Molecular Characterization of Yellow Mosaic Virus Resistance

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Molecular characterization of yellow mosaic

virus resistance in cowpea (Vigna

unguiculata L.Walp.)

By

Tran Dinh Gioi2004BS1D

Thesis submitted to the Chaudhary Charan Singh

Haryana Agricultural University in partial fulfilment

of the requirements for the degree of

DOCTOR OF PHILOSOPHYDOCTOR OF PHILOSOPHY

ININ

BIOTECHNOLOGY AND MOLECULAR BIOLOGYBIOTECHNOLOGY AND MOLECULAR BIOLOGY

DEPARTMENT OF BIOTECHNOLOGY & MOLECULAR BIOLOGY

COLLEGE OF BASIC SCIENCES AND HUMANITIES

CCS, HARYANA AGRICULTURAL UNIVERSITY

HISAR 125004 (INDIA)


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2008

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CERTIFICATE - I

This is to certify that this thesis entitled Molecular

Characterization of Cowpea Yellow Mosaic Virus in Cowpea

(Vigna unguiculata L.Walp.), submitted for the degree of Doctor

of Philosophy, in the subject of Biotechnology and Molecular

Biology to the Chaudhary Charan Singh Haryana Agricultural

University, is a bonafide research work carried out by Tran Dinh

Gioi under my supervision and that no part of this research project

has been submitted for any other degree.

The assistance and help received during the course of

investigation have been fully acknowledge

(Dr. Kamla Chaudhary)

Major Advisor (Professor)Deptt. of Biotechnology & Molecular

BiologyCCS HAU, Hisar 125 004 (India)

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CERTIFICATE - II

This is to certify that this thesis entitled, " Molecular

Characterization of Cowpea Yellow Mosaic Virus in Cowpea

(Vigna unguiculata L.Walp.) submitted by Tran Dinh Gioi to the

Chaudhary Charan Singh Haryana Agricultural University, in partial

ful filment of the requirements for the degree of Doctor of

Philosophy in the subject of Biotechnology and Molecular

Biology has been approved by the Student's Advisory Committee

after an oral examination on the same.

MAJOR ADVISOR

HEAD OF THE DEPARTMENT

DEAN, POST-GRADUATE STUDIES

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ACKNOWLEDGEMENT

In the last three and half years of accumulation in the study

and research, it is the immense pleasure reaching this stage of

releasing my thesis. From which I would like to express my profound

gratitude and sincere thanks to all people who have been contributed

to my success.

With the gratefulness and respectability, I express my deep

sense of regard and unforgettable indebtedness to my major advisor

Dr. (Ms) K. Chaudhary, Professor, Department of Biotechnology and

Molecular Biology for her invaluable guidance, constant

encouragement and suggestions during the course of investigation and

in preparation of thesis manuscript. Her constant encouragement andsympathetic understanding at every step is much appreciated. I will

never forget the valuable suggestions given by her, which I never

expected from anyone other than my parents.

I take this opportunity to express my deepest sense of

gratitude towards my co-advisor, Dr. K. S. Boora, Professor,

Department of Biotechnology and Molecular Biology, for his keen

interest, learned counsel and sublime suggestions during the course

of investigation because without his untiring help this research wouldnot have been possible. His positive attitude and unstinted

advisement made my studies and research more interesting.

It is my privilege to express my profound sense of gratitude

and indebtedness to my advisory committee, Dr. A.S Yadav, Sr.

scientist, Genetics Department, Dr. (Mrs) Indra Hooda, Proffessor,

Pathology Department, Dr. R. S. Khatri, Ass. Professor, Forage

section, Plant Breeding Department, for their constructive help,

stimulating suggestions and encouragement.I express my esteem and deep sense of gratitude to Prof. V.K.

Chowdhary, Dean and Head, Department of Biotechnology and

Molecular Biology for providing necessary facilities during the course

of investigation.

I would like to express sincerely and profoundly my

thankfulness to entire professors and scientists of the Biotechnology

and Molecular Biology Department for their teaching to impart the

scientific knowledge. I will never forget those who have given to methe key to open the treasure of knowledge. I also record my cordial

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thanks to entire officials and staffs of BMB Department for the help

and cooperation in the time of my study.

It gives me immense pleasure to record my gratitude to the

Vietnamese and Indian governments, especially ICCR, HAU and the

Cuu Long Delta Rice Research Institute for providing financial

support and encouragements in completion of my studies in Chaudhary

Charan Singh, Haryana Agriculrural University, Hisar, Haryana, India.

I do acknowledge my deeply sense of gratefulness to leaders

and scientists: Prof. Dr. Nguyen Van Luat, Prof. Dr. Bui Ba Bong, Prof.

Dr. Bui Chi Buu, Ass. Prof. Dr. Nguyen Thi Lang, Ass. Prof. Dr. Pham

Van Du, Dr. Nguyen Thi Loc, Dr. Bui Thi Thanh Tam, ect. of the Cuu

Long Delta Rice Research Institute, Ministry of Agriculture and Rural

Development, Vietnam.Words in my vocabulary are too less and inappropriate to

express my innermost feeling and sincere appreciation to all of my

friends, especially Aditi Gualati, Harish Dhingra, Urvasi, Poonam

Sharma, Anshu Bajaj, Shardul Shanker, Zerihun Demrew, Rochika,

Poonam Yadav and all Vietnamese students/colleagues: Mr. N.C.

Thanh, Mr. D.V. Tam, Mrs, T.T.K. Trang, Mss. N.T.Q. Thuan, Mrs.

T.T.M. Hanh, Mss. N.T.N. Truc, Mrs V.T.T. Hang, Mr. N.V. Phong, Mr.

N.V. Khiem, Mr. V.T. Khang, Mr. D.H. Duc, Mr. N.L. Thang, Mr.P.H.Lam, Mr. N.T. Hieu, Mr. D.H. Son, Mr. D.Q. Hung, Mr. N.X. Thang,

Mr. P.D. Tuan, Mr. B.V. Thu, Mr. Rajdeep Singh, and to all my

classmates for their help and cooperation.

Last but not the least, no words of mine can adequately express

my indebtedness to my respected parents my parents in-law, my

brothers and sisters, especially, my wife Nguyen Thi Pha and my son

Tran Minh Quang for their love, affection, inspiration, patience,

encouragement, well wishes and help throughout the course of study.Sometimes silence is the only language in which I can express

my regards to those whose names I forget to mention in this

endeavour.

Finally, Iwould like to thank all whose direct and indirect

support helped me completing in my thesis in time.

Dated: May 2008 TRAN DINH GIOI

Place : Hisar

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CHAPTER-I

INTRODUCTION

Cowpea, Vigna unguiculata (L.) Walp. is an important grain legume crop

in developing countries of the tropics and subtropics, especially in sub-

saharan Africa, Asia, Central and South America (Singh et al., 1997). Its

value lies in its high protein content (23-29%, with potential for perhaps

35%); and its ability to fix atmospheric nitrogen, which allows it to grow

on, and improve poor soils (Steele, 1972). Cowpea is cultivated for its

seed (shelled green or dried), pods and/or leaves, which are consumed in

fresh form as green vegetables, while snacks and main meal dishes are

prepared from the dried grain. All the plant parts used for food are

nutritious, making it extremely valuable where many people cannot afford

protein foods such as meat and fish. The rest of the cowpea plant, after

pods are harvested, is also used as a nutritious livestock fodder. Cowpea

seed is a nutritious component in the human diet and livestock feed. It is

a well-balanced vegetarian diet with low-fat, high-complex carbohydrate,

and moderate protein characteristics of the edible portion (Bubenheim et

al., 1990). The protein in cowpea seed is rich in the amino acids, lysine

and tryptophan, compared to cereal grains. Therefore, cowpea seed is

valued as a nutritional supplement to cereals and an extender of animal

proteins. Cowpea also has the ability to be intercropped with cereals such

as millet and sorghum.

Cowpea is considerable as one of the most widely adapted and versatile

crop which can tolerate to high temperatures and drought compared to

other crop species. Drought resistance is one reason that cowpea is such

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an important crop in many underdeveloped parts of the world. One of the

more remarkable things about cowpea is that it thrives in dry

environments; it can produce the dry grain yield of up to 1000 kg/ha in a

Sahelian environment with only 181 mm of rainfall and high evaporative

demand (Hall and patel, 1985). It is estimated that cowpea is now

cultivated on at least 12.5 million hectares, with an annual production of

over 3 million tons worldwide (Singh et al. 1997). In India cowpea is

mainly cultivated for fodder, green manure and soil improving cover crop.

Green pods of cowpea are used as vegetable in Northern Indian States

whereas in West Bengal, Tamil Nadu, Andhra Pradesh, Kerala and

Maharashtra cowpea is cultivated as a pulse crop.

The crop productivity is greatly affected by a numbers of biotic factors

such as fungi, bacteria and viruses. Viral diseases are considered to be a

major limiting factor for the production of cowpea in the tropical and sub-

tropical countries (Mali and Thottappilly, 1986). More than 20 viruses are

reported from various cowpea-growing areas worldwide (Thottappilly andRossell, 1985). Among these viruses, cowpea yellow mosaic virus

(CYMV) is the most serious disease of cowpea. It may cause 80-100 %

yield reductions (Chant, 1960; Shoyinka, 1974; Gilmer et al., 1974; and

Williams, 1977). Cowpea yellow mosaic virus also affected seriously in

vegetative parts of the plant (Bashir et al., 2002). It may cause 14 to 54 %

decrease in plant height 30 to 95 % decrease in dry stem weight of

cowpea and mung bean (Ilyas, 1999).

Microsatellites or simple sequence repeats (SSR) are DNA sequences

with repeat lengths of a few base pairs. Variation in the number of repeats

can be detected with PCR by developing primers for the conserved DNA

sequence flanking the SSR. As molecular markers, SSR combine many

desirable marker properties including high levels of polymorphism and

information content, unambiguous designation of alleles, even dispersal,

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selective neutrality, high reproducibility, co-dominance, and rapid and

simple genotyping assays. Microsatellites have become the molecular

markers of choice for a wide range of applications in genetic mapping

and genome analysis (Chen et al., 1997; Li et al., 2000), genotype

identification and variety protection (Senior et al., 1998), seed purity

evaluation and germplasm conservation (Brown et al., 1996), diversity

studies (Xiao et al., 1996), paternity determination and pedigree analysis

(Ayres et al., 1997; Bowers et al., 1999; van de Ven and McNicol, 1996),

gene and quantitative trait locus analysis (Blair and McCouch, 1997; Koh

et al., 1996), and marker-assisted breeding (Ayres et al., 1997; Weising

et al., 1998). For identification of molecular markers linked to

agronomically important genes, SSR was also the best choice in

compared to RAPD and AFLP in a more polymorphic information or more

cost effective manner, respectively (Lee 1995; Kelly and Miklas 1998;

Young 1999). The development and use of molecular marker

technologies has also facilitated the subsequent cloning and

characterization of disease, insect, and pest resistance genes from avariety of plant species (Hammond-Kosack and Jones 1997; Ronald

1998; Meyers et al. 1999). Therefore, this study was done with the

following objectives:

1. To investigate the genetic basis of cowpea yellow mosaic virus

resistance in cowpea using microsatellites markers.

2. To tag microsatellite markers linked to cowpea yellow mosaic virus

resistance in cowpea.

3. To identify quantitative trait loci (QTLs) for resistance to cowpea

yellow mosaic virus in cowpea.

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CHAPTER-II

REVIEW OF LITERATURE

Cowpea has a number of common names, including southern peas,

blackeye peas, and crowder pea. However, they are all the species Vigna

unguiculata (L.) Walp., which in older references may be identified as

Vigna sinensis (L.). It is classified in Vigna genus and unguiculata species

whose chromosome numbers were counted from root tips of 192 cultivars

and lines including wild forms, cultivated forms from 42 countries revealed

2n=22 (Faris, 1964). Mukherjee (1968) conducted a critical study of

panchytene chromosomes of V unguiculata and described each of 11

bivalents. He found that the complement consisted of a short (19m), 7

medium (26-36 m), and 3 long (41-45m) chromosomes. The

chromosomes were not distributed uniformly along the chromosome

arms.

Cowpea diseases induced by species of pathogens belonging to various

pathogenic groups (fungi, bacteria, viruses, nematodes, and parasitic

flowering plants) constitute one of the most important constraints to

profitable cowpea production in all agro-ecological zones where the crop

is cultivated. Genes conferring resistance to these pathogens have been

isolated from a variety of plant species, including almost all of the

agronomically important grasses and legumes (Baker et al. 1997;

Gebhardt 1997; Hammond-Kosack and Jones 1997). The products of the

resistance (R) genes have been suggested to act as receptors that

specifically bind ligands encoded by the corresponding pathogen

avirulence factors in a gene-for-gene recognition process (Baker et al.

1997; Hammond-Kosack and Jones 1997). The R-gene product factor

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complex is thought to initiate a series of signaling cascades within the cell

leading to disease resistance. Among the downstream cellular events that

characterize the resistant state are rapid oxidative bursts, cell wall

strengthening, the induction of defense gene expression, and rapid cell

death at the site of infection (Morel and Dangl 1997).

2.1 GENETICS OF PLANT VIRUS RESISTANCE

The study of plant resistance genes (R genes), plant genes in which

genetic variability occurs that alters the plants suitability as a host, also

raises many fundamental questions regarding the molecular, biochemical,

cellular, and physiological mechanisms involved in the plant-virus

interaction and the evolution of these interactions in natural and

agricultural ecosystems. Over the past decade, the cloning and analysis

of numerous plant R genes (Hanson et al., 2000 and Martin et al., 2003)

have stimulated attempts to develop unifying theories about mechanisms

of resistance and susceptibility, and co-evolution of plant pathogens andtheir hosts. The focus has been mainly on monogenic dominant

resistance to fungal and bacterial pathogens (Hanson et al., 2000);

however, there is clear evidence that common mechanisms can be

involved in virus resistance.

2.1.1 Types of Resistance

Resistance to disease of plants has historically been divided into two

major categories (Fraser, 1990): non-host resistance and host resistance.

The former, which encompasses the case where all genotypes within a

plant species show resistance or fail to be infected by a particular virus,

specifically signifies the state where genetic polymorphism for

susceptibility to a particular virus has not been identified in a host taxon.

Clearly, most plant species are resistant to most plant viruses.

Susceptibility is the exception to the more general condition of resistance

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or failure to infect. Although underlying mechanisms of non-host

resistance to viruses are largely unknown and are likely as diverse for

viruses as they are for other classes of plant pathogens (Mysore and Ryu,

2004), improved understanding of the ways in which infection fails in

these interactions may be particularly important for breakthroughs in the

development of plants with durable broad-spectrum disease resistance.

Host resistance to plant viruses has been more thoroughly investigated,

at least in part because, unlike non-host resistance, it is genetically

accessible. This general case, termed host resistance, specific

resistance, genotypic resistance, or cultivar resistance, occurs when

genetic polymorphism for susceptibility is observed in the plant taxon, i.e.,

some genotypes show heritable resistance to a particular virus whereas

other genotypes in the same gene pool are susceptible. In resistant

individuals, the virus may or may not multiply to some extent, but spread

of the pathogen through the plant is demonstrably restricted relative to

susceptible hosts, and disease symptoms generally are highly localizedor are not evident.

The distinction between resistance to the pathogen and resistance to the

disease is important to articulate. Resistance to the pathogen typically

leads to resistance to the disease; however, resistant responses involving

necrosis can sometimes be very dramatic, even lethal, e.g., the I gene in

Phaseolus vulgaris for resistance to Bean common mosaic virus (Collmer

et al., 2000). In the case of resistance to disease symptoms or tolerance

to the disease, the virus may move through the host in a manner that is

indistinguishable from that in susceptible hosts, but disease symptoms

are not observed. If the response is heritable, these plants are said to be

tolerant to the disease, although they may be fully susceptible to the

pathogen. This host response is very prevalent in nature, and has been

used to considerable benefit in some crops, e.g., the control of Cucumber

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mosaic virus (CMV) in cucumber, even though the genetic control of this

response is typically difficult to study (Fraser, 1990 and Roger, 2002).

The genetics of tolerant responses are not considered further due to the

complexity of the biology and relative lack of information.

More recently, a third important category of host resistance has been

identified, systemic acquired resistance (SAR). This response can be

activated in many plant species by diverse pathogens that cause necrotic

cell death (Ross, 1961), resulting in diminished susceptibility to later

pathogen attack. Virus-induced gene silencing, another induced defense

mechanism to virus disease, has also been reviewed recently

(Baulcombe, 2004).

Transgenic approaches to plant virus resistance have been widely

explored since the earliest experiments where by transgenic tobacco

plants expressing Tomato Mosaic Virus (TMV) coat protein (CP) were

challenged with TMV and shown to be resistant (Goldbach et al., 2003;Roger, 2002 and Rudolph et al, 2003). It is now possible to engineer

resistance and tolerance to plant viruses using transgenes derived from a

wide range of organisms including plant-derived natural R genes,

pathogen-derived transgenes, and even non-plant and non-pathogen-

derived transgenes. The issues related to the creation and deployment of

genetically engineered resistance in crop breeding has been recently

reviewed (Dunwell, 2000; Nap et al., 2003 and Tepfer, 2002).

2.1.2 Genetics of Virus Resistance in Nature

The first step in the study of genetics of viral resistance is to determine

whether the resistant response is inherited, if so, the number of genes

involved and their mode of inheritance. More than 80% of reported viral

resistance is mono-genically controlled; the remainder shows oligogenic

or polygenic control. Only slightly more than half of all reported

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monogenic resistance traits show dominant inheritance. In most but not

all (Fraser, 1986) cases, dominance has been reported as complete. The

heterozygote may show a clearly different response from that of the

homozygote; however this is rarely checked carefully in inheritance

studies. Where incomplete dominance is observed, there are important

implications for mechanisms that may involve gene dosage effects. The

relatively high proportion of recessive viral R genes is in marked contrast

to fungal or bacterial resistance where most reported resistance is

dominant.

Dominant resistance is often, although not always, associated with the

hypersensitive response (HR) (Fraser, 1986), possibly due to the frequent

use of HR as a diagnostic indicator for field resistance by plant breeders.

HR, induced by specific recognition of the virus, localizes virus spread by

rapid programmed cell death surrounding the infection site, which results

in visible necrotic local lesions. HR-mediated resistance is a common

resistance mechanism for viruses and for other plant pathogens. Becausethe extent of visible HR may be affected by gene dosage (Collmer et al.,

2000), genetic background, environmental conditions such as

temperature, and viral genotype, etc., schemes that classify or name virus

R genes based on presence or absence of HR may obscure genetic

relationships.

In contrast to dominant R genes, many recessive R genes appear to

function at the single cell level or affect cell-to-cell movement. More than

half of the recessive R genes identified to date confer resistance to

potyviruses, members of the largest and perhaps the most economically

destructive family of plant viruses (Shukla et al, 1994). In general,

considerably less is known regarding the mechanisms that account for

recessively inherited resistance mechanisms. Several recessive R genes

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have recently been cloned and/or characterized (Gao et al., 2004; Kang

et al., 2005; Nicaise et al., 2003; Ruffel et al, 2002; Wicker et al, 2005).

2.1.3 Natural Resistance Mechanisms

To complete their life cycles, viruses undergo a multistep process that

includes entry into plant cells, uncoating of nucleic acid, translation of viral

proteins, replication of viral nucleic acid, assembly of progeny virions,

cell-to-cell movement, systemic movement, and plant-to-plant movement

(Carvalho and Lazarowitz, 2004). Plant viruses typically initiate infection

by penetrating through the plant cell wall into a living cell through wounds

caused by mechanical abrasion or by vectors such as insects and

nematodes. Unlike animal viruses, there are no known specific

mechanisms for entry of plant viruses into plant cells (ShawJ, 1999).

When virus particles enter a susceptible plant cell, the genome is

released from the capsid, typically in the plant cytoplasm. Although not

yet comprehensively analyzed, current work suggests this uncoating

process is not host-specific. e.g.. TMV and Tobacco yellow mottle viruswere uncoated in both host and non host plants (Kiho et al., 1972 and

Matthews and Witz, 1985). Once the genome becomes available, it can

be translated from mRNAs to give early viral products such as viral

replicase and other virus-specific proteins. Here after the virus faces

various constraints imposed by the host and also requires the

involvement of many host proteins, typically diverted for function in the

viral infection cycle.

Successful infection of a plant by a virus therefore requires a series of

compatible interactions between the host and a limited number of viral

gene products. Absence of a necessary host factor or mutation to

incompatibility has long been postulated to account for recessively

inherited disease resistance in plants, termed passive resistance by

Fraser (1986, 1990).

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Figure 1 (A) Possible virus resistance mechanisms showing dominant or

recessive inheritance contrasted with a susceptible interaction. (B) Stages

of a viral infection cycle with points of potential host interference identified

as resistance targets (Fraser, 1990).

2.1.3.1 Cellular Resistance to Plant Viruses

Resistance at the single cell level may be characterized as a state where

virus replication does not occur, or occurs at essentially undetectable

levels in inoculated cells. This type of resistance has been termed

extreme resistance (ER), cellular resistance: or immunity (Fraser,

1986 and Fraser, 1990). A classical example of this type of resistance is

observed when Vigna tin guiculata is challenged with the Comovirus

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move from the initially infected cells to adjoining cells, eventually resulting

in systemic infection. An important class of host response to viral infection

is apparent when the virus appears to establish infection in one or a few

cells, but cannot move beyond the initial focus of infection. Resistance at

this level can result from either failure of interactions between plant and

viral factors necessary for cell-to-cell movement, or from active host

defense responses that rapidly limit virus spread.

Most plant virus genomes code for one or more movement proteins,

which are required for viral cell-to-cell movement. Based on their primary

structure, movement proteins can be divided into several superfamilies,

one of which is the "30K" superfamily, related to the Tobacco mosaic

virus movement protein (Melcher, 2000). Within this 30K superfamily, two

basic mechanisms for cell-to-cell movement have been proposed

(Lazarowitz and Beachy, 1999). Tobacco mosaic virus movement protein

typifies one mechanism whereby the movement protein modifies

plasmodesmata, allowing viral RNA-movement protein complexes tomove from cell to cell. The other type of movement, best known from

Cowpea mosaic virus movement protein, is the tubule-guided movement

of mature virus particles through drastically modified plasmodesmata.

Cell-to-cell movement of CPMV occurs through tubular structures, built-up

from the viral movement protein, that replace the desmotubule (ER

portion inside the plasmodesmata) and through which mature virions are

transported from one cell into the adjacent ones (Fig. 2; Wellink & van

Kammen, 1989 and van Lent et al., 1990).

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Fig. 2 Model of the cell-to-cell movement mechanism of CPMV. In this

model a CPMV-infected cell is depicted, from which virus particles aretraveling to the neighbouring (uninfected) cell through a modified

plasmodesma.

As described above for viral replication and translation, intra-and

intercellular viral movement also requires both virus-encoded components

and specific host factors (Carrington et al., 1996 and Lazarowitz and

Beachy, 1999). With respect to intercellular movement, it is well

established that movement proteins (MP), identified for most families of

plant viruses (Deom et al., 1992; Gilbertson and Lucas, 1996; Mahajan et

al., 1998 and Santa Cruz, 1999), perform dedicated functions required for

cell-to-cell movement by modifying pre-existing pathways in the plant for

macro-molecular movement such that viral material can translocate

between plant cells (Carrington et al., 1996 and Lazarowitz, 2002). In the

case of potyviruses, which do not encode a dedicated MP, the movement

functions have been allocated to several proteins, including CP, HC-Pro,

the cylindrical inclusion (CI) protein, and the genome-linked protein (VPg)

(Revers et al., 1999). In mutant viruses defective in these proteins,

movement from the initially infected cell to adjacent non-infected cells did

not occur.

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A number of mutations in host genes are known that prevent cell-to-cell

movement of plant viruses. The Arabidopsis cum1 and cum2mutations

inhibit CMV movement (Yoshii et al., 1998a and Yoshii et al., 1998b). In

protoplasts prepared from plants homozygous for these alleles, CMV

RNA and CP accumulate to wild-type levels, but the accumulation of the

CMV 3a protein, necessary for cell-to-cell movement of the virus, is

strongly reduced.

The HR also serves to disrupt cell-to-cell movement of plant viruses.

Recognition of the viral elicitor results in the induction of a cascade of

host defense responses that include oxidative H2O2 bursts and up-

regulation of hydrolytic enzymes, PR proteins, and callose and lignin

biosynthesis. As a consequence, viral movement may be limited to a

small number of cells, illustrated by such classic examples as the tobacco

N gene (Otsuki et al., 1972) and the tomato Tm-2 and Tm-22 alleles

(Motoyoshi and Oshima, 1975). Protoplasts isolated from the plants

carrying these R genes allowed replication of TMV; no cell death wasobserved. Despite the strong correlation of HR and disease resistance,

necrotic cell death is now thought to be an ancillary con-sequence of the

resistant response, not necessary for pathogen suppression.

Furthermore, when HRT was introgressed into Col-1, most of the HRT-

transformed plants developed HR upon TCV infection, yet the virus

spread systemically without systemic necrosis (Cooley et al., 2000).

2.1.3.3 Resistance to Long-Distance Movement

In susceptible hosts, plant viruses that do not show tissue restrictions

move from the mesophyll via bundle sheath cells, phloem parenchyma,

and companion cells into phloem sieve elements (SE) where they are

translocated, then unloaded at a remote site from which further infection

will occur (Carrington et al., 1996, Santa Cruz, 1999). This pathway is

typically part of an elaborate symplastic network in plants through which

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viruses establish systemic infection (Lucas et al., 1995). Plasmodesmata,

elaborate and highly regulated structures with which viruses interact for

both cell-to-cell and long-distance movement, provide symplastic

connectivity between the epidermal/ mesophyll cells and cells within the

vasculature, including sieve elements (Carrington et al., 1996; Lucas and

Gilbertson, 1994 and Santa Cruz, 1999). Entry into the SE-companion

cell complex is currently thought to be the most significant barrier to long-

distance movement (Ding et al., 1998 and Wintermantel et al., 1997).

Once present in a companion cell, a virus potentially has direct access to

the sieve tube, the conducting element of the phloem that serves as the

pathway for both nutrient and virus transport throughout the plant

(Carvalho and Lazarowitz, 2004).

Virus particles loaded in the phloem apparently follow the same pathway

as photo-assimilates and other solutes, albeit not necessarily via strictly

passive processes (Murphy, 2002 and Santa Cruz, 1999). Most plant

viruses require CP for long-distance movement, independent of anyrequirement for CP in cell-to-cell movement. Analysis of CP mutants for a

number of viruses including TMV suggests that CP is essential for entry

into and/or spread through sieve elements (Carvalho and Lazarowitz,

2004 and Lazarowitz and Beachy, 1999). Some DNA viruses also require

CP for long-distance movement (Boulton et al., 1989), although other

white fly transmitted geminiviruses do not require CP for systemic

infection (Gardiner et al., 1988). Phloem-limited viruses, e.g., Luteovirus,

are typically limited to phloem parenchyma, companion cells, and SE, and

apparently lack the ability to exit phloem tissue (Taliansky and Barker,

1999) or possibly to infect non-phloem tissue (Barker et al., 2001). A few

viruses, most notably members of the Sobemovirus genus, use xylem for

long-distance movement. The mechanisms of viral interaction with xylem

are largely unknown (Carvalho and Lazarowitz, 2004 and Moreno et al.,

2004).

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Cowpea mosaic virus represents a large group of different plant viruses,

including comoviruses (van Lent et al., 1990, 1991), nepoviruses

(Wieczorek & Sanfaon, 1993; Ritzenthaler et al., 1995), caulimoviruses

(Perbal et al., 1993) and tospoviruses (Storms et al., 1995), which employ

the tubule guided movement mechanism of virions. By means of a

surgical isolation procedure for leaf parts and pinpoint-inoculation of virus

it was demonstrated that CPMV can be loaded into the phloem of both

major veins and minor veins to establish systemic infection of the upper

leaves. Three possible routes for entry of virus into leaf veins have been

suggested (Ding et al., 1998; Nelson & Van Bel, 1998). Viruses could

enter the veins at the vein terminus, a gap at a vein branch or the side of

a vein. The successful systemic invasion of cowpea after pinpoint-

inoculation of isolated midveins suggests that CPMV is able to approach

and enter the phloem stream directly from the surrounding parenchyma

tissues.

2.2 GENETIC BASIS OF VIRAL DISEASE RESISTANCE IN COWPEA

As soon as Mendels work was rediscovered, Biffen (1905) illustrated that

disease resistance may be inherited in accordance with Mendelian laws,

and the genetic basic for breeding disease resistant varieties was

developed. From that many resistant genes were discovered in a wide

range of crops. Many genes resistance to virus diseases were identified

in cowpea, such as bean yellow mosaic virus resistance controlled by a

single recessive gene (Reeder et al., 1972), cowpea chlorotic mottle virus

resistance controlled by a single recessive gene (Rogers et al., 1973),

cowpea mottle virus controlled by single dominant gene (Bliss and

Robertson, 1971), cowpea severe mosaic virus controlled by a single

recessive gene (Mendoza et al, 1989), and cucumber mosaic virus

resistance controlled by a single dominant gene (Sinclair and walker,

1955).

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Many other viruses virulence in cowpea were described in detail with their

distinct symptoms and genetic base resistance

2.2.1 Cowpea Aphid-Borne Mosaic Virus (CAbMV)

The cowpea plants infected with CAbMV show variable amounts of dark

green vein banding, leaf distortion, blistering and stunting (Bock and

Conti, 1974; Boswell and Gibbs, 1983). The first symptoms of the virus

when carried with seed appear on first trifoliate as a fine vein clearing and

irregular mosaic (Tsuchizaki et al., 1970; Ladipo, 1977; Ata et al., 1982).

About 15-87 per cent yield reduction was reported due to infection of

CAbMV (Kaiser and Mossahebi, 1975) and complete loss of an irrigated

crop in northern Nigeria was tentatively attributed to an aphid-borne virus

disease (Raheja and Leleji, 1974). The gene of CAbMV resistance was

reported controlled by a single dominant gene (Ramiah and

Narayanaswamy, 1983)

2.2.2 Blackeye Cowpea Mosaic Virus (BICMV)

Blackeye cowpea mosaic virus produces both local and systemic

symptoms on blackeye cowpea. Local symptoms include large reddish

lesions spreading along the veins. Systemic symptoms are severe

mottling, distortion, yellowing, mosaic and vein necrosis. Lima et al.

(1979) reported that BICMV causes mottling or mosaic symptoms in

different cultivars of cowpea. The other symptoms are systemic mosaic

(Boswell and Gibbs, 1983) and vein banding mosaic (Chang, 1983).

Murphy et al (1987) reported that BICMV had developed systemic mosaic

with distortion of leaflets and stunting of the plants. BICMV is a member

ofpotyvirus group. It was reported that BICMV resistance is controlled by

a single recessive gene (Taiwo et al, 1981; Walker and Chambliss, 1981;

and Melton et al, 1987)

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2.2.3 Southern Bean Mosaic Virus-Cowpea Strain (SBMV-CS)

The cowpea strain of southern bean mosaic virus (SBMV-CS) produced

different types of symptoms in cowpea. They include mosaic, vein

clearing, leaf distortion, stunting, chlorosis, distinct chlorotic spots, early

senescence, generalized necrosis, necrotic local lesions and spindled

plants (OHair et aL, 1981). Kuhn et al. (1986) and Hobbs & Kuhn (1987)

reported symptoms of SBMV-CS in different cultivars of cowpea as leaf

chlorosis, leaf distortion, mosaic, mottling, stunting and systemic necrosis.

SBMV-CS is a member of sobemovirus group. Southern bean mosaic

virus resistance was reported with several hypotheses which controlled

by a single dominant gene (Brantley and Kuhn, 1970), a single recessive

gene (Hobbs et al, 1983), two recessive genes (Melton et al, 1987), and

three genes with incomplete dominance (Melton et al, 1987).

2.2.4 Legume Yellow Mosaic Virus (LYMV)

Yellow mosaic disease in India was first reported by Vasudeva (1942)

particularly from Punjab state and later from Maharashtra (Capoor et aL,

1947), Tamil Nadu, Gujrat, Uttar Pradesh, Punjab, Haryana and

Rajasthan (Nariani and Kandaswamy, 1961; Govindaswamy et al., 1970;

Khatri and Chenulu, 1970; Sharma and Varma, 1975).

LYMV causes typical mosaic symptoms in cowpea (Smith, 1924; Dale,

1949; Chant, 1959). Smith (1972) reported that LYMV caused chlorotic

lesions (2-4 nm dia.) on inoculated leaves of several cowpea cultivars. In

certain cases, these lesions may be in the form of alternating light and

dark green rings. When leaves are inoculated before attaining full size,

the local lesions tend to coalesce. The next leaf to unfold usually shows

pronounced vein clearing which changes, as the leaves expand to a fine

grained mosaic of numerous dark green islands on a pale green

background. The leaves developing subsequently show irregular

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yellowish and dark green mottling accompanied by blistering of the

laminae. Lima and Nelson (1977) reported that LYMV causes mosaic and

leaf distortion on cowpea. The cowpea mosaic virus causes mottling,

mosaic, leaf distortion, systemic necrosis, chlorosis and plant death.

Shankar et al. (1973) observed that the cowpea mosaic disease produced

mosaic, mottling, banding and vein clearing symptoms on certain cultivars

of cowpea. Genetically isolated begomoviruses of YLMV was

investigated by Qazi et al. (2007)

The yield reduction due to LYMV varies from 60 to 100 per cent (Gilmer

et al., 1974). The virus belongs to the Geminiviridae group. Legume

yellow mosaic virus resistance was reported due to a single dominant

gene (Ouattara and Chambliss, 1991), and another un-allelic single

recessive gene reported by Raj and Patel (1979)

2.2.5 Cowpea Mottle Virus (CMeV)

Cowpea plants with conspicuous symptoms of bright mosaic, vein-

banding, distortion of leaves and often stunting of the whole plant were

widely found during the rainy season around Abidjan. The primary leaves

of cowpea developed diffuse chlorotic lesions 3-5 days after inoculation,

often followed by veinal necrosis and detachment of inoculated leaves.

Systemic symptoms which appeared 7-9 days after inoculation on young

leaves included chlorosis, veinal mottle, yellow mosaic, and sometimes

distortion. The entire plant was stunted. The virus is easily transmitted by

mechanical inoculation (Thouvenel, 1988), and two species of beetle

Monolepta tenuicornis Jacoby and Medythia quaterna Fairmaire

(Coleoptera; Chrysomelidae) were reported capable of transmitting the

virus to cowpea (Thouvenel, 1990). Cowpea mottle virus (CMeV) was first

described by Shoyinka et al. (1978) in Nigeria and, until now, only known

from that country, some 3000 km to the east of Ivory Coast. The resistant

gene was reported controlled by single dominant gene (Bliss andRobertson, 1971)

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.2.2.6 Cowpea Yellow Mosaic Virus (CYMV)

Chan (1959) first described the properties, symptom, and host range of

CYMV. Bliss and Robertson (1971) reported that CYMV caused varied

symptoms differ with cowpea variety. Systemic symptoms in susceptible

varieties range from an inconspicuous light green mottle to a distinct

yellow mosaic, leaf distortion with significantly reduced growth and pre-

mature death of plant. The first symptoms of yellow mosaic are

manifested by its damage to the host plant cells causing yellow specks

and spots on the leaves (Verma et al., 1991). The leaves emerging from

the apex show bright yellow patches interspersed by green areas. Later

on the specks coalesce and form bigger spots with yellow area. In severe

cases whole leaves become yellow and these symptoms later appear on

pods also leading to the formation of shriveled grains. The infected plants

also become stunted in growth. The size of the pods and seed reduced.

The gene for cowpea yellow mosaic virus resistance was reported

controlled by a single dominant gene (Bliss and Robertson, 1971; and

Kumar et al., 1994). Dixielee variety is resistant to CYMV due to the

dominant gene Ymr. In addition, tolerance reaction to CYMV was also

reported due to the contribution of three additive loci and the tolerance

variety (Alabunch) was probably homozygous for the three genes (Bliss

and Robertson, 1971). The Ymr resistance gene segregates

independently of the three tolerance genes. The presence of the Ymrdominant allele masked the effects of the three additive loci, with tolerant

and susceptible plants being seen only when the resistance gene was

homozygous recessive (ymr/ymr). The virus belongs to the comovirus

group. A weak serological relationship is reported between cowpea

mosaic virus and some other viruses of the genus Comovirus i.e. cowpea

severe mosaic virus (Swaans & Van Kammen, 1973); bean pod mottle

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virus (Agrawal & Maat, 1964); red clover mottle virus (Agrawal, 1964);

broad bean true mosaic virus (Jones & Barker, 1976).

Cowpea yellow mosaic virus was reported to be transmitted by various

beetles with biting mouthparts. In Africa the chrysomelid beetle Ootheca

mutabilis is an efficient vector (Chant, 1959; Bock, 1971) but

Paraluperodes quaternus (Chrysomelidae) and Nematocerus acerbus

(Curculionidae) were also found to transmit the virus (Whitney & Gilmer,

1974). Jansen & Staples (1971) listed Cerotoma trifurcata, Diabrotica

balteata, D. undecimpunctata howardi, D. virgifera and Acalymma

vittatum (all chrysomelid beetles) as vectors. The transmission is

characterised by short acquisition and inoculation access periods and an

apparent lack of a latent period (Gergerich and Scott, 1996). Beetle

vectors may remain viruliferous for 1-2 to more than 8 days depending on

the species (Chant, 1959; Jansen & Staples, 1971). Transmission

efficiency and retention of infectivity are correlated with the amount of

vector feeding (Jansen & Staples, 1971). Whitney & Gilmer (1974)reported also transmission by white fly Bemisia tabaci Genn. (Ahmad,

1978), and by two species of grasshoppers (Cantotops spissus spissus

and Zonocerus variegatus) as well as two species of thrips, foliage thrips

(Sericothrips occipilatis Hood) and flower bud thrips Megalurothrips

sjostedtiTryb (Whitney and Gilmer, 1974; Allen and Damme, 1981).

Major, monogenic resistance genes are attractive to the breeder because

they are easy to manipulate, and can be rapidly introgressed into

susceptible materials through simple backcrossing (Kelly and Miklas,

1999). Nonspecific, polygenic resistance would be more durable, but its

deployment creates a major challenge for the breeder since epistasis and

environmental variability often mask this type of resistance. The

disadvantage of major genes is that the resultant resistance can easily be

overcome by new, virulent insect biotypes (Yencho et al., 2000). These

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The phenotype of most morphological markers can only be determined

at the whole plant level; whereas molecular loci can be assayed at

whole plant, tissue and cellular level.

Allele frequency tends to be much higher at molecular loci compared

with morphologioal markers.

Morphological markers tend to be associated with undesirable

phenotypic effect.

Alleles at morphological loci interact in a dominant recessive manner

that limits the identification of heterozygous genotypes. Molecular loci

exhibit a codominant mode of inheritance that allows the genotypic

identification of individuals in segregating populations.

Fewer epistatic or pleiotropic effects are observed with molecular

markers than with morphological markers.

With these advantages of molecular markers, a large number of

polymorphic markers can be generated and monitored in a single cross.

Therefore, a large progeny in a breeding programme can be screened

easily at an early generation by using molecular markers. Molecular

markers can be divided into two categories biochemical (storage proteins

and isozymes) and molecular (DNA) markers.

2.3.1 Protein Markers

Protein markers, including seed storage proteins, structural proteins, and

isozymes were among the first group of molecular markers exploited forgenetic diversity assessment and genetic linkage map development. They

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unguiculata and other species of genus Vigna, finding that V. unguiculata

was relatively closer to V. vexillata (subgenus Plectotropis) than other

species belonging to genus Vigna Study the seed globulins of ten Vigna

species, Rao et al. (1992) performed SDS electrophoresis to separate

and observe their polymorphism. Both inter and intraspecific variation,

thus observed allowed the identification of the ten Vigna spp. analysed.

Isozymes have also been used to manipulate quantitatively determined

characters (Stuberet al., 1987). However, the paucity of isozyrne loci and

other limitations of protein markers often restrict their utility (Hash and

Bramel-Cox, 2000).

A huge amount of the genome does not code for genes, which can be

used as protein markers.

Different biochemical procedures are required to visualize allelic

differences for enzymes having different functions, and

Many proteins are several post-transcriptional steps removed from

underlying DNA sequence polymorphism and thus can mask variation

present at that level.

2.3.2 DNA Markers

DNA molecular markers were defined as DNA sequences that are

characteristic of an individual, a group of individuals, of species, even of

systematic groups. They are extremely useful for individual and varietalidentification, the establishment of phylogenetic relationship, population

genetics and for marker assisted selection. Most points on molecular

marker based genetic linkage maps are anonymous DNA polymorphisms

and do not correspond to any gene of known function. However, some

molecular markers (including coding DNA and expressed sequence tag

markers, as well as isozyme markers) do pinpoint individual genes.

Anonymous DNA markers are generated by a wide variety of techniques,

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differing in their reliability, difficulty, expense, and nature of polymorphism

that they detect. Because of these differences, they also vary greatly in

their stability for various uses. DNA markers may be hybridization based

(FLLP) or PCR based (RAPD, AFLP, SSRs etc.). DNA markers may

detect single locus, oligo-locus, or multiple locus differences and markers

detected may be inherited in a presence/absence, dominant, or co-

dominant.

2.3.2.1 Hybridization Based (probe) Marker:

The Restriction Fragment Length Polymorphism (RFLP) technique

consists of DNA isolation from a suitable set of plants, digestion of the

DNA with restriction enzyme, separation of the restricted fragments by

agarose gel electrophoresis, transfer of the separated restriction

fragments to a filter membrane by a method known as Southern Blotting

(Southern, 1975), detection of individual restriction fragment by nucleic

acid hybridization with labeled cloned probe, and scoring of RFLPs by

direct observation of auto radiogram.

2.3.2.2 PCR Based Markers:

Polymerase chain reaction (PCR) is a procedure for the in vitro enzymatic

amplification of a specific segment of DNA (Mullis and Faloona, 1987).

This technique has certain advantages over RFLP. PCR methods are

rapid, need very little quantities of target DNA, avoid the need for radio

labeling, blotting and hybridization steps and are more amenable to

automation.

The polymerase chain reaction has been used to develop several DNA

markers systems. Three strategies primarily have been employed in the

development of PCR based marker systems. These include:

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Markers are amplified using single primers in PCR where marker

system diversity results from variation in the length and/or sequence of

primers, and where anchor nucleotides are present at 5 (or) 3 termini

of primers e.g. RAPDs, DAFs, SSR anchored PCR.

Markers that are selectively amplified with two primers in PCR such

that their selectivity comes from the presence of two to four random

basic at the 3 ends of primers that anneal to the target DNA during the

PCR (AFLP)

Markers amplified using two primers in PCR commonly require cloning

and/or sequencing for the construction of specific primers. In this case

variation in marker technology result from differences in the target

DNA sequence present between two primers e.g. STRs, AMP-FLP,

and SSRs,

Randomly Amplified Polymorphic DNA (RAPD)

RAPD markers are generated by PCR amplification of random genomic

DNA segments with single primers (usually 10 nucleotides long) of

arbitrary sequence (William et al., 1990). The primers are generated with

at least 60 per cent G + C content to ensure effective annealing and with

sequences that are not capable of internal pairing that can produce PCR

artifacts. This technique can be developed without knowledge of any

specific target DNA and can detect several loci simultaneously so it is

useful for polygenic studies. Amplification products can be separated by

electrophoresis on agarose or polyacrylamide gels and visualized by

staining with ethidium bromide or silver. RAPDs are usually dominant

markers with polymorphisms between individuals defined as the presence

or absence of a particular RAPD band. Therefore, RAPDs have

limitations in their use as markers for mapping, which can be overcome to

some extent by selecting those markers that are linked in coupling.

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Powell (1992) gave the advantage of RAPDs over conventional RFLP

technology, which includes:

Requirement for a small amount of genomic DNA (25-100 ng

per reaction) compared to 5-10 g for RFLP analysis.

An ethidium bromide based detection system.

Many primers can be screened on a single PCR run (Gale and

Witcombe, 1992).

RAPD may provide markers in regions of the genome

inaccessible to RFLP analysis due to presence of repetitive

DNA sequences (Williams et al., 1990).

Sequence Characterized Amplified Regions (SCARs)

Michelmore et al. (1991) and Paran and Michelmore (1993) introduced

SCARs for amplification of specific locus wherein the RAPD marker

termini are sequenced and longer primers (22-24 nucleotides bases long)

are designed. Hence, these PCR-based secondary markers are detected

with two primers homologous to sequenced ends of a RAPD marker.

They amplify a single band with high reproducibility. Many are

codominant and digesting the PCR product with restriction enzymes

having four-nucleotide binding sites can increase their polymorphism.

SCARs are advantageous over RAPD markers due to the following

reasons:

They detect only single, genetically defined loci.

Their amplification is less sensitive to reaction conditions.

They can potentially be converted into co-dominant marker that will

increase the available information in a MAS program.

They are not aware of the presence introns that could eliminate the

priming sites.

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Scoring results obtained by SCARs are more straightforward than

other PCR based markers.

Consequently, SCAR markers offer the most practical method for

screening numerous samples in a time and labour-saving manners, being

accurate, feasible to use and cost efficient (Kasai etal., 2000).

Adam-Blondon et al. (1994) constructed four pairs of near isogenic lines

(NILs) in which the Are gene (dominant gene conferring resistance to

anthracnose in common bean) was introgressed into different genetic

backgrounds. Five RAPDs and four RFLPs were found to discriminate

between the resistant and the susceptible members of NILs. The most

tightly linked RAPD marker RoH 20 (450 bp amplified fragment) was used

to generate a pair of SCAR primers SCH 20-1 and SCH 20-2, which

specifically amplified 450 bp band.

Ohmori et al. (1996) cloned and sequenced six RAPD fragments tightlylinked to Tm-1 gene, which confers tomato mosaic virus (TMV) resistance

in tomato. These co-dominant markers were useful for differentiating

heterozygotes from both types of homozygotes. Similar studies were

conducted in tomato by Chague et al. (1996). Bulked segregant analysis

was used to identify two RAPD markers linked to Sw- 5 gene for

resistance to tomato spotted wilt viruses (TSWV). One of these markers

was used to develop a SCAR marker and another was stabilized into a

pseudo SCAR marker for further marker-assisted plant breeding studies.

DNA Amplification Fingerprinting (DAF):

DAF is quite similar to RAPD but DNA amplification is achieved using one

or more arbitrary primers 5-6 nucleotides in length. DAF generates a

complex and more detailed pattern when separated on a polyacrylamide

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gel which is visualised using the highly sensitive silver staining method

(Caetano-Anolles et al., 1991).

Microsatellites or Simple Sequence Repeats (SSRs):

Simple Sequence Repeats (SSRs) or Microsatellites are co-dominant

markers that are routinely used in many industrial and academic labs.

Microsatellites are the most widely used markers, occur at high frequency

and appear to be distributed throughout the genome of higher plants.

These are DNA sequences that consist of two to five nucleotide core units

such as (AT)n, (CTT)n and (ATGT)n, which are tandemly repeated. The

regions flanking the microsatellites are generally conserved among

genotypes of the same species, allowing the selection of PCR primers

that will amplify the intervening SSR in all genotypes. Variation in the

number of tandem repeats, n, results in different PCR product lengths.

These repeats are highly polymorphic even among closely related

cultivars, due to mutations causing variations in the number of repeating

units. They detect a large number of alleles; level of heterozygosity ishigh and follows Mendelian inheritance (Wu and Tanksley, 1993). Unlike

the other PCR- based marker techniques, microsatellites markers are

rapidly becoming the predominant type of DNA markers used by human

geneticists for linkage map developed (Hudson et al; 1995) and for

identification of individuals (Hammond et al; 1994) while plant geneticists

still rely on restriction fragment length polymorphism (RFLP) and random

amplified polymorphic markers (RAPD). Investigators in many plant

species have begun to develop and use SSR markers in a wide range of

plant species. For assessment of genetic diversity among cultivars and

their wild relative a variety of molecular markers have been used in past

(Karp et al, 1998). However microsateIites have been considered to be

the markers of choice and their utility for this purpose has been

demonstrated in many crops including Soybean, maize, wheat, rice,

sorghum, barley, sunflower, potato etc. by different workers.

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The use of SSR markers involves the isolation of SSR-containing DNA

clones from enriched genomic DNA libraries; synthesizing primer sets to

amplify the SSR contained region, and mapping SSR loci that are

polymorphic. Although many improved procedures are now available to

construct SSR-enriched libraries and to subsequently sequencing positive

clones, the isolation of SSRs is still a time consuming and expensive

process. The cost of developing a substantial number of robust SSR

makers for use in genotyping applications involving thousands of

individuals is often prohibitive. Moreover, even in the dense maps

containing many SSRs, there are many regions of the map that are

completely devoid of any SSR marker. Although they are abundant and

may occur with a frequency of one SSR for every 30-kb region of plant

genome, the realization of that density on a genetic map has not been

achieved yet in any crop species. Some SSRs can also be identified by

searching EST databases. As these SSRs are likely to be within or

adjacent to coding sequences, they may be less polymorphic than SSRsderived from non-coding regions.

In most plant species the level of polymorphism with microsatellites is

considerable higher than found with RFLP markers. SSRs are reported to

exhibit high level of length polymorphism with as many as 37 alleles in

barley (Saghai-Maroof et a! 1994) and 26 alleles in soybean (Rongwen et

a! 1995). The high number of alleles per locus, precise allele identification

through the use of allelic ladders and the accurate comparison of data

make SSR markers one of the most informative techniques for genome

mapping, DNA fingerprinting and population studies (Taramino and

Tingey, 1996). Other microsatellites based markers e.g. STMS

(Sequence tagged microsatellite site), ISSR (inter- simple sequence

repeats) and RAMPs (Random amplified microsatellite polymorphism)

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etc. have also been used for cultivar identification and for assessment of

genetic diversity in several plant system (Wolff et al, 1993).

Inter-Simple Sequence Repeats (ISSR):

ISSR involves amplification of DNA segments present at an amplifiable

distance in between two identical microsatellite repeat regions oriented in

opposite direction. The technique uses microsatellites as primers in a

single primer PCR reaction targeting multiple genomic loci to amplify

mainly inter simple sequence repeats of different sizes. The microsatellite

repeats used as primers for ISSRs can be di-nucleotide, tri-nucleotide,

tetranucleotide or penta-nucleotide. The primers used can be either

unanchored (Meyer et al., 1993; Gupta et al., 1994; Wu et al., 1994) or

more usually anchored at 3` or 5` end with 1 to 4 degenerate bases

extended into the flanking sequences (Zietkiewicz et al., 1994). ISSRs

use longer primers (1530 mers) as compared to RAPD primers (10

mers), which permit the subsequent use of high annealing temperature

leading to higher stringency. The annealing temperature depends on the

GC content of the primer used and ranges from 45 to 65 oC. The amplified

products are usually 2002000 bp long and amenable to detection by

both agarose and polyacrylamide gel electrophoresis.

ISSRs exhibit the specificity of microsatellite markers, but need no

sequence information for primer synthesis enjoying the advantage of

random markers (Joshi et al., 2000). The technique is simple, quick, and

the use of radioactivity is not essential. ISSR markers usually show high

polymorphism (Kojima et al., 1998) although the level of polymorphism

has been shown to vary with the detection method used. Polyacrylamide

gel electrophoresis (PAGE) in combination with radioactivity was shown

to be most sensitive, followed by PAGE with AgNO 3 staining and thenagarose gel with EtBr system of detection. Like RAPDs, reproducibility,

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AFLP is a PCR-based technology for marker-assisted breeding and

genotyping. AFLP represents a significant breakthrough compared to the

currently available methods in terms of facility, precision, flexibility, speed

and cost. Essentially, AFLP enables the generation of thousands of DNA

markers from a genome of any complexity and without prior knowledge of

the genomes structure or sequence.

AFLP involves the amplification of small restriction fragments, obtained by

cleaving genomic DNA with restriction enzymes, to produce high

resolution DNA "fingerprinting" patterns on denaturing polyacrylamide

gels (Vos et al., 1995). The rationale of the AFLP technique is based on

the use of specifically designed PCR primers which selectively amplify a

small subset of restriction fragments, or "markers", out of a complex

mixture comprising as many as several million different fragments. The

products of the reaction can be visualised by conventional DNA staining

or DNA labelling procedures using either radioactive or non-radioactive

methods.

AFLP is an extremely flexible technology which offers multiple

applications in the field of crop breeding and plant genome analysis,

especially in the fields of genotying, marker-assisted breeding and plant

genome analysis.

Single-Strand Conformation Polymorphism (SSCP)

Single-strand conformation polymorphism is technically simple and

sensitive. It can have mutation detection efficiency 100%. The technique

relies on the variation in electrophoretic mobility of secondary structures

formed by single stranded DNA. Fragments of different primary structures

i.e. DNA samples usually PCR products are denatured by heat and or

chemical denaturants and electrophoresed into a non-denaturing gel. As

the ssDNA moves from denaturing to non-denaturing conditions, intra-

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strand base pairing causes folding of the fragments with stable

conformations, and mobility differences among them can be detected

under the appropriate electrophoretic conditions. DNA fragments 100-

400 bp in length are most appropriate as the efficiency of mutation

detection decreases outside this range. It is currently used in diagnostics

of inherited diseases in humans, but is not well developed for crop

applications.

Sequence-Tagged Site (STS):

STS was first developed by Olsen et al. (1989) as DNA landmarks in the

physical mapping of the human genome, and latter adopted in plants.

STS is a short, unique sequence whose exact sequence is found

nowhere else in the genome. Two or more clones containing the same

STS must overlap and the overlap must include STS. Any clone that can

be sequenced may be used as STS provided it contains a unique

sequence. In plants, STS is characterized by a pair of PCR primers that

are designed by sequencing either an RFLP probe representing a

mapped low copy number sequence (Blake et al., 1996) or AFLP

fragments. Although conversion of AFLP markers into STS markers is a

technical challenge and often frustrating in polyploids such as hexaploid

wheat (Shan et al., 1999; Prins et al., 2001), it has been successful in

several crops (Meksem et al., 1995, 2001; Qu et al., 1998; Shan et al.,

1999; Decousset et al., 2000; Parker and Langridge, 2000; Prins et al.,

2001; Guo et al., 2003). The primers designed on the basis of a RAPD

have also sometimes been referred to as STSs (Naik et al., 1998),

although they should be more appropriately called SCARs. STS markers

are co-dominant, highly reproducible, suitable for high throughput and

automation, and technically simple for use (Reamon-Buttner and Jung,

2000).

Expressed sequence tags (EST)

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Messenger RNA (mRNA) was converted to complementary DNA (cDNA)

which represented only expressed DNA sequence or expressed gene. A

few hundred nucleotides from either the 5' or 3' end of these expressed

genes can be sequenced to create 5' expressed sequence tags (5' ESTs)

and 3' ESTs, respectively (Jongeneel, 2000). A 5' EST is obtained from

the portion of a transcript (exons) that usually codes for a protein. These

regions tend to be conserved across species and do not change much

within a gene family. The 3' ESTs are likely to fall within non-coding

(introns) or untranslated regions (UTRs), and therefore tend to exhibit

less cross-species conservation than do coding sequences. The

challenge associated with identifying genes from genomic sequences

varies among organisms and is dependent upon genome size as well as

the presence or absence of introns, which are the intervening DNA

sequences interrupting the protein coding sequence of a gene.

Single Nucleotide Polymorphisms (SNP)

Single nucleotide polymorpisms (SNPs) are DNA sequence variations

between individuals which are the most common form of DNA

polymorphisms in a genome. Since these are the most abundant

variations in a genome and thus have the potential of providing the

highest map resolution, a large amount of SNP data is available in

humans, but very limited data are available on SNPs in plants. Detection

of SNPs does not require DNA fragment length measurement, thus

allowing one to design high throughput, automatic assays, without

separating DNA by size. While SSRs can often represent many alleles,

SNPs are biallelic in nature. SNP discovery approaches such as re-

sequencing or data mining enable the identification of insertion deletion

(indel) polymorphisms. These indels can be treated as biallelic markers

and can be utilized for genetic mapping and diagnostics. The

mechanisms that generate indel polymorphisms are still largelyspeculative. Insertion and deletion can occur by unequal crossing over or

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provide information for selection of parents in conventional breeding. The

genetic diversity in cultivated cowpea has been assessedon the basis of

morphological and physiological traits (Ehlers and Hall, 1996;Fery, 1985),

allozymes (Panella and Gepts, 1992;

Pasquet, 1993, 1999; Vaillancourt et

al., 1993), seed storage proteins (Fotso et al., 1994), chloroplast DNA

polymorphism (Vaillancourt and Weeden, 1992), restriction fragment

length polymorphisms (RFLP) (Fatokun et al., 1993), amplified fragment

length polymorphisms (AFLP) (Fatokun et al., 1997), simple sequence

repeat (SSR) (Li et al., 2001), and random amplified polymorphic DNA

(RAPD) (Mignouna et al., 1998).

Pasquet (1996) evaluated cowpea gene pool organization on the basis of

morphological and isoenzymatic data. Morphologically analysis, cultivated

cowpea can be split up into two groups, well characterized by their ovule

numbers and their photosensitivity, with fairly primitive and fairly evolved

forms in each group. These two morphophysiological groups are,

however, difficult to distinguish isoenzymatically; all of the cultivar groupshave the same most common alleles for each isozyme.

Determining genetic similarities and relationships among cowpea

breeding lines and cultivars by microsatellite markers, Li et al (2001)

observed 90 cowpea lines grams shared an average of 44% similarity. A

large group of 47 cowpea lines shared over 45% similarity on the

dendrogram. The microsatellite markers were also highly polymorphic in

cowpea. They could be used in germplasm conservation and analysis,

not only for breeding lines and cultivars but also for the wild cowpea

species and otherVigna species.

Degenerate oligonucleotides designed to recognize conserved coding

regions within the nucleotide binding site (NBS) and hydrophobic region

of known resistance (R) genes from various plant species were used to

target PCR to amplify resistance gene analogs (RGAs) from a cowpea

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(Vigna unguiculata L.Walp.) cultivar resistant to Striga gesnerioides

(Gowda et al, 2002). The nucleotide sequence of fifty different cloned

fragments was determined and their predicted amino acid sequences

compared to each other and to the amino acid sequence encoded by

known resistance genes, and RGAs from other plant species. Cluster

analysis identified five different classes of RGAs in cowpea. Gel blot

analysis revealed that each class recognized a different subset of loci in

the cowpea genome. Several of the RGAs were associated with

restriction fragment length polymorphisms, which allowed them to be

placed on the cowpea genomic map.

The efficiency of RAPD, AFLP, and SAMPL marker systems was

investigated to detect genetic polymorphism in cowpea landraces (Vigna

unguiculata subsp. unguiculata L. Walp.) (Tosti and Negri, 2002). Each

marker system was able to discriminate among the materials analysed,

but a clear distinction between all the local varieties was only obtained

with AFLP and SAMPL markers. The average diversity index was quitesimilar for each marker system, but owing to the differences in the

effective multiplex ratio values the marker index was higher for the AFLP

and SAMPL systems than for the RAPD system. The AFLP and SAMPL

techniques appear to be more useful than the RAPD technique in the

analysis of limited genetic diversity among the cowpea landraces tested.

The significant correlations of SAMPL similarity and cophenetic matrices

with those of the other markers, and the lower number of primer

combinations required, indicate that this technique is the most valuable.

The low genetic similarity detected among landraces suggests that all the

cowpea landraces should be maintained on the respective farms from

which they came.

Fall et al. (2003) studied the genetic diversity of cultivated Senegalese

varieties using physiology trait based on nitrogen fixation and genotypic

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analysis utilized by RAPD observed the polymorphisms of RAPD can be

used to reorganize the cowpea germplasm in order to eliminate the

putative duplicates, and to identify elite varieties. The polymorphic data

showed that some DNA fragments could be specific to the higher or lower

nitrogen fixing varieties suggesting that some genes could govern the

higher nitrogen fixation character in cowpea. These findings also provide

an alternative avenue for understanding the biological nitrogen fixation

process and the genetic identification of parent plants in a breeding

program.

Genetic diversity of cultivated cowpeas and their wild types was reported

that wild accessions were more diverse than domesticated cowpeas, wild

cowpeas were more diverse in eastern than in western Africa, and a

unique domestication event in cowpea in the northern Africa was

suggested by Coulibaly et al. (2002) and Fana et al. (2004). The AFLP

technique was reported superiority over isozymes resided in its ability to

uncover variation both within domesticated and wild cowpea, and shouldbe a powerful tool once additional wild material becomes available

(Coulibaly et al., 2002). As isozymes and AFLP markers, although with a

larger number of markers, RAPD data confirmed the single domestication

hypothesis, the gap between wild and domesticated cowpea, and the

widespread introgression phenomena between wild and domesticated

cowpea (Fana et al., 2004).

Pandey and Dhanasekar (2004) studied morphological features and

inheritance of foliaceous stipules of primary leaves in Cowpea (Vigna

unguiculata). The stipules have been recognized as an important

morphological character for identification of species or varieties.

In 1992, Vaillancourt and Weeden discovered a very important mutation

for studying cowpea evolution and domestication. A loss of a BamHI

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restriction site in chloroplast DNA characterized all domesticated

accessions and a few wild (Vigna unguiculata ssp. unguiculata var.

spontanea) accessions. In order to screen a larger number of accessions,

Feleke et al. (2006) screened 54 domesticated cowpea accessions and

130 accessions from the wild progenitor using PCR RFLP or direct PCR

methods. The use of s13.3/BamHI haplotype specific primers developed

for chloroplast DNA was a key element to further evaluate the various

domestication hypotheses. The absence of haplotype 0 was confirmed

within domesticated accessions, including primitive landraces from

cultivar-groups Biflora and Textilis, suggesting that this mutation occurred

prior to domestication. However, 40 var. spontanea accessions

distributed from Senegal to Tanzania and South Africa showed haplotype

1. Whereas this marker could not be used to identify a precise center of

origin, it did highlight the widely distributed cowpea crop-weed complex.

Its very high frequency in West Africa could be interpreted as a result of

either genetic swamping of the wild/weedy gene pool by the domesticated

cowpea gene pool or as the result of domestication by ethnic groupsfocusing primarily on cowpea as fodder.

2.4.2 Markers Linked to Disease Resistance Gene in Cowpea

The local reactions of primary leaves were used as morphological trait to

recognize resistant gene in cowpea (Robertson, 1965). Varieties that

gave no reaction or developed necrotic lesion when inoculated with

CYMV were immune from infection and were therefore resistant; those

that developed chlorotic lesions became systemically infected and

therefore susceptible to infection.

Since the number of morphological traits are limited and affected by

environment condition, molecular markers are recently best of choice

complementation with conventional segregation analysis to identifydisease resistance loci in the plant genome.

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Fatokun et al. (1993) used RFLP analysis of nuclear Sequences to study

the genetic relationships in 18 species belonging to four subgenera of

genus Vigna and higher amount of variation was found in species from

Africa as compared to those from Asia.

A highly sensitive reverse transcription-polymerase chain reaction (RT-

PCR) was used to detect the presence of cowpea mottle carmovirus

(CPMeV) in germ plasm of Vigna spp (Gillaspie et al., 1999), and the

presence of cowpea aphid-borne mosaic virus (CABMV) in peanut

(Gillaspie et al. 2001) instead of ELISA techniques. The RT-PCR method

was up to 105 times more sensitive than direct antigen coating enzyme-

linked immunoadsorbent assay (DAC-ELISA) in detecting CPMoV, and

was ten times more sensitive than enzyme-linked immunoadsorbent

assay (ELISA) in detecting CABMV.

Based on bulked segregant analysis described by Michelmore et al.(1991), identification of AFLP markers linked to resistance of cowpea to

parasitic weed disease (Striga gesnerioides) was carried out by

Ouedraogo et al. (2001). Three AFLP markers were identified that are

tightly linked to resistance reaction to S. gesneroides race 1 (a single

dominant gene, designated Rsg21) with the distance of 2.6 cM, 0.9 cM,

and 0.9 cM, respectively; and six AFLP markers linked to resistance

reaction to S. gesneroides race 3 (a single dominant gene, designated

Rsg43) with the distance of 10.1 cM, 4.1 cM, 2.7 cM, 3.6 cM, 3.6 cM,

and 5.1 cM.

Marker-assisted selection (MAS) was applied in breeding cowpea for

resistance to the parasitic weed Striga gesnerioldes (WilId.) using AFLP

and ALFP-derived SCAR markers (Boukar et al., 2004). An F2 population

developed from the cross between a resistance breeding line (1T93K-

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693-2) and the susceptible cultivar 1AR1696 was characterized for

resistance against race 3 of S. gesnerioldes for genetic analysis and

molecular mapping. 1T93K-693-2 was found to have a single dominant

gene for resistance. Four AFLP markers, designated E-ACTIM-CTC115,

E-ACTIM-CAC115, E-ACAIM-CAG108 and E-AAGJE-CTA1, were

identified and mapped 3.2, 4.8, 13.5 and 23.0 cM, respectively, from Rsgl,

a gene in 1T93K-693-2 that gives resistance to race 3 (or Nigerian strain)

ofS. gesnerioldes. The first two markers were validated in a second F2

population developed from crossing the same resistant parent with

Kamboinse local, a different susceptible cultivar. The AFLP fragment

from marker combination E-ACTIM-CAC, which is linked in coupling with

Rsgl was cloned, sequenced, and converted into a sequence

characterized amplified region (SCAR) marker named

SEACTMCACX3/85, which is codominant and useful in breeding

programs.

A new marker system, targeted region amplified polymorphism (TRAP),has been utilized for mapping and tagging disease resistance traits in

common bean (Phaseolus vulgaris L.) (Miklas et al., 2006). Most widely

used marker types, random amplified polymorphic DNA (RAPD) and

amplified fragment length polymorphisms (AFLP), for linkage mapping in

bean are located randomly throughout the genome and associate with

particular traits by chance. The new marker system, TRAP, uses

expressed sequence information and a bioinformatics approach to

generate polymorphic markers around targeted candidate gene

sequences. TRAP markers were amplified by fixed primers designed

against sequenced expressed sequence tag (EST) associated with

disease resistance in the Compositae Genomics database or against

sequenced resistance gene analog (RGA) from common bean.

Seventeen of 85 TRAP markers located in the BAT 93/Jalo EEP558 core

mapping population mapped in the vicinity of R genes. Six of 21 TRAP

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markers generated in the Dorado/XAN 176 mapping population were

linked with newly identified QTL, two conditioning resistance to ashy stem

blight (14% and 16% of the phenotypic variation explained, R2), and one

each conferring resistance to Bean golden yellow mosaic virus (BGYMV)

(15%) and common bacterial blight (30%). The TRAP marker system has

potential for mapping regions of the common bean genome linked with

disease resistance.

A single incompletely dominant gene was suggested controlled clover

yellow vein virus (ClYVV) elicits lethal tip necrosis in pea after observing

ratios of necrosis, mosaic with slight stem necrosis, and mosaic fit the

expected 1:2:1 ratio from F2 population of a cross between PI 118501 and

PI 226564 (Ravelo et al., 2007). This locus in pea, conferring necrosis

induction to ClYVV infection, was designated Cyn1 (Clover yellow vein

virus-induced necrosis). A linkage analysis using 100 recombinant inbred

lines derived from a cross of PI 118501 and PI 226564 demonstrated that

Cyn1 was located 7.5 cM from the SSR marker AD174 on linkage groupIII.

2.4.3 Genetic Mapping in Cowpea

Genetic maps of cowpea have been established by Fatokun et al. (1992,

1993), Menancio-Hautea et al. (1993), Menendez et al. (1997), Ubi et al.

(2000) and Ouedraogo et al. (2002). Of these, the latter, building on the

earlier version developed by Menendez et al.(1997), is the most current

and complete map. This map was established in the recombinant inbred

population IT84S-2049 x 524B (n=94) developed by the Bean/Cowpea

Collaborative Research Support Program (CRSP) project at the

University of California, Riverside. IT84S-2049 is an advanced breeding

line developed at IITA in Nigeria for multiple disease and pest resistance

and has resistance to several races of blackeye cowpea mosaic virus

(B1CMV) and to virulent root-knot nematodes in California (Menendez et

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al., 1997). Line 524B is a black-eyed cowpea that shows resistance to

Fusarium wilt and was developed at the University of California,

Riverside, from a cross between cultivars CB5 and CB3, which

encompasses the genetic variability that was available in cowpea

cultivars in California.

As many studies suggested domesticated cowpea consists of a single

gene pool (Coulibaly et al., 2002; Fana et al. 2004). The genetic diversity

in this gene pool for RFLPs was limited and alternative markers have

been pursued, including RAPDs (Menendez et al., 1997) and AFLPs

(Ouedraogo et al., 2002), which detect a larger number of polymorphic

loci. The current map of cowpea consists of 11 Linkage groups (LGs)

spanning a total of 2670 cM, with an average distance of approximately 6

cM between markers. It includes 242 AFLP, 18 disease or pest

resistance-related markers (Ouedraogo et al., 2002) and 133 RAPD, 39

RFLP, and 25 AFLP markers from the original map of Menendez et al.

(1997) for a total of 441 markers, of which 432 were assigned to a LG.Among these markers loci, genes for a number of biochemical and

phenotypic traits have been located on this map. These include C, a

general color factor, and P, for purple pod color, on LG4, a 35 kDa

dehydrin protein, implicated in chilling tolerance during emergence (LG2;

Ismail et al., 1999), and markers for resistance to Striga gesnerioides

races 1 and 3 (LG1 and LG6), cowpea mosaic virus (CPMV) and cowpea

severe mosaic virus (CPSMV) (two distinct loci on LG2), B1CMV (LG8),

southern bean mosaic virus (SBMV) (LG6), Fusarium wilt (LG6, distinct

from the previous locus), and root-knot nematodes (Rk on LG1)

(Ouedraogo et al., 2002). Resistance gene c

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