Download Four Genes Affecting Seed Traits in Soybeans Map to Linkage

Four Genes Affecting Seed Traits in
Soybeans Map to Linkage Group F
Z. Chen and R. C. Shoemaker
To map members of a family of genes affecting seed traits, the genotype T311
and cultivars Raiden and Keburi were used to develop two F2 populations; Raiden
3 T311 and Keburi 3 T311. T311 is a shriveled seed mutant and progeny of the
cross between breeding lines AP2 and P2180. Raiden contains a null allele in the
Gy4 gene, and thus lacks the glycinin subunit A5A4B3. Keburi contains a null allele
in the Cgy1 gene, and thus lacks the a9 subunit of b-conglycinin. The objectives
of this research were to genetically map the location of the genes conferring the
shriveled seed phenotype of T311 and the a’ subunit of b-conglycinin from Keburi
and to confirm the genetic location of Gy4. Genetic analysis showed that the
shriveled seed trait is inherited as a single recessive gene with maternal effect.
Linkage analysis mapped the shriveled seed gene (Shr) and the Cgy1 gene to
linkage group F. Other seed trait genes, Gy5 (glycinin subunit) and B1 (seed coat
bloom), were previously mapped to this linkage group. The significance of the
cluster of these seed genes was discussed. This research also confirmed the
previous mapping of Gy4.
From the Department of Agronomy and Interdepartmental Genetics Program, Iowa State University, Ames,
Iowa (Chen) and the USDA-ARS, Corn Insect and Crop
Genetics Research Unit, Department of Agronomy and
Department of Zoology/Genetics, Iowa State University,
Ames, IA 50011 (Shoemaker). Joint publication of the
USDA-ARS-Corn Insect and Crop Genetics Research
Unit and journal paper no. J-17366 of the Iowa Agriculture and Home Economics Experiment Station, Ames,
Iowa (project no. 3236). Names are necessary to report
factually on the available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no
approval of the product to the exclusion of others that
may also be available. The authors thank Ms. Cindy
Clark for helping with growth chamber and field work.
q 1998 The American Genetic Association 89:211–215
Storage protein accounts for approximately 40% of seed dry weight in soybean [Glycine max. ( L.) Merr.] ( Nielsen 1995; Wilson
1987). These proteins include glycinins
and b-conglycinins. Glycinin is composed
of products of five genes, Gy1–Gy5 ( Nielsen 1995). Gy1 and Gy2 are tightly linked
within one DNA domain ( Nielsen et al.
1989). However, the Gy1-Gy2, Gy3, Gy4, and
Gy5 domains segregate independently of
one another (Cho et al. 1989). Diers et al.
(1994) have mapped Gy4 and Gy5 to linkage groups O and F, respectively.
b-conglycinin is composed of a9, a, and
b subunits, encoded by the genes Cgy1,Cgy2,
and Cgy3, respectively ( Lelievre et al. 1992;
Nielsen 1995). b-conglycinin genes constitute a supergene family, with some genes
clustering ( Harada et al. 1989). Genetic
analysis showed that Cgy2 is tightly linked
to Cgy3, whereas Cgy1 segregates independently of the Cgy2 and Cgy3 genes ( Davies
et al. 1985). The Cgy1 gene also has been
reported to segregate independently of
the Gy4 gene ( Kitamura et al. 1984). Taken
together, these results suggest that the
eight major storage protein genes could
be distributed among four to six chromosomes or independent chromosomal segments.
The genotype Keburi lacks the a9 subunit of b-conglycinin (encoded by Cgy1)
( Kitamura et al. 1984). The genotype Raiden lacks the A5A4B3 subunits of glycinin,
which are encoded by Gy4 ( Kitamura et al.
1984; Staswick and Nielsen 1983). The absence of these protein subunits can be
used for mapping the Cgy1 and Gy4 genes.
T311 is a shriveled seed mutant. This trait
is inherited as a single recessive allele,
with partial penetrance and incomplete
expressivity ( Honeycutt et al. 1989b). The
shriveled seed trait is associated with
seed reserve deposition (Chen et al., unpublished data; Honeycutt et al. 1989a).
For instance, the b subunit was not easily
detected in severely shriveled seeds; development of protein bodies and deposition of crystals in protein bodies were altered and starch broke down more slowly
in shriveled seeds.
The objectives of this research were to
genetically map Shr (shriveled seed) and
Cgy1 (a9 subunit of b-conglycinin), and to
confirm Gy4 (A5A4B3 subunit of glycinin)
( Diers et al. 1994) in soybean. We found
that the Shr gene was linked with Cgy1 and
that they were located on linkage group F
with two other previously mapped seed
trait genes, Gy5 (A3B4 subunit of glycinin)
and B1 (seed coat bloom) (Palmer and Kilen 1987). The map position of Gy4 was
confirmed on linkage group O.
211
Materials and Methods
Genetic Material
Soybean genotypes T311, Raiden, and Keburi were used in this study. T311 is a
shriveled seed mutant spontaneously derived from a cross between breeding lines
AP2 and P2180. Raiden contains a null allele in the Gy4 gene and lacks glycinin subunit A5A4B3. Keburi contains a null allele in
the Cgy1 gene and lacks the a9 subunit of
b-conglycinin. The crosses Raiden 3 T311
and Keburi 3 T311, and their reciprocals,
were made in the greenhouse during the
winter of 1994. Because the genotype of
T311 is w1w1 (white flower) and Raiden
and Keburi are W1W1 (purple flower), hybridization was confirmed by checking the
segregation of hypocotyl color of 15 F2
seedlings derived from each F1 plant
(Palmer and Kilen 1987). The seeds derived from confirmed crosses of Raiden 3
T311 and Keburi 3 T311 were used to develop two independent F2 populations.
Ninety-two F2 plants from the cross Raiden
3 T311 and 94 F2 plants from the cross
Keburi 3 T311 as well as the parental
plants were grown in growth chambers at
268C/208C (day/night) and a 15 h photoperiod for 4 weeks, then at 348C/208C and
a 12 h photoperiod until maturity. Mature
seeds from each plant were harvested and
stored. Ten or more F3 plants derived from
each F2 plant were grown in the field to
develop F2-derived lines in F3 generation
during the summer of 1996.
RFLP Analysis
The DNA used to determine the genotypes
of F2 individuals was extracted from leaves
harvested from seven or more greenhouse-grown F3 seedlings derived from
each selfed F2 plant. DNA extraction,
Southern blotting, and hybridization were
performed as described by Keim et al.
(1989). Two hundred forty-eight mapped
clones covering all soybean linkage
groups at intervals of less than 20 cM were
initially screened with five restriction enzymes (HindIII, EcoRI, EcoRV, DraI, and
TaqI) to detect polymorphisms between
Raiden and T311 and between Keburi and
T311. Among them, 87 clones for Raiden
versus T311 and 90 clones for Keburi versus T311 were polymorphic with at least
one of the five restriction enzymes. When
the seed trait genes were mapped, markers flanking the loci were assayed for polymorphism with additional restriction enzymes: HaeIII, XbaI, BamHI, and SphI. SSR
markers flanking the loci also were tested
212 The Journal of Heredity 1998:89(3)
for polymorphisms (Akkaya et al. 1995),
but no polymorphisms were observed.
The MapMaker program ( Lander et al.
1987) was used to construct the linkage
map using the RFLP data and the phenotype data of shriveled seeds and storage
proteins. A LOD score of 3.0 was used as
the lower limit for accepting linkage between two markers. The JoinMap program, version 2.0 (Stam and Van Ooijen
1995) was used to integrate the phenotypic markers and the markers mapped in
this study into the USDA-ARS/ISU soybean
linkage maps (Shoemaker et al. 1995). The
critical LOD score for linkage group was
3.0.
Shriveled Seed Phenotype Scoring
Seeds from parental plants, F2 plants and
F3 plants were classified as shriveled or
round. The phenotypes of F2 plants grown
in the growth chambers were classified as
shriveled or round based on the percentage of shriveled seed relative to the parental values. That is, a plant bearing more
than 60% shriveled seeds was classified as
shriveled (mutant), while a plant bearing
less than 40% shriveled seeds was classified as round (normal). The phenotype of
each F3 plant was scored as shriveled or
round based on the presence or absence
of any shriveled seed among 30 inspected
seeds. If the phenotype of all plants in an
F2-derived line was round, the F2 plant was
considered to be ShrShr. If the phenotype
of all plants in an F2-derived line was shriveled, the F2 plant was considered to be
shrshr. If some of the plants in an F2-derived line were shriveled and some were
round, the F2 plant was considered to be
heterozygous. Lines in which F2 scores
and F3 scores conflicted and F2-derived
lines containing fewer than six plants were
eliminated from the analyses. Therefore,
62 F2 plants for the Raiden 3 T311 population and 65 F2 plants for the Keburi 3
T311 population were used for linkage
analysis.
Storage Protein Analysis
Cotyledon tissue (;3 mg) from six or
more F3 seeds representing each F2 plant
were extracted individually with 100 ml extraction buffer containing 50 mM Tris, 2%
SDS, 10 mM b-mercaptoethanol, and 5 M
urea for 20 min, then centrifuged at
10,0003 g for 10 min. A total of 40 ml of
supernatant was added to 20 ml tracking
dye buffer. Twenty-five microliters of the
mixture was loaded onto a denaturing gradient gel. Gel composition, electrophoresis conditions, staining, and destaining
Table 1. Phenotypes of F 1 seeds of the reciprocal
crosses of T311, Raiden, and Keburi
F1 seed phenotypes
Crosses
Female 3 Male
Round
seeds
Shriveled
seeds
T311 3 Raiden
Raiden 3 T311
T311 3 Keburi
Keburi 3 T311
3
16
0
34
24
0
27
0
Percentage
of
shriveled
seeds
88
0
100
0
Data indicate a material effect on expression of shriveled seed.
were performed as described by Diers et
al. (1994). F2 plant genotypes in the Keburi
3 T311 population were determined by
scoring for the presence or absence of the
a9 subunit of b-conglycinin in F3 seeds derived from each F2 plant. That is, if all tested F3 seeds possessed the a9 subunit, the
F2 plant genotype was considered to be
Cgy1Cgy1. If all tested F3 seeds lacked the
a9 subunit, the F2 plant genotype was considered to be cgy1cgy1. If the F3 seeds segregated for the presence or absence of the
a9 subunit, the F2 plant genotype was considered to be heterozygous. Similarly the
F2 plant genotypes in the Raiden 3 T311
population were determined by scoring
for the presence or absence of the A4 subunit.
Results and Discussion
Inheritance of the Shriveled Seed Trait
F1 seeds from the crosses T311 3 Raiden
and T311 3 Keburi exhibited 88% and 100
% shriveling, respectively, while no F1
seeds from the reciprocal crosses Raiden
3 T311 and Keburi 3 T311 were shriveled
under either greenhouse or field conditions ( Table 1). A maternal effect on seed
shriveling is not surprising since the supply of carbohydrates, nutrients, and water
provided by the plant to the developing
seeds as well as the structure of the seed
coat and pod could affect seed development and deposition of seed reserve
( Bewley and Black 1994).
A single recessive gene conditions the
shriveled seed trait. In the Raiden 3 T311
population, the genotypes of F2 plants segregated 19 ShrShr:32 Shrshr:15 shrshr. In
the Keburi 3 T311 population, F2 plants
segregated as 16 ShrShr:35 Shrshr:14 shrshr.
The segregations of Shr in the Raiden 3
T311 and Keburi 3 T311 populations thus
fit a single locus model with a segregation
ratio 1:2:1 (x2 5 0.55 and 0.51, P 5 .76 and
.77, respectively). These results agree with
those of Honeycutt et al. (1989b).
Figure 1. Gel profiles of soybean storage proteins isolated from (A) Raiden, T311, and F2:3 progeny seeds and
(B) Keburi, T311, and F2:3 progeny seeds. First lane is molecular weight standard. A4 5 A4 subunit of glycinin; a’
5 a9 subunit of b-conglycinin.
Mapping of Shr, Cgy1, and Gy4
The Gy4 gene encodes the A5, A4, and B3
glycinin subunits ( Nielsen 1995). Because
the A4 subunit is easy to detect ( Figure
1A), and the absence of this subunit is diagnostic for gy4 , this subunit was used for
scoring F3 seeds to determine F2 plant genotypes. In the Raiden 3 T311 population,
F2 plants segregated 20 Gy4Gy4:45 Gy4gy4:
26 gy4gy4. The a9 subunit encoded by Cgy1
is missing in Keburi ( Figure 1B). By scoring for the presence or absence of the a9
subunit in F3 seeds, F2 plants were classified as cgy1cgy1, Cgy1cgy1, or Cgy1Cgy1.
The segregation ratio was 18 Cgy1Cgy1:50
Cgy1cgy1:25 cgy1cgy1 in the Keburi 3 T311
population. Therefore the segregations of
Gy4 in the Raiden 3 T311 population and
Cgy1 in the Keburi 3 T311 population fit a
single locus model with a segregation ratio
1:2:1 (x2 5 0.80 and 1.86, P 5 .67 and .39,
respectively).
Initially RFLP probes were screened
with five restriction enzymes to detect
polymorphisms between Keburi, Raiden,
and T311. Eighty-seven clones for Raiden
versus T311 and 90 clones for Keburi versus T311 were polymorphic with at least
one of the five enzymes. The preliminary
linkage analysis with the population of
Raiden 3 T311 showed that Shr was linked
to RFLP markers A708Dp1 and K644Dp1 on
linkage group F with LOD values of 12.6
and 6.0, respectively. Further analysis
found that B212Tp1 and K007Hp2 also were
linked to Shr ( Figure 2A). Linkage analysis
with the Keburi 3 T311 population confirmed that Shr was linked to A708Dp1 and
B212Tp1 with LOD values of 4.7 and 1.6,
respectively. Markers B148Vp1, K644Dp1,
A186Tp1, and A806Dp1 also mapped to
their corresponding positions (reference
to USDA-ARS/ISU soybean linkage map;
Shoemaker et al. 1995) ( Figure. 2B). Therefore linkage analyses with two independent populations positioned the Shr gene
onto linkage group F ( Figure 2A,B).
In the Keburi 3 T311 population, Cgy1
mapped 19.2 cM from the Shr gene with a
LOD value of 4.7 ( Figure 2B). In the Raiden
3 T311 population, Gy4 linked to the
markers A882Dp1 and K265Vp2 on linkage
group O with LOD values of 3.0 and 5.8,
respectively ( Figure 2C). Previously in the
G. max (A81–356022) 3 G. soja (PI 468916)
population, Gy4 was mapped 19.2 cM from
marker A882Dp1 on linkage group O using
a Gy4 cDNA clone ( Diers et al. 1994).
The deficiency of the a9 subunit of bconglycinin in Keburi is caused by a deletion in the Cgy1 locus ( Ladin et al. 1984).
This is the only known functional gene of
the a9 subunit within the Keburi genome.
However, Southern hybridization studies
and characterization of isolated genomic
clones showed that there are at least 15
distinct b-conglycinin gene sequences in
the genome ( Harada et al. 1989). These
genes are located on six distinct DNA
regions and three of these regions include
a cluster of two or more genes ( Harada et
al. 1989). Because we have mapped the
storage protein ‘‘nulls’’ for this protein
subunit, we have obviously mapped the
location of the functional gene for the subunit.
To date, the seed storage protein
genes—Cgy1 (this study) and Gy5 ( Diers
et al. 1994)—the shriveled seed gene—Shr
(this study)—B1 (seed coat bloom) ( Diers
et al. 1992, 1994), and one QTL for seed
weight (Mian et al. 1996) have been
mapped onto soybean linkage group F. Using combined data from the various populations, except for data from the QTL
study, the JoinMap program (Stam and
Van Ooijen 1995) was used to create a consensus map of linkage group F showing
the relative positions of these genes ( Figure 2D).
Chen and Shoemaker • Genes Affecting Seed Traits 213
jor histocompatibility complex genes and
other genes involved in immune response
are clustered on the short arm of chromosome 6 (Abdulkadir et al. 1995; Gerachty et al. 1996).
A group of genes with related function
constitutes a multigene family. Members
of a multigene family often reside in close
proximity on a chromosome ( Li and Graur
1991). Such linkage has been proposed to
help preserve the structural motifs necessary to function in a pathway ( Kanazin
et al. 1996). Duplication of genes followed
by multiple recombination or mutation
events generates additional informational
raw material for selection ( DeScenzo et al.
1994) and positive selection favors nonredundant, independent functions of duplicated genes (Pickett and Meeks-Wagner
1995). Although storage protein genes
could be distributed among several chromosomes or chromosomal segments in
soybean, linkage group F represents an important location for these genes and also
for other seed trait genes. The finding that
genes associated with normal seed development are clustered on one linkage
group provides information about soybean genome organization and evolution
and may provide clues about evolution of
storage protein gene families in soybean.
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Corresponding Editor: Reid G. Palmer
Chen and Shoemaker • Genes Affecting Seed Traits 215