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REVIEW OF LITERATURE

2.1. GREENGRAM:

2.1.1.Green gram as a crop:

Green gram or mung bean is the third most important pulse crop in the

Indian subcontinent. It is common in Southeast Asia, Central Africa, the

warmer parts of China and the United States (Walde et al.2005).It is

harvested almost exclusively for pulse crop or dry grains. Green gram

contains approximately 23.86‐27%protein, 1.15%fat, 62.62%carbohydrates,

16.3% fiber, 6.60% total sugars, 9.05%water (USDA,2009, El‐Adawy 2000).It

contributes to small holder income, as a higher-value crop than cereals, and

to diet, as a cost-effective source of protein that accounts for approximately

15% of protein intake. Moreover, green gram offers natural soil maintenance

benefits through nitrogen-fixing, which improves yields of cereals through

crop rotation, and can also result in savings for smallholder farmers from

less fertilizer use. Green gram may also be sown as an intercrop or as a

green manure or cover crop. The transformation of green gram from a

marginal to a major crop has brought many benefits to Asia (Table 2.1).

Table-2.1: Summary of the impact of improved green gram in Asia (1984-2006).

Areas of impact Estimated impact

Adoption of improved varieties Percent of total mungbean area 50 to 95% Area covered 2,932,000 ha Number of farmers 1,466,000a

Average growth rate of area -0.1 to 22% Increase in production 35% (800,000 t)b

Average annual growth rate for production - -0.3 to 23.5% Yield increase 28 to 55% (187 to 426 kg/ha) Average annual growth rate for yield -0.1 to 1.7%

Increase in income due to Increase in yield 28 to 55% Increase in paddy yield with rice-wheat mungbean rotation

$148,700,000c

Economic benefit due to improved health of anemic women, enhancing productivity

$3,500,000 to 4,000,000/countryd

Increase in consumption of mungbean 22 to 66% Anemic children benefited 1,500,000e

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a Assuming an average land holding of 2 ha per farmer across different

countries.

b Production in Asia increased from 2.3 million tons in 1985 to 3.1 million

tons in 2000.

c Rice-wheat-mungbean rotations gave 450 kg/ha higher paddy yield than

rice-wheat-fallow rotation. It is assumed that the whole 2.932 million ha

were in rotation.

d It is assumed that the average is the same as that observed in Pakistan.

e Assuming that an average farm family has two children across the countries.

The major beneficiaries of the intervention are the poor, especially children

and women, whose diets lacked much-needed protein and iron. Both urban

and rural consumers now have access to improved quality green gram

available at reasonable cost. The crop offers the potential for a new income

stream for small-scale farmers.

Green gram is an erect or sub-erect herb, 0.5-1.3m tall. Flower is pale

yellow. The seed color exhibits a wide range of variations from yellow,

greenish yellow, light green, shiny green, dark green, dull green, black,

brown, and green mottled with black. Pod color is either black, brown or

pale gray when mature. 100 seeds weight is 3-7g.

Tomooka et al.,(1991,1992a) revealed the geographical distribution of growth

types, seed characters and protein types in green gram landraces collected

from throughout Asia. In the South and West Asia, green gram strains

characterized by small seeds with various seed color including black, brown

and green mottled with black, which show diverse growth habit and protein

types, were distributed. In the Southeast Asian countries, green gram

strains characterized by various sized seed with shiny green seed testa,

which show tall plants with high branching habit, late maturity, and simple

protein type composition were distributed. In East Asia, green gram strains

characterized by medium-sized dull green seed testa, which show short

plants with an early maturity, low-branching habit and relatively diverse

(similar to that of West Asia) protein types, were distributed. The

inflorescence is an axillary raceme, with 10 to 25 pale-yellow flowers, 1 1/2

to 2 cm long, and clustered at the top. The flower consists of one standard,

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two wings and two keel petals; stigma surrounded by a staminal column of

nine stamens and a free tenth stamen (Poehlman, 1991). Dehiscence

occurred in the afternoon before the flower opens the following morning. The

flower fades the same afternoon. The observations on green gram made at

Pusa, showed that pollen grains came out from the anthers between 9 to 10

p.m. in the bud stage before opening of the flower completing the act of

pollination in the night. Though green gram is a predominantly self-

pollinated crop although the natural cross-pollination is not absolutely

impossible (Narasimham, 1929).Only about half of the flowers (64 percent)

open to permit possible cross-pollination. No case of natural outcrossing

was reported there (Bose, 1932). Van Rheenen (1964) reported 4 to 5 %

natural outcrossing in green gram. A small amount of natural cross

pollination in green gram is virtually capable of enhancing genetic variation

(Dana, 1969). Natural cross pollination of 0.5 % has been reported by Empig

et al. (1970). Bhadra and Shill (1986) reported 0.91 to 3.10 % outcrossing in

green gram, depending upon the distance between the pollen source and the

recipient.

The generic epithet Vigna was named after Dominico Vigna, professor of

Botany at Pisa (1609-14) and the specific name came from a Latin word

radius means “radiating in every direction from the centre” like the spokes of

a wheel. The specific name actually indicates the position of pods (fruits) on

peduncle. Chatterjee and Randhawa (1952) included different common

names for green gram like mung, moong, mungo, greengram, goldengram,

Chickasaw pea and Oregon pea. Bundo and yaenari are also two of the

common names of green gram.

The present taxonomy of green gram is like this :

Kingdom:Plantae→Sub-division:Spermatophyta→Division:

Magnoliophyta→Class:Magnoliopsida→Sub-class:Rosidae→Order:

Fabales→Family:Fabacae→Sub-family:Papilionaceae→Genus:Vigna

Savi→Sub-genus: Ceratotropis (Piper) Verdcourt →Species: radiata.

Green gram is an annual food legume belonging to the

subgenus Ceratotropis in the genus Vigna. The genus Vigna, together with

the closely related genus Phaseolus, forms a complex taxonomic group, so

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called Phaseolus-Vigna complex. Verdcourt (1970) proposed a very restricted

concept of Phaseolus, limiting it exclusively to those American species with a

tightly coiled style and pollen grains lacking course reticulation, hence,

promoting significantly the concept of Vigna. According to his proposal,

green gram and its relatives (which is now recognized as the

subgenus Ceratotropis) were transferred to the genus Vigna from the

genus Phaseolus. In 1973, this change of classification of green gram was

adopted by the United States Department of Agriculture (Gunn, 1973). This

was also supported by pollen grain studies, seed protein electrophoresis

studies (Sahai and Rana, 1977; Renganayaki et al., 1987), serological

evidence (Chrispeels and Baumgartner, 1978), leaf and pollen morphology

(Subramanian, 1979), cross-inoculation studies (Sahai and Rana, 1980),

and seed protein immunochemical studies (Turkova & Klozova,1985).

Marechal et al. (1978) followed Verdcourt and presented a monograph on

the Phaseolus-Vigna complex. Their taxonomic system is generally accepted

now. Three botanical varieties were recognized in their

monograph. V.radiata var.radiata is cultivated form (green gram or

mungbean), var.sublobata is the wild ancestral form of green gram, and

var.setulosa is also wild form which distribute in India, Indonesia, southern

China. 2n=22.

Green gram has been considered to have been domesticated in India (Vavilov

1926). His theory has been supported by other authors based on the

morphological diversity (Singh et al. 1974), existence of wild and weedy

types (Chandel 1984), and archaeological remains (Jain and Mehra 1980) of

green gram in India. Two wild forms of green gram are Vigna radiata (L.)

Wilczek var. sublobata (Roxb.) Verdc. and Vigna radiata (L.) Wilczek var.

setulosa (Dalz.) Ohwi and Ohashi. Previously wild forms of green gram were

recognized as Phaseolus sublobatus. According to Verdcourt, V. radiata var.

sublobata Roxb. –“is undoubtedly the wild form of mung”. Wild forms of

green gram, V.radiata var.sublobata show a wide area of distribution,

stretching from Central and East Africa, Madagascar, through Asia, New

Guinea, to North and East Australia.

Tomooka et al. (1992a) examined the variations of seed proteins in green

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gram landraces from Asia, and proposed the regions of protein type diversity

and two dissemination pathways in green gram. According to their study,

the region of protein type diversity is found in West Asia (Afghanistan-Iran-

Iraq area) rather than in India. Judging from the geographical distribution of

protein types, green gram may have spread mainly to the east by two routes.

One route is from India to Southeast Asia; strains consisting of a few protein

types were disseminated by this route. Another dissemination pathway is

known as the Silk Road. By this route, strains spread from West Asia or

India to China and Taiwan via the Silk Road, not by the route from

Southeast Asia.

Wild form of green gram are scattered all over India, specially in the

undulated plateau regions and foot hills of the Himalayas, the Eastern

Ghats and the Shahyadris. They occur upto 1000 metre height of almost all

hilly regions. Sublobata has a wide distribution in coastal region of East

Africa and Madagascar, throughout Tropical Asia, Indonesia and Northern

Australia. Prof. S. Dana of Bidhan Chandra Krishi Viswavidyalaya (BCKV),

Mohanpur, Nadia, West Bengal, collected the wild green gram, Vigna radiata

var.sublobata (Roxb.) Verdc., lines from Shiwalik range, near Chandigarh,

PLX – 270 by the courtesy of Plant International Division, IARI in 1972 and

1979 and from near Neral railway station, Kulaba, Maharastra in 1992. He

cultivated those wild green gram lines at Kalyani and Haringhata complex of

BCKV.

Few wild populations of green gram are equal to or even superior to the

cultivated variety in respect of photosynthetic efficiency, protein content and

seed weight. These can be selectively used as a potential donor of desirable

traits to improve cultivated green gram (Ignacimuthu and Babu, 1987). The

wild germplasms of green gram have a high content of most of the essential

amino acids. It ranges from 38.3 to 42.2 g / 100 g of protein (Babu et al.,

1988).

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2.1.2. Production and productivity:

In India, green gram is cultivated in 3 million hectare with the production of

about 1.1 million tonnes. The coverage and production is highest in the

world, but the productivity is very low (400 kg/ha) as compared to China

(1154 kg/ha). More than 80 cultivars have been released so far in India

(AVRDC, 2004). While the areas for cereals and other pulses have decreased,

that for green gram has doubled in the last two decades with an annual rate

of 2.5%. This growth may very likely continue since green gram’s short-

growth duration (60 days) makes it suitable for the various cropping

systems. Green gram has been transformed from a marginal to a relatively

important crop and, in the process, has contributed to improving rural

household income, expanding employment opportunities, diversifying diets,

increasing nutritional security, and enhancing soil fertility in Asia. Since the

1980s, improved green gram has led farmers to plant the crop on more and

more land (fig.2.1). Today, improved green gram varieties occupy almost 90

percent of the area under green gram cultivation in Pakistan and Thailand,

85 percent in China, and around 50 percent in Bangladesh and Myanmar,

accounting for almost 3 million hectares of land. Overall in Asia, production

of green gram varieties increased from 2.3 million tons in 1985 to 3.1 million

tons in 2000. Globally, improved green gram constitutes more than 25

percent of worldwide green gram production. Moreover, green gram

production is raising the yields of farmers’ other crops by improving the

productivity of the soil. Including green gram in the rice–wheat crop rotation

system enriches the soil and breaks soil fatigue caused by cereal–cereal

rotations. Farmers in Punjab who grow green gram as well as paddy rice

have found their paddy rice yields, and their incomes, rising.

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Fig.2.1: Estimated area under green gram before and after intervention

Estimated global production of green gram is 2.5–3 mt from about 5 million

ha. Annual green gram production worldwide is around 2.5 to 3.0 million

metric tons harvested from about 5.0 million ha.

2.1.3. Constraints:

The yield of green gram is stagnant over years because of some major

constraints. The narrow genetic variability in the primary gene pools, the

limited gene pool of the cultivated species of Vigna has restricted the

conventional plant breeding programme to improve the yield. For producing

the crop types, which combine the high productivity quality, and resistance

to disease and pest, it has become necessary to widen the gene pools of the

cultivated species through interspecific hybridization. This would be

enabling the interspecific gene transfer, which may lead to the additional

sources of variation for the desirable characters (Bharathi et al., 2006).

Besides, has non-synchronous maturity, pod shattering, pod dropping and

viviparous germination (particularly in kharif season), anti-nutritional

factors like Trypsin inhibitor, Haemagluteline, saponin, etc.. Some of the

genotypes have trailing growth habit. High yielding and protein rich

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genotypes for diversified environmental conditions, including biotic and

abiotic stresses are unavailable. One of the barriers to the improvement of

green gram is the tested germplasm revealed low to moderate variability,

both in morphological and molecular level (Chattopadhyay et al.,2005,

2008). High yielding genotypes that are resistant to green gram yellow

mosaic virus and to bruchid are not available.

2.2. GREENGRAM AND THEIR RELATIVES:

Vigna is a large tropical genus consisting 82 described species distributed

among 6 subgenera. The subgenera are Ceratotropis, Haydonia, Lasiospron,

Plectotropis, Sigmoidotropis and Vigna. Within the subgenera in the genus

Vigna especially subgenus Ceratotopis has its center of species diversity in

Asia (Tomooka et al., 2006).

The subgenus Ceratotropis consists of 21 species of which eight are

domesticated including Moth bean [V. aconitifolia (Jacq.) Maréchal], Azuki

bean [V. angularis (Willd.) Ohwi& Ohashi], Black gram [V. mungo (L.)

Hepper], Mungbean [V. radiata (L.) Wilczek], Creole bean [V. reflexo-pilosa

Hayata var. glabra) (Maréchal, Mascherpa & Stainer) N. Tomooka &

Maxted], Jungli bean [V. trilobata (L.) Verdc], Toapée (Thai) [V. trinervia

(Heyne ex Wall) Tateishi & Maxted], and Rice bean [V. umbellata (Thunb.)

Ohwi& Ohashi]. The five most important domesticated species are

mothbean, azuki bean, rice bean, black gram and green gram (Tomooka et

al., 2006).The subgenus Ceratotropis in the genus Vigna is widely

distributed from the Himalayan highlands to South, South-East and East

Asia.

The taxonomy of Asian Vigna was described based on several traits, such as

seedling characteristics, size of floral parts, and habitat. Three groups

within Ceratotropis have been recognized as sections, section Angulares

(Azuki bean group), section Ceratotropis (Mungbean group) and section

Aconitifoliae (Intermediate between azuki and mungbean group).The

characters were taxonomically informative in the subgenus Ceratotropis with

a high degree of speciation. The species of the Subgenus Ceratotropis have

flowers colored various shades of yellow, but are never purple, violet, blue or

white as is often found in other Vigna subgenera .

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Asian Vigna were classified as Phaseolus (Verdcourt 1970). The genus Vigna

consists of over 90 species in six subgenera (fig.2.2). It seems likely that

Vigna first evolved in Africa because major species radiation of the genus

Vigna has occurred in Africa where the subgenera Haydonia, Plectotropis,

and Vigna are found (Vaillancourt et al., 1993). In addition, molecular

studies suggest tha Vigna may have evolved from the African genus Wajira

as it is basal compared to Vigna and Phaseolus (Thulin et al,. 2004).

Chloroplast and nuclear genes have enabled molecular clock divergence

dates to be estimated for legumes including Phaseolus and Vigna (Lavin et

al., 2004, 2005; Thulin et al., 2004). Chloroplast DNA (Lavin et al., 2004,

2005; Thulin et al., 2004) and nuclear DNA studies (Lavin et al., 2004) have

compared many species of the entire genus Vigna and related genera; these

studies have provided a new interpretation of the relationships in the genus

Vigna compared to studies based mainly on morphological traits. Molecular

studies suggest that Vigna subgenus Haydonia of Africa may be the most

primitive and well-diverged group within Vigna. Vigna subgenus

Sigmoidotropis of the New World is more closely related to Phaseolus than to

the other subgenera of the genus Vigna (Vaillancourt et al., 1993).

Genetic analyses show that interspecies divergence in section Aconitifoliae is

greatest. The comparison of genomic DNA sequence data between subgenus

Ceratotropis and Vigna suggests section Aconitifoliae is the ancestral section

in subgenus Ceratrotropis. Species in the section Angulares are least

diverged and probably derived from species in section Aconitifoliae via

section Ceratrotropis. Section Ceratrotropis is intermediate both

morphologically and in terms of interspecies diversity and has two distinct

phylogenetic lineages each containing one cultigen, V. radiata and V.mungo,

and one wild species. All these species share several morphological traits,

such as epigeal germination and first and second leaves being narrowly

elliptic to ovate and lacking a petiole. This group of species in the subgenus

Ceratotropis, was given the section name Ceratotropis by Tomooka et al.

(2006). The close relationship between green gram (V. radiata) and black

gram (V.mungo) has contributed to the confusion surrounding their

presumed progenitors, both of which occur in India and which have

morphological similarities. Even so, broader stipules, pale yellow flowers,

more ovules per pod, a spreading pod with short brown hairs, and a non

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arillate hilum, as well as chemical and molecular characters, distinguish

cultivated and wild green gram from cultivated and wild black gram.

The gene pools of green gram consist of a primary gene pool of domesticated

V. radiata and its presumed wild progenitor var. sublobata (fig.2.3). The

secondary gene pool of green gram consists of the other species in section

Ceratotropis, V. mungo, V. subramanian, V. grandiflora, as well as V.

stipulacea (Section Aconitifolia), V. tenuicaulis, V. trinervia, and V. umbellata

(Section Angulares). Other species in sections Aconitifolia and Angulares are

within the tertiary gene pool. There have been many reports of interspecific

hybridization involving green gram (Dana and Karmakar 1990). Results of

interspecific hybridization suggests that green gram and related Asian Vigna

have more complexity to genome structure than indicated by just two

groups for the 21 species in the subgenus Ceratotropis.

Besides the cultivated species of Vigna the wild species are Vigna trilobata,

Vigna grandis, Vigna dalzalliana, Vigna vexillata, Vigna radiata Var.

sublobata, and Vigna mungo Var. silvestris.

Fig.2.2: The relationships, geographical distribution, and approximate

number of species in the six subgenera in the genus Vigna and closely

related genera Phaseolus and Wajira. Approximate species number in

each genus and subgenus is shown. Sections within subgenus Vigna and

subgenus Ceratotropis are shown.

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Fig.2.3: Gene pools of major Vigna crops. Gene pool 1 (GP-1)

constitutes the biological species. Gene pool 2 (GP-2) includes these

species that cross with GP-1 with at least some fertility; Gene pool 3

(GP-3) includes those species where gene transfer requires radical

techniques. (a) Azuki bean. (1) Species in section Angulares. Some of

the species in the section Ceratotropis have not yet been examined for

their cross compatibility relationships with azuki bean and therefore

this section is tentatively classified as GP-3. (b) Mungbean. (1) This

species is in section Aconitifolia. (2) These species are in section

Ceratotropis. (3) These species are in section Angulares. (c) Cowpea [(a)

and (b)updated from Tomooka et al. (2005) and Vaughan et al. (2005)]

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2.3. MOLECULAR MARKERS OF Vigna AND OTHER LEGUMES:

Genetic marker can be divided into three general groups; morphological,

cytological, and molecular. Molecular markers or DNA markers have become

important in the genetic characterization and improvement of many crop

species. They have been used to identify the genetic region or different

alleles of loci on chromosomes. They have contributed the assessment of

biodiversity and understanding of phylogenetic relations. Molecular markers

can be divided into two general categories, depending on whether they are

based on restriction fragment length polymorphisms (RFLP) or on

polymerase chain reaction(PCR). PCR-based markers can be further sub-

divided into two groups, viz. dominant marker and co-dominant markers.

Dominant markers include amplified fragment length polymorphisms (AFLP)

and random amplified polymorphic DNA (RAPD). Co-dominant markers

include simple sequence repeats (SSR), single nucleotide polymorphisms

(SNP), and insertion deletions (InDel).

Due to a lack of sufficient molecular markers, understanding the degree of

genetic variability in green gram has been greatly limited, which represents

a great challenge for successful improvement of this crop( Ali and Kumar

2005, Kumar and Ali 2006). Green gram encounters multiple stresses

during their lifecycle. To ensure stable productivity, it is necessary to

develop varieties resistant to more than one stress. The introgression of

several genes to develop superior genotypes is expected to perform better

against incidence of multiple stresses and/or diseases. Sometimes, negative

linkage between the genes controlling resistance to different stresses and

agronomic and quality parameters makes it problematic to combine them in

the background of high-yielding genotypes using conventional methods of

breeding (Chaturvedi et al., 1998). Pyramiding multiple genes is challenging

and time-consuming; in modern era when there is inaccessibility of space

for field evaluation; the selected few polymerase chain reaction (PCR) based

primers can be employed.

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2.3.1.Hybridization-based markers:

RFLP has been commonly used as informative molecular marker in mapping

of crop plant genomes. RFLPs are useful markers because of their

codominant properties, and thus can distinguish between homozygosity and

heterozygosity. RFLP analysis utilizes the difference in nucleotide sequences

at specific sites recognized and cut by restriction Enzymes, and then

separated according to size under electrophoresis. Individual DNA fragments

are identified by labeled probes specific to certain sequences. The presence

of particular alleles at these loci is detected by length polymorphisms caused

by mutations that have led to loss or gain of a restriction site between

genotypes. However, a limitation of RFLP is that it requires a large amount

of good quality DNA for analysis. This technique is also time consuming and

expensive, making it less suitable for large-scale screening programs in

plant breeding.

2.3.2. PCR-based markers:

PCR-based markers use amplification of target DNA sequences by the

polymerase chain reaction (PCR) in vitro. PCR based markers have

advantages over RFLP as the assay requires comparatively little DNA. It can

generate a large number of polymorphic markers quickly without the need to

develop libraries. Genetic markers based on PCR include, amplified fragment

length polymorphisms (AFLP), inter simple sequence repeats (ISSR), random

amplified polymorphic DNA (RAPD), sequence tagged sites (STS), simple

sequence repeats (SSR), and single nucleotide polymorphisms (SNP). The

cost of generating such markers is moderate.

Amplified Fragment Length Polymorphisms (AFLP)

AFLP is a sequence-arbitrary method which amplifies of DNA fragments

generated by specific restriction enzymes and oligonucleotide adapters

containing few variable nucleotide bases (Vos et al., 1995). The method was

developed from RFLP combined with RAPD techniques. In this technique,

genomic DNA is first digested with one or two restriction endonuclease.

Next, an adapter of known sequence is ligated to the ends of the digested

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genomic DNA. Amplification is carried out using primers with sequence

specificity for the adapter. The primer(s) also contains one or more base at

their 3’ ends which provide amplification selectivity by limiting the number

of perfect sequence matches between the primer and pool of available

adapter/DNA templates. The resulting amplification products are typically

observed by limiting the number of primers concentrations, followed by

fragment separation on acrylamide gels. The strengths of this method are

the very high multiplex ratio and genotyping throughput while no marker

development work is needed. However, AFLP primer screening is often

necessary to identify optimal primer specificities and combinations. Then it

can be utilized in DNA fingerprint, genetic mapping, and gene tagging

(Powell et al., 1996; Mohan et al., 1997; Cato et al., 1999; Tar’an et al., 2002;

Kelly et al., 2003; Peters et al., 2004).

Random Amplified Polymorphic DNA (RAPD) & Inter Simple Sequence

Repeat (ISSR)

Random amplified polymorphic DNA (RAPD) was the first arbitrarily primed

PCR markers methodology developed (Williams et al.1990) and is still widely

used. A number of modifications have been made to the technique,

predominantly in primer length (8-12 bp) and detection methodology. RAPD

markers are advantageous, since they are easy to generate, rapid,

multilocus and do not require radioactivity. This PCR-based technique

requires arbitrary short oligonucleotide primers targeting unknown

sequences in the genome, usually resulting in presence/absence

polymorphism. However, questions have arisen regarding their reliability.

RAPD and ISSR markers have already been used with great success in

parentage determination and hybrid identification in many crops (Wang et

al.,1994, Crockett et al., 2000). Ability of those molecular marker systems to

identify hybrids or heterozygotes, during marker-assisted selection is yet to

be tested in green gram. The use of RAPD, ISSR markers in generating DNA

fingerprint profile and differentiating genotypes was found a great success in

many cereal crops, like rice (Ko et al., 1994), pearl millet (Chowdari et al.,

1998), wheat (Prasad et al., 2000) and barley (Fernandez et al., 2002). By

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the similar fashion, PCR has a tremendous potentiality to offer the pulse

geneticists a simple, rapid and reliable method for genotype identification

and recognition of lines that could contribute genetic diversity as well as in

varietal release. Kaga et al., (1996) found the largest intra-specific variation

in Vigna radiata with its wild forms (Vigna radiata var. sublobata) through

RAPD analysis.

Earlier it was reported that mungbean genome was one of the small plant

genomes with a haploid DNA content of about 0.48 pg or 4.7 x 108 base pair

(Murray et al.1979). Most interestingly, only 35 % of the genome is repetitive

and 46 % of the single copy sequences was more than 6700 bp long. Limited

research efforts have been made to understand the green gram genome

structure and genetic diversity as compared to other legumes (Humphry et

al.,2002). Although few reports are available on the use of molecular

markers such as RAPD and microsatellite for estimation of genetic

variability of green gram cultivars (Lakhanpaul et al. 2000; Kumar et al.

2002a; Betal et al., 2004) but their numbers are very limited. Using various

informative and semi-informative PCR-based molecular markers and

considering the absence of overlapping sequences, researchers could still

able to cover only 0.1 % of the total genome of green gram. Chattopadhyay et

al., (2005) used molecular markers like RAPD and ISSR to assess the genetic

diversity among selected germplasms of green gram comprising varieties,

landraces and the wild accessions. In Vigna species, SSR markers have been

developed in cowpeas [V. unguiculata (L.) Walpers] (Li et al., 2001),

mungbean (Kumar et al.2002b) and azuki bean (Wang et al.2004).

Seven out of ninety three designed primer pairs were found to amplify

polymorphic microsatellite loci in green gram which are currently being

utilized for diversity assessment within the green gram germplasm collection

(1400 accessions) of the Rural Development Administration (RDA) Gene

Bank of Korea (Gwag et al., 2006). Through EcoTILLING, ten primer sets

produced a total of 157 DNA polymorphisms when using V. radiata var.

sublobata as the reference in a collection of green gram (Barkley et al.,

2008). Chattopadhyay et al. (2008) identified some highly informative

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primers which could be utilized in generating useful molecular descriptors

for fingerprinting of green gram genotypes from Indian subcontinent.

Simple Sequence Repeats (SSR)

Microsatellites are simple sequence repeats (SSRs) of 2-6 nucleotides.

Microsatellites have been detected within the genomes of every eukaryotes

so far analyzed, and are often found at frequencies much higher than would

be predicted purely on the basis of base composition, although the

frequency of microsatellites varies between species. They are abundant,

dispersed throughout the genome and show higher level of polymorphism

than most other genetic markers. These features, coupled with their ease of

detection, have made them useful molecular markers. Their potential for

automation and their inheritance in a co-dominant manner are additional

advantages when compared with other types of molecular markers

(Goldstein and Schlötterer,2001; Holton,2001). However, SSR requires

sequence information and are relatively expensive to develop.

SSR markers have been developed for plant genomes, including common

bean, azuki bean (Wang et al., 2004) and mungbean (Kumar et al., 2002a,

2002b; Gwag et al., 2006). Microsatellite analysis has bean used to study

genetic diversity in various legume species including green gram (Yu et

al.,1999; Kumar et al., 2002a, 2002b; Gwag et al., 2006), yardlong bean

(Phansak et al., 2005), cowpea (Li et al., 2001). Very few SSR markers have

been developed for green gram and thus there is no genome map that can

resolve the 11 linkage groups. SSR marker libraries have been developed for

azuki bean (V.angularis) (Wang et al., 2004) and these have been used to

produce a genome map for azuki bean (Han et al., 2005). Azuki SSR

markers have also been used to produce a genome map of black gram (V.

mungo) that has resolved the 11 linkage groups for this species (Chaitieng et

al., 2006). Due to their phylogenetic relationship, azuki bean SSR markers

are likely to be useful for detecting polymorphism in green gram.

Restriction fragment length polymorphisms (RFLPs) and SSRs or

microsatellites are widely employed to construct linkage maps and mapping

of agronomically important traits in many crop plants because they are

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highly informative and reproducible. To make progress in genome analysis

of azuki bean, a linkage map was constructed onto eleven linkage groups

using SSRs, AFLPs and RFLPs (Han et al., 2005). With the use of SSR

primers, RFLP probes, AFLP loci and one morphological marker, the first

genetic linkage map of black gram was constructed. The results of

comparative genome mapping between black gram and azuki bean showed

that the linkage order of the markers was highly conserved. However

inversions, insertions, deletions / duplications and a translocation were

detected between black gram and azuki bean linkage maps. This study

suggested that the azuki bean SSR markers could be widely used for Asian

Vigna spp. and the black gram genetic linkage map would assist in Vigna

crop improvement (Chaitieng et al., 2006).

Microsatellite or simple sequence repeat (SSR) markers have been

successfully used for genomic mapping, DNA fingerprinting, and marker-

assisted selection in green gram and other related species. Xu et al. (2011)

report the first genetic map of asparagus bean based on SNP and SSR

markers. The map consists of 375 loci mapped onto 11 linkage groups (LGs),

with 191 loci detected by SNP markers and 184 loci by SSR markers. This

work provides the basis for mapping and functional analysis of genes/QTLs

of particular interest in asparagus bean, as well as for comparative

genomics study of green gram at the subspecies level. Yu et al. (2000) report

the first successful assignment of 15 SSR markers to the Phaseolus vulgaris

molecular linkage map. A total of 37 SSR primer pairs were developed and

tested for amplification. Sixteen of the SSRs polymorphic to the parental

lines were analyzed for segregation and 15 of them were assigned to seven

different linkage groups, indicating a widespread distribution throughout

the bean genome.

2.5. LINKAGE MAP OF Vigna

Two genome maps have been developed in black gram (Chaitieng et al.

2006). These maps show linkage order of markers is highly conserved

between black gram and azuki bean. These maps were developed using

simple sequence repeat (SSR) markers from azuki bean as well as restriction

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21

fragment length polymorphism (RFLP), amplified fragment length

polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and

intersimple sequence repeat (ISSR) markers. However, both maps had

significant gaps that may require development of specific molecular markers

for black gram.

The development of linkage maps for cowpea has concentrated on identifying

the location of QTLs for various agronomically important traits. Genome

maps that have used one cultivated and one wild plant have focused on

identifying morphoagronomic traits (Fatokun et al., 1992; Ubi et al., 2000)

and aphid resistance (Myers et al., 1996). However, the most detailed genetic

maps for cowpea have been developed from a cross between two cultivated

parents that have complementary useful traits such as resistance to

different races of Striga, cowpea mosaic virus, root-knot nematode virus,

and Fusarium wild (Mene´ndez et al. 1997; Oue´draogo et al., 2002a, b).

Rice bean is not a major cultivated legume; however, it is locally important

in parts for South and Southeast Asia. It produces profuse numbers of pods.

Its major interest to Asian Vigna specialists is its useful source of resistance

to pests and diseases. Resistance to green gram mosaic virus, one of the

most devastating diseases of this crop, has been found in rice bean. It is

also a source of bruchid resistance (Tomooka et al. 2000). Consequently a

genetic map of this species has been developed (Isemura et al. 2007b).

Domestication-related traits have also been analyzed in a cross between

cultivated and wild rice bean.

It is only recently that a genetic map of green gram with the expected 11

linkage groups of this species has been reported (Isemura et al. 2008; table-

2.2). A comparison across the genus Vigna with regard to a QTL related to

the important domestication-related trait seed size is shown in fig.2.4

An increasing number of genome resources are now available for Vigna. The

National Institute of Agrobiological Sciences (NIAS) has developed SSR

markers for azuki bean that have been used successfully in mapping other

Asian Vigna (Chaitieng et al. 2006; Gupta et al. 2008; Somta et al. 2008b).

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Fig.2.4 : A major QTL for seed weight on linkage group 1 of azuki bean

compared with QTL for seed weight at a similar genomic position in

other Vigna species. Of Vigna species analyzed to date only black gram

did not have a QTL for seed size at a similar location to azuki bean.

References: Rice bean, mungbean 1 (Isemura et al. 2008), mungbean 2

(Fatokun et al. 1992, Menacio-Hautea et al. 1993), azuki bean (Isemura

et al. 2007a), black gram (Chaitieng et al. 2006), cowpea (Fatokun et

al. 1992).

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Table –

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2.2 : Vigna genome linkage maps

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24

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25

2.6. SYNTENY IN Vigna SPECIES:

2.6.1.Vigna and other genera :

The efforts have been made to understand the comparative genome

organization among Vigna and related cultigens. Comparisons of genome

maps of V. radiata with V. unguiculata and Phaseolus vulgaris have revealed

conserved blocks of considerable size some containing loci for important

traits. The comparison with P. vulgaris showed that average size of

conserved blocks is about 36.6cMwith the longest being 103.5 cM (Menacio-

Hautea et al., 1993; Boutin et al., 1995). Hence, there is a scope for

understanding genome organization of the genus Vigna by comparison with

better developed genome maps in other related species.

Soybean genome map is one of the best-developed genome maps among

legumes. Comparison of V. radiata and Glycine max revealed a different type

of genome organization than the comparison of Glycine max and Phaseolus

vulgaris. Conserved linkage blocks are smaller and are highly scattered in

the V. radiata comparison compared to P. vulgaris). Comparative mapping of

V. radiata and Lablab purpureus (hyacinth pea), both belonging to subtribe

Phaseolinae, revealed that the order of markers is highly conserved and

enabled suggestions of which linkage group belonged on the same

chromosome in lablab and green gram (Humphrey et al. 2002). Surprisingly,

the results suggest that green gram shares a higher level of genome

organization with lablab than taxonomically more closely related species in

the subgenus Ceratotropis (V. angularis and V. umbellata). However, while

green gram and lablab maintain the same marker order, they have

accumulated a large number of deletions/duplications after divergence

(Humphrey et al. 2002). Despite the incompleteness of the genetic map data,

comparisons between Phaseolus vulgaris and V. radiata and Arabidopsis

have enabled a reconstruction of a proposed ancestral DNA segment in the

present genome of soybean (Lee et al. 2001).

2.6.2. Within the genus Vigna :

The comparison of cowpea and green gram linkage maps revealed that 90%

(48 out of 53) RFLP probes hybridized with both species. Though marker

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26

order was often similar, distances between markers varied. Ten regions of

the linkage maps of these two cultigens showed syntenic association

(Menacio-Hautea et al. 1993). A specific study of the genetics of seed weight

resulted in finding quantitative trait loci (QTLs) that accounted for 52.7 and

49.7% of the variation for this trait in cowpea and green gram, respectively

(Fatokun et al. 1992).

2.6.3. Within Vigna subgenus Ceratotropis :

Two interspecific linkage maps (rice bean × azuki bean and azuki bean × V.

nakashimae) when compared revealed seven conserved linkage blocks (size

range 7–115 cM; Kaga et al. 2000). Comparison of the rice bean × azuki

bean linkage map with the mung bean linkage map of Menacio-Hautea et al.

(1993) revealed 16 conserved segments without regions of inversion and

translocation (size range 2–95 cM; Kaga et al. 2000). This study enhances

the chance of orthologous linkage groups in the different maps to be

proposed.

2.7 BRUCHID LINKED MOLECULAR MARKERS IN Vigna :

2.7.1. Bruchid :

Green gram production in our country is constrained by an array of

destructive pests, a notable group of which are the storage pests. Among the

most destructive pests which affect green gram production and marketing

are bruchids, belonging to the genus Callosobruchus (Coleoptera:

Bruchidae). The most important of the bruchid beetles are Callosobruchus

chinensis (L.) and C. maculatus (F.) (Southgate, 1979). C. chinensis is the

most devastating pest of stored Vigna spp. in East Asia (Lin, 1981; Shinoda

and Yoshida, 1984). These insects attack stored seeds, and an infestation

can result in major losses. No resistant commercial cultivars are available to

date, and currently the insects are controlled by the use of insecticides. This

type of preventive measure has two main disadvantages however,

application of the insecticides adds extra costs to the green gram industry,

and residual insecticides are a major concern. For these reasons, bruchid

resistance has been one of the main breeding objectives in green

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27

gram improvement. To effectively breed a bruchid resistant cultivar,

breeders have actively sought sources of bruchid resistance (Fuji et al.

1989).

Infestation may start in the field and is generally carried over until the seeds

are stored. Insects breed throughout the year as long as seeds are available.

The weevil can complete four generations in a year under optimum storage

conditions (Kim and Choi, 1987). Bruchid beetles first infest Vigna species

in the field where the adult female lays eggs on young pods, hatched larvae

bore through the pod wall and feed within the seeds (Southgate, 1979).

When the crop is harvested and stored, the bruchid continues feeding and

eventually comes out as an adult and causes secondary infestation, which

can result in total destruction of a seed lot within a period of 3-4 months

(Banto and Sanchez, 1972).

In storage, the adult female bruchids deposit eggs on the seed coat of the

dry seed. The larvae bore into the seed and hollow out the interior as they

feed. After pupation, the adults emerge, leaving holes where they exit, and

deposit eggs on sound seeds starting a new life cycle. The cycle from egg to

adult requires only three to four weeks. If uninterrupted, infestation may

continue until all seeds in the storage area are destroyed.

Density pressure plays a role in emergence of bruchids (Murai and Fujii,

1970). One female weevil can lay about 15 eggs per day (Chun and Ryoo,

1992) and at 30º C one generation can be completed in 21 days.

Eggs of C. chinensis hatch approximately one week after oviposition and

larvae burrow through their egg shell’s underside and into the cotyledons.

The shell serves as a cover over the entrance hole; pupation occurs about

three weeks after feeding with adults emerging about a week later. Males

emerge one to two days before the females (Shinoda and Yoshida, 1984).

Kim and Choi (1987) found that C. chinensis had an optimum oviposition

temperature of 25 to 30 º C. Kim and Choi (1987) opined that C. chinensis

preferred azuki bean followed by mungbean for oviposition.

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2.7.2. Markers linked with bruchid:

Hence without appropriate preventive and eradicative control measures

bruchids have the potential to cause complete loss of the seed harvest

within a period of three to four months ( Banto and Sanchez, 1972). No

resistant commercial cultivars are available to date, and currently the

insects are controlled by the use of insecticides. The use of resistant

varieties has been the most cost effective and environment friendly means of

mitigating pest damage to green gram. However breeding for bruchid

resistance has been hampered by the lack of diverse and effective resistance

within the green gram species (Talekar and Lin 1981). Development of insect

resistant green gram would reduce the loss of green gram by insect

predation.

To effectively breed a bruchid resistant cultivar, breeders have actively

sought sources of bruchid resistance (Fuji et al. 1989).Reeves (1993)

reported that with the help of RFLP technology, efficiency of transferring

resistance to bruchids could be improved. Santalla et al., (1998) found some

informative RAPD primers of green gram. Miyagi et al. (2004) constructed

green gram BAC libraries and applied in developing PCR-based and locus-

specific markers ( STSbr 1 and STSbr 2) for linked closely with this major

locus conferring bruchid resistance. STSbr 1 generated a co-dominant

marker, while STSbr 2 generated a dominant marker. Two co-dominant PCR

markers were identified and recommended for marker assisted selection of

bruchid resistance in green gram during marker-assisted selection (Cheng et

al., 2005) using the bulked segregant analysis (BSA) method.

Ma et al. (2005) used amplified fragment length polymorphism (AFLP)

technique and collected polymorphic bands from polyacrylamide gel and

conducted PCR and thereafter, they performed sequence alignment

facilitating further studies on AFLP molecular marker research, conversion

and gene cloning of bruchid resistance gene.

Chen et al., (2007) identified ten randomly amplified polymorphic DNA

(RAPD) markers associated with the bruchid resistance gene (Br) in

recombinant lines of green gram accession TC 1966 [Vigna sublobata (Roxb.)

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29

Bairig.] through bulked segregant analysis. They cloned four closely linked

RAPDs and transformed into sequence characterized amplified region

(SCAR) and cleaved amplified polymorphism (CAP) markers. Through

transformation of these RAPDs into CAPs, they successfully obtained co-

dominant markers for bruchid resistance.

A linkage map for bruchid resistance gene (Br) and vignatic acid gene (Va)

using RFLP markers in green gram was constructed by Kaga and Ishimoto

(1998). A major QTL underlying bruchid resistance to C. chinensis in V.

umbellata has been found in an interspecific mapping population of V.

umbellata –V. angularis cross (Kaga et al., 2000). Somta et al. (2008a) found

Callosobruchus spp. resistance in V. nepalensis Tateishi and Maxted, a

species that was cross compatible with azuki bean. They detected seven

quantitative trait loci (QTLs) for bruchid resistance; five for resistance to C.

chinensis and two for resistance to C. maculatus. QTLs on linkage group (LG)

1 and LG 2 for bruchid resistance to C. chinensis co-localized with seed size

QTLs suggesting that incremental increase in seed size accompanied

susceptibility to C. chinensis. Sun et al. (2008) determined the genetic

distance between the two markers, namely OPC-06 and STSbr 2, and the

bruchid resistant gene 11.0 and 5.8 cM respectively which would benefit for

marker assisted selection in breeding programmes of green gram.

Fujii and Miyazaki (1987) suggested a two-step interspecific hybridization of

a weevil-resistant Vigna sublobata race II cultivar to green gram and then

azuki bean to the V. sublobata-mungbean cross to transfer the resistance

gene(s) to azuki bean The single dominant gene Br in green gram accession

TC1966 (Kitamura et al., 1988) has been mapped with molecular markers

(Young et al., 1992, Kaga and Ishimoto, 1998) and successfully used in a

breeding programme (Tomooka et al., 1992b). Kaga and Ishimoto (1998)

incorporated the Br gene from a wild green gram that conferred the

resistance to infestation by the azuki bean weevil and cowpea weevil into a

susceptible cultivar. Transfer of bruchid resistance from V. umbellata to

susceptible azuki bean is difficult due to cross incompatibility. As an

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30

alternative approach to gene transfer, the use of a bridging species, has

been proposed (Tomoka et al., 2000).

The segregation study of a cross between a mungbean yellow mosaic virus

(MYMV) resistant cultivar of V. mungo and C. maculatus resistant Trombay

wild urdbean (V. mungo var. silvestris) indicated the presence of two

dominant duplicate genes for resistance to C. maculatus and the bruchid

resistance of the seeds was due to antibiosis (Souframanien and

Gopalakrishna, 2007). Somta et al. (2007) found that resistance to bruchids

in green gram seeds was controlled by maternal plant genotype and by a

major gene. They observed the presence of modifiers and they also

concluded that the resistance was dominant at varying degrees of

expressivity. Sun et al. (2008) found that the bruchid resistance in V2709, a

green gram cultivar with bruchid resistance from India, was controlled by

one dominant gene called Br 2.