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CHAPTER 2
Soybean (Glycine max (L.) Merr.)
Ram J. Singh, Randall L. Nelson, and Gyuhwa Chung
CONTENTS
2.1
2.2
2.3
2.4
2.5
Introduction.............................................................................................................................14
Domestication and Dissemination of Soybean ......................................................................15
Botany.....................................................................................................................................17
2.3.1 Taxonomy ...................................................................................................................17
2.3.2 Morphology ................................................................................................................17
Gene Pools of Soybean ..........................................................................................................20
2.4.1 Soybean GP-1.............................................................................................................20
2.4.2 Soybean GP-2.............................................................................................................21
2.4.3 Soybean GP-3.............................................................................................................21
Cytogenetics ...........................................................................................................................22
2.5.1 Evolution of the Glycine Genome .............................................................................22
2.5.2 Genomic Relationships among Diploid Species........................................................26
2.5.2.1 Genome Designation...................................................................................27
2.5.2.2 Classical Taxonomy ....................................................................................27
2.5.2.3 Crossing Affinity .........................................................................................27
2.5.2.4 Chromosome Pairing...................................................................................27
2.5.2.5 Molecular Methods .....................................................................................30
2.5.3 Polyploid Complexes of G. tabacina and G. tomentella ..........................................30
2.5.3.1 Glycine tabacina (2n = 80).........................................................................31
2.5.3.2 Glycine tomentella (2n = 78, 80) ...............................................................31
2.5.4 Chromosomal Aberrations: Structural Changes.........................................................32
2.5.5 Chromosomal Aberrations: Numerical Changes .......................................................32
2.5.5.1 Autopolyploidy............................................................................................32
2.5.5.2 Aneuploidy ..................................................................................................32
2.5.5.2.1 Primary Trisomics.....................................................................32
2.5.5.2.2 Monosomics ..............................................................................33
2.5.5.2.3 Tetrasomics ...............................................................................34
2.5.6 Linkage Mapping........................................................................................................34
2.5.6.1 Chromosome Map.......................................................................................34
2.5.6.2 Classical Genetic Linkage Map..................................................................34
2.5.6.3 Molecular Linkage Map..............................................................................35
13
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
2.6
Soybean Germplasm Enhancement........................................................................................35
2.6.1 Conventional Breeding ...............................................................................................35
2.6.2 Interspecific Hybridization .........................................................................................36
2.6.3 Intersubgeneric Hybridization ....................................................................................37
2.6.4 Mutation Breeding......................................................................................................38
2.6.5 Biotechnology.............................................................................................................40
2.6.5.1 Plant Regeneration from Callus and Cell Suspension Cultures ................40
2.6.5.2 Protoplasts Culture ......................................................................................41
2.6.5.3 Genetic Transformation...............................................................................41
2.6.6 Potential to Produce Hybrid Soybeans ......................................................................42
2.7 Summary.................................................................................................................................42
References ........................................................................................................................................43
2.1 INTRODUCTION
The soybean (Glycine max (L.) Merr.; 2n = 40) is an economically important leguminous seed
crop for feed and food products that are rich in seed protein (40%) and oil (20%). Soybean is
ranked number one in world production in the international trade markets among the major oil
crops, such as cottonseed, groundnut (peanut), sunflower seed, rapeseed, linseed, sesame seed, and
safflower (see Chapter 1). Soybean is widely grown in the U.S., Brazil, Argentina, China, and India.
In the past 45 years (1961 to 2004), the U.S. has been the leader in soybean production, and
currently produces more than half (53%) of the world production. Soybean yield per hectare has
increased over 60% in China and Brazil, over 30% in Argentina and the U.S., but has remained
unchanged in India (Figure 2.1; FAO STAT, 2004).
The enormous economic value of the soybean was realized in the first two decades of the 20th
century. Osborne and Mendel (1917) demonstrated experimentally that heated soybean meal promoted growth in rat at normal rate, which was in contrast with raw soybean meal. This study
resulted in the establishment of soybean processing industries in the U.S. Mr. A.E. Staley Sr. laid
the foundation for operational soybean processing facilities in Decatur, IL, in 1922 (Windish, 1981).
Soybean is processed worldwide for oil and meal and has great influence on other oilseeds in the
international trade.
Soybean Yield (Mt) per Hectare
3.00
Yield (Mt/Ha)
2.50
2.00
1.50
1.00
2003
2001
1999
1997
1995
1993
1991
1989
1987
1985
1983
1981
1979
1977
1975
1973
1971
1969
1967
1965
1963
0.00
1961
0.50
Year
Argentina
Figure 2.1
Brazil
China
India
USA
A graphic representation of soybean yield (Mt/ha) in the U.S., Argentina, Brazil, China, and India
from 1961 to 2004. (From www.FAOSTAT.org.)
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SOYBEAN (GLYCINE MAX (L.) MERR.)
AU: Please
introduce
RFLP and
SSR.
15
Soybean is not considered a model plant for cytogenetic studies because of the large number
of chromosomes (2n = 40), the small and similar chromosome sizes (1.42 to 2.84 μm), and the
lack of morphological distinguishing landmarks (Singh, 2003). Using primarily RFLP and SSR
loci, a linkage map with 20 linkage groups has been developed, but not all linkage groups have
been associated with the respective chromosomes. A pachytene karyogram (Singh and Hymowitz,
1988) and a set of possible 20 primary trisomics (Xu et al., 2002c) have been established. However,
primary trisomics seeds are not available. (Hymowitz-Nelson, communication). Cytogenetics of
the soybean is far behind that of maize, barley, rice, wheat, tomato, and others (Singh, 2003).
The objective of this chapter is to present information on the origin and domestication of the
soybean, the maintenance and use of Glycine genetic resources, and the available genetic and
cytogenetic tools for exploiting available germplasm for soybean improvement.
2.2 DOMESTICATION AND DISSEMINATION OF SOYBEAN
Soybean was domesticated from the wild soybean, G. soja Sieb. & Zucc. (formerly G. formosana
Hosokawa; G. ussuriensis Regal & Maack (Fukuda, 1933; Hermann, 1962)), which is an annual
weedy-form climber whose pods contain black seeds that shatter at maturity. G. soja grows wild in
China, far eastern Russia, the Korean peninsula, Taiwan, and Japan (Hymowitz, 1970; Singh and
Hymowitz, 1999). The wild soybean seed has a wide range of protein concentration (31 to 52%),
similar to that of soybean, but is generally much lower in oil (9 to 12%) (Hymowitz et al., 1972).
The cultivated soybean and its progenitor G. soja belong to the subgenus Soja (Moench.) F.J. Herm.,
and both are cross compatible, contain 2n = 40 chromosomes, and produce vigorous fertile intermediate F1 hybrids (Fukuda, 1933; Palmer et al., 1987; Singh and Hymowitz, 1988, 1989).
Based on linguistic, geographical, and historical literature, the soybean was likely domesticated
during or prior to the Shang dynasty, which ruled China between 1700 to 1100 B.C. (Gai, 1997; Gai
and Guo, 2001; Hymowitz and Newell, 1981; Qiu et al., 1999). There is no definitive research that
establishes the location of the domestication of soybean. Different authors have indicated that the
soybean may have been domesticated in northeastern China (Fukuda, 1933), the middle and lower
Yellow River Valley of central China (Hymowitz and Newell, 1981; Xu, 1986; Zhou et al., 1998),
southern China (Wang et al., 1973; Gai et al., 1999, 2000; Shimamoto et al., 1998), in a corridor
from southwest to northeast China, which included Sichuan, Shaanxi, Shanxi, Hebei, and Shandong
provinces (Zhou et al., 1999), or simultaneously at multiple centers (Lu, 1977; Dong et al., 2001).
Soybean is reported to have come to the Korean peninsula around the fourth or fifth century
B.C.E. (Kim, 1993). Kihara (1969) reported that rice (Oryza sativa L.) was introduced to Japan on
Kyushu Island around the third century B.C.E. and that soybean arrived in Japan about the same
time as rice. It is possible that soybean in Japan could have come from either Korea or China.
Using cluster analyses of DNA marker data from primitive germplasm from China, Japan, and the
Republic of Korea, Li and Nelson (2001) showed that accessions from Japan and the Republic of
Korea were clearly distinct from Chinese accessions, but not from each other, and were less diverse
than the accessions from China. Because soybean was introduced into Japan and Korea centuries
earlier than to any other country, these data provide justification that they be considered the
secondary center of diversity.
It is difficult to find any published history about the spread of soybean to other Asian countries,
but soybean may have been cultivated in Vietnam, Indonesia, India, and Nepal longer than other
areas of southeastern and central Asia. These, plus other Asian countries, may be considered the
tertiary center of diversity.
Missionaries and sailors brought the soybean to Europe from China and Japan. Soybean was
grown in 1740 in botanical gardens in France and in 1790 in the Royal Botanical Garden, Kew,
England. Soybean was cultivated in several European countries, but the acreage was very limited
(Morse, 1927). Soybean was introduced to North America from China by Samuel Bowen in 1765
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
210,000,000
200,000,000
190,000,000
180,000,000
170,000,000
160,000,000
150,000,000
140,000,000
130,000,000
120,000,000
110,000,000
100,000,000
90,000,000
80,000,000
70,000,000
60,000,000
50,000,000
40,000,000
30,000,000
20,000,000
10,000,000
0
Hectares
Metric Tons
1961
1962
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Hectares & Metric Tons
World Soy Production & Area 1961 - 2004
Years
Figure 2.2
A graphic representation of world soybean production and area under cultivation from 1961 to
2004. (From www.FAOSTAT.org.)
and was planted in Greenwich, located at Thunderbolt, a few miles east of Savannah, GA (Hymowitz
and Harlan, 1983). Since 1765, soybean has been introduced into the U.S. several times by seed
dealers, merchants, plant explorations, and various individuals, but the oldest extant cultivar in the
U.S. was not introduced until ca. 1880 (Bernard et al., 1987). Seed and Plant Introduction was
established in 1898 within the U.S. Department of Agriculture (USDA) and initiated the introduction
of a large number of soybean accessions from Asian countries (Morse, 1927). This facilitated
centralized plant introduction activities and preserved records of imported accessions. William J.
Morse and P.H. Dorsett conducted plant exploration trips to China, Japan, India, and Korea to
enhance the U.S. germplasm resources. They collected more than 5000 of the 8300 soybean
introductions documented between 1989 and 1949 (Bernard et al., 1987). When the USDA Soybean
Germplasm Collection was established in 1949, only 1677 accessions had been preserved in the
collections of individual soybean scientists. Before 1945, soybean in the U.S. was used as much
for forage as for grain. During World War II, the emphasis of soybean production and utilization
shifted to a source of oil.
Argentina, Brazil, China, India, and the U.S. are currently the five largest producers of soybeans
in the world. According to Food and Agriculture Organization (FAO) statistics (http://faostat.fao.org/),
these five countries had approximately 90% of the harvested area and produced approximately 93%
of the total crop in each year between 1961 and 2004 (Figure 2.2 and Figure 2.3). The consistency
of these figures belies the dramatic changes that have occurred in total area harvested, total
production, and the distribution of that production among the five leading countries. In 1961, China
had 42% and the U.S. 46% of nearly 24 million ha harvested. The other three countries had 1%
or less. The U.S. produced 69% and China 23% of the 23.8 million metric tons. Over the next 20
years, soybean production continued to expand in the U.S. and declined slightly in China. In 1973,
60% of the area harvested and 71% of the production was in the U.S., and only 20% of the area
and 14% of the production was in China. Brazil increased from 1 to 10% of the area harvested
between 1961 and 1973. Total area harvested in 1973 was 33.8 million ha, and that produced
55.9 million metric tons of soybeans. In 2004, the area harvested and total soybean production
both reached new highs, with 91.4 million ha and 189.3 million metric tons (Figure 2.3). The
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SOYBEAN (GLYCINE MAX (L.) MERR.)
17
Average Annual Soy Production and Area per Decade
(1960 - 2004)
200,000,000
Hectares & Metric Tons
180,000,000
Area
160,000,000
Production
140,000,000
120,000,000
100,000,000
80,000,000
60,000,000
40,000,000
20,000,000
0
1960s
1970s
1980s
1990s
2000s
Decades
Figure 2.3
A graphic representation of average annual soybean production and area per decade (1960 to 2004).
(From www.FAOSTAT.org.)
distribution of the harvested area has also changed significantly (Figure 2.2 and Figure 2.3).
Only 33% of the harvested area is in the U.S. Brazil and Argentina have 24 and 16%,
respectively. With 11% of current world total, China has nearly the same amount of harvested
area as it did in 1961. India, like South America, has greatly increased its production area to
8% of the world total.
2.3 BOTANY
2.3.1
Taxonomy
The taxonomy of the soybean is as follows:
Order
Family
Subfamily
Tribe
Subtribe
Genus
Subgenus
Species
2.3.2
Fabales
Fabaceae (Leguminosae)
Papilionoideae
Phaseoleae
Glycininae
Glycine Willd.
Soja (Moench) F.J. Herm.
Glycine max (L.) Merr.
Morphology
Soybean is an annual plant. It exhibits taproot growth initially, followed later by a large number
of secondary roots. Roots establish a symbiotic relationship with the bacterium (Bradyrhizobium
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
wings
androecium
standard
keel
gynoecium
B
sepals
A
anthers
stigma
style
filament
ovary
C
Insert page
number.
AU: Please
provide a reference for.
Insert page
number.
Figure 2.4
D
(See color insert following page xxx.) Reproductive organs (identified) of soybean: (A) complete
mature flower, (B) mature androecium and gynoecium, (C) a mature gynoecium with stigma, style,
and ovary, and (D) mature anthers with five anthers on longer filament (outer whorl), four anthers
on shorter filament (inner whorl), and one free anther always below the stigma.
japonicum) through formation of root nodules. Soybean has four different types of leaves: the seed
(first pair of simple cotyledons leaves; epigeal germination), simple primary leaves, pinnately
trifoliolate leaves, and prophylls (a pair of 1-mm-long simple leaves at the base of each lateral
branch) (see Lersten and Carlson (2004) for a detailed description of vegetative morphology).
There are two loci that are known to control stem termination (Dt1 and Dt2) (Woodworth,
1933; Bernard, 1972). With the determinate stem type (dt1) there is usually little growth in stem
length after flowering with blunt stem termination and a terminal raceme, whereas with the indeterminate stem type (Dt1) stem elongation and node production continue after flowering, producing
a longer, more tapered main stem and branches. There is considerable variation in stem growth
within each of these two types, with time of flowering and time of maturity having major effects
on stem morphology. An intermediate phenotype is conditioned by the Dt2 genotype and is called
semideterminate (Bernard, 1972). Thompson et al. (1997) identified a third allele (dt1-t) at the Dt1
locus. It produces a phenotype for plant height that is similar to Dt2 but with fewer main stem
nodes and larger terminal leaflets.
Soybean plants enter into the reproductive stage following vegetative growth. Axilary buds
develop into clusters of 2 to 35 flowers. From 20 to 80% of the flowers abscise. The earliest and
latest flowers produced generally abort most often. Soybean has a typical papilionaceous flower
with a tubular calyx of five unequal sepals, and a five-parted corolla. The corolla consists of a
standard (posterior banner) petal, two lateral wings, and two anterior keel petals contacting each
other but not fused (Figure 2.4A*). Stamens are clustered around the stigma, ensuring selfpollination (Figure 2.4B). The gynoecium constitutes an ovary, style, and stigma (Figure 2.4C). As
many as four ovules appear in the ovary. Nine stamens are arranged in two whorls; the outer and
inner whorls contain five and four stamens, respectively. The two whorls of nine stamens align
themselves into a single whorl on a staminal tube. The larger and older stamens alternate with the
smaller and younger stamens in sequence around the developing gynoecium. The single (10th
* Color figures follow page xxx.
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SOYBEAN (GLYCINE MAX (L.) MERR.)
Figure 2.5
19
Mature pods of soybean; pods containing one, two, and three seeds (left to right).
stamen) free stamen is the last to appear (Figure 2.4D). Soybean is highly self-pollinated with
natural crossing usually less than 1% because stamens are elevated so that the anthers form a ring
around the stigma; thus, pollen is shed directly on the stigma, ensuring self-pollination (Carlson
and Lersten, 2004).
Pollen is shed on the stigma. Pollen tubes travel through style and enter into the filiform
apparatus. The pollen tube tip bursts and releases two sperm nuclei. One sperm nucleus fuses with
the egg and forms a zygote, while the second sperm unites with the secondary nucleus, forming
an endosperm. Mature seeds develop from 30 to 50 days after fertilization. They are devoid of
endosperm and contain two large fleshy cotyledons, a plumule with two well-developed primary
leaves enclosing one trifoliolate leaf primordium, a hypocotyl-radicle axis, a micropyle, a hilum
with central fissure, and a raphe (see Carlson and Lersten, 2004).
The inflorescence of each node of soybean plant may develop into 1 to more than 20 pods.
A plant may have up to 400 pods. The soybean pod is similar to that of other legumes. A pod
usually contains 1 to 3 seeds and rarely 4 seeds (Figure 2.5), except for plants that have the have
the na allele that produces narrow leaflets and a much higher proportion of 4-seeded pods.
Since soybean was introduced into the U.S. from several geographical regions of East Asia,
the response to the adopted country was extremely variable. Morse (1927) realized the problem
and developed the concept of relative maturity groups based on critical day length. He identified
five soybean maturity groups (MGs) (1 = southern through 5 = northern) in the soybean-growing
regions of the U.S. Morse et al. (1949) reclassified the maturity groups of soybean and divided
varieties into nine maturity groups (0 through VIII). Maturity group 0 and I cultivars were those
adapted to the northern part of the country, reverse of the previous classification of Morse
(1927). Succeeding maturity groups contained cultivars adapted to areas farther south, with
those of group VIII suited for the Gulf Coast region. Now, 13 (000 to X) maturity groups have
been identified for the appropriate latitude at which maximum commercial yield is produced
(Figure 2.6).
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
Figure 2.6
Maturity groups (MGs) of soybean cultivars relative to the area in which they are cultivated. (From
Fehr, W.R., in Oil Crop of the World, Röbbelen, G. et al., Eds., McGraw-Hill, New York, 1989, pp.
283–300. With permission.)
2.4 GENE POOLS OF SOYBEAN
Harlan and de Wet (1971) developed the concept of three — primary (GP-1), secondary (GP-2),
and tertiary (GP-3) — gene pools based on the success rate of hybridization among species. The
clear understanding of taxonomic and evolutionary relationships between cultigen and its wild
relatives is a prerequisite for the exploitation of primary, secondary, and tertiary gene pools.
2.4.1
Soybean GP-1
Soybean GP-1 consists of biological species that can be crossed to produce vigorous hybrids
that exhibit normal meiotic chromosome pairing and possess total seed fertility. Gene segregation
is normal and gene exchange is generally easy. Based on this definition, all soybean (G. max)
germplasm and the wild soybean, G. soja Sieb. & Zucc., are included in GP-1. Both species have
2n = 40 chromosomes, hybridize easily, and produce normal fertile F1 hybrids; meiotic chromosome
pairing is normal, but may differ by a reciprocal translocation (Palmer et al., 1987; Singh and
Hymowitz, 1988) or by an inversion (Ahmad et al., 1977). Skvortzow (1927) characterized a distinct
species Glycine gracilis Skvortzow; however, subsequent cytogenetic research demonstrated that
this species is a hybrid derivative of G. max and G. soja (Fukuda, 1933; Karasawa, 1952; Singh
and Hymowitz, 1989), and chloroplast DNA variation suggested that subgenus Soja precludes
G. gracilis as an independent species (Shoemaker et al., 1986). However, Chen and Nelson (2004)
found that former G. gracilis accessions in the USDA Soybean Germplasm Collection are distinct
from both G. max and G. soja based on either morphogical data or SSR allelic diversity.
The largest collection of soybean germplasm is the National Crop Gene Bank in Beijing, China,
with 25,034 accessions of G. max and 6172 accessions of wild soybean (http://icgr.caas.net.cn/
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SOYBEAN (GLYCINE MAX (L.) MERR.)
21
Tertiary gene pool
GP-3
26 wild perennial species
Secondary gene pool
GP-2
unknown
Glycine soja
Primary gene pool
GP-1
soybean cultivars
land races
Secondary gene pool
GP-2
unknown
Tertiary gene pool
GP-3
26 wild perennial species
Figure 2.7
AU: Please
provide a reference for.
Gene pools of the soybean based on the classification of Harlan and de Wet, (1971). Primary gene
pool (GP-1) contains land races and its wild annual progenitor Glycine soja. Secondary gene pool
(GP-2) is not identified. Tertiary gene pool (GP-3) constitutes 26 perennial wild species.
cgris_english.html). The USDA Soybean Germplasm Collection at Urbana, IL, is the second largest
collection, with 18,567 accessions of G. max and 1117 accessions of G. soja (http://www.ars-grin.gov/
cgi-bin/npgs/html/site_holding.pl?SOY). More than 70 countries maintain soybean germplasm
collections, and the total world collection exceeds 170,000 accessions, but the number of duplicates
is unknown (Carter et al., 2004; IPGRI, 2005).
2.4.2
Soybean GP-2
GP-2 species can hybridize with GP-1 easily, and F1 plants exhibit at least some seed fertility
(Harlan and de Wet, 1971). G. max is without GP-2 because no known species has such a relationship
with soybean (Figure 2.7). It is possible that species in the soybean GP-2 exist in Southeast Asia,
where the Glycine genus may have originated. It is merely a speculation, and extensive plant
exploration in this part of the world is required to validate this assumption.
2.4.3
Soybean GP-3
GP-3 is the extreme outer limit of potential genetic resources. Hybrids between GP-1 and GP-3
are anomalous, lethal, or completely sterile, and gene transfer is not possible or requires radical
techniques (Harlan and de Wet, 1971). Based on this definition, GP-3 includes the 26 wild perennial
species of the subgenus Glycine. These species are indigenous to Australia and are geographically
isolated from G. max and G. soja (Figure 2.8). Table 2.1 shows species, 2n chromosome number,
nuclear and plastome genomes, and geographical distribution of the Glycine species. Only three
species (G. argyrea, G. canescens, and G. tomentella) have been hybridized with soybean. The
USDA Soybean Germplasm Collection maintains 919 accessions of the 16 wild perennial species
(http://www.ars-grin.gov/cgi-bin/npgs/html/site_holding.pl?SOY).
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
Soybean and wild
annual Glycine soja
common ancestor of the
genus Glycine with n=x=10 is
unknown
Twenty six perennial wild
Glycine species
Figure 2.8
A geographical map showing the home of Glycine; the common progenitor (2n = 2x = 20) of G. soja
and soybean (annual) and 26 wild species (perennial) is unknown. It may be extinct or not yet
identified. Soybean is domesticated in China from G. soja, and 26 wild perennial Glycine species
were not domesticated in Australia.
2.5 CYTOGENETICS
Cytogenetics of soybean has lagged behind that of the other economically important crops,
such as maize, wheat, rice, barley, tomato, and faba bean. Soybean is not considered a model crop
for cytogenetic studies because it contains a high chromosome number (2n = 40; Karpechenko,
1925; verified by Fukuda, 1933; Veatch, 1934) and small and symmetrical chromosome size (1.42 to
2.84 μm; Sen and Vidyabhusan, 1960), lacks morphological landmarks by Giemsa C-banding
technique (Ladizinsky et al., 1979), and only one pair of nucleolus organizer chromosomes is
occasionally visible (Figure 2.9). Yanagisawa et al. (1991) separated 40 soybean mitotic metaphase
chromosomes into five groups (A, B, C, D, E) by using a chromosome image analyzing system
(CHIAS). Group A included a pair of nucleolus organizer (satellite) chromosomes, group B included
two submedian chromosomes with a gap at the center of the long-arm contraction, and groups C,
D, and E consisted 10, 14, and 12 chromosomes, respectively. However, pachytene chromosomes
exhibit defined euchromatin and heterochromatin differentiation (Singh and Hymowitz, 1988).
Heterochromatin is distributed proximal to and on either side of the centromeres on the long and
short arms, and 6 of the 20 short arms are very heterochromatic (Figure 2.10 and Figure 2.11).
It is interesting to note that 36% of the soybean genome is heterochromatic, which is higher than
that observed (29%) for tomato (Barton, 1950). This latter feature makes soybean pachytene
chromosomes unique (Singh, 2003).
2.5.1
Evolution of the Glycine Genome
The basic chromosome number x = 10 has been proposed for G. max (Darlington and Wylie,
1955). Based on this proposal, Singh (2003) hypothesized a putative ancestor with 2n = 20
chromosomes for the genus Glycine and carrying at least one pair of nucleolus organizer regions
(NORs). Although such a progenitor is currently unknown, it would be most likely found in
Southeast Asia (Cambodia, Laos, and Vietnam). Whether tetraploization (2n = 4x = 40) involved auto(spontaneous chromosomes doubling) or allo- (interspecific hybridization followed by chromosome
G. curvata Tindale
G. cyrotoloba Tindale
G. falcata Benth.
G. gracei B.E. Pfeil and Craven
G. hirticaulis Tindale and Craven
G. lactovirens Tindale and Craven
G. latifolia (Benth.) Newell and Hymowitz
G. latrobeana (Meissn.) Benth.
G. microphylla (Benth.) Tindale
G. montis-douglas B.E. Pfeil and Craven
G. peratosa B.E. Pfeil and Tindale
G. pescadrensis Hayata
G. pindanica Tindale and Craven
G. pullenii B. Pfeil, Tindale and Craven
G. rubiginosa Tindale and B.E. Pfeil
G. stenophita B. Pfeil and Tindale
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. G. syndetika B.E. Pfeil and Craven
24. G. dolichocarpa Tateishi and Ohashi
25. G. tabacina (Labill.) Benth.
Tindale and Craven
G. aphyonota B. Pfeil
G. arenaria Tindale
G. argyrea Tindale
G. canescens F.J. Hermann
G. clandestina Wendl.
2.
3.
4.
5.
6.
AU: Please introduce all
abbreviations in a table
note. 1. G. albicans
Mol.
Group
of the Genus Glycine Willd.a
Species
AU: There
is no
footnote
Table
2.1
Taxonomy
a. Please provide or
delete.
40
80
40
80
40
40
40
40
40
80
40
40
40
40
40
40
80
40
40
40
40
40
40
40
40
40
40
2n
D 1A
B2
BB1, BB2, B1B2
I1
B1
A3
B
?
A5
AB1
H2
H3
A4
B3
C1
C
F
?
H1, (??)
I
I3
H
A2
A
A1
Cb
PI Number
?
B
B
A
B
A
B
?
A
A
A
A
A
B
C
C
A
?
A, (A)
A
A
A
A
A
A
373990
373992
441000
440954
378705
440996
595818
IL1246
IL943
IL1247
378709
483196
440956
505166
440962
505179
505204
505151
440932
440958
Subgenus Glycine
Genome
Symbol
Na
1317
1314
1300
2916
1433
2951
2599
1874
2600
1849
1184
1155
3124
2876
1956
2720
1697
1385
1867
2049
2589
1305
1420
1853
1126
G Number
Aust.: Q
Taiwan
Aust.: Q, NSW
Aust.: Q, NSW, V, SA
West-central and south Pacific
islands
Aust.: Q
Aust.: Q, NSW
Aust.: Q, NT, WA
Aust.: NT
Aust.: NT
Aust.: NT
Aust.: WA
Aust.: Q, NSW
Aust.: V, SA, T
Aust.: Q, NSW, V, SA, T
Aust.: NT
Aust.: WA
Aust.: Q, NSW Taiwan, Japan
Aust.: WA
Aust.: WA
Aust.: NSW, SA, WA
Aust.: Q, NSW
(Japan ??)
Aust.: WA
Aust.: WA
Aust.: WA
Aust.: Q
Aust.: Q, NSW, V, SA, NT, WA
Aust.: Q, NSW, V, SA, T
Distribution
continued
Provenance
of 378705
is probably
not Japan
Atypical western
slopes ssp.
Comment
3639_C002.fm Page 23 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
23
G. max
PE: Okay that author uses color
to make distinctions in table, or
Note:
J.J. Doyle,
should
he useA,
superscript
let- personal communication;
ters or
asterisks
instead?
red,
A.H.D.
Brown; isozyme groups are in parentheses.
A
A
A
A
A
A
A
A
A
A
Cb
330961
483219
440998
505222
505294
505203
441001
509501
505286
441005
PI Number
G1
Note changes
G
G
51762
Subgenus Soja (Moench) F. J. Hermann
DH2
40
80
T4
DD2
G
80
T3
E
D
H2
D2
D 3E
AE
EH2
DA6
40
38
40
40
40
78
78
78
80
D1, D2
D3
D5B
D5A
T1
T5
T6
T2
2n
Genome
Symbol
Na
1348
1927
1858
1749
1943
1303
1133
1487
1945
1188
G Number
China, Japan, Russia, Korea,
Taiwan
Cultigen; worldwide
Aust.: Q
Aust.: Q, WA, PNG
Aust.: WA
Aust.: WA, NT
Aust.: Q, NSW. PNG
Aust.: NSW
Aust.: WA
Aust.: Q
Taiwan
Aust.: Q, NT, WA, PNG
Timor
Aust.: Q, NT, WA
Philippines, Taiwan
Distribution
Comment
24
G. soja
26. G. tomentella Hayata
Species
Mol.
Group
Table 2.1 (continued) Taxonomy of the Genus Glycine Willd.
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
3639_C002.fm Page 25 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
Figure 2.9
25
A mitotic metaphase cell of soybean with 2n = 40 chromosomes. One pair of chromosomes
containing a nucleolus organizer region (NOR) can be distinguished, while 38 chromosomes are
almost similar.
Pachytene Chromosomes of Soybean
K
7
D2
9
17
16
A2
6
8
5
18
11
C1
N
4
G
15
A1
14
3
L
19
2
D1a
1
Figure 2.10
10
12
F
H
13
20
The pachytene chromosome complement of G. max × G. soja F1 hybrid. Each figure shows a
different chromosome, 1 to 20. Arrows indicate centromere location. The letter above the number
represents the molecular linkage group. (From Singh, R.J. and Hymowitz, T., Theor. Appl. Genet.,
76, 705–711, 1988. With kind permission of Springer Science and Business Media.)
3639_C002.fm Page 26 Wednesday, July 19, 2006 1:09 PM
26
GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
Kinetochore
Figure 2.11
Proposed idiogram, based on Figure 2.7, of the pachytene chromosomes of the soybean. Arrow
indicates centromere location. (From Singh, R.J. and Hymowitz, T., Theor. Appl. Genet., 76,
705–711, 1988. With kind permission of Springer Science and Business Media.)
doubling) polyploidy of the progenitor species, and whether it occurred prior to dissemination or
after, cannot be substantiated experimentally, because we do not know where the progenitor of the
genus Glycine originated. The progenitor of the wild perennial species of the subgenus Glycine
radiated out into several morphotypes depending on the growing conditions in the Australian
subcontinent. These species have never been domesticated and remained as wild perennials.
In contrast, the pathway of migration from a common progenitor to China is assumed: wild perennial
(2n = 4x = 40; unknown extinct) wild annual (2n = 4x = 40; G. soja) soybean (2n = 4x = 40;
G. max) (Figure 2.8). All species of the genus Glycine exhibit diploid-like meiosis and are inbreeders
(Singh and Hymowitz, 1985a).
Allopolyploidization probably played a key role in the speciation of the genus Glycine. This
implies that the 40-chromosome Glycine species and 80-chromosome G. tabacina and G. tomentella
are allotetraploid and allooctoploid, respectively (Singh and Hymowitz, 1985b). Meiotic pairing in
haploid (2n = 20; range = 0II to 5II) soybean (Crane et al., 1982) and in interspecific hybrids (Singh
and Hymowitz, 1985b, 1985c; Singh et al., 1988; Grant et al., 1984), fluorescent in situ hybridization
(Singh et al., 2001; Pagel et al., 2004), and molecular studies (Shoemaker et al., 1996) elucidate
that soybean is of tetraploid origin.
2.5.2
Genomic Relationships among Diploid Species
Understanding the genomic relationships among species is important to systematists, evolutionary biologists, cytogeneticists, molecular biologists, and plant breeders. The taxonomic nomenclature of species and their evolutionary relationships can be refined by cytogenetic evidence such
as chromosome morphology, crossability, hybrid viability, meiotic chromosome pairing, and
molecular (isozymes and nuclear, chloroplast, and mitochondrial DNA markers) approaches. Thus,
phylogenetic relationships among species can be understood more precisely by a multidisciplinary
approach rather than through reliance on a single technique (Singh, 2003). Broué et al. (1977)
established, for the first time, phylogenetic relationships among four species of the subgenus
Glycine by starch gel electrophoresis. Extensive plant exploration, cytogenetic, and molecular
studies currently have identified 26 species in the subgenus Glycine, while only 6 species were
recognized by Broué et al. (1977).
3639_C002.fm Page 27 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
27
A
Insert page
number.
Figure 2.12
B
C
(See color insert following page xxx.) Morphology of three Glycine species grown in the
greenhouse: (A) G. tomentella (2n = 78); (B) G. cyrtoloba (2n = 40), showing the characteristic
curved pod (arrow); and (C) G. falcata (2n = 40), showing the characteristic falcate trait of pods.
2.5.2.1 Genome Designation
AU: Please
indicate
which 1998
reference.
The genomes of diploid Glycine species are assigned capital-letter symbols according to the
degree of chromosome homology between species in F1 hybrids (Kihara and Lilienfeld, 1932).
Similar letter symbols are designated for species with interspecific F1 hybrids that show normal
chromosome pairing. Placing a subscript after the letter indicates minor chromosome differentiation,
such as inversions or translocations. Highly differentiated species are designated by different letter
symbols, because their hybrids exhibit highly irregular chromosome pairing and hybrids are completely sterile. Singh and Hymowitz (1985b, 1985c) conceived assigning genome symbols to
Glycine species based on cytogenetic results. Molecular methods helped to assign genome symbols
to those species where cytogenetic information was not obtained (Singh et al., 1992a, 1998;
Kollipara et al., 1993, 1995, 1997; Doyle et al., 2002; Brown et al., 2002; Table 2.1).
2.5.2.2 Classical Taxonomy
Classical taxonomy has played a major role in the identification and nomenclature of new species
in the subgenus genus Glycine (Table 2.1). G. clandestina (2n = 40) has been observed to be a
morphologically highly variable species (Hermann, 1962). Stems of wild perennial species are twining,
climbing (Figure 2.12A*), or procumbent and exhibit morphologically distinct traits. G. cyrtoloba
and G. curvata contain curved pods (Figure 2.12B), G. microphylla, G. latifolia, and G. tabacina
carry adventitious roots, and falcate pods (Figure 2.12C) are a unique trait for G. falcata. Table 2.1
contains 26 wild perennial species of the subgenus Glycine and 2 species of the subgenus Soja.
2.5.2.3 Crossing Affinity
Crossability rate is an excellent indirect measure for estimating the degree of genomic relationship between parental species. Interspecific crosses involving parental species with similar genomes
usually set normal pods and seeds, while in crosses between genomically dissimilar species, seed
abortion is common and hybrids are sterile (Singh, 2003).
2.5.2.4 Chromosome Pairing
AU: "inferring" meant?
Insert page
number.
The degree of chromosome pairing in interspecific hybrids provides an important cytogenetic
context for interfering phylogenetic relationships among diploid species, enhances our understanding
* Color figures follow page xxx.
AU: Please
check wording: "subgenus genus."
3639_C002.fm Page 28 Wednesday, July 19, 2006 1:09 PM
28
GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
A
B
Figure 2.13
Meiosis in intragenomic F1 hybrid of G. latifolia (B1B1; 2n = 40) × G. microphylla (BB; 2n = 40).
(A) Metaphase I, showing 20 bivalents. (From Singh, R.J., unpublished result.) (B) Anaphase I,
showing a chromatin bridge and an acentric fragment (paracentric inversion) in an interspecific
hybrid of G. clandestina (A1A1; 2n = 40) × G. canescens (AA; 2n = 40). (From Singh, R.J. et al.,
Genome, 30, 166–176, 1988. With permission.)
of evolution of the genus, and provides information about the ancestral species. Generally, species
with similar genomes exhibit complete or almost complete chromosome pairing (intragenomic
chromosome pairing) in their hybrid (Figure 2.13A). Sometimes, species differ by chromosomal
interchanges or by paracentric inversion (Figure 2.13B). Based on classical taxonomy, G. soja and
G. max are different species (Hermann, 1962). However, both species carry 2n = 40 chromosomes,
hybridize readily, produce viable, vigorous, and fertile hybrids, and sometimes differ by a reciprocal
translocation (Karasawa, 1936; Palmer et al., 1987; Singh and Hymowitz, 1988) or by a paracentric
inversion (Ahmad et al., 1977). Therefore, G. soja and G. max are now assigned genome symbols
G and G1, respectively. In the genus Glycine, all F1 plants from crosses among A- (G. canescens,
G. argyrea, and G. clandestina) and B- (G. microphylla, G. latifolia, and G. tabacina) genome
species display 20 bivalents in the majority of sporocytes.
The extent of chromosome association in the hybrids of genomically dissimilar species elucidates structural homology in the parental chromosomes, and hence furnishes evidence regarding
the progenitor species (Singh, 2003). Usually the F1 generated from genomically unlike parents
(different biological species) are germinated through in vitro techniques. In general, hybrids are
weak, slow in vegetative and reproductive growth, and sterile. In the subgenus Glycine, A and
B genome species hybrids show an average chromosome association of 19.7I + 10.2II (A3 × B1)
and 20.9I + 9.5II (A × B1) (Figure 2.14). This suggests strongly that one genome is common in A
and B genome species. Furthermore, the common genome may be the progenitor species with
2n = 20 chromosomes. Hybrid seed inviability, seedling lethality, and vegetative lethality are common occurrences in intergenomic crosses (Singh et al., 1988). G. cyrtoloba (C genome) and
G. curvata (C1 genome) contain only a curved pod, a distinct morphological trait that distinguishes
these species from other species (Tindale, 1984). These species also express two pairs of nucleolus
3639_C002.fm Page 29 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
Figure 2.14
29
Meiosis in intergenomic F1 hybrid of G. latifolia (B1B1; 2n = 40) ∞ G. canescens (AA; 2n = 40);
metaphase I, showing 20 univalents + 10 bivalents. (From Singh, R.J. and Hymowitz, T., Theor.
Appl. Genet., 71, 221–230, 1985. With kind permission of Springer Science and Business Media.)
A
B
Figure 2.15
Meiosis in interspecific Glycine hybrids. (A) Metaphase I, showing 31 univalents + 4 bivalents in
G. tomentella (2n = 38; EE) × G. canescens (2n = 40; AA). (B) Metaphase I, showing 40 univalents
in G. clandestina (A1A1; 2n = 40) × G. canescens (AA; 2n = 40) × G. falcata (FF; 2n = 40). (From
Singh, R.J. et al., Genome, 30, 166–176, 1988. With permission.)
organizer chromosomes at mitotic metaphase by Feulgen staining and by fluorescent in situ hybridization (FISH) (Singh et al., 2001), while other species of the genus Glycine express one pair; it
is feasible that the second pair is either silent or has lost its NOR activity.
Variable (semihomologous-homoeologous) and minimal chromosome pairing are common in
intergenomic F1 hybrids. A wrong conclusion can be drawn if genome designation of species is
based on classical taxonomy. For example, aneudiploid (2n = 38) G. tomentella is morphologically
similar to 40-, 78-, and 80-chromosome tomentellas. By contrast, limited chromosome pairing was
observed between 40-chromosome G. tomentella and G. canescens (A genome) (Figure 2.15A).
G. falcata is morphologically distinct among 23 wild perennial species of the genus Glycine and
3639_C002.fm Page 30 Wednesday, July 19, 2006 1:09 PM
30
GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
2 species of subgenus G. soja. Chromosome pairing results (B1 × F, 37.8I + 1.1II; A × F,
38.7I + 0.6II) support the uniqueness of genome (F) of G. falcata because it showed the minimum
chromosome synapsis with A and B genomes (Figure 2.15B). Similar observation was recorded
by Doyle et al. (1996).
2.5.2.5 Molecular Methods
AU: Please
introduce
AFLP and
RAPD.
During the past decade, literature on genomic relationships (plant phylogenetic relationships)
has been dominated by molecular data, including nuclear (RFLP, AFLP, RAPD, SSR) sequences
variation in the gene, such as the internal transcribed spacer (ITS) region of rDNA, extranuclear
(chloroplast and mitochondrial) DNA variation, and genomic in situ hybridization (GISH) by
multicolor FISH. This latter approach is extremely powerful, where production of interspecific or
intergeneric hybrids is not feasible by conventional methods (Singh, 2003). Molecular tools verified
cytogenetic results that G. max and G. soja are genomically similar (Doyle, 1988; Zhu et al., 1995;
Kollipara et al., 1993, 1995, 1997). The sequence divergence between G. soja and G. max was
0.2% (Kollipara et al., 1997). Kollipara et al. (1997) determined phylogenetic relationships among
18 species of the genus Glycine and two species of the subgenus Soja from nucleotide sequence
variation in the ITS region of nuclear ribosomal DNA. This study helped to assign a genome symbol
to five species: H to Glycine arenaria, H1 to G. hirticaulis, H2 to G. pindanica, I to G. albicans,
and I1 to G. lactovirens. The cytogenetic relationship of these five species is unavailable, as they
are difficult to grow in the greenhouse at Urbana, IL, and verified by histone, H3-D gene sequences
and genomes were assigned to G. aphyonota (I3), G. peratosa (A5), G. pullenii (H3), and
G. stenophita (B3) (Brown et al., 2002; Doyle et al., 2002; A.H.D. Brown and J.J. Doyle, personal
communication, 2004). The ITS region (nrDNA) is a multigene family. However, in the soybean,
the nrDNA is mapped to a single locus on the short arm of chromosome 13 based on the location
of the nucleolus organizer region by pachytene chromosome analysis (Singh and Hymowitz, 1988)
and also by FISH using ITS as a probe (Singh et al., 2001). The wild perennial Glycine species
also contain one pair of NOR chromosome, like those in the soybean, except for G. curvata and
G. cyrtoloba, which have two NOR chromosomes (Singh et al., 2001).
Of the 26 wild perennial Glycine species, G. tomentella is unique because it constitutes four
cytotypes (2n = 38, 40, 78, 80). Aneudiploid (2n = 38) G. tomentella is distributed in a restricted
region of Queensland. The diploid (2n = 40) cytotype is distributed in Queensland, Northern
Territory, Western Australia, and Papua New Guinea. The isozyme banding pattern grouped aneudiploid into two isozyme groups (D1 and D2) and the diploid form into six isozyme (D3A, D3B,
D3C, D4, D5, and D6) groups (Doyle and Brown, 1985). Cytogenetics revealed that D1 and
D2 isozyme groups carry a similar genome, and Singh et al. (1988) assigned the E genome symbol.
The D4 isozyme group G. tomentella contains PI441000 (D3 genome) and has close affinity
cytogenetically with A genome species (Singh et al., 1988; Grant et al., 1984). Although the
D4 isozyme group is morphologically distinct from the A genome species, it does have long and
narrow leaves and a longer pod length, which is a characteristic feature of A genome species that
distinguishes it from other diploid G. tomentella accessions (Kollipara et al., 1998). Based on DNA
sequence variation at the single-copy nuclear locus histone H3-D, Brown et al. (2002) also grouped
D4 isozyme accessions with A genome species; D4 isozyme group G. tomentella should be
designated the A6 genome symbol. They also separated the D5 isozyme group into D5A and D5B.
2.5.3
Polyploid Complexes of G. tabacina and G. tomentella
Of the 26 species of the subgenus Glycine, G. hirticaulis, G. tabacina, and G. tomentella contain
2n = 40 and 2n = 80 chromosomes. Furthermore, G. tomentella consists of aneudiploid (2n = 38)
and aneutetraploid (2n = 78) accessions. Tetraploid G. hirticaulis, described by Tindale and Craven
(1988), has restricted geographical distribution. Tateishi and Ohashi (1992) described G. dolichocarpa
3639_C002.fm Page 31 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
AU: Please
doublecheck use of
"to."
31
Tateishi and Ohashi to the 80-chromosome morphological variant G. tomentella of Taiwan. On the
other hand, G. tomentella (2n = 78) is found in Australia and Papua New Guinea and G. tomentella
(2n = 80) is distributed in Australia, Papua New Guinea, the Philippines, Timor Island of Indonesia,
and Taiwan. Tetraploid tabacinas and tomentellas are allopolyploid complexes of multiple origins
(Singh and Hymowitz, 1985c; Singh et al., 1989, 1994; Hsing et al., 2001; Doyle et al., 2002;
Rauscher et al., 2004). Classification of the polyploid G. tabacina and G. tomentella accessions
into discretely defined, reproductively isolated groups using various methods (morphological,
cytogenetic, biochemical, and molecular) helps us to better understand the origin of the species
complex (Kollipara et al., 1994).
AU: Please
provide a reference for.
2.5.3.1 Glycine tabacina (2n = 80)
AU: Please
indicate
1992a or
1992b.
Diploid G. tabacina is indigenous to Australia, while tetraploid (2n = 80) cytotype is found
sympatrically with diploids in Australia and in the islands of the south Pacific (New Caledonia,
Vanuatu, Fiji, Tonga, Niue) and west-central Pacific (Taiwan, Ryuku, Marianas) (Singh et al.,
1992b). Morphological observations (Costanza and Hymowitz, 1987), cytogenetic investigation
(Singh et al., 1987, 1992), and molecular studies (Doyle et al., 1990a, 1990b, 1999) have shown
two distinct groups in the 80-chromosome G. tabacina. It is an allopolyploid complex and is of
multiple origins. The one group contains adventitious roots (with adventitious roots (WAR)), while
the other group lacks adventitious roots (no adventitious roots (NAR)). All the intraspecific F1
hybrids within each group showed normal meiosis and complete fertility. However, F1 hybrids
between the groups were sterile owing to disturbed meiosis. At metaphase I, a model chromosome
association of 40I + 20II was recorded (Singh et al., 1987). This indicates that both groups have
one genome in common and differ for the second genome.
Singh et al. (1992b) proposed, based on cytogenetics, that the 80-chromosome G. tabacina (NAR)
is a complex, probably synthesized from the A genome (G. canescens, G. clandestina, G. argyrea,
D4 isozyme group G. tomentella), and G. tabacina (WAR) evolved through segmental allopolyploidy from the B genome (G. latifolia, G. mictophylla, G. tabacina). Doyle et al. (1999b) suggested,
based on sequencing of histone H3-D locus, the multiple origins with gene exchange among lineage
increases the genetic base of a polyploid and helps in better colonization of polyploid G. tabacina
relative to its diploid progenitors. Hybridization is unlikely in a highly self-inbreeder in nature;
however, F1 hybrids among B genome species are completely fertile (Putievsky and Broué, 1979;
Newell and Hymowitz, 1983; Grant et al., 1984; Singh and Hymowitz, 1985c). Since B genome
species are sympatric (Doyle et al., 1999a), adventitious root trait is controlled by a recessive gene
(Singh et al., 1987) and Bowman–Birk inhibitor (BBI) is present in A genome species, including
80-chromosome G. tabacina (NAR), but absent in B genome species and 80-chromosome
G. tabacina (WAR). This suggests segmental allotetraploid origin of 80-chromosome G. tabacina
(WAR) and true allotetraploid origin of 80-chromosome G. tabacina (NAR) (Singh et al., 1992b).
2.5.3.2 Glycine tomentella (2n = 78, 80)
AU: Please
provide a reference for.
Diploid-like meiosis, isozyme banding patterns among the accessions and meiotic pairing in
intraspecific and interspecific F1 hybrids, wide geographical distribution, and aggressive and vigorous
growth habits suggest that 78- and 80-chromosome tomentellas are of allopolyploid origin and are
polyploid complex (Putievsky and Broué, 1979; Newell and Hymowitz, 1983; Grant et al., 1984; Singh
and Hymowitz, 1985a, 1985b, 1985c; Singh et al., 1987, 1989, 1994; Doyle et al., 1986). Morphologically, four cytotypes (2n = 38, 40, 78, 80) are indistinguishable. This suggests that one or both ancestors
of 78- and 80- chromosome tomentellas may be 38- and 40- chromosome G. tomentella.
The isozyme banding pattern revealed three groups (T1, T5, T6) in aneutetraploid and three
groups (T2, T3, T4) in tetraploid G. tomentella (Doyle and Brown, 1985, 1989; Doyle et al., 1986).
Cytogenetics, biochemical, and molecular methods verified isozyme results and clearly demonstrated
AU: Please
specify
1999a or
1999b.
3639_C002.fm Page 32 Wednesday, July 19, 2006 1:09 PM
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
the distinct reproductively isolated genomic groups in aneutetraploid and tetraploid tomentellas
(Kollipara et al., 1994). This strongly supports three distinct isozyme and genomic groups for
aneutetraploid T1 (D3E genome), T5 (AE genome), and T6 (D1E genome) and tetraploid T2
(D1A genome), T3 (D1D2 genome), and T4 (D1D3 genome) G. tomentella. Various groups within
78- and 80-chromosome tomentellas originated in Australia by allopolyploidization, most likely
through multiple independent events (Kollipara et al., 1994; Doyle et al., 1999b).
2.5.4
Chromosomal Aberrations: Structural Changes
Chromosomal structural changes such as deficiencies, duplications, interchanges, and inversions
have not been systematically produced, identified, and used in physical genetic mapping in soybean.
An interchange of spontaneous origin in soybean (Sadanaga and Grindeland, 1984) has been used to
locate the w1 (white flower) locus on the satellite chromosome (chromosome 13) in soybean. Palmer
et al. (1987) surveyed 56 G. soja accessions from China and the Soviet Union, which also included
PI81762 studied by Singh and Hymowitz (1988). They concluded that these accessions have a single
similar or identical interchange. Singh and Hymowitz (1988) examined an interspecific F1 hybrid of
soybean ∞ PI81762 and observed that one quadrivalent was always associated with the nucleolus.
Inversions (paracentric and pericentric) are neither produced nor used in physical mapping of
soybean genome. Study has been limited to identifying a paracentric inversion in the soybean ×
G. soja hybrid (Ahmad et al., 1977; Palmer et al., 2000). Wild perennial Glycine species with
similar genomes are differentiated by a paracentric inversion (Singh, 2003), as the majority of
sporocytes show normal pairing at metaphase I, but at anaphase I a chromatin bridge and an acentric
fragment are observed.
2.5.5
Chromosomal Aberrations: Numerical Changes
2.5.5.1 Autopolyploidy
Haploid (Crane et al., 1982), triploid (Chen and Palmer, 1985), and tetraploid soybean plants have
been reported. Haploid and triploid are completely sterile, and tetraploid soybean produces few one
or two large seeded pods. Tetraploid soybean has no commercial value. A tetraploid × diploid cross
has failed to produce an autotriploid, an excellent source for producing primary trisomics (Singh,
2003). However, Chen and Palmer (1985) identified autotriploid from the progenies of male-sterile
lines, but the derived autotriploid was not used to produce primary trisomics. Xu et al. (2000a)
found a hypertriploid (2n = 3x + 1 = 61) plant from a cross T31 (a homozygous recessive glabrous
(pp)) × T190-47-3 (an unidentified primary trisomic). The hypertriploid plant produced 98 selfed
seeds, and the chromosome number ranged from 2n = 50 to 69. The chromosome number in
hypertriploid ∞ diploid seeds ranged from 2n = 44 to 56.These lines were not used to produce
primary trisomics.
2.5.5.2 Aneuploidy
2.5.5.2.1 Primary Trisomics
AU: Please
specify
2000a,
2000b, or
2000c.
Primary trisomics in soybean (an individual with normal chromosome complements plus an
extra complete chromosome; 2n = 2x + 1 = 41) have been isolated from the progenies of asynaptic
and desynaptic mutants (Palmer, 1976; Gwyn et al., 1985; Xu et al., 2000). Four primary (2n = 41)
trisomics (Tri A, B, C, and D) examined by Gwyn et al. (1985) were similar to the diploid (2n = 40),
and this was attributed to the polyploid nature of soybean (Palmer, 1976). Skorupska et al. (1989)
identified Tri S that contained three satellite chromosomes. Singh and Hymowitz (1988) developed
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SOYBEAN (GLYCINE MAX (L.) MERR.)
33
A
B
Figure 2.16
Meiotic chromosome configurations at metaphase I in Triplo 20 of the soybean. (A) A cell with 1III
(arrow) + 19II. (B) A cell with 20II + 1I. (From Xu, S.J. et al., Crop Sci., 40, 1543–1551, 2000.
With permission.)
a chromosome map for soybean by pachytene chromosome analysis. Singh and Hymowitz (1991)
identified, by using a pachytene chromosome, Tri A as Triplo 5, Tri C as Triplo 1, Tri D as Triplo 4,
and Tri S as Triplo 13. Xu et al. (2000c) verified the identification of these four primary trisomics.
They isolated and tentatively identified 16 additional primaries from 37 aneuploid lines (2n = 41,
42, 43). These aneuploid lines originated from the progenies of asynaptic and desynaptic mutants
that were supplied by Reid Palmer. Triplo 1 contains the longest chromosome and Triplo 20 the
smallest chromosome.
At metaphase I of meiosis, a majority of microsporocytes in primary trisomics exhibit either
1III + 19II (Figure 2.16A) or 20II + 1I (Figure 2.16B). The average female transmission of
20 soybean primary trisomics was 42%, with a range of 27 (triplo 20) to 59% (triplo 9). The female
transmission rate has been estimated from the hybrid population (Xu et al., 2000). This may be
the reason for the high female transmission rate of the extra chromosome in primary trisomics of
soybean; heterozygosity often favors the higher female transmission rate (Singh, 2003).
Three marker genes, Eu1 (seed urease), Lx1 (lipoxygenase 1), and P2 (puberulent), were located
on chromosomes 5, 13, and 20, respectively. Zou et al. (2003b) associated yellow leaf mutant y10
to chromosome 3 by the primary trisomics method.
2.5.5.2.2 Monosomics
In soybean, monosomics (2n – 1 = 39; an individual lacking one chromosome is called
monosomic) have been isolated in the progenies of Triplo 3 and Triplo 6 and were designated
as mono-3 and mono-6 (Xu et al., 2000b). Morphologically, mono-3 was smaller with reduced
vigor while mono-6 was similar to the disomics. Female transmission in mono-3 was 6.5%, while
mono-6 was not transmitted among 105 S1 plants. Skorupska and Palmer (1987) reported two
monosomic plants among 94 S1 KS-6 progeny. The monosomics were not identified for a
particular chromosome. The transmission rate in monosomics is sporadic in soybean. It was
concluded that monosomics in soybean are viable and fertile and can be produced; however, no
systematic effort is being made to isolate monosomics in this economically important crop.
AU: Please
specify
2000a,
2000b, or
2000c.
3639_C002.fm Page 34 Wednesday, July 19, 2006 1:09 PM
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
Figure 2.17
A mitotic metaphase cell of soybean tetrasomic for chromosome 13 showing four SAT chromosomes (arrows). (From Singh, R.J., Plant Cytogenetics, 2nd ed., CRC Press, Boca Raton, FL,
2003. With permission.)
2.5.5.2.3 Tetrasomics
In soybean, tetrasomics (2n + 2 = 42), an individual carrying two extra chromosomes in addition
to its normal somatic chromosome complement (Figure 2.17), are identified in low frequencies
from the selfed progenies of primaries (2n = 41) (R.J. Singh, unpublished results). Soybean
tetrasomics are viable and, compared to their counterpart, primary trisomics, are slow in vegetative
and reproductive growth and partially to completely fertile. Primary trisomics are morphologically
indistinguishable except triplo 1, 13, and 17, but the extra two chromosomes cause morphological
characteristic modification in tetrasomics. Gwyn and Palmer (1989) observed, based on morphological measurement, that tetrasomics and double trisomics (2n + 1 + 1) could be distinguished
accurately from their disomics sibs. Tetrasomics mostly breed true, and occasionally related
trisomics (2n = 41) and diploids (2n = 40) are identified. Most primary trisomics plants are produced
from tetrasomics ∞ disomics crosses. Tetrasomics in the soybean are unique cytogenetic stocks, as
they are unviable in diploid crops such as maize, barley, rice, and tomato. The occurrence of
tetrasomics suggests that soybean is an ancient tetraploid but behaves like a true diploid.
2.5.6
AU: Please
rewrite for
sense.
Linkage Mapping
Chromosome, genetic, and cytogenetic maps in the model economically important crops like
rice, maize, barley, wheat, and tomato were developed first, and molecular maps followed. By
contrast, several molecular maps have been developed first in the soybean and are now being
associated with the chromosomes by primary trisomics.
2.5.6.1 Chromosome Map
Although the precise chromosome number of soybean was determined in 1925 (Karpechenko,
1925), the chromosome map was not developed based on somatic metaphase chromosomes because
chromosomes are symmetrical and only a pair of nucleolus organizer chromosomes is identified
in one of the best chromosome spreads. Singh and Hymowitz (1988) constructed a chromosome
map of soybean by using pachytene chromosomes (Figure 2.10). This pioneering research has set
the stage to produce all possible primary trisomics in the soybean (Xu et al., 2000c).
2.5.6.2 Classical Genetic Linkage Map
A genetic linkage map with 20 linkage groups, designated the Classical Genetic Linkage
Map (CGLM) of soybean, has been proposed (Palmer et al., 2004). The CGLM linkage groups
2, 3, 12, 13, 15, 16, 18, 20, and (21?); each has two qualitative trait loci. Thus, the genetic
3639_C002.fm Page 35 Wednesday, July 19, 2006 1:09 PM
SOYBEAN (GLYCINE MAX (L.) MERR.)
35
linkage map of soybean is not saturated with classical markers compared to other economical
important crops.
2.5.6.3 Molecular Linkage Map
In soybean, several types of DNA markers have been used to develop molecular linkage groups
(MLGs) (Cregan et al., 1999; Shoemaker et al., 2004). Cregan et al. (1999) and Song et al. (2004)
integrated several molecular linkage maps into one map with 20 molecular linkage groups. Song
et al. (2004) used the JoinMap software program to integrate the data from five soybean mapping
populations (Minsoy × Noir 1, Minsoy × Archer, Archer × Noir 1, Clark × Harosoy, A81-3560022
× PI468916). The integrated genetic map spanned 2523.6 cM of Kosambi map distance across
20 linkage groups and consists of 1849 markers, including 1015 SSRs, 709 RFLPs, 73 RAPDs,
24 classical traits, 6 AFLPs, 10 isozymes, and 12 others. However, MLGs are not all associated
with the individual soybean chromosomes. By using SSR markers from 20 MLGs and primary
trisomics, Zou et al. (2003a) found the following relationships between chromosomes and MLGs:
MLG
CGLM
Chromosome
A1
A2
B1
B2
C1
C2
D1a + Q
D1b + W
D2
E
F
G
H
I
J
K
L
M
N
O
—
07, 09
—
17
21
01
03
11
20
14
08, 13
18
20
04
19
02, 12
05
—
10
15
05
08
—
—
14
—
01
—
17
—
13
18
—
20
—
9
19
—
03
—
Note: MLG = molecular linkage group; CLGM = classical
linkage genetic map.
Nine MLGs have not been associated with the chromosomes. Segregation distortion is common
using primary trisomics and SSR markers. This may be due to the preferential selection of gametes
containing certain genotypes. Soybean scientists are focusing on sequencing the soybean genome,
but no major effort is under way to develop a universal cytogenetic map for the soybean, as has
been accomplished in other crops.
2.6 SOYBEAN GERMPLASM ENHANCEMENT
2.6.1
Conventional Breeding
Only a small fraction of the genetic diversity available is currently used in soybean breeding
worldwide. The two oldest and largest national breeding programs in the world are in China and
the U.S. The genetic base of soybean in the U.S. and Canada is narrow (Gizlice et al., 1996; Burton,
3639_C002.fm Page 36 Wednesday, July 19, 2006 1:09 PM
36
AU: Please
check year. If
correct,
please provide a reference for.
GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
1997; Singh and Hymowitz, 1999). Based on pedigrees of North American public soybean cultivars
released from 1947 through 1988, 6 ancestral lines supply 60% of the genetic base for U.S. soybean
production, and an additional 16 progenitors provide another 30% (Gizlice et al., 1996). Analyses
of DNA markers indicate that these ancestral lines are quite diverse (Thompson and Nelson, 1998)
and contain as much diversity as a relatively large set of exotic introductions that have been used
in recent population development and selection for yield improvement (Brown-Guedira et al., 2000;
Kisha et al., 1998; Li et al., 2001). Efforts to expand the genetic base of soybean production in the
U.S. are not new (Hartwig, 1973), but recent results have been more promising. Improved germplasm has been released that contained 50% exotic parentage by pedigree and exceeded the yield
of the best cultivars in regional testing, and another released line that is derived from 100% exotic
parentage equaled the best cultivar in regional testing (Nelson and Johnson, 2006).
The genetic base of soybean breeding in China is much larger than in the U.S. The pedigrees
of 651 Chinese soybean cultivars released from 1923 to 1995 contain 339 ancestors, and as many
as 190 ancestral lines contributed 80% of the genetic base of production (Cui et al., 2000). Based
on both pedigrees (Cui et al., 2000) and DNA markers (Li et al., 2001), the genetic bases of the
three major soybean-growing regions of China — northeastern, central, and southern — are distinct
and could be considered independent gene pools. The initiation or expansion of many breeding
programs in southern China and the intentional efforts to broaden the genetic base of soybean
cultivars in northern China have resulted in the incorporation of much new germplasm in the past
25 years (Carter et al., 2004).
Despite the apparent genetic limitation of a narrow genetic base for world soybean production,
soybean breeding has continued to make significant progress. Analyzing data collected from
60 years of cooperative regional tests in the production area of the U.S. and Canada, Wilcox (2001)
concluded that annual rates of yield improvement in kg ha–1 were 21.6 (MG 00), 25.8 (MG 0),
30.4 (MG I), 29.3 (MG II), 30.6 (MG III), and 29.5 (MG IV). Rates of yield improvement in recent
years were equal to or greater than those in earlier years. In 1961 the world average yield was
1.13 Mt/ha. In 2004, that had been raised to 2.23 Mt/ha (Figure 2.2). There are a variety of breeding
procedures that have been and are currently being used (Orf et al., 2004), but the use of single seed
descent and winter nurseries has had a major impact by greatly reducing the time from hybridization
to yield testing. Increased mechanization and computerization have also increased the efficiencies
of most breeding programs.
There is still a large discrepancy among mean yields of the top producing countries. The average
yield in Argentina, Brazil, and the U.S. from 2000 to 2005 was nearly 2.6 Mt/ha. During the same
period, the average yield in China was 1.7 Mt/ha and in India 0.9 Mt/ha (Figure 2.1). These values
represent not only the genetic potential of the cultivars within each country, but also differences
in environmental conditions. Lack of inputs, marginal soils, and complex cropping systems can
reduce yield.
Soybean contains several antinutritional factors. Kunitz trypsin inhibitor protein is one of the
major antinutritional elements present in raw mature soybeans. Kunitz (1945) isolated, identified,
and crystallized the protein that inhibited the proteolytic action of trypsin, which is commonly
known as Kunitz trypsin inhibitor (SBTI-A2). Treating the moist seed with heat destroys antinutritional factors. A mutant without SBTI-A2 has been identified (Singh et al., 1969), and Bernard
et al. (1991) registered L81-4590 as a Kunitz soybean (registration 271, PI542044) cultivar for
commercial production.
2.6.2
Interspecific Hybridization
Soybean breeders have not fully exploited the wealth of genetic diversity from exotic germplasm, including soybean’s progenitor G. soja (Singh and Hymowitz, 1999; Carter et al., 2004).
G. soja may be an excellent source of genetic variability, although it harbors several undesirable
genetic traits, for example, vining, lodging susceptibility, lack of complete leaf abscission, seed
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37
shattering, and small black seed coat (Carpenter and Fehr, 1986; Carter et al., 2004). However,
G. soja has been shown to be more genetically diverse than G. max (Choi et al., 1999; Li and
Nelson, 2001), and the undesirable traits can be separated from the desirable traits during the course
of selection in successive backcross generations and perhaps through marker-assisted selection.
Attempts to broaden the genetic base of soybeans by utilizing G. soja were reported by Hartwig
(1973), Ertle and Fehr (1985), Carpenter and Fehr (1986), and Carter et al. (2004). Hartwig (1973)
reported highly productive and high protein lines derived from soybean and G. soja hybrids. Ertle
and Fehr (1985) concluded that introgression of G. soja germplasm into the two soybean cultivars
was not an effective method for increasing their yield potential. To obtain a relatively high frequency
of useful segregates for cultivar development, three backcrosses to the soybean were preferred.
However, small-seeded (seed of <100 mg) cultivars, such as ‘SS201’, ‘SS202’ (Fehr et al., 1990a,
1990b), and ‘Pearl’ (Carter et al., 1995), have been developed where G. soja was used as a nonrecurrent
parent. The small-seeded cultivars are used for sprouts and the fermented Japanese product natto.
Qian et al. (1996) have recorded the accessions of G. soja that are potential sources of additional
genes that restrict nodulation of soybean with specific strains of Bradyrhizobium. They concluded
that introgression of such genes could result in soybean cultivars that exclude some of the indigenous
strains and become nodulated with commercial strains that are more efficient in fixing nitrogen.
Since 1970, soybean production increased rapidly in Brazil and Argentina, and the initial varietal
improvement program was from the germplasm introduced from the U.S. and other countries.
2.6.3
Intersubgeneric Hybridization
The 26 wild perennial species of the subgenus Glycine have not been exploited in soybean
breeding programs. These species are extremely diverse morphologically, cytologically, and
genomically, grow in very diverse climatic and soil conditions, and have a wide geographical
distribution (Singh and Hymowitz, 1999). Wild perennial Glycine species has great potential for
soybean improvement. They are a rich source of agronomically useful genes, such as resistance
to soybean rust (Phakopsora pachyrhizi Sydow), soybean brown spot (Septoria glycines Hemmi.),
powdery mildew (Microsphaera diffusa Cke. & Pk.), phytophthora root rot (Phytophthora sojae
H.J. Kaufmann & J.W. Gerdemann), white mold (Sclerotinia sclerotiorum (Lib. De Bary)), sudden
death syndrome (Fusarium solani (Mart.) Sacc.), tobacco ring spot, yellow mosaic virus, alfalfa
mosaic virus, and soybean cyst nematode (SCN) (Heterodera glycines Ichinohe), and tolerance
to certain herbicides and salt (Singh and Hymowitz, 1999).
Soybean rust is one of the major soybean diseases in China, Thailand, India, Australia, and
Taiwan. A significant reduction (80%) in yield may be caused by the pathogen (Hartman, 1996).
Soybean rust has been reported in Puerto Rico and Brazil (Bonde and Peterson, 1996). Killgore
(1996) reported soybean rust on vegetable soybeans grown on the islands of Kauai, Oahu, and
Hawaii. This suggests that soybean rust is a great threat to mainland U.S. soybean production.
Significant yield loss (>10%) is predicted in nearly all soybean-growing areas. However, the greatest
loss (up to 50%) could occur in the Mississippi delta and the southeastern coastal areas (Yang,
1996). On November 10, 2004, the U.S. Department of Agriculture announced the presence of
soybean rust on soybean leaf samples taken from two plots associated with a Louisiana State
University research farm on November 6. This was the first instance of soybean rust to be found
in the continental U.S. (http://www.asa-cssa-sssa.org/soybean_rust.html).
Several researchers have attempted to hybridize wild perennial Glycine species with the soybean,
but only a few sterile intersubgeneric F1 hybrid combinations have been reported (Newell et al.,
1987; Singh et al., 1999). Thus far, only Singh et al. (1990, 1993) have successfully produced
backcross-derived fertile progenies from the soybean and a wild perennial, Glycine tomentella
(2n = 78). Monosomic alien addition lines (MAALs) and modified diploid (2n = 40) lines are being
isolated and identified (Singh et al., 1998a). The modified diploid lines could be screened for pests
and pathogens. Riggs et al. (1998) reported the introgression of SCN resistance from G. tomentella
3639_C002.fm Page 38 Wednesday, July 19, 2006 1:09 PM
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
into modified derived diploid soybean lines. This study sets the stage for the exploitation of perennial
germplasm to broaden the genetic base of the cultivated soybean.
An extensive wide hybridization between soybean and 78-chromosome G. tomentella has been
reinitiated by the authors. Several combinations of F1 hybrids involving four soybean cultivars
(Dwight, Ina, Macon, and IA 3010) and two 78-chromosome G. tomentella (PI441001, PI441008)
have been produced. Amphidiploid and backcross generations are produced in a systematic way
(R.J. Singh, unpublished results).
2.6.4
AU: What
does "Nr."
stand for? If
"number,"
can delete.
Mutation Breeding
Mutation breeding in soybeans has lagged behind that of other economically important crops.
Micke et al. (1985) compiled information on cultivars produced using induced mutations. They
listed 17 soybean cultivars developed by various mutagens: 10 cultivars from China (Hei Noun 4,
5, 7, 8, 16, and 26; Mu Shi 6; and Tai Nung 1(R) and 2(R); and Tie Feng 18); 3 cultivars from
Japan (Nanbushirome, Raiden, and Raiko); and one cultivar from each of Bulgaria (Boriana),
Algeria (Cerag Nr. 1), Korea (KEX-2), and the former U.S.S.R. (Universal I). All cultivars from
Japan were high yielding, because they were resistant to nematodes. Cultivar KEX-2 from Korea
was earlier maturing (11 days) with a higher yield (ca. 16%) and larger seed.
Karmakar and Bhatnagar (1996) listed 43 soybean cultivars released in India from 1969 to
1993. Three cultivars (Birsa Soy1, VL Soy1, and NRC2) were developed by mutagenesis, five
cultivars (Bragg, Lee, Improved Pelican, Hardee, and Monetta) were direct introductions from the
U.S., and the remaining cultivars were selected from introductions and single crosses (two parents).
Buss (1983) isolated a recessive genetic male-sterile (gms) line from a M3 generation of ‘Essex’
soybean that had been irradiated with neutrons. Allelism tests revealed that the gms line inherited
independently from ms1, ms2, ms3, and ms4. Thus, the newly identified ‘Essex’ gms gene was
assigned the symbol ms5.
By using chemical mutagenesis (ethyl methanesulfonate (EMS), N-nitroso-N-methylurea
(NMU), or ethyl nitrosourea (ENU)), Sebastian and Chaleff (1987) and Sebastian et al. (1989)
isolated soybean lines with increased tolerance for sulfonylurea herbicides. Sebastian and Chaleff
(1987) identified four single recessive genes. Allele tests revealed that each mutation resided at one
of three loci (hs1, hs2, or hs3). They observed, in biochemical studies, that the mutants did not
contain an altered form of acetolactate synthase (the site of action of sulfonylurea herbicide). In
subsequent studies, Sebastian et al. (1989) identified a monogenic semidominant mutant that was
nonallelic to the hs1, hs2, and hs3 genes. They assigned the gene symbol Als1 to the line that was
resistant to the action of sulfonylurea herbicide.
Carroll et al. (1985) mutagenized soybean seeds of cv. Bragg with EMS. They isolated
15 independent nitrate-tolerant symbiotic (nts) mutants from 2500 M2 families. Mutant nts382 was
studied extensively. In the presence of KNO3, nts382 produced six times more nodules than those
observed in control ‘Bragg’ grown under identical culture conditions. Song et al. (1995) evaluated
yield, N2 fixation, and the effects on cereal crops grown subsequent to the harvest of intermediate
supernodulating (two times), extreme supernodulating (six times), and nonnodulating mutants of
‘Bragg’, genotypes derived from the mutants, and commercial cultivars. The experiment was
conducted for 6 years at two locations. The results were as follows: (1) the supernodulators and
‘Manark’ were similar, with values 13 to 21% above those for ‘Centaur’; (2) in the plots fertilized
with nitrogen, the supernodulators exhibited higher activity than the commercial cultivars, including
‘Manark’; (3) grain yield of the supernodulators was either the same or up to 25% less than those
of ‘Bragg’ and ‘Centaur’; and (4) oats and barley sown immediately after soybean harvest produced
significantly greater yields than after commercial soybean cultivars.
Soybean seed oil is the major vegetable oil (53%) among oilseed crops produced in the world
(Figure 1.1). Genetic studies have elucidated that oil synthesis in soybean is determined largely by
the genotype of the maternal plants, because the oil content of F1 plants was not significantly
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Table 2.2 Changes in Fatty Acid Content in Soybean Produced through Mutagensis
Fatty
Acids
Palmitic
Content
Level
High
Low
Stearic
High
Oleic
High
Linolenic
Low
High
Low
AU: Is this
the source of
table, or just
a reference?
Mutagen
Reference
Ethyl methanesulfonate
Wilcox and Cavins, 1990;
Fehr et al., 1991
Fehr et al., 1991
Wilcox and Cavins, 1990
Fehr et al., 1991
Hammond and Fehr, 1983b
Rahman et al., 1997
Brossman and Wilcox, 1984
Rahman et al., 1996
Hammond and Fehr, 1983b
Brossman and Wilcox, 1984
Takagi et al., 1989
Hammond and Fehr, 1983a;
Brossman and Wilcox, 1984
N-nitroso-N-methyl-urea
Ethyl methanesulfonate
N-nitroso-N-methyl-urea
Sodium azide
X-rays
Ethyl methanesulfonate
X-rays
Sodium azide
Ethyl methanesulfonate
X-ray
Ethyl methanesulfonate
Table 2.3 Fatty Acid Composition of Mutants A5 and A6 and Their parents, FA9525 and
FA8077 (Hammond and Fehr, 1983a, 1983b)
Mutagen
EMS
Sodium azide
Genotype
A5
FA9525
A6
FA8077
Palmitic
16:0
9.3
9.3
8.0
8.4
Fatty Acid (%)
Stearic
Oleic
Linoleic
18:0
18:2
18:2
3.9
3.1
28.1
4.4
39.8
39.1
19.8
42.8
42.9
42.9
35.5
36.7
Linolenic
18:3
4.1
6.3
6.6
7.6
Arachidic
2.0
<1.0
different from those of selfed seeds of the female parent (Singh and Hadley, 1968). Similarly, fatty
acid composition in soybean seed is determined by the maternal parent (Hammond et al., 1972).
Breeding efforts to increase soybean oil above approximately 20% have been unsuccessful, because
oil content and seed yield have a negative relationship (Burton, 1985).
Soybean breeders initiated programs to improve soybean oil quality (Table 2.2). The principal
fatty acids in soybean oil are palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic
(18:3). A common soybean cultivar contains 11% palmitic, 3% stearic, 22% oleic, 56% linoleic,
and 8% linolenic acid (Wilcox, 1985). The high linolenic acid content (7 to 9%) is associated with
poor flavor (fishy, painty, grassy, melony) stability in soybean oil (Dutton et al., 1951). Mounts
et al. (1988) analyzed the fatty acid composition of more than 5000 soybean samples from both
northern and southern soybean germplasm collections and identified one line, PI361099B, with
low linolenic (4.2%) and normal oleic acid content. The low linolenic acid content of PI361088B
remained stable regardless of environmental conditions, which suggests that soybean germplasm
lacks a strain with a linolenic acid content of 3% or less (Hammond and Fehr, 1975).
Mutagenesis has been an excellent tool for creating variability for fatty acid content in soybeans.
From the M4 generation, following the use of EMS, Hammond and Fehr (1983a) selected a line,
designated A5, that contained an average of 4.1% linolenic acid, while the parent (FA9525) contained
6.3%; the content of other fatty acids remained unchanged (Table 2.3). They also isolated a line with
an elevated stearic acid (28.1%) content and a marked reduction in oleic acid (19.8%), designated
A6, from an M2 population of sodium azide-treated seeds of FA8077 (Hammond and Fehr, 1983b;
Table 3.3). Wilcox et al. (1984) identified a genetically stable low linolenic acid (3.4%) mutant from
ca. 15,000 M2 plants, where seeds of soybean cv. Century had been treated with EMS. The linolenic
acid content of seeds in the M2 populations ranged from 3.4 to 11.1%, and for ‘Century’, it ranged
from 6.6 to 9.4%. In contrast, Takagi et al. (1989) developed a line with high linolenic acid content
(18.4%) by treating seeds of cv. Bay with x-ray irradiation. ‘Bay’ contains 9.4% linolenic acid.
3639_C002.fm Page 40 Wednesday, July 19, 2006 1:09 PM
40
AU: Please
provide a reference for.
GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
Linolenic acid is essential in the mammalian diet. In some mammals, lack of linolenic acid causes
skin lesions, lowered learning ability, stunted growth, and mental retardation (Coscina et al., 1986).
Wilcox and Cavins (1987) assigned gene symbols for linolenic acid content: Fan Fan for high
levels (= 7.2 ± 0.11%), Fan fan for intermediate levels (= 5.2 ± 0.07%), and fan fan for low levels
(= 3.2 ± 0.13%). They observed that linolenic acid content was controlled by the genotype of the
embryo rather than by the genotype of the maternal parent. Rahman et al. (1994) observed no
maternal and cytoplasmic effects for linolenic acid content. It has also been demonstrated that low
linolenic acid content is a quantitative trait (Fehr et al., 1992).
Palmitic (16:0) and stearic (18:0) acids are the two main saturated fatty acids in the soybean.
Fehr et al. (1991) produced a mutant containing reduced palmitic acid content by treating soybean
cv. A1937 with NMU. Low palmitic acid content was controlled by two different alleles at two
different loci. They assigned the genotypes fap1 fap1 and fapx fapx. This line contains 44 g kg–1
palmitic acid, the lowest content known in soybean.
Wilcox and Cavins (1990) isolated two mutants, C1726 (registration GP-116; PI532833) and
C1727 (registration GP-117; PI532834) from cv. Century by EMS treatment. Mutant C1726 contained 8.5% palmitic acid, and mutant C1727 contained 17.2% palmitic acid, while ‘Century’ had
11.2%. Both mutants bred true for low and high palmitic acid content. Genetic studies revealed
that alleles from two independent loci segregated for palmitic acid percentage and that the gene
action was additive. The gene symbol fap1 was assigned to an allele in C1726 that acts to lower
the palmitic acid level in soybean oil, and fap2 was assigned to an allele in C1727 that acts to
increase the palmitic acid level (Erickson et al., 1988). A reduction in palmitic acid content improves
the quality of oil. An elevated palmitic acid content enhances its use in the production of food
products, such as shortening and margarine (Schnebly et al., 1994).
Rahman et al. (1996) examined the genetics of mutants with high oleic acid content (M11 and
M23) produced by x-ray irradiation. Low oleic acid content in cv. Bay was partially dominant to
the high oleic acid content in mutant M23, but completely dominant to the high oleic acid content
in mutant M11. An inverse relationship between oleic and linolenic acid content in both mutants
was recorded. Oil with high levels of oleic acid is less susceptible to oxidative changes during
refining, storage, and frying (Miller et al., 1987).
Mutagenesis can sometimes be used to break the linkage between two closely linked genes.
The grassy-beany flavor in soybeans and soybean products is caused by lipoxygenases. Three
soybean lipoxygenases (L1, L2, and L3) have been characterized. L1 and L2 are linked. Hajika
et al. (1995) isolated a line without L1, L2, and L3 liposygenases by -ray irradiation. Soybean
plants lacking lipoxygenases showed normal plant growth and yield. The production of soybeans
without lipoxygenases is cost-effective, because heat treatment to inactivate these enzymes will not
be required in the processing of soybean food products. Thus, mutation breeding provides an
alternative method to wide hybridization and biotechnology.
2.6.5
Biotechnology
Conventional plant breeding has failed to revolutionize gains in soybean yield. Biotechnology
is considered an innovative science with which to broaden the genetic base of crops by overcoming
the genetic barriers in extremely distant crosses. Biotechnological methods include somaclonal
variation, cybrids, and recombinant DNA technology. Thus, genetic engineering is one of the
alternatives for developing high-yielding soybeans with high protein and oil content, resistance to
pests and pathogens, and tolerance to herbicides.
2.6.5.1 Plant Regeneration from Callus and Cell Suspension Cultures
Soybeans have received attention from tissue culture scientists of both public and proprietary
institutes in order to generate normal soybean plants with increased genetic variability. However,
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soybean has been one of the most recalcitrant plant species as far as plant regeneration from tissue
cultures is concerned (Lippmann and Lippmann, 1984).
Ranch et al. (1985) first achieved a controlled somatic embryogenesis system for initiation,
proliferation, and fertile plant regeneration using Murashige and Skoog (1962; MS) medium with
2,4-dichlorophenoxyacetic acid (2,4-D). Barwale et al. (1986) also regenerated plants via embryogenesis and shoot organogenesis by changing medium composition; embryogenesis resulted when
explants were planted on MS medium containing naphthalene acetic acid (NAA), while the addition
of benzylaminopurine (BAP) with a high concentration of MS minor salts resulted in organogenesis.
However, these regeneration systems are relatively inefficient, complicated, and genotype dependent. A simple, efficient, and more rapid regeneration procedure through somatic embryogenesis
(Komatsuda et al., 1992; Samoylov et al., 1998) and shoot organogenesis (Shetty et al., 1992;
Reichert et al., 2003) has been established. The regeneration efficiency from soybean cell cultures
is highly dependent on genotype regardless of the protocol routes (Komatsuda and Ohyama, 1988;
Hofmann et al., 2004), but genotype-independent regeneration protocols have also been reported
(Bailey et al., 1993; Tomlin et al., 2002; Reichert et al., 2003; Sairam et al., 2003). It is concluded
that the early maturity genotypes, immature embryos, and MS-based medium are ideal factors for
plant regeneration in soybean tissue culture. Morphological variants in soybean have been obtained
through cell and tissue culture (Graybosch et al., 1987; Bailey et al., 1993), and although these
research efforts failed to deliver high-yielding soybeans, methodologies were developed to regenerate complete soybean plants, a prerequisite for genetic transformation.
Soybean has been regenerated by suspension cultures. Christianson et al. (1983) regenerated
through embryogenesis from immature embryo-derived cell cultures at very low frequency. Soon
after, Li et al. (1985) reported a regeneration system by single cells derived from the frozen
immature embryos. Suspension cultures are potentially useful for the application of modern
biotechnologies to soybean improvement, particularly for the selection of mutant cell lines, if
cultures were totipotent.
Haploid induction through anther culture is a useful tool for the production of homogeneous
plants; microspore-derived whole plant production has not yet been reported in soybean, except
callus development from anthers cultured on several media modifications (de Moraes, 2004).
2.6.5.2 Protoplasts Culture
Protoplasts provide techniques for genetic manipulation and plant improvement programs at
the cellular level, in particular the induction of somaclonal variation, somatic hybridization, and
transformation. Because of economic significance of soybean, researchers have long sought to
improve and optimize the protoplast culture system. Plant regeneration from soybean protoplasts
has been difficult. There are only few reports where success has been achieved in plant regeneration
from soybean protoplasts. Wei and Xu (1988) first established a routine plant regeneration system
from immature cotyledon protoplasts.
2.6.5.3 Genetic Transformation
AU: Please
provide a reference for.
Foreign genes of economic importance can be delivered into soybeans by Agrobacterium
(Hinchee et al., 1988; Olhoft et al., 2001) and particle bombardment (Sato et al., 1993; Kinney,
1996; reviewed by Finer et al., 1995; Christou, 1997; Furutani and Hidaka, 2004). Padgette et al.
(1995) reported a stable glyphosate-tolerant soybean line (known as Roundup Ready®) that had
been developed using the Agrobacterium-mediated gene transfer method.
Kinney (1996) produced a high oleic acid content (84%) soybean through particle bombardmentmediated transformation. The high oleic acid-containing transgenic soybean lines were stable over
a number of different environments during a single growing season and were competitive in terms
of yield with the parental commercial soybean line.
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provide a reference for.
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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT
High lysine (up to 12%) soybeans lines have been produced by transformation. Normal soybeans
contain about 6% lysine. The high lysine trait was stable in R2 and R3 seeds. Soybean transformants
with a lysine content higher than 15% carried wrinkled seed coat and exhibited poor germination
(Falco et al., 1995).
Soybean transformation methods are not routinely reproducible (Christou, 1997). Soybean
transformations are often sterile, and sterility is attributed mostly to chromosomal aberrations (Singh
et al., 1998c). Frequently, unexpected segregations and low expression or disappearance of foreign
genes have been observed. Genes may be physically present but may be poorly expressed or totally
lost in subsequent generations. This may be explained by the poorly understood phenomenon of
cosuppression or gene silencing (Stam et al., 1997).
2.6.6
AU:
"escapes"
correct here?
Potential to Produce Hybrid Soybeans
Attempts to produce commercial hybrid soybean cultivars have not succeeded because (1 a good
system of producing male-sterile plants is generally not available; (2) soybean pollen must be carried
by insect vectors and soybean flowers are generally unattractive to these insects, so even on malesterile plants seed set is often low; and (3) the difficulty in producing hybrids greatly limits the parental
combinations that can be tested in order to find commercially acceptable heterosis. Patent 4,545,146
(October 8, 1985) has been granted for hybrid soybean production (Davis, 1985). The methodology
remains on the books, but its application in hybrid soybean production has not been realized.
Sun et al. (1997) isolated a stable, cytoplasmic-nuclear male-sterile soybean line (cms; A line)
and its maintainer (B line) from an interspecific hybrid between G. max and G. soja. Average pollen
sterility in all BC4 plants was about 98%, and the parallel crosses showed that the female was
normal. This system has been used to develop experimental hybrid cultivars.
Several genic (nuclear) male-sterile (gms) soybean lines (ms1 through ms9) are available, and
this literature was summarized by Palmer et al. (2004). Jin et al. (1997) recently identified a gms
mutant not allelic to any previously described soybean gms lines. Male-sterile lines can be used
to produce hybrid seeds. More than 99% of the seed set on monogenic ms1 ms1 male-sterile plants
is the result of natural crossing (Brim, 1973).
Distinguishing morphological markers that are visible in seedlings and tightly linked with gms
would facilitate early identification of gms plants (Skorupska and Palmer, 1989). Skorupska and
Palmer (1989) recorded close linkage between the w1 locus (white flower and green hypocotyls)
and the ms6 locus. By utilizing w1 ms6 genetic stock, Lewers et al. (1996) suggested a cosegregation
method for hybrid soybean production: purple hypocotyl seedlings are removed shortly after
germination, leaving only male-sterile plants, and escapes and recombinants with purple flower are
removed manually at flowering. They used the terms traditional and dilution to describe the methods
for hybrid soybean seed production. The cosegregation method produced higher seed yield, better
efficiency, and equal or better seed quality than the traditional and dilution methods. The cosegregation method may be used for male-sterile-facilitated selection, and the cyclic mating system and
marker-assisted recurrent selection (Lewers and Palmer, 1997) for cultivar development.
The degree of heterosis is an important issue in hybrid soybean production. Nelson and Bernard
(1984) examined 27 hybrid combinations. Five hybrids yielded 13 to 19% more than their better
parent in at least one season. Manjarrez-Sandoval et al. (1997) recorded heterosis of yield as high
as 11% across locations. This may justify resources for breeding hybrid soybean cultivars, but the
continual improvement of inbred cultivars will make this a difficult goal to achieve.
2.7 SUMMARY
World soybean production has doubled in the past 20 years to over 200 million metric tons
in 2004. This increase was made possible with over a 70% increase in the area harvested and a
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30% increase in yield. Despite this enormous increase, demand has kept pace with supply. There
is every indication that demand for soybean products will continue to increase for both oil and
protein from commodity soybeans and products from specialty cultivars. Meeting this demand will
be a challenge for breeders and geneticists that will require innovation in technology and an
expansion of the genetic resources that are employed in developing improved cultivars. Accomplishing this goal will require significant cooperation among a wide variety of scientists in both
public institutions and commercial companies, and finding ways of overcoming legal barriers that
prevent access to needed genetic resources.
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