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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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