
Data from the two tests in the greenhouse and four environments in the field were analyzed by PROC GLM of SAS (SAS Institute, 2002) and a significance threshold of 
= 0.05. Data from the field tests were analyzed within and across the four environments. Nontransformed Pi and Pf values are presented in the tables; however, before analysis they were transformed to log10(x + 1) values and the Rf values transformed to log10[(Pf + 1)/(Pi + 1)] to normalize the variance (Chen et al., 2001b; Wang et al., 2000; MacGuidwin et al., 1995). The log10 transformation has been used in SCN population studies to better represent nematode reproduction in the field. The (x + 1) equation was used for the Rf (Pf/Pi) value to prevent a Pi of zero in the denominator. To measure the effect of rhg1 and surrounding genetic regions, means of NILs predicted to have the resistant or susceptible allele on the basis of linked markers were compared by PROC GLM. This analysis was done for SCN egg numbers and agronomic traits. The phenotypic correlations between SCN reproduction and agronomic traits across environments were calculated by PROC CORR of SAS using the means of NILs.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SR-2 (S22-C3 x S42-M1) Field Tests
The analysis across all lines in the SR-2 population revealed significant (P < 0.05) differences among environments and NILs across environments for all traits with the exception of Pi. In addition, NIL by environment interactions were significant for yield and lodging. Marker analysis showed that a region at least 4.5 cM and as great as 9.2 cM surrounding rhg1 was segregating in the population. More detailed information on how much genome surrounding rhg1 in this population and the BR-2 population is given in Kopisch-Obuch et al. (2005). Height, maturity, lodging, and yield were significantly associated with segregation of the marker CTA in the NIL population across environments, but differences for height, maturity, and lodging were small (Table 2). Near isogenic lines homozygous for the resistance allele tended to be taller, mature later, lodge more, and yield greater than lines homozygous for the susceptibility allele. These effects were significant in all environments for maturity and lodging in three out of four environments for yield and in two out of four environments for height.
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SCN reproduction in the greenhouse was significantly (P < 0.05) correlated with height (r = –0.59), maturity (r = –0.84), lodging (r = –0.63), yield (r = –0.87), Pf (r = 0.76), and Rf (r = 0.60) in the field. Yield was significantly correlated with height (r = 0.60), maturity (r = 0.87), lodging (r = 0.55), Pf (r = –0.65), and Rf (r = –0.50) in the field.
BR-2 (Bell x Colfax) Field Tests
Like the SR-2 population, the analysis across lines in the BR-2 population revealed significant differences among environments and NILs across environments for all traits except for Pi. In addition, genotype x environment interactions were significant for yield, maturity, Pf, and Rf. Marker analysis showed that a region surrounding rhg1 that was at least 9.1 cM and potentially as large as 18.2 cM was segregating in the population. Across all environments, lodging was significantly associated with segregation in the NIL population for the marker Satt309, but this difference was small and no significant associations were found for height, maturity, and yield (Table 3). Within environments, Satt309 was significantly associated with lodging in three of the four environments, yield in one environment, and no significant association was found for height and maturity.
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SCN reproduction in the greenhouse for BR-2 was significantly (P < 0.05) correlated with lodging (r = –0.36), Pf (r = 0.76), and Rf (r = 0.72) in the field. Yield was significantly correlated with height only (r = 0.76) in the field.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We did not detect significant differences in Pf between the homozygous resistant and susceptible NILs at either location in 2002. We believe that this was largely due to our taking final soil samples too early. Because of greater than average precipitation and cool temperatures in May of 2002 (Peters, 2002), neither location was planted until 4 June. Therefore, when we collected the Pf samples, there was not the typical 60-d period between planting and R1 that normally occurs in Illinois. The Pf samples in 2002 were taken at the R3 growth stage for SR-2 and the R5 growth stage for BR-2, which was, on average, 46 d after Pi samples. This only allowed for one generation of reproduction to occur between sampling dates, which was before the completion of the logarithmic increase of SCN populations that occurs during the first 60 d after planting. During the first generation of the growing season, SCN eggs and juveniles decline in the soil as they infect and accumulate in the soybean root continuously over time (Bonner and Schmitt, 1985; Schmitt and Ferris, 1998). Continuous infection leads to overlapping generations that mature at different times during the growing season and when soil samples are taken at the wrong time, juveniles and eggs in the soil will be low. In retrospect, we should have waited to take the final samples after two generations of SCN reproduction. In contrast, in 2003 when homozygous resistant and susceptible NILs were significantly different for Pf and much higher Rf values were observed, there was 70 d on average between Pi and Pf.
The HG Type test on field SCN isolates revealed a HG Type 2.5.7 isolate for Urbana in 2003. The PI 88788 source of resistance gives only partial resistance to a HG Type 2.5.7 isolate (Table 1). This is revealed by the significant increase in egg numbers between Pi and Pf in NILs homozygous for the resistance allele at this environment for both populations. In all other environments, the SCN egg numbers decreased between Pi and Pf for the homozygous resistant NILs, except in one environment where there was a very minor increase in egg numbers.
Near isogenic lines predicted to be homozygous resistant at rhg1 were significantly higher yielding than homozygous susceptible NILs across environments for SR-2 but not BR-2. This difference in SR-2 was significant for each environment with the exception of Newton in 2002, where the Pi was very low [mean <150 SCN eggs (100 cm3 soil)–1]. Previous research by Chen et al. (2001a) also indicated that SCN resistance does not give a yield response when Pi is low. An explanation for the lack of yield response in BR-2 is the early maturity of NILs in this population. These NILs were grown out of their range of adaptation and on average matured about 20 d earlier and yielded 30% less than NILs in SR-2. This low yield potential of the NILs in BR-2 could have reduced the beneficial effect of resistance. Remarkably, the NILs homozygous for the susceptibility allele in BR-2 significantly out-yielded the NILs homozygous for the resistance allele at Urbana in 2002 (Table 3). This difference may be due to linkage between rhg1 and undesirable genes. BR-2 was tested by Kopisch-Obuch et al. (2005) in field locations with low SCN pressure. Although they did not observe the same trend for this population in their study, they observed a similar trend in a different NIL population derived from the same cross.
Our results contribute further to literature on SCN reproduction by showing the effect of rhg1 on reducing SCN reproduction. In addition, we have shown that in some environments, rhg1 may have a positive impact on seed yield.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication August 6, 2004.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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