Asian Vigna Genome Research
Kaga Akito1, Takehisa Isemura1, Takehiko Shimizu2, Prakit Somta3, Peerasak Srinives3,
Satoshi Tabata4, Norihiko Tomooka1 and Duncan Vaughan1
1. National Institute of Agrobiological Sciences , Tsukuba, Ibaraki, 305-8602, Japan
2. Institute of Society for Techno-Innovation of Agriculture , Forestry and Fisheries (STAFF ), Kamiyokoba
Ippaizuka 446-1, Tsukuba, Ibaraki 305-0854, Japan
3. Kasetsart University , Nakhon Pathom 73140, Thailand.
4. Kazusa DNA Research Institute , Kazusa Kamatari 2-6-7, Kisarazu, Chiba 292-0818, Japan

Abstract
To make progress in genome analysis of the Asian Vigna cultigens, genetic linkage map
for azuki bean (Vigna angularis ), mungbean (V. radiata ), black gram (V. mungo ) and rice bean
(V. umbellata ), among six fully domesticated Vigna species in Asia, have been constructed using
mapping populations between cultigens and their presumed wild ancestors mainly based on azuki
bean genomic SSR markers. Newly developed cowpea genomic SSR markers and soybean EST-SSR
markers have been integrated into the mungbean linkage map. Simultaneously, we have constructeda detailed comparative genome map across four Asian Vigna species based on these linkage maps.
Comparison of the order of common azuki bean SSR markers and RFLP markers on the linkage maps
allowed detection of high level macro-synteny between genomes of the four Asian Vigna species.
The azuki bean, mungbean, black gram and rice bean have been domesticated from 4 different
wild species in different locations in Asia. These cultigens have parallel remarkable morphologicaland physiological changes the so-called domestication syndrome. The traits related to thedomestication syndrome are controlled by several major and some minor QTLs. These QTLs have atendency to be clustered and non-randomly distributed across the genome. Based on the comparative
genome map, the genomic regions of the QTL clusters in mungbean and black gram are different
from those of azuki bean and rice bean. Many orthologous QTLs for seed size, seed shattering andplant height were identified. In contrast, different sets of alleles on QTLs controlling seed dormancy
and flowering time appears to be used in each species. The present studies on comparative analysisof domestication or adaptation traits that played a major role on the evolutionally changes from wild
relative into crop offer further evolutionary potential for Asian Vigna cultigens.
The Asian Vigna comparative map is being used to develop comparative map between Asian
Vigna and soybean. Preliminary comparative approaches using sequence information of azuki bean,
cowpea and soybean SSR markers on the mungbean linkage map could infer presumed syntenic
regions between mungbean and soybean. Although much more information is required to test colinearity of markers, segmentations of soybean linkage block are frequently observed at most ofmungbean linkage groups. Further efforts are need to make steady progress on the establishment of agenomic base for the Asian Vigna by collaboration in order to utilize gene and sequence informationof soybean in Asian Vigna through comparative genome analysis.

Importance of the development of genomic resources and collaborative initiatives for the Asian
Vigna
The genus Vigna is a large genus with about
90 species distributed worldwide in warm and tropical regions. The genus includes 13 cultigens of thesecowpea, mungbean and azuki bean, are the mostimportant. Perhaps due to Vigna cultigens being
mainly crops in the developing world molecularmarker and genomic studies have lagged behind
other major crops. However, there has recently been
considerable progress in cowpea genome studies.
Cowpea Genome Initiative was set up in September
2000 by funding from the Kirkhouse Trust, a UKbased charity, and now provides cowpea sequence
database (http://cowpeagenomics.med.virginia.edu/)from University of Virginia (Timko et al . 2008).
A database for cowpea physical map will be avail-able from University of California (http://phymap.
ucdavis.edu:8080/cowpea/), as an outcome of the
CGIAR Generation Challenge Program which has
been supported since May 2007. This group is preparing a consensus genetic map containing more than
1000 SNP markers to generate a BAC based cowpea
physical map. Cowpea EST database is also available from University of California (http://harvest.ucr.
edu/). This research is supported by USDA Plant Genome program earlier and CGIAR Generation Challenge Program now.
We frequently use phrase “Asian Vigna ” conspicuously discriminate species originated in Asia
from species in Africa such as cowpea or “African
Vigna ”. Asian Vigna and African Vigna belong to
two different subgenera, Ceratotropis and Vigna ,respectively. Azuki bean (V. angularis ), mungbean(V. radiata ), black gram (V. mungo ) and rice bean (V.

- 33 -

umbellata ) are taken up as the four major domesticated Asian Vigna in the present paper. Besides of thesedomesticated pulses, moth bean (V. aconitifolia ) andcreole bean (V. reflexo-pilosa var. glabra ) have been
fully domesticated and several other Vigna species
are being domesticated or cultivated across Asia.
Comparative studies of these independently domesticated crop from different species such as understanding the evolutionary changes from wild species
to crops might offer possible further evolutionarychanges of current crop.
The potential use of genetic diversity in thewild species of Asian Vigna has been other focus
of our studies. The Asian Vigna show wide adaptation in relation to the climates in which they grow,from cool and wet temperate to hot and dry tropicalclimates. Germplasm collection of Asian Vigna has
resulted in a comprehensive collection of germplasm
from several countries in Asia conserved in the
Genebank at NIAS. These wild species, collectedfrom various natural habitats, possess potentially useful traits related to environmental stress resistances
and insect and disease pest resistances. In spite of therich genetic resources available for the Asian Vigna ,establishment of a germplasm evaluation system andgenomic tools have lagged behind other crop groups.
Genomic studies on Asian Vigna are proceeding
gradually but as they are not mandated crops of the

CG system have not received the research attention
their importance deserves. International collaborative
research on Asian Vigna will be a powerful motivation to discover and utilize efficiently genes responsible for important economic and adaptive traits.
Development of molecular marker and genetic
linkage map of the Asian Vigna
To make progress in genome analysis of the
Asian Vigna cultigens we have developed azuki bean
SSR markers using a new method in plants. The SSR
library construction involved an oligo-primed second-strand synthesis enrichment procedure on single
strand genomic library (Wang et al . 2002). The enriched library enabled low redundant SSR containing
clones to be identified and SSR primers revealed ahigh percentage of successful single-locus amplification. These SSR markers have successfully involvedin trait mapping (Chaitieng et al . 2006; Isemura et al .
2007; Gupta et al . 2008; Somta et al . 2008; Kaga et
al . 2008) and evaluation of other Asian Vigna germplasm (Sangiri et al . 2007; Gupta and Gopalakrishna2009). The SSR marker information is available fromthe NIAS Genebank database(http://www.gene.affrc.go.jp/databases-marker_information_en.php).

Azuki bean linkage map : The azuki bean SSR

Fig.1. Consensus genetic linkage map of azuki bean (Vigna angularis ). Azuki bean SSR,
AFLP and RFLP markers are indicated by red, black and blue, respectively.
- 34 -

markers in addition to AFLP and RFLP markers
from other legumes were used to develop the mostsaturated genome map for Asian Vigna (Han etal. 2005). This map has 205 SSR, 93 RFLP and
187 AFLP markers. Further, we integrated marker
information from three different mapping populations, (V. nepalensis x V. angularis ) x V. angularis
BC1F1 population (Han et al . 2005), V. angularisvar. angularis x V. angularis var. nipponensis F2
population (Kaga et al . 2008) and (V. angularisx V. nepalensis ) x V. angularis BC1F1 population(Kaga unpublished), into single consensus linkage
map. Currently this map consists of 896 markers
and spans a total length of 854 cM. The average
distance between SSR markers is 3.1 cM and this is
the standard linkage map for azuki bean (Fig.1).

Black gram linkage map : The first genome
map was developed by Chaiteng et al . (2006) using the azuki bean SSR and RFLP makers fromother legumes. Recently a more saturated map
consisting of 254 AFLP, 47 SSR, 86 RAPD and 41
ISSR markers is reported by Gupta et al . (2008).
Relatively lower amplification or polymorphismsof the azuki bean SSR markers in black gram thanthe other Asian Vigna has force the use PCR-based
anonymous dominant markers. Development of
SSR markers is prerequisite to obtain more informative markers for black gram.
Rice bean linkage map : Isemura et al . (2010)
developed the first genome map for rice bean byusing 172 azuki bean SSR markers, 103 AFLP
markers, 55 SSR markers from other legumes. The
linkage map has been successfully resolved into 11
linkage groups. Among the 55 SSR markers, 44 are
newly designed cowpea SSR markers. Since gene
Thresher sequence database, containing mostly
gene-rich genomic sequence, has become available from http://cowpeagenomics.med.virginia.
edu/CGKB/ (Chen et al . 2007), we expected the
SSR containing 30,877 sequences deposited in the
database might provide a good marker resources
for Asian Vigna . After removal of redundant sequences, 6,580 sequences can be used to design
SSR primer pairs. Approximately 480 cowpea SSR
markers containing mainly di-nucleotide repeat
with more than 25 repeat have been tested for amplification and polymorphisms among the parentsof the rice bean mapping population, but only halfof the markers produce a single reliable PCR product, suggesting the cowpea genome sequence iswell diverged from rice bean. Only 44 cowpea SSR
markers have been integrated into rice bean linkagemap.

Mungbean linkage map : Isemura et al . (paper in preparation) have constructed a linkage mapthat initially resolved into 11 linkage groups. The
map has been constructed with 141 SSR markers
from azuki bean, 30 SSR markers from mungbean,
196 EST-SSR makers from soybean, 59 cowpea
SSR markers described above and 9 common bean
SSR markers. All of the markers on the map arehighly informative because these have sequence
information. Although stable and single locus PCRamplification of 7,000 soybean EST-SSR markers
developed by Hisano et al . (2007) was very difficult in Asian Vigna , 196 of 7000 markers have successfully mapped. These soybean EST-SSR markers are very useful for identifying syntenic regions
such that new candidate EST-SSRs were able to filla large gap at one problematic linkage group, 4, of
mungbean.
Genome synteny among Asian Vigna
Comparison order of common azuki bean
SSR and RFLP markers on the linkage maps constructed above with each other allowed the detection of a high level macro-synteny between the
genomes of four different Asian Vigna . The highlevel synteny ensures that chromosome nomenclature of Asian Vigna species other than cowpea
can be standardized by cytogenetical studies to- 35 -

Fig.2. Comparative linkage map among four
major Asian Vigna species. Numbers
aside circumferential map indicatename of linkage group.

determine chromosome length and is certainly usefulfor future research. Major difference is a chromosomaltranslocation where a block on linkage group 1 in blackgram, the most divergent species among four species,
has moved to linkage group 10 (Fig.2, orange). Azuki
bean SSR markers that can be compared are gradually
reduced as genetic divergence increased from azuki
bean, several inversions (Fig.2, blue) and many changes
on genetic distance (Fig.2, yellow) are inferred in somespecies. Azuki bean and rice bean belongs to the same
section Angulares of the subgenus Ceratotropis and their
speciation appears to be recent as interspecific genetic
divergence is not so great. As expected, rice bean shareda highly conserved genome organization with azuki
bean as well as highly successful transferability of azuki
bean SSR markers and supported well results of previous cytogenetical studies. Mungbean belongs to section
Ceratotropis , including black gram, but showed genome
similarity to azuki bean and rice bean in the section Angulares rather than black gram. The comparative map
provides adequate syntenic context of domestication
related traits among Asian Vigna discussed below.
Domestication syndrome of Asian Vigna
Four major Asian Vigna cultigens, azuki bean,
mungbean, black gram and rice bean, have been domesticated from 4 different wild species in different locationof Asia (Fig. 3).

tates comparison of major QTLs for the domestication
related traits across these species.
Previous genetic studies have revealed that traits
related to the domestication syndrome in various crops
are controlled by several major and some minor genes
with non-random distribution across genomes (Gepts
2004). A similar situation exists in the four Asian Vigna .
QTLs for various domestication or adaptation related
traits of azuki bean were clustered on linkage groups 1, 4,
7 and 9. Such clusters were found at similar locations on
rice bean linkage groups 1 and 7. The genomic regionsof the QTL clusters in mungbean and black gram weredifferent from azuki bean and rice bean. These are distributed mainly on mungbean linkage groups 2 and 6 andon the black gram linkage groups 8 and 9. Further studies are required to determine whether QTLs for traits
related to the domestication syndrome are clustered at
locations that have highly suppressed recombination orby pleiotropic effects of some key gene with large effect. Since pericentrometric genome regions have been
elucidated in the soybean genome, alignment Vigna
genome with soybean will be very useful to clarify theformer hypothesis.
Distribution of QTL locations on the Asian Vigna
comparative map are shown for seed size, seed dormancy and seed shattering (Fig. 4), and plant height andSoybean

Azuki

Rice bean
MungbeanBlack gram

These cultigens have parallel morphological andFig 3. The putative domestication center of major
legumes in Asia. Right and left of seed picturesindicate the cultigen and its presumed wild
ancestor, respectively.
physiological changes such as the increase in organ sizeof various plant parts, loss of dormancy and loss of pod
shattering, the domestication syndrome (Hammer 1984).
We have been collected QTL information on the domestication or adaptation related traits in azuki bean (Kagaet al . 2008, Isemura et al . 2007), mungbean (Isemura etal . paper in preparation), black gram (Chaitieng et al .
2010) and rice bean (Isemura et al . 2010), and now thesedetailed comparative maps of four Asian Vigna facili- 36 -Fig.4.Distribution of major QTLs for seed
size, shattering and seed dormancy on acomparative map for Asian Vigna .

Fig.5. Distribution of major QTLs forflowering and plant height oncomparative map for Asian Vigna.
flowering (Fig. 5). Despite the limitations of QTL information results from a single specific cross combinationin each species and the extent of morphological andphysiological differences between cultigens and the wild
ancestors are not the same across four species, some
QTLs were identified in all species and some were specific to one or several species.
Among variation between cultigens and their wild
ancestors, seed size is conspicuous and the extent differ
across four species. For example, mungbean has seeds
approximately 5 fold as heavier than its wild ancestor,
and azuki bean has seeds approximately 10 fold as heavier than the wild ancestor. However, there was no difference in the number of QTL between mungbean andazuki bean. Seed size QTL, probably species specific,
with a large increase effect was identified in rice bean
on linkage group 4 and in black gram on linkage group 8.
In contrast, orthologous QTLs across all four cultigens
and three cultigens were found on linkage groups 2 and 1,
respectively.
Seed shattering is an important characteristics toprevent seed loss before harvesting. Orthologous QTL
with a large effect for loss of seed shattering for azuki
bean and rice bean was found in linkage group 7. Seedshattering of mungbean is controlled by partially thesame QTL with minor effect and another QTL on linkage group 1. In contrast, black gram had a different QTL
with minor effect on the linkage group 5. Most seedshattering QTL were clustered with QTL for pod length.
Large variation in pod length are observed among thefour wild ancestors; pod length of wild azuki bean andwild rice bean are longer than those of wild mungbean
and wild black gram. Each wild species may have
evolved different physiological mechanism and/or genefor pod dehiscence.
Seed dormancy QTLs were completely different
among the Vigna species. The parallel phenomena ofloss of dormancy appear to involve different genes inmungbean, black gram and azuki bean. There are different seasonal changes of climate where cultivated andwild species are distributed; mungbean and black gram(dry and rainy season) and azuki bean (cold and rainy
warm season), might be one of factors that resulted in
the different physiological mechanisms underlie seed
dormancy in Vigna .
A different set of alleles on QTL controlling flowering time appears to be used in each species. Since thepresumed wild ancestors of mungbean and black gramare distributed in lower latitudes, different genes fromazuki bean distribute in higher latitude might have involved different photothermal responses which are important for flowering response. However, an orthologous
QTL between mungbean and azuki bean was identified
on linkage group 4. Since significant progress in theidentification of major genes which control flowering
time in soybean, has been made, comparative analysis
of a conserved syntenic region between soybean andAsian Vigna will bring about greater understanding of
the flowering mechanism in Asian Vigna .
The wild ancestors of Asian Vigna possess an indeterminate habit in which the plant continues to growin length throughout its life whereas most domesticatedAsian Vigna have a determinant growth habit. Only
domesticated rice bean has indeterminate growth habit.
Two orthologous QTLs for plant height on the linkage
group 2 and 9 were found in mungbean, black gram andazuki bean mapping populations and the allele of thecultigens reduced plant height, suggesting that these two
QTL might be important for transition of plant architecture into determinate habit.
Morphological and physiological changes fromwild ancestors into cultigens might be consciously selected by the farmer preference and their agricultural activities. However, it is interesting that several orthologous
genes underlying domestication appear to have been unconsciously selected across four Asian Vigna based on
an interplay of photoperiod sensitivities (long and shortday), seasonal changes of climate (dry, rainy, hot andcold), etc. In addition, the present studies on comparative analysis of domestication or adaptation traits that
played a major role on the evolutionally changes fromwild species to crop offer further evolutionary potential
to Asian Vigna cultigens. These four major cultigens are- 37 -

Table 1. Presumed syntenic regions between mungbean and soybean. Each mungbean linkage
group is divided into three parts. Letter in parenthesis and after inequality sign indicate
soybean linkage group with second highest score based on similarity search andchanges to another linkage groups in the middle of partition, respectively.
Mungbeanlinkage group(MLG)

Top of MLG

Middle of MLG

Bottom of MLG

No.of
makerscompared

1
2
3
4
5
6
7
8
9
10
11

A1(A2) > M
C2(C1)
B2(D1b)G(A2) > L
E or Knd
E or F > F or H
I or ON
D1a
L

A1 > A2 > J
C2(C1)
B2(D1b)
J > L(N)E or K > L(N)B1 or Hnd
I or Ond
D1a > K or B1
L>K

A1 > D2(E,F)
E(K)
B2(D2)ndG
B1 or Hnd
O > D1bA2(M)K or B1
K > K(M)

36
30
24
21
23
13
14
21
10
13
17

Corresponding soybean linkage groupwithin each others secondary genepool, so verification of this hypothesis will be possible by the crossbreeding. For example, introduction of azuki bean
QTLs controlling plant height on linkage group 2 and9 into rice bean and introduction of rice bean QTL
controlling seed size on linkage group 4 into azuki
bean will assist breeding varieties with determinate
habit in rice bean and increased seed size in azuki
beans.
Genome synteny between Asian Vigna and soybean
Soybean EST-SSR markers have been integrated into the Vigna linkage maps to efficiently
utilize soybean sequence data via synteny. Since
December 2008, the chromosome-scale assembly of
the soybean genome, Glyma 1.0, has been available
from Soybean Genome Project, DoE Joint Genome
Institute (http://www.phytozome.net/soybean.php).
Initially, synteny was examined comparing locations
196 soybean EST-SSR markers on the mungbean
linkage map with the soybean linkage map. However, the number of mapped markers common to boththe mungbean and the soybean linkage map was inadequate to find syntenic region by direct comparison. Many integrated Vigna SSR markers, azuki bean
and cowpea SSR, are expected to provide sequence
data that can identify similar soybean chromosomal
location as well as soybean EST-SSR markers. Alternatively, similarity search using blastn program witha cut off E-value of E-20 between the Glyma 1.0 sequence database and sequence of the mapped Vigna
SSR markers was performed and location of the top
hit sequence from 171 Vigna SSR markers has been
- 38 -

used for comparison.
The result was very complex all query sequences from each marker matched to several soybean chromosomes within the database and mungbean linkage group was composed of several soybean
chromosomes. Since extensive sequence differences
exist between soybean and Vigna so far as genomic
sequences around SSR compared, similarity searchwith a cut off E-value of E-4, a lower threshold, increased to 222 marker comparisons between mungbean and soybean linkage groups.
Table 1 indicates presumed syntenic regions
between mungbean and soybean. This shows similarsoybean linkage groups with a highest and second
highest sequence similarity, but further comparison
using more information may alter this result. On
mungbean linkage group 5, 6, 7, 8 and 10, we could
not determine single syntenic soybean linkage group
because two soybean similar chromosome blocks,
may be paralogous blocks, that revealed top hit withthe sequences around Vigna SSR tend to jumble together. Although much more information is required
to test co-linearity, segmentations of soybean linkage
block are also observed for most of the mungbean
linkage groups. Only one mungbean linkage grouprevealed high similarity to a soybean linkage group(B2), mungbean linkage group 3. When multiple
comparisons that include second hit information are
tested, the top to central part of mungbean linkage
group 3 is similar to soybean linkage group D1b.
Such segmental duplication among soybean chromosomes has been reported by Shoemaker et al . (2006)
and Hisano et al . (2007).
Other instances are A1 and A2, C1 and C2,

B1and H and these are supported in mungbean linkage group 1, 2 and 6, respectively. Soybean is wellknown as a diploid species having 20 chromosomes,
but these chromosomes are thought to be derived
from at least two rounds of genome wide duplication and many rearrangements, a paleopolyploid
species (Shoemaker et al . 2006, Walling et al . 2006).
These events might prevent finding clear syntenic
relationships between mungbean and soybean as
was observed between soybean and the model legume, Lotus japonicus (Hisano et al . 2007). In future,extensive investigation into distribution and extent
of paralogous genomic segments within soybean
genome sequence is essential to utilize soybean sequence information simply in other legumes.
Future perspectives
We plan to combine genome map informationof four different Asian Vigna into single Vigna consensus map to integrate marker information bridging the soybean sequence. The soybean sequence
information has the potential to serve as a landmark
for gene identification through comparative genome
analysis. However, it might be too complex for direct
use of such information in marker development in
the Asian Vigna . Since a high level of macro-synteny
between the genomes of four different Asian Vigna
are observed, development of the genomic foundation with sequence information, for example in azuki
bean as a representative species, would be an effective approach for various Vigna species. There is a
growing tendency for sequencing various organisms
by read length improvements of next generation
sequencing technologies. As with the success of thesoybean genome sequence assembly based on information of a high density linkage map and FPC (Finger
Printed Contigs) of BAC clones, there is a need tomake steady progress on the establishment genomic
base by collaboration until embarking on genome sequencing of Asian Vigna . To this end use of gene andsequence information from soybean in Asian Vigna
through comparative genome analysis will be mosthelpful.
Acknowledgement
This research was supported by Grant-in-Aid
for Scientific Research Japan (KAKENHI) of Society
for the Promotion of Science (JSPS) and Genebank
project of the National Institute of Agrobiological
Sciences (NIAS) of Japan.
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