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CROP BREEDING, GENETICS & CYTOLOGY

Genetic and Agronomic Evaluation of wp-m in Soybean

^a Univ. of Illinois at Urbana-Champaign, Dep. of Crop Sciences, 1102 S. Goodwin, Urbana, IL 61801 USA

cnickell{at}uiuc.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Transposable element systems have been proposed to explain instability in floral pigmentation of several plant species, including soybean [Glycine max (L.) Merr.]. Soybean lines with chimeric (purple and pink sectored) flowers are hypothesized to contain wp-m, an active transposable element that is able to excise from the wp locus during morphological development. The objectives of this research were (i) to determine the inheritance of the chimeric flower phenotype when crossed to stable pink or purple flowered revertant lines and (ii) to determine the effect of wp on agronomic traits in stable flowered lines derived from wp-m. Chimeric flowers crossed to pink flowered revertant (wp*) lines produced four F₂ populations with unusual segregation ratios of 52 pink, three purple, and two chimeric flowered plants. Crossing chimeric flowers to revertant purple flowered (Wp*) lines resulted in F₂ populations that did not have the chimeric flower phenotype evident. In the agronomic evaluations, stable wp* lines were later in maturity and averaged 4 g kg^-1 higher in protein content and 3 g kg^-1 lower in oil content than Wp* lines. The data suggest wp acts in a pleiotropic manner to influence protein synthesis, as purple flowered revertant lines from a pink flower source had lower protein content than sister lines with wp*. Pink flowered lines derived from a purple flower source had higher levels of protein than sister lines with Wp*. The influence of wp on the anthocyanin pathway, plant morphology, and protein accumulation is a unique phenomenon that has not been reported in other plant species.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
TRANSPOSABLE ELEMENT SYSTEMS have been well characterized in maize (Zea mays L.) since their initial discovery by Barbara McClintock in 1944 (Peterson, 1987; Fedoroff, 1988; Vodkin, 1989). A transposable element in Antirrhinum majus is one of the few systems in a plant species other than maize that has been characterized at the genetic and molecular level. In soybean, transposable element systems have been proposed for Y18-m (variegated leaves), r-m (chimeric seed coat), and w4-m (purple or white chimeric flowers) (Vodkin, 1994). However, the molecular characterization of transposable element presence has only been characterized for Tgm1, an element that blocks the expression of lectin (Rhodes and Vodkin, 1988).

A mutation to the wp locus (wp-m) in soybean is thought to cause pink and purple flowers on the same plant or chimeric (pink and purple sectored) flowers at different nodes of the same plant (Johnson et al., 1998). Lines derived from LN89-5320-8-53 were observed to undergo flower phenotype switching from one generation to the next (Johnson et al., 1998). Lines that were purple flowered in one generation changed to pink flowered in the next. Other lines that were pink flowered in one generation reverted to purple flowered in the next. Since these lines were F_6:10, and therefore highly homogenous, this observation suggests the presence of an active transposable element system. The exact mechanism of transposition in wp-m materials and whether this system is similar to the currently identified transposable element systems in other species has yet to be elucidated.

A difference between w4-m and wp-m involves the successful transfer of the chimeric flower trait through crossing. The w4-m element remains active when crossed to different genetic backgrounds (Groose and Palmer, 1990). Johnson et al. (1998) concluded that the wp-m chimeric flower trait appears to become inactivated, or silenced, in the event of crossing. It is not understood if certain genetic factors are required at the wp locus in order for the chimeric flower phenotype to be expressed. We determined if the chimeric flower phenotype would be expressed or become inactivated when crossed to stable pink (wp*) or purple (Wp*) flowered revertant lines derived from LN89-5320-8-53.

In addition, other lines derived from LN89-5320-8-53 have been identified to be stable for flower color across several generations of observation. These lines are genetically identical except for the wp locus and therefore offer the opportunity to further characterize the effect of wp on agronomic characteristics. The objectives of this research were (i) to determine the inheritance of wp-m when crossed to wp* and Wp* lines developed from the chimeric flower line and (ii) to report the effect of wp on agronomic traits in stable flowered lines derived from LN89-5320-8-53.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Crossing and Genetic Analysis
In 1996, 25 lines with chimeric flowers were grown as a mutability test at the University of Illinois Crop Sciences Research and Education Center, Urbana-Champaign, IL (Table 1) . These lines were derived as single plants from LN89-5320-8-53, a plant row that exhibited plants with chimeric flowers, or plants with pink and purple flowers at different nodes. LN89-5320-8-53 originated from remnant seed of LN89-5320-8, an F₅ plant row from the cross [(`Sherman' (McBlain et al., 1987) x Asgrow A2943) x `Elgin 87' (Fehr et al., 1988)] that segregated for purple or pink flowers.

Agronomic Evaluations
The agronomic evaluation of stable flowered lines derived from the chimeric flower source consisted of two different field tests. The first test was conducted in 1996 using F_6:10 progeny lines of LN89-5320-8-53. Individual plants with chimeric flowers were tagged and harvested separately. In 1997, approximately 45 seeds from each selected plant were grown in 1.3-m plant rows, with 76-cm between-row spacing. Of the 733 observation rows from selected chimeric plants, 58 rows were homogeneous for either purple or pink flowers. Seeds were bulked within plant rows that were homogeneous for flower color. In 1998, a yield test of the 58 homogeneous lines was grown in a randomized complete block design as four-row plots, with two replications in two different environments of the University of Illinois Research and Education Center. An environment is defined as the year and field location where the test was grown. Environment 1 was in 1998 at the south farm in Urbana, on a Flanagan silt loam (fine, smectitic, mesic Aquic Argiudoll) soil. Environment 2 was in 1998 at the Cruse farm in Champaign, also on a Flanagan silt loam soil. Flower color was recorded to confirm that every plant in both replications of each entry was homozygous for either purple or pink flowers, as determined in 1997.

An agronomic evaluation of lines that were stable for pink or purple flowers across six generations was examined to provide additional information regarding the influence of wp on agronomic traits. This test, grown in 1996 and 1998, contained 15 lines derived from LN89-5320-8 and 13 lines derived from LN89-5320-8-53. The 28 lines were grown in four-row plots with two replications at four environments of the University of Illinois Research and Education Center. Environment 1-was in 1996 at the south farm in Urbana, on a Flanagan silt loam soil. Environment 2 was in 1996 at the Cruse farm in Champaign, on a Flanagan silt loam soil. Environment 3 was in 1998 at the South Grein farm, on a Dana silt loam (fine-silty, mixed, superactive, mesic Oxyaquic Argiudoll). Environment 4 was in 1998 at the Cruse farm, on a Flanagan silt loam soil. Flower color was confirmed to be either homogeneous purple or homogeneous pink for all plants in each line during both years of testing.

For both environments, the agronomic traits measured were yield (kg ha^-1), maturity (date when 95% of the plants had mature pod color), height to top node (cm), lodging (scored: 1 = all plants erect to 5 = all plants prostrate), and seed quality (scored: 1 = good quality to 5 = poor quality). A 100-seed sample of each replicate was analyzed for protein and oil content by infrared reflectance (Rinne et al., 1975) at the USDA-ARS National Center for Agricultural Utilization Research in Peoria, IL. Data from agronomic tests were subject to analysis of variance (ANOVA) using the PROC GLM and PROC MIXED functions of SAS (Littell et al., 1996). In the materials examined, flower color and entries (nested within flower color) were considered as fixed effects, while environments, replications (nested within environments), and environment x entry (nested within flower color) were considered random effects. Means were separated at the 0.05 probability level using the least significant difference. Significance was determined by using an F test as described by McIntosh (1983), and the numerator and denominator degrees of freedom were approximated as described by Satterthwaite (1946).

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Crossing and Genetic Evaluation
The crossing of chimeric flowers to stable pink flower (wp*) lines had a higher percentage of successful crosses than crosses to stable purple flower (Wp*) lines. Of the 25 combinations crossing chimeric flowers to wp* lines, 66 F₁ seed were obtained (52.8% success rate). Of the 20 combinations crossing chimeric flowers onto Wp* lines, only 23 F₁ seed were obtained (23.0% success rate). In addition, it was observed that several of these crosses that initially appeared to be successful aborted seed or entire pods later in development. These data suggest that unique molecular factors may be associated with wpwp genotypes. The absence of these unknown factors in other lines may prevent or silence the expression of the chimeric flower phenotype. This observation is supported by Johnson et al. (1998), who reported that the wp-m flower trait was not transmitted when crossed to cultivars Jack (Nickell et al., 1990), Kenwood (Cianzio et al., 1990), and different Clark isolines (Bernard, 1974).

The F₁ seed of the cross wp* x wp-m produced 43 pink flowered plants, five purple flowered plants, and four chimeric flowered plants. All 43 F₂ populations derived from the 43 pink flowered F₁ plants had only pink flowered progeny (Hegstad, 1999). This would suggest that in these populations, the wp-m trait was inactivated, or the wp-m element was not present in the pollen grain that fertilized the egg.

From the cross wp* x wp-m, populations LHX96302-1 and LHX96302-2 had purple flowered F₁ plants and were homogeneous for purple flowers in the F₂ generation (Table 1). Approximately 320 plants were observed for these two F₂ populations. In order to obtain all purple flowered F₂ plants, both Wp loci must be present due to transposase-mediated excision of both alleles or some mechanism whereby both alleles are affected. This type of activation would restore the alleles to Wp, producing homozygous purple flowered F₂ plants.

Three F₁ plants were purple flowered as a result of crossing wp* x wp-m. These populations (LHX96309, LHX96316, and LHX96320-2) segregated approximately 3:1 (purple flowered/pink flowered) (Table 1). This would be expected if the F₁ were heterozygous at the Wp locus, suggesting that the wp-m element inserted into a different gene or was inactive upon crossing. In these populations, these data suggest that the pollen carried a Wp* allele. It is possible that the wp-m element excised from the wp locus of the male parent and inserted into a different location in the genome. This type of action would restore Wp and purple flower color. Alternatively, a switching of wp-m to Wp* that does not involve element excision potentially may be involved.

In crossing wp* x wp-m, four F₁ plants had chimeric flowers. The segregation ratios of F₂ populations LHX96314-2, LHX96319-3, LHX96321-1 and LHX-96322 were approximately 445:24:17 (pink/purple/chimeric flowered) (Table 1). The ratios for these populations are not the 1:2:1 ratio that would be expected if the chimeric trait were controlled by a single gene following Mendelian inheritance. The unusual inheritance of this trait and the relatively low numbers of chimeric flowers observed provide additional evidence that the chimeric flower is controlled by a transposable element, or some other novel switching mechanism.

The crosses of Wp* x wp-m produced 16 F₁ plants that were purple flowered, and seven F₁ plants that were sterile (Hegstad, 1999). No chimeric flowered plants were detected in the F₁ of these crosses. The F₂ populations LHX96328, LHX96336, and LHX96342 segregated 3:1 (purple flowered/pink flowered) (Table 2). This would be expected if the F₁ plants were heterozygous at the Wp locus. These data suggest that the wp-m that was crossed in the F₁ was most likely integrated into the Wp locus and would be expressed as the recessive pink phenotype. The element is most likely defective because the F₂ population segregated 3:1, as expected if a nonautonomous element was present. This type of action may have occurred in the original pink flowered phenotypes detected by Stephens and Nickell (1991). The wp-m element would be integrated into the Wp locus to alter anthocyanin biosynthesis, resulting in pink flowers that would be inherited as a stable recessive allele in subsequent generations.

Agronomic Evaluations
Soybean lines derived from LN89-5320-8-53 were inbred to the F_6:10 generation and were expected to be highly homogeneous. Of the selected chimeric flowered plants in the 1996 mutability test, 58 lines were derived that were stable for flower color in 1997 and 1998. A range of the means for the selected lines in two sampled environments was variable for yield (2721–3816 kg ha^-1), maturity (25 August–16 September), and height (61–91 cm) (Hegstad, 1999). This high level of variability was unexpected because the lines were inbred for an additional two generations from single plants derived from LN89-5320-8-53.

When mean square estimates for agronomic traits were examined for the test with 58 entries, the environment was significant at the 0.01 level for yield, lodging, height, seed quality, protein, and oil, and at the 0.05 level for maturity (Table 3) . Lines with purple flowers were significantly different from pink flowered lines at the 0.01 level for maturity, height, seed quality, protein, and oil (Table 3). Overall, pink flowered lines were later in maturity, taller, and had lower seed quality scores compared with purple flowered lines (Hegstad, 1999). In addition, pink flowered lines were higher in protein content and lower in oil content than purple flowered lines. These data are unusual, as later-maturing lines have generally been correlated with higher oil content (Miller and Fehr, 1979).

The data from source lines -45, -49, -66, and -150 (derived from LN89-5320-8-53) suggest the wp allele is involved in a pleiotropic effect on protein synthesis (Table 4) . These lines were originally classified as homogeneous for pink flowers in 1992 (Johnson et al., 1998). Revertant purple flowered lines from these sources are hypothesized to result from the excision of the wp-m element to restore Wp, or due to a unique expression mechanism. The majority of lines that were stable for pink flowers were significantly higher (LSD = 0.05) in protein content than revertant stable purple flowered lines from the same source (Table 4). These data suggest the wp allele is involved in a pleiotropic interaction to increase protein accumulation.

An agronomic evaluation of 28 lines that were stable for pink or purple flowers across six generations was examined to provide additional information regarding the influence of wp on agronomic traits. The mean values of the lines at four environments across 2 yr were variable for yield (2177–3319 kg ha^-1), maturity (31 August–20 September), and height (60–91 cm) (Hegstad, 1999). The large degree of variation in this test was unusual and similar to the level of variation found in the evaluation with 58 entries stable for flower color across only two generations.

When mean square estimates for agronomic traits were examined for the test with 28 entries, the environment was significant at the 0.01 level for all agronomic traits measured (Table 5) . The flower color was significantly different at the 0.01 level for yield, maturity, protein, and oil, and at the 0.05 level for height and seed quality (Table 5). In general, for this evaluation, pink flowered lines were lower in yield, later in maturity, had higher protein, and lower oil content than purple flowered lines (Hegstad, 1999).

Combining information from the two agronomic evaluations, there was a significant difference (LSD = 0.05) for protein and oil when the mean values of pink flowered lines were compared with the mean values of purple flowered lines. In both evaluations, wp* lines were 4 g kg^-1 higher in protein content and 3 g kg^-1 lower in oil content than Wp* lines (Table 6) . The ranges of values for wp* lines were consistently higher for protein and lower for oil than Wp* lines. These data confirmed the results of Stephens et al. (1993).

Progeny lines derived from LN89-5320-8 and LN89-5320-8-53 had an unusually high amount of variability for several different agronomic characteristics. The agronomic data provide strong evidence to suggest wp exerts a pleiotropic effect to increase protein content. The lines used in this study should be highly homogeneous, as they were derived from F_6:10 families of the single plant row LN89-5320-8-53. In this study, the majority of purple flowered revertant lines derived from a pink flowered source were lower in protein content than wp* sister lines. In addition, several pink flowered lines derived from a purple flowered source were significantly higher for protein content than Wp* sister lines. The effect on the anthocyanin pathway, plant morphology, and protein biosynthesis by wp is unique and has not been documented in other plant species.

	ACKNOWLEDGMENTS

 
The authors would like to acknowledge Dr. German Bollero for his assistance with SAS programs necessary for the agronomic data analysis.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Contribution from the Illinois Agric. Exp. Stn., Urbana, IL. Research supported by the Illinois Soybean Program Operating Board.

Received for publication May 10, 1999.

	REFERENCES

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