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CROP BREEDING, GENETICS & CYTOLOGY

Molecular Marker Analysis of Seed Size in Soybean

^a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
^b USDA-ARS, Corn Insect and Crop Genetics Res. Unit, Iowa State Univ., Ames, IA 50011

* Corresponding author (wfehr{at}iastate.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Seed size is an important attribute of soybean [Glycine max (L.) Merr.] for some food uses. The objectives of this study were to identify simple-sequence-repeat (SSR) markers associated with quantitative trait loci for seed size (SSQTL) and to compare the effectiveness of phenotypic selection and marker-assisted selection for seed size among individual F₂ plants. Three small-seeded lines were crossed to parents with normal seed size to form three two-parent populations. The parents of the populations were screened with 178 SSR markers to identify polymorphism. Population 1 (Pop 1) had 75 polymorphic SSR markers covering 1306 centimorgans (cM), Pop 2 had 70 covering 1143 cM, and Pop 3 had 82 covering 1237 cM. Seed size of each population was determined with 100 F₂ plants grown at Isabela, Puerto Rico, and their F₂–derived lines grown in two replications at three environments. Single-factor analysis of variance and multiple regression were used to determine significant marker-SSQTL associations. Population 1 had 12 markers that individually accounted for 8.1 to 14.9% of the variation for seed size combined across environments, Pop 2 had 16 markers that individually accounted for 7.8 to 36.5% of the variation, and Pop 3 had 22 markers that individually accounted for 8.6 to 28.8% of the variation. Three marker loci that had significant SSQTL associations in this study also were significant in previous research, and 13 markers had unique SSQTL associations. The relative effectiveness of phenotypic and marker-assisted selection among F₂ plants varied for the three populations. Averaged across the three populations, phenotypic selection for seed size was as effective and less expensive than marker-assisted selection.

Abbreviations: cM, centimorgan • MAS, marker-assisted selection • PCR, polymerase chain reaction • PE/ABI, Perkin-Elmer Applied Biosystems • Pop 1, Population 1 • Pop 2, Population 2 • Pop 3, Population 3 • SSQTL, quantitative trait loci for seed size • SSR, simple sequence repeat

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
SEED SIZE IS AN IMPORTANT TRAIT for production of some specialty soy food products, including tofu, natto, miso, and edamame. Seed size of G. max is quantitatively inherited and ranges from 40 to 550 mg seed^-1 (Hartwig, 1973). Plant breeders select for improved yield and other desirable agronomic traits in developing soybean cultivars with different seed sizes for various food products. The traditional method of soybean breeding involves artificial hybridization to develop genetic variability followed by self-fertilization and phenotypic selection for seed size among the offspring. Molecular markers may improve traditional methods of breeding for seed size by increasing the reliability with which desirable progeny are selected.

Molecular marker associations with SSQTL of soybean have been reported. Mian et al. (1996) developed two G. max populations utilizing parent lines with normal seed size. They identified 16 independent marker loci that were significantly associated with SSQTL that together explained 73 to 74% of the phenotypic variation in each of the two populations. None of their marker loci was significantly associated with SSQTL across both populations. Twelve of the 16 marker loci were significantly associated with SSQTL in all environments, three were significant in two environments, and one was significant in only one environment. Maughan et al. (1996) developed a population by crossing a G. max line with a seed size of 240 mg seed^-1 to an accession of wild soybean (G. soja Siebold & Zucc.) with a seed size of 15 mg seed^-1. Three molecular markers were associated with SSQTL in F₂ plants that explained 50% of the phenotypic variation, while five markers were associated with SSQTL for F_2:3 lines that explained 60% of the variation. Mansur et al. (1996) observed three molecular markers that explained 23.1% of the variation for seed size among F_2:7 lines developed from the cross between ‘Minsoy’ (130 mg seed^-1) and ‘Noir 1’ (140 mg seed^-1). Orf et al. (1999) found seven marker loci that accounted for 50% of the variation for seed size among F_2:7 lines in the cross of Noir 1 x ‘Archer’, seven in a Minsoy x Noir 1 population that accounted for 50%, and two in a Minsoy x Archer population that accounted for 12% of the variation. Only one molecular marker was significantly associated with SSQTL in the three populations. This study was conducted to identify SSR markers associated with SSQTL and to compare the effectiveness of phenotypic selection and marker-assisted selection (MAS) for seed size among individual F₂ plants.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Three single-cross populations were developed from six G. max cultivars and lines for this study. The parents with normal seed size were ‘S12-49’, developed by the Northrup King Co., Washington, IA, and A96-492041 and A96-492058, developed by Iowa State University. The small-seeded parents, A97-775019, A97-775006, and A97-775026, were developed by Iowa State University. Population 1 was from the cross A97-775019 x A96-492041, Pop 2 from A97-775006 x S12-49, and Pop 3 from A97-775026 x A96-492058. The crosses were made in March 1998 at Iowa State University–University of Puerto Rico soybean breeding nursery in Isabela, Puerto Rico. The F₁ seeds were planted in May 1998 at the Agronomy Research Center near Ames, IA. Pubescence color was used to confirm that F₁ plants of the populations originated from hybrid seed. The F₂ and parent seed of each population were planted in February 1999 at Isabela. The soil type is a Coto clay (very-fine, kaolinitic, isohyperthermic Typic Eutrustox). The 200 F₂ seeds of each population and 40 seeds of each parent were planted 0.15 m apart in rows 1.02 m wide under artificial lights to extend the daylength for increased seed production. The F₂ and parent plants were harvested individually. Seed size of 10 random parent plants and 100 random F₂ plants from each of the populations was measured in milligrams per seed by dividing the weight of all the seeds by the number of seeds.

For each population, a set of the 100 F_2:3 lines derived from the F₂ plants and 10 entries of each of the parents were evaluated in 1999 as a separate experiment. The 120 entries in a set were planted in a randomized complete-block design with two replications on 24 May 1999 at the Burkey Farm and on 26 May 1999 at the Agronomy Research Center near Ames, IA. The soil type at both locations is a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludolls). The entries were grown in single-row plots 0.76 m long with 1.02 m between rows and a 1.07-m alley between the ends of the plots. The seeding rate was 12 seeds per plot. The plots were harvested in bulk with a self-propelled combine. After harvest, a random sample of F₄ seeds from the F₂–derived lines and seeds of the parents from one replication were used to plant the three sets of 120 entries on 1 Nov. 1999 at Puerto Rico. Each set was planted under natural daylength conditions in two replications of a randomized complete-block design. Each plot was a single row 0.61 m long with 1.02 m between rows and a 0.3-m alley between the ends of the plots. The seeding rate was 16 seeds per plot. The plots were harvested by hand and threshed in bulk with a stationary belt thresher. Seed size was measured by weighing 400 random seeds from each plot in the three environments.

A 15- to 20-g sample of leaf material was collected at Puerto Rico from at least 10 F₄ plants of each F₂–derived line and 10 plants of each parent. The leaf samples were placed in a plastic bag with an identification card and kept on ice until they were frozen in liquid nitrogen and dried in a vacuum for 24 h. The dried leaf samples were stored at -20°C until DNA extraction. Dried leaf tissue was placed in 50-mL screw-cap polypropylene tubes containing 4 g of 1.5-mL glass beads. The leaf material was ground into a powder by agitation with a paint shaker. DNA was extracted from each sample using the CTAB (cetyltrimethyl ammonium bromide) protocol of Keim et al. (1988).

A total of 178 SSRs was used to evaluate the six parents of the three populations. Population 1 had 75, Pop 2 had 70, and Pop 3 had 82 polymorphic markers. Each SSR marker had been mapped in soybean (Cregan et al., 1999). For each population, there was an average of four markers in each of the 20 linkage groups. Multiplex sets of nine markers were constructed based on the forward primer label and the allele size of the different markers as described by Narvel et al. (2000).

The multiplex sets were used to determine the marker alleles of the F₂–derived lines. All reagents, instruments, and software for the marker analysis were obtained from Perkin-Elmer Applied Biosystems (PE/ABI, Foster City, CA). The final polymerase chain reaction (PCR) volume was 10 µL and consisted of 30 ng genomic DNA, 0.8 µL of 25 mM magnesium chloride, 0.8 µL of 10 mM dNTPs, 0.2 µL (1.0 unit) of AmpliTaq Gold DNA polymerase, 1.0 µL of GeneAmp tenfold PCR Buffer II, 1.0 µL of 5 pM forward/reverse primer, and 5.7 µL of sterile water. The quantity of primer used in each reaction was chosen to optimize PCR. Polymerase chain reaction was conducted with GeneAmp thermocyclers (PE/ABI) model 9600 or 9700. The PCR procedure was 95°C for 10 min followed by 35 cycles of 95°C for 25 s, 58°C for 25 s, and 72°C for 25 s, followed by a final extension at 72°C for 60 min (Narvel et al., 2000). A 1.5-µL sample from each PCR run was submitted to the DNA Facility at Iowa State University for analysis with a PE/ABI model 377 automated DNA sequencer. Electrophoresis was performed at 3000 V for 2 h. Data were collected using the DNA Sequencing Collection software version 2.5 (PE/ABI) and analyzed with GENESCAN Prism software version 2.1 (PE/ABI). Simple-sequence-repeat-allele sizes were automatically estimated by GENOTYPER software version 2.0 (PE/ABI). Allele sizes not identified automatically were estimated manually from the electropherogram peaks.

The lines were scored based on the marker genotype of the parents. Lines that possessed a homozygous allele derived from the parent with normal seed size were scored as 0, lines that possessed alleles from both parents were scored as 1, and lines that possessed a homozygous allele derived from the small-seeded parent were scored as 2.

MAPMAKER/EXP v. 3.0 (Lander et al., 1987; Lincoln et al., 1992) was used to test marker pairs for evidence of linkage, and two-point recombination values were calculated by maximum likelihood at a minimum LOD of 3.0 and a maximum recombination frequency of = 0.50 using the GROUP command. The order of each group was determined using either the COMPARE or THREE POINT commands, and loci orders were confirmed using the RIPPLE command. Linkage maps were created using the Haldane map function.

Single-factor analysis of variance (GLM) was used to associate polymorphic markers with SSQTL (SAS Institute, 1992). Significant SSQTL associations for each population were identified when a marker at an individual environment was significant at P 0.01 or significant at P 0.05 across multiple environments. Interval mapping was not used because individual linkage groups were not fully saturated and many markers were unlinked (Lander and Botstein, 1989). Two-way analyses of variance were used to test for digenic interactions between markers significantly associated with SSQTL and all other marker loci using the program EPISTACY (Holland, 1998). Significant marker loci were combined in a multiple-locus regression model (REG) to determine their combined effect (SAS Institute, 1992).

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
There were significant (P < 0.01) differences in seed size among the three environments at which the F₂–derived lines were evaluated (Table 1). The mean seed size of the six parents was 102 mg seed^-1 at the Agronomy Research Center, 101 mg seed^-1 at the Burkey Farm, and 137 mg seed^-1 at Puerto Rico. Significant differences were present among the F₂–derived lines of each population at the three environments and combined across environments. The genotype x environment interactions were significant for each population. None of the F₂–derived lines had the same seed size as either of their parents based on the means combined across environments, except for one line in Pop 1. The failure to recover lines with seed sizes similar to the parents was consistent with segregation reported for other small-seeded x normal-size soybean crosses (Weber, 1950; Buhr, 1976; Carpenter and Fehr, 1986; Johnson et al., 2001). The broad-sense heritabilities for the three populations ranged from 0.45 to 0.85 on the plot basis and from 0.76 to 0.93 on the entry-mean basis, which were consistent with previous heritability estimates for small-seeded x normal-size crosses (Bravo et al., 1980; Leroy et al., 1991; Johnson et al., 2001).

In Pop 1, 12 SSRs had significant associations with SSQTL in one or more environments (Table 2). The 12 marker loci were on nine linkage groups. Four of the markers (Satt409, Satt322, Satt045, and Satt510) were significantly associated with SSQTL in each of the four environments and five markers (Satt070, Satt002, Satt154, Satt185, and Satt273) were significant in more than one environment. The marker loci individually explained 8.1 to 14.9% of the variation for seed size combined across environments according to results derived from the single-factor analysis of variance. Multiple-locus regression identified eight markers that were significantly associated with SSQTL at one or more environments (Table 3). Six of the eight marker loci marginally contributed 3.0 to 14.8% of the variation after accounting for the other marker loci in the model and together explained 44.2% of the total variation for seed size combined across environments. The small-seeded parent A97-775019 contributed alleles for small seed at five loci (Satt409, Satt070, Satt002, Satt154, and Satt510) and for large seed at three loci (Satt322, Satt001, and Satt273) (Table 2). The normal-size parent A96-492041 contributed alleles for small seed at three loci (Satt322, Satt001, and Satt273) and for large seed at five loci (Satt409, Satt070, Satt002, Satt154, and Satt510). The parent alleles associated with Satt185 and Satt045 produced the same seed size in the homozygous condition, but a larger size when combined in the heterozygotes.

Satt187 in Pop 3 and Satt277 and Sat_099 in Pop 2 were marker loci previously found to be associated with SSQTL in soybean populations by Orf et al. (1999). In both their study and ours, a small-seeded parent had an allele at Satt187 and Sat_099 that decreased seed size. For Satt277, the small-seeded parent in their population contributed an allele for large seed, whereas it contributed an allele for small seed in our study. The difference between the studies may be due to the limited seed-size difference between the two parents in their population or different parent alleles at the identified SSQTL in the two studies.

Across the three populations, Satt409 on linkage group A2; Satt304, Satt070, Sct_094, and Satt534 on linkage group B2; Satt565 on linkage group C1; Satt322 and Satt227 on linkage group C2; Sctt008, Satt154, and Satt135 on linkage group D2; Satt185 and Satt045 on linkage group E; Satt510, HSP176, Satt114, Satt334, and Satt072 on linkage group F; Satt431 on linkage group J; Satt001 and Satt273 on linkage group K; and Satt166, Satt006, Satt143, and Satt373 on linkage group L were within 1.4 to 36.4 cM of marker loci identified in previous studies (Mansur et al., 1996; Maughan et al., 1996; Mian et al., 1996; Orf et al., 1999; Sebolt et al., 2000). Thirteen marker loci located on linkage groups D1A, D1B, H, M, N, and O represent unique SSQTL associations not previously identified in other studies (Tables 2, 4, and 5). No significant marker loci were associated with seed size on linkage groups A1, B1, G, and I.

The effectiveness of MAS for small and large seed size using the molecular markers associated with the trait was compared with traditional phenotypic selection (PS) and an index based on ranking F₂ plants by MAS and PS. All significant markers identified using single-factor analysis of variance or multiple-locus regression were used to determine the MAS score for the F₂ plants and the F₂–derived lines. All lines were scored based on their marker genotype. Individuals that possessed a homozygous allele associated with large seed size were scored as 1, individuals that possessed both alleles were scored as 0, and individuals that possessed a homozygous allele associated with small seed size were scored as +1. Selection was practiced among the 100 F₂ plants of each population separately and among the 300 plants of the three populations without regard to the population from which they originated. The number of plants selected was 20% (Tables 6 and 7). The selection percentage was based on the marker score at which there was a separation among groups of lines. For PS and MAS, F₂ plants and F₂–derived lines were sorted based on their seed size and marker score. The plants and lines with the smallest and largest seed size and marker score were selected. For the index selection method, the F₂ plants and F₂–derived lines were given a rank score for PS and for MAS. The rank scores for each individual were added to determine the index score. F₂ plants and F₂–derived lines were sorted based on their index score, and the plants and lines with the smallest and largest index score were selected.

The three methods varied in effectiveness across populations for selection of F₂ plants with small and large seed (Tables 6 and 7). All the methods were more effective than random selection. The method of choice for evaluation of individual plants from a population would primarily depend on the cost of conducting each method. There was not an advantage for index selection; therefore, the cost of conducting both phenotypic and marker selection could not be justified. The cost of phenotypic selection was estimated to be US $0.35 per plant, including harvesting and threshing the plants and counting and weighing their seeds to determine mg seed^-1. The current cost of MAS was estimated to be a minimum of US $0.75 per plant, which assumed that six multiplexed markers run on one lane were used for each plant. On the basis of these estimates, phenotypic selection for seed size in soybean would be preferred.

	ACKNOWLEDGMENTS

 
Some of the molecular markers used in the study were kindly provided by H.R. Boerma, Dep. of Crop and Soil Sciences, and R.S. Hussey, Dep. of Plant Pathology, Univ. of Georgia. Facilities and thermocyclers were generously provided by G.F. Polking, DNA Sequencing and Synthesis Facility, Iowa State Univ.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Journal Paper No. J-19645 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA. Project No. 3732 and supported by the Hatch Act, State of Iowa, Iowa Soybean Promotion Board, and Raymond F. Baker Center for Plant Breeding.

Received for publication December 31, 2001.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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