introduction

Piyaporn Phansak1, Watcharin Soonsuwan1, James E. Specht1, George L. Graef1, Perry B. Cregan2, and David L. Hyten2 1 Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68583-0915

2 Soybean Genomics and Improvement Laboratory, USDA-ARS, BARC-West, Beltsville, MD 20705-2350

INTRODUCTION Ritchie (2003) identified 52 Glycine max germplasm accessions of exceptionally high seed protein in MG 000, 00, 0, I, II, III & IV, and mated these to seven high-yielding cultivars of an equivalent MG. She eventually obtained 41 F2 populations averaging ~120 F2 plants each. Only the F2:3 seed progenies within the highest and lowest deciles of the seed protein distribution in each population were genotyped, and then only with SSR markers linked to four seed protein QTLs known to have a large additive effect. Using a selective genotyping protocol reduces genotyping costs in marker-QTL linkage studies, since only the highly informative F2 individuals in the high and low phenotypic tails are genotyped (Lander and Botstein, 1989). See Lebowitz et al. (1987) for more details about selective genotyping and its statistical power. In her SSR-based phenotypic tail analyses, Ritchie (2003) discovered that 85%, 30%, 9%, and 20% of the 41 populations segregated for the respective QTLs located on LG-I, LG-E, and LG-H (top & bottom). The high-protein allele of the LG-I QTL was detected in 35 of the 41 accessions. The phenotypic tail analysis of Ritchie (2003) had limitations – just four genomic regions were examined (i.e., using SSRs linked to the four known QTLs), the average population size of just 120 F2 plants restricted the statistical power of QTL detection (i.e., 1 minus the imputed Type II error probability), and only 41 of the 52 accessions were evaluated.

OBJECTIVES The purpose of the research described in this poster was to repeat that selective genotyping experiment with the 52 high protein accessions, but this time doubling the F2 population size to 240 plants (thereby gaining more statistical power) and selectively genotyping the decile fractions with ~500 SNP markers distributed over the 20 chromosomes comprising the soybean genome. Our primary objective was to determine if the high protein phenotypes of these accessions was attributable to the high protein alleles at known QTLs or possibly yet undiscovered QTLs.

RESULTS & DISCUSSIONIn this research, the 22 lowest and 22 highest protein F2:3 progenies selected from ~220 total

progeny in 48 of 52 populations (4 were lost) were genotyped with 1536 SNPs distributed over the 20 chromosomes of the soybean genome (Fig. 1). Here, we report on the QTL analyses which have now been completed on 48 populations. Obviously, all 1536 SNPs were not parentally polymorphic, but we expected approximately 30-50% (460-770) of the SNPs to be polymorphic. About 400-500 SNPs segregated in nearly every population.

Fig. 2 right depicts the QTL scans produced by R/qtl in each of the last 28 populations (See author for handout for the first 20 populations. In the first 20 populations, 20 protein QTLs with LOD scores greater than 3.0 were detected in seven linkage groups. Of particular interest are the six populations for which Ritchie (2003) did not detect allelic segregation at four known protein QTLs, plus the 11 populations she was unable to evaluate). In the last 28 populations, 43 protein QTLs were detected in 12 linkage groups. QTL on chromosome 20 was detected in all but three of our populations. Our decile-based selective genotyping protocol with a 220 F2 population size, assuming a phenotypic standard deviation estimate of 3.0% protein (Ritchie, 2003), has a statistical power of nearly 80% for detecting QTLs with an additive effect of ≥ 1.2 percentage units in protein, and ca. 20% for an additive effect of ≥ 0.8 percentage units.

Figure 2: Protein QTLs identified in the soybean genome by standard interval mapping using the maximum likelihood estimation method of R/qtl. A solid horizontal line denotes the threshold LOD score at the 95 percentile of the genome-wide maximum LOD scores obtained with 1000 stratified permutation replicates.

Although Ritchie (2003) found no evidence of allelic segregation for the known protein QTLs on chromosomes 20 (I),15 (E), & 12 (Htop, Hbot) in six of her 41 populations, we detected QTLs on LG-I (1139) & LG-E (1143) due to our greater statistical power. Of the 11 populations she did not test, we detected QTLs in six. We confirmed LG-I QTL segregation in 25 of 28 populations. Over all 48 populations, statistically significant seed protein QTLs were detected on chromosomes 2, 3, 4, 5, 6, 7, 8, 10, 14, 15, 16, 18 and 20. The QTLs we detected on chromosome 2, 7, 10, 14, 16 & 18 are not currently listed in SoyBase. For improving the seed protein content in high yielding soybean cultivars, accessions homozygous for the high protein allele at these new protein QTLs may be useful to soybean breeders.

CONCLUSIONS

We thank the United Soybean Board (Project 8212) and the Nebraska Soybean Board for providing the funding for this research.

ACKNOWLEDGEMENTS

REFERENCES Broman, K.W., H. Wu, Ś. Sen, and G.A. Churchill. 2003. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19:889-890.Hyten, D.L., I. Choi, Q. Song, J.E. Specht, T.E. Carter, Jr., R.C. Shoemaker, E. Hwang, L.K. Matukumalli, R.L., and P.B.Cregan. 2010. A high density integrated genetic linkage map of soybean and the development of a 1,536 Universal Soy Linkage Panel for QTL mapping. Crop Sci. 960-968.Lander, E. and D. Botstein. 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 134:943-945. Lander, E., P. Green, J. Abrahamson, A. Barlow, M. Daly, S. Lincoln, and L. Newburg. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174-181.Lebowitz, R.J., M. Soller, and J.S. Beckmann. 1987. Trait-based analyses for the detection of linkage between marker loci and quantitative trait loci in crosses between inbred lines. Theor . Appl. Genet. 73:556-562. Lincoln, S., M. Daly, and E. Lander. 1993. Constructing genetic maps with MAPMAKER/EXP version 3.0: a tutorial and reference manual: pp 97.Ritchie, R. A. 2003. High-protein plant introductions: selective genotyping to detect soybean protein QTL. M.S.thesis. Univ. of Nebraska, Lincoln.

MATERIALS & METHODS

Soybean Genetic Map - USLP 1.0 - 1536 SNP Markers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

JEB2FHB1OKA2MC2A1C1ND1bD1a D2 G L I

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147

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124120 120

108

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92

119

107101

113

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Chromosome Number

Gen

etic

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tanc

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M)

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(Old) Linkage Group Name

SNP

Figure 1: The soybean genetic map USLP 1.0 with 1536 SNP markers (Hyten et al., 2010).

Verify hybridity with SSR Markers

P1 normal protein (AA) x P2 high protein (BB)

5-10 plants F1 (AB)

F2 (1 AA :2 AB :1 BB)

Harvest F2:3 seed from each F2 plant.

Field Planting: 300 F2 Plants per cross + 20 plants of each Parent + F1s. Collect and store leaf sample from each plant for DNA extraction

Seed Protein and Oil Analysis: Near Infrared Transmittance Analysis of 240 F2plant progeny, parents and F1s. Select the 22 highest and 22 lowest seed protein progeny.

Analysis with the USLP 1.02304 total plants genotyped with 1536 SNP markers

SELECTIVE GENOTYPING: Extract DNA from stored leaf tissue. Analyze the 22 highest and the 22 lowest seed protein lines with the Universal Soy Linkage Panel 1.0

Detection of Soybean Seed Protein QTLs Using Selective Genotyping

0

1

2

3

4

5

P1001 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20

Simple Interval Analysis (Maximum Likelihood Estimation Method)

4.29

0

1

2

3

4

5

6

P1002 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.64

0

2

4

6

P1003 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.84

0.0

0.5

1.0

1.5

2.0

2.5

3.0

P1004 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.68

0

1

2

3

4

5

6

P1005 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.13

0

1

2

3

4

P1006 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.16

0

1

2

3

4

P1007 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.66

0

1

2

3

4

5

6

P1009 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.79

0

1

2

3

4

P1026 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20

Simple Interval Analysis (Maximum Likelihood Estimation Method)4.08

0

1

2

3

4

5

P1027 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.9

0

1

2

3

4

P1039 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


0

1

2

3

4

5

P1040 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.87

0

1

2

3

4

P1041 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.08

0

1

2

3

P1042 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


0.0

0.5

1.0

1.5

2.0

2.5

P1043 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


0

1

2

3

4

5

6

P1044 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.15

0.0

0.5

1.0

1.5

2.0

2.5

3.0

P1054 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.64

0

1

2

3

4

P1055 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


0

1

2

3

P1056 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.12

0

1

2

3

4

5

6

P1057 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.8

0

1

2

3

4

P1058 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.3

0

2

4

6

8

P1075 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.02

0

1

2

3

4

5

P1098 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.86

0

1

2

3

4

5

P1109 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.68

0

2

4

6

8

P1111 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


4.7

0.0

0.5

1.0

1.5

2.0

P1145 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


0

2

4

6

8

P2211 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.1

LINKAGE MAPPING AND QTL ANALYSIS: The program R/qtl (Broman et. al, 2003) was used for determining genetic linkage and QTL analysis.

0

2

4

6

8

10

12

P2213 Seed Protein

Chromosome

LOD

Sco

re

1 3 5 7 9 11 13 15 17 192 4 6 8 10 12 14 16 18 20


3.76

introduction

Documents

f2 populations

high protein accessions

high seed protein

seed protein qtls

highest protein f2

high protein alleles

high protein phenotypes

highprotein allele