Genetic Diversity and Its Use in Soybean
Abe JunHokkaido University

Introduction
Cultivated and wild soybeans form a primarygene pool (Hymowitz, 2004). Both usually produce
fertile hybrids, and there is no marked obstacle togene exchange, although the sterility due to abnormal chromosome pairing has often been observed.
Molecular assays have revealed that genetic variability of the cultivated soybean is low compared tothat of the wild soybean (Xu et al . 2002; Hyten etal . 2006). As suggested by the results of chloroplastand mitochondorial genomes analyses, however, theintrogressions from the wild to the cultivated germplasm most likely has occurred repeatedly in various
regions of East Asia in the past (Xu et al . 2002). Thisfinding may predict that the cultivated soybean possesses a relatively rich genetic variability in contrast
to a general trend that the diversity in cultivatedcrops that is limited by the bottle-neck that occurredin the domestication process (Tanksley and McCouch, 1996). The most easily-accessible and usefulgenetic resources for soybean breeding may be landraces/local varieties that have been established invarious regions of Asia. Here, I present an example
that shows diverse genetic variability embedded in asingle locus controlling important adaptative traits insoybean.
Genetic base of flowering under long days in soybean
Soybean is cultivated in a wide range oflatitudes from the equator to 50o north. This wide
adaptability most likely has been created by genetic
diversity at a large number of the major genes andquantitative trait loci (QTL) controlling flowering
behavior. Soybean is basically a short-day plant, andsoybean cultivars adapted to high latitude environments possess insensitivity to photoperiod. Four
major loci, E1 , E3 , E4 and E7 , are known to be involved in the control of this insensitivity, particularly
to long-day (LD) conditions (Buzzell1971; Buzzell
and Voldeng 1980; Cober et a l. 1996; Cober andVoldeng 2001a; 2001b). Soybean plants also responddifferentially to light quality controlled artificiallyby fluorescent and incandescent lamps with different
red-to-far red quantum (R:FR) ratios (Cober et al .

1996). The E3 locus was first identified with the useof fluorescent lamps to extend day length; the e3e3
recessive homozygote can initiate flowering under
LD conditions when the day length is extended to
20 h using fluorescent lamps with a high R:FR ratio(Buzzell 1971). The E4 locus was identified by extending the natural daylength to 20 h with incandescent lamps with a low R:FR ratio (Buzzell and Voldeng 1980). A recessive allele at the E4 locus can notsimply confer insensitivity to LD conditions inducedby both fluorescent and incandescent lamps, but isnecessary for plants homozygous for the e3 allele toflower under the LD condition with a low R:FR ratio(Buzzell and Voldeng 1980; Saindon et al . 1989; Cober et al . 1996). The E1 and E7 loci are also involvedin the control of the insensitivity to artificiallyinduced LD conditions in the e3 and e4 background(Cober et al . 1996; Cober and Voldeng 2001b). Usingnear isogenic lines (NILs) for those maturity genes,
Cober et al . (1996) found different responses to eachphotoperiod-sensitivity gene to 20-h LD conditions
with different R:FR ratios, suggesting that some ofthese genes may belong to the phytochrome family.
Classification of ILD-insensitive cultivars basedon isozyme and SSR markers
We evaluated the responses of soybean accessions introduced from various countries of East
Asia to incandescent-induced long daylength (ILD)
where the natural day length was extended to 20 hwith incandescent lamps. We defined the ability ofplants to initiate flowering under ILD without any
delay relative to natural daylength condition as ILDinsensitive. A total of 29 accessions were determined
as ILD-insensitive. They were further classified into
several groups, based on the result of UPGMA for
genotypes at 7 isozyme and 9 SSR markers. There
are three clearly-separated groups (Fig.1). The firstgroup (designated group I) mostly consists of thelandraces that are adapted to the cool summer ofnorthern and northeastern Hokkaido of Japan and
Sakhalin, where the frost-free season is limited toless than 130 d. The second group (designated groupII) consists of the landraces that have been cultivatedas a short-season crop in wide areas of Japan and- 91 -

Group I(FER)Group II(SK)Group III

(FER)
(NEC)
(FER)
(FER)

Fig. 1. Classification of ILD insensitive soybean accessions in East Asia, based on genotypes at
Fig. 1. Classification of ILD insensitive soybean accessions in East Asia, based onseven isozyme
andatninemarkers.
Northeast
Russia, SK;
genotypes
sevenSSR
isozymeand nineNEC;
SSR markers.
NEC; China,
NortheastFER;
China,Far-east
FER;
Far-east
South Koreadata)
South Korea(AbeRussia,
et al . SK;
unpublished
the Korean peninsula. The third group (designatedgroup III) consists of the landraces collected in Iwate
Prefecture of northern Japan. All of the accessions
within each group had almost identical genotype.
Another two accessions collected in Iwate Prefecture
formed a different clade, which are connected to theabove three groups. The accessions from North-East
China and Far-East Russia are loosely related to eachother, and form the fifth group.
The identity of the genetic mechanisms underlying the ILD insensitivity was tested between thecultivar groups I and II. The F2 progeny of the crossbetween Miharudaizu (group I) and Sakamotowase(group II) exhibited transgressive segregation toward ILD-insensitive late flowering, suggesting thatboth possessed different genetic systems (Abe et al .,1998). From the genetic analyses for NILs for the
ILD insensitivity, Abe et al (2003) determined the
genotypes at the three loci, E1 , E3 and E4 , of Miharudaizu and Sakamotowase as E1E1e3e3e4e4 ande1e1e3e3E4E4 , respectively. The genotype of Miharudaizu was therefore the same as the double recessive genotype for E3 and E4 , which was determinedin previous studies to condition ILD-insensitivity(Buzzell, 1971; Buzzell and Voldeng, 1980). However, the genotype of Sakamotowase was the sameas a Harosoy NIL for the e3 allele, which develops- 92 -

no flower buds under the ILD treatment. Therefore,a novel gene(s) may be needed for Sakamotowase toinitiate flowering under ILD. The test crosses with
the Harosoy isoline were then carried out to identify
the novel gene for the photoperiod-insensitivity of
Sakamotowase. By marker-assisted analyses, we
detected a major QTL for insensitivity near a SSR
marker in linkage group C2 and a minor QTL in
linkage group L. It was estimated from the positionof tagging marker that the novel gene may be an allele at the E1 locus or a gene tightly linked to the E1
locus (Liu and Abe, 2009).
Identification of the maturity gene E4 as a paralog of phytochrome A
As suggested by Cober et al . (1996), the E4gene appears to encode phytochrome A protein. We
isolated two phyA genes designated as GmPHYA1
and GmPHYA2 from the soybean genome to test theassociation between phyA genes and soybean maturity genes. Analysis of the GmPHYA2 gene from thephotoperiod insensitive lines carrying the recessive
allele e4 revealed an insertion of a retrotransposonin exon 1 of the gene, which resulted in dysfunctionof the gene (Fig.2A and 2B; Liu et al . 2008). Genetic
mapping allocated GmPHYA1 and GmPHYA2 intohomoeologous regions of linkage groups O and I,

A

E4
SORE1

e4

B

Insertion of SORE1

C

e4

Stop codon

Accessions

E4

Fig. 2. The dysfunctional allele at the E4 locus and PCR amplification using allele-specific primers.
A) the e4 allele possesses a TY1/copya-like retrotransposon, SORE1, in the first exon of GmPHYA2.
B) Insertion of SORE1 that causes a premature stop codon at the e4 allele.
C) Identification of accessions carrying the e4 allele by PCR. (Liu et al . 2008)
respectively, suggesting that GmPHYA1 and GmPHYA2 are homoeologs which resulted from ancientchromosomal duplications and rearrangements insoybean. Of these, GmPHYA2 was mapped at theposition corresponding to the E4 locus previously
mapped (Abe et al. 2003), and cosegregated with the
ILD insensitivity. Taken together, the E4 locus encodes the GmPHYA2 protein, and the dysfunctional
allele due to the insertion of retrotransposon conditions the ILD-insensitivity.
The phytochrome A protein is involved in various developmental processes that are regulated bydifferent R:FR ratios, such as germination, de-etiolation, early-neighbor detection, shade avoidance, resetting of the circadian clock, and flowering (CASAL
et al . 1997). Studies using mutants deficient in phytochromes in Arabidopsis, pea and rice have revealed
different roles of phyA and phyB in the de-etiolation
responses to different light conditions (Weller et
al . 1997; Neff and Chory 1998; Takano et al . 2001;
2005). phyA and phyB are involved in de-etiolationof seedlings under FR-light and R-light conditions,
respectively. The phyA mutants of Arabidopsis, pea
and rice show a complete loss of the de-etiolation response under the continuous FR-light condition. Like
the phyA null mutants of these species, the e4 allele
impaired the de-etiolation response to the continuous
FR-light condition. Plants homozygous for the e4 al-lele produced significantly elongated hypocotyls under continuous FR light, when compared with thosegrown under continuous R light, whereas plants
homozygous for the E4 allele exhibited similar hypocotyl growth under both FR and R light (Fig. 3A;
Liu et al . 2008). The similar elongated internodes
are also observed in higher nodes of plants (Fig. 3B).
However, the phyA function of the e4 allele was lostpartially, not completely, possibly due to the presence of another copy, GmPHYA1 . The presence of
duplicated copies of phyA genes may account for thegeneration of photoperiod insensitivity, while protecting against the deleterious effects of mutation (Liuet al . 2008). This is in contrast to a complete loss ofthe de-etiolation response under the continuous FRlight condition that is observed in the phyA mutantsof Arabidopsis, pea and rice, in which the phyA geneis present as a single copy gene (Weller et al . 1997;
Neff and Chory 1998; Takano et al . 2001; Weller et
al . 2001; Takano et al . 2005).
Distribution of soybean cultivars carrying the
SORE-1 -inserted GmPHYA2 allele
Inactivation of GmPHYA2 , which constitutes
the e4 allele that confers insensitivity to a long-day
condition, is caused by the insertion of SORE-1 inexon 1 of the gene (Liu et a l. 2008; Kanazawa et al .
2009). Based on these findings, we hypothesized that- 93 -

B

A
Hypocotyl length (cm)

16

a

a

14

b

12

c

10

c

c

8
6

Dark

Red

Far-red

Dark

E4E4

Red

Far-red

e4e4

E4/E4

C

e4/e4

1. Harosoy-e3 (E4)
2. Kamaishi 17
3. Tsukue 4
4. Otome wase
1

2

3

4

5. Harosoy-e3e4 (e4)5Fig.3. E4 and e4 alleles exhibit different photomorphogenesis under long day.
A) De-etiolation response under dark, red light and far-red light, Different alphabets indicate
statistically significant differences at a level of 5 %B) Plant morphology of Harosoy isolines for E4 and e4
C) Plant morphology of various ILD-insensitive soybean accessions carrying different
dysfunctional alleles.(Liu et al .2008 Abe et al . unpublished data)
the insertion of SORE-1 in exon 1 of GmPHYA2 is
one of the major genetic changes that allowed distribution of soybean in high latitude regions. To test
this hypothesis, we analyzed the presence or absenceof SORE-1 at this locus in various cultivated andwild soybean accessions.
PCR that amplifies a region encompassing a
portion of SORE-1 and its flanking genomic region(Fig.2C) was performied using DNA isolated from332 cultivated soybean accessions introduced fromvarious East Asian countries that cover a wide rangeof latitude and include regions where cultivatedsoybean originated (Kanazawa et al . 2009). We also
analyzed 85 wild soybean (ssp. soja ) accessions thatwere collected from natural populations in various
regions of Japan. While no plants that harbor SORE1 at the locus were found in wild soybean lines
examined, the SORE-1 insertion at the locus was
detected in 10 accessions of cultivated soybean, nineof which were distributed in Hokkaido, Japan (Fig.
4). Most of the nine accessions belong to the group
I (Fig.1). A remaining accession (Col/Aomori/1981/
L145) is a collection from Aomori Prefecture, thenearest prefecture to Hokkaido Island. All these ac-

cessions had an ILD-insensitive and early-maturing
habit. In addition, the historical record indicates thata local variety named ‘Ohyachi’, an introduction by
an immigrant from the North East region of Japan,
enabled the expansion of soybean cultivation into
inland, northern and eastern areas of Hokkaido withharsher environments in the late 19 century, where
different named landraces, as those of group I, hadbeen established. These results are consistent with
the notion that disruption of GmPHYA2 by the insertion of SORE-1 contributed to the expansion ofcultivated region of soybean toward higher latitude
regions (Kanazawa et al . 2009).
Independent mutations that result in dysfunctional alleles at the E4 loci
It remains unsolved what mechanisms are involved in the genetic controls for ILD insensitivity
for other accessions. We determined the sequencesof two phyA genes for representative accessions ofgroups. No non-synonimous mutation was detected
for GmPHYA1 among the accessions sequenced. On
the other hand, sequencing the GmPHYA2 gene forthese accessions revealed four additional recessive- 94 -

Fig. 4. Geographical distribution of the e4 allele in cultivated soybeans in East
Asia. Figures indicate the numbers of accessions carrying the e4 allele
per those of total accessions tested in each region (Kanazawa et al .
2009).

B

E4

e4

A

e4-otoe4-kame4-kes3081
3270

e4-tsu1339

692

e4, SORE1

2656

A

C

B
Regulatory
element

NH2-TERMINALHKL

COOH-TERMINAL

e4-SORE1
e4-otoe4-tsue4-kame4-kesE4

Fig. 5. Independent mutations at the E4 locus that produced premature stop codons andtruncated proteins in the ILD-insensitive accessions.
A) Positions of single-base or two-base deletions in the exons 1 and 2 ofGmPHYA2 gene.
B) Domain structure of phytochrome A gene and the deduced amino acid regionstranslated from the mutant alleles(Abe et al . unpublished data).
- 95 -

alleles, e4-kes (in Keshung from northeast China),
e4-kam , e4-tsuk and e4-oto (all from Iwate Prefecture), all of which exhibited single-base or twobase deletions in the first and second exons, resulting in premature truncated proteins (Fig. 5A). PCR
analyses using allele-specific primers further revealed that all of the ILD-insensitive accessions ingroup III carried the e4-kam allele, and three accessions from Far-east Russia carried the e4-kes allele
as did the Chinese accession Keshung. Therefore,
all of the ILD accessions except for the accessionsof group II possessed dysfunctional alleles at theE4 locus. These accessions possessed the recessive
alleles at both the E3 and E4 loci to condition the
ILD insensitivity. A swapping experiment for thedomains of phyA and phyB indicated that the NH2terminal domain and core region for regulatory
activity possessed important functions for de-etiolation responses for both Arabidopsis phytochrome
A and B genes (Quail, 1997). The deduced aminoacids differ in length among the alleles detected
at the E4 locus (Fig. 5B). In particular, the e4-kes
and e4-kam alleles are expected to produce aminoacids that cover the regions important for the deetiolation function. In fact, the accession Kamaishi-17 having the e4-kam allele showed almost thesame internode growths as plants having the E4
allele under LD (Fig. 3C). Different GmPHYA2 alleles may regulate responses of soybeans to photoenvironments, such as photoperiod-sensitivity andphotomorphogenesis, differently. The alleles from
Kamaishi-17 and Keshung at the E4 locus might
therefore make it possible for the soybean cultivars
to flower under long day without defective morphogenesis.

Concluding remarks

Soybean is considered to be a paleopolyploid species with a complex genome (reviewed by
Shoemaker et al . 2006). Most of genes possess theirhomoeologous partners, like the two phytochrome
A genes, GmPHYA1 and GmPHYA2 . The genetic
redundancy may have generated genetic materials
through the accumulation of mutations, which havebeen subjected to selections by farmers. The duplicated genes may have a potential to acquire a novel
function via sub-functionalization and/or neofunctionalization of genes and to adjust the phenotype to the appropriate levels by the change of genedosages as caused by the null allele at either of thetwo loci. Molecular dissection, together with phenotypic evaluation of diverse genetic resources fortargeted traits and their detailed characterization,
may enable us to construct a new breeding strategy
for improvements of yield, quality and adaptation
in soybean.

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