Agronomic, Genetic and Molecular Characterization of MYMIV-Tolerant
Mutant Lines of Vigna mungo
The Mungbean Yellow Mosaic India Virus (MYMIV), transmitted
through Bemisia tabaci causes severe damage in several grain legumes.
Three mutant MYMIV-tolerant lines, namely, VM 1, VM 4 and VM 6, along
with the susceptible Vigna mungo cultivar T9 were characterized.
The objective of this study was to evaluate these three MYMIV-tolerant
lines in comparison to the susceptible cv. T9 on the performance of eight
agro-morphological traits. The four genotypes were grown in a randomized
complete block design and combined analysis of variance over two years
was carried out. The analysis of variance for individual years showed
significant differences between these two genotypes for two traits in
two consecutive years. However, combined analysis of variance over two
years showed that except for few traits, there were no significant differences
in other major traits amongst the genotypes. Genetic control of MYMIV-resistance
was re-evaluated and confirmed a monogenic recessive nature. The molecular
analysis revealed defect in the NB-ARC domain of putative disease resistance
(R) gene in the susceptible cv.T9. While NB-ARC domains of all the MYMIV-tolerant
mutant lines have common functional motifs. Presumably, the susceptibility
of cultivar T9 is due to the limitation in transcript formation for the
R-gene, which otherwise is a high yielding superior cultivar. Therefore,
MYMIV-tolerant lines may prove useful to the plant breeders for further
improvement towards sustainable agriculture.
Vigna mungo (urdbean) is one of the most popular pulses in South
East Asia and a substantial source of dietary protein. Mungbean yellow
mosaic India virus (MYMIV; Mayo, 2005) is a Begmovirus transmitted through
the white fly, Bemisia tabaci Genn. (Nariani, 1960; Honda et
al., 1983). It causes significant yield loss for many legume seeds,
not only, Vigna mungo, but also, V. radiata and Glycine
max throughout the South-Asian countries. Depending on the severity
of the disease the yield penalty may reach up to cent percent (Basak et
al., 2004). And an annual loss of 300 million US$ due to this viral
infection to the leguminous crop has been projected.
Several attempts have been made in improving this leguminous crop in
India. At the G.B. Plant University of Agriculture and Technology, V.
radiata var. sublobata and V. mungo var. silvistris,
the wild progenitors of mungbean and urdbean, were used to improve yield
components and to incorporate resistance to MYMV (Singh, 1981). A genetic
study involving crosses of MYMV-resistant and susceptible urdbean and
mungbean (Singh, 1980, 1981; Verma and Singh, 1986) and soybean (Singh,
1988) showed two recessive genes were governed disease resistance and
susceptibility to be dominant over resistance. Interspecific transfer
of MYMV-resistance from V. mungo to V. radiata was initiated
at the Punjab Agriculture University, India (Gill et al., 1983).
The resistant character is inherited independently of the seed color and
maturity (Singh, 1988). Dana (1966) attempted crossing between V. mungo
(Synonym. Phaseolus mungo) and V. radiata (Synonym. Phaseolus
aureus) to induce MYMV resistance in V. mungo (P. mungo),
but none of the resistant progeny lines sustained the resistance trait.
According to him MYMV-resistance breaks down within 10 years in the gangetic
plains of Bengal, India (Dana S, personal communication). On the contrary,
we are maintaining the mutant MYMIV (previously known as MYMV)-tolerant
lines for over 24 years. However, considering the importance of Vignas
as a pulse crop, development of MYMIV-resistant varieties is of prime
importance for stabilizing the yield levels for sustainable agriculture.
Additionally, introduction of virus resistant pulses in the farmer`s field
would also reduce the insecticide application. In our laboratory we are
maintaining six MYMIV-resistant mutant lines (VM1 to VM6) of V. mungo
through selfing since 1984. These mutant lines were derived from a MYMIV-susceptible
cultivar of V. mungo, T9, which has wide adaptability and higher
agronomic yield, but, the crop production often suffers due to the prevalence
of the viral disease. These mutant line-seeds were inbred for five generations
prior to any experimentation. Of these, VM 1, VM 4 and VM 6 were tested
at Indian Agricultural Research Institute (New Delhi, India) through the
Agroinfection method (by infecting with T-DNA containing viral coat protein
genes; Jacob et al., 2003) and the virus-tolerant nature of these
lines were confirmed. The objective of this study was to evaluate performance
of the three MYMIV- tolerant lines in comparison to the MYMIV-susceptible
cultivar T9 based on eight agro-morphological traits under the prevailing
climatic condition of Madhyamgram, North 24-paraganas, Kolkata, India;
to determine inheritance pattern of MYMIV resistance and to investigate
probable cause of gain-in-function mutants from the MYMIV-susceptible
cultivar T9 at the molecular level.
MATERIALS AND METHODS
Plant Materials and Experimental Design
Vigna mungo L. Heppar cv. T9, a MYMIV-susceptible but agronomically
superior cultivar, was collected from the Behrampur Pulses and Oilseeds
Research Station (West Bengal, India). Three MYMIV-tolerant lines, VM
1, VM 4 and VM 6 and T9 were grown at the Madhyamgram Experimental Farm
(22°41` N, 88° 27` E, Bose Institute, Kolkata, India) for two
years (February to May, 2000 and 2001) following the Randomized Complete
Block Design (RCBD) with three replications/year and five plants/replication.
Combined analysis of variance of the RCBD experiments over two years was
carried out with data collected on eight agro-morphological and yield
traits following the procedure given by Gomez and Gomez (1984). The traits
were: plant height, branches/plant, pods/plant, pod length, seeds/pod,
100-seed weight, seed yield/plant and seeds/plant.
Development of Segregating Populations for MYMIV-Reaction
The susceptible cultivar T9 (female) was crossed with the resistant
line, VM6 (male), F1s produced and F2 populations
were raised along with the parental lines and a resistant check.
Phenotyping the Population Segregating for MYMIV-Reaction
The lines involved in the crosses and the F1s were screened
for MYMIV-reaction under field epiphytotic condition with abundant white
fly population during February to May 2003.
The parental lines and the F2 populations were screened from
July-September 2003, both under natural field condition and artificial/forced
feeding conditions. Data on MYMIV-reaction under natural epiphytotic condition
was obtained from 312 F2 plants and analysed. For forced feeding,
white flies were collected from the plants and confined on a susceptible
plant showing typical MYMIV symptoms for 24 h using a small, transparent
glass cage with a spring cap. The same cage with the flies was attached
to a healthy plant and the viruliferous insects were allowed to feed on
a leaf for 24 h. After acquisition feeding, the flies were used for 3-5
transfers for inoculation feeding. Following this protocol a total number
of 484 F2 plants were forced inoculated and the MYMIV-reaction
Mean value of each character over five randomly selected plants in
each replication was computed. The Analysis of Variance (ANOVA) table
was prepared following statistical analysis (Gomez and Gomez, 1984) to
find out the mean sum of square from which the Least Significance Difference
(LSD) of different genotypes for each character was computed. F-tests
of homogeneity of error variance of all traits were applied to combine
the values of two consecutive years. For the calculation of segregation
pattern of MYMIV-reaction on F2 individuals under natural condition
and forced inoculated condition Chi-square test was employed to determine
the probability (P) in accepting the hypothesis (expected ratio).
Genomic DNA Isolation and Detection of Polymorphism Between MYMIV-Tolerant
Lines, VM1-VM6 and T9
The genomic DNA were isolated from six MYMIV-tolerant lines and cv.
T9 following the method described by Basak et al. (2004). Initially
several operon primers were tried but most of the cases monomorphic profiles
generated. Subsequently, we have designed degenerate primers from the
conserved motifs of NB-ARC domain of plant disease resistance gene in
members of Fabaceae (Pal et al., 2007), referred to as Resistance
Gene Analog (RGA). Isolated genomic DNA were PCR amplified with 175 RGA
primer combinations (Table 1) and the amplification
conditions were followed as described by Basak et al. (2004).
Cloning of Polymorphic Fragments and Sequence Analysis
Two amplified polymorphic fragments were cloned separately using pGEM-T
easy vector kit (Promega, USA) following the supplied protocol and sequenced
using a ABI Prism 3100 automated DNA sequencer. The polymorphic markers
were named as Vigna mungo resistance gene homolog of VM6 (VMYR6) and Vigna
mungo susceptible allele 1 (VMYS1). Nucleotide sequence and in silico
translated peptide-sequence similarities between the VMYR6 and VMYS1 and
other published sequences were determined by screening the GenBank non-redundant
database using the computer program NBLAST (Basic Local Alignment Search
Tool, Altschul et al., 1997). To ascertain the presence of ORF
and conserved domain(s) in VMYR6 and VMYS1, the NCBI ORF finder (http://www.ncbi.nlm.nih.gov/projects/gorf/)
and conserved domain (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)
databases were also searched.
Sequence Alignment and Database Search
Sequences (VMYR6 and VMYS1) were aligned using CLUSTALW (ver 1.83)
(Thompson et al., 1994) with the default settings of gap opening
penalty (10.0) and gap extension penalty (0.1). The Gannet 250 protein
weight matrix has been used.
||RGA primers and respective annealing temperatures
Agro-Morphological Traits of MYMIV-Susceptible and -Tolerant Genotype
Significant differences between the MYMIV-susceptible and -tolerant
genotypes for characters like, branches/plant, pods/plant and seeds/pod
were evident, while no significant differences were noted for rest of
the traits among individuals of these genotypes (Table 2).
The mean and LSD values for the eight agro-morphological traits are shown
in Table 2. The results of the analysis of variance
of the RCBD experiments for individual years and the combined analysis
of variance over two years are shown in Table 3. The
analysis of variance for individual years showed significant differences
amongst the genotypes for two different traits in two subsequent years
(Table 3). In the first year, seeds/plant was significantly
different at 1% level. In the second year, branches/plant was significantly
different at the 5% level. However, combined analysis by F-test for homogeneity
of error variances revealed that all traits, except for branches/plant
(significant at 5 and 1% due to the genotype), pods/plant and seeds/pod
(significant at 5% due to the genotype), were homogeneous amongst the
MYMIV-tolerant lines and the susceptible cultivar T9 (Table
3). The tolerant lines and the susceptible T9 cultivar have since
been used for crossing to generate populations segregating for MYMIV-reaction
(Basak et al., 2004).
||The mean of the eight agro-morphological traits of the
MYMIV-susceptible cultivar T9 and -tolerant lines, VM 1, VM 4 and
||Analysis of variance of the RCBD experiments for individual
years and the combined analysis of variance over two years
|*and **Significant at 5 and 1% levels, respectively
Inheritance of MYMIV-Resistance Trait
In this research, the inheritance of MYMIV-resistance in crosses of
V. mungo was studied. Segregation into tolerant and susceptible
individuals in progenies of the cross between tolerant line, VM6, with
the susceptible cv. T9 showed expression of one R gene in the tolerant
individuals studied both under natural and artificial screening conditions
and the data indicates that segregation fits into 3:1 ratio. The segregating
pattern showed MYMIV-resistance to be monogenic recessive (Table
4). Data on MYMIV-reaction under natural epiphytotic condition from
F2 plants are shown in Table 4 and analyzed,
which corroborated with monogenic recessive control.
Generation of Polymorphism Between MYMIV-Tolerant and -Susceptible
Out of 175 pairs of RGA primer combinations used so far, most of the
primer combinations produced monomorphic amplification profiles (Fig.
1A-G). While, only the combination of RGA-1F-CG (5`-AGTTTATAATTCGATTGCT-3`)
and RGA-1R (5`-ACTACGATTCAAGACG TCCT-3`) generated one polymorphic fragment
in all the 6 MYMIV-tolerant lines (Fig. 1H), whereas,
no amplification product was obtained in the MYMIV-susceptible cultivar
T9 of V. mungo. While, a degenerate primer RGA-1F-TG (5`-AGTTTATAATTTGATTGCT-3`)
with RGA-1R generated one polymorphic fragment only in cv. T9 of V.
mungo but not in MYMIV-tolerant genotypes (Fig. 1I).
||Monomorphic (A-G) and polymorphic (H, I) amplification
profiles generated from genomic DNA of susceptible cv. T9 and six
MYMIV-tolerant mutant lines VM1-VM6, employing different RGA primer
combinations: RGA 11 F-G/RGA 10 R-AA, RGA 2 F-TC/RGA 2 R, RGA 8 F-G/RGA
8 R, RGA 11 F-G/RGA 10 R-AG, RGA 10 F-G/RGA 10 R-GA, RGA 6 F/RGA 6
R, RGA 11 F-G/RGA 10 R-GG, RGA 1 F-CG/RGA 1 R and RGA 1 F-TG/RGA 1
||(A) Pairwise nucleotide sequence alignment between VMYR1
and VMYS6 using CLASTALW software. Reported conserved motifs of NB-ARC
domain are highlighted and (B) Nucleotide sequence of VMYS1 and in
silico translated aminoacid sequences showing the presence of
stop codons within the ORF. * = Denotes to the identity, : = Strongly
similar, . = Weakly similar
||MYMIV-reaction of individuals of F2 segregating
population under Natural Condition (NC) and Forced-Inoculat (FI) conditions
|an: No. of samples
Analysis of Nucleotide Sequences of Marker Fragments
Sequence analysis followed by sequence alignment of VMYR6 and VMYS1
showed sequence similarity (Fig. 2A) and these sequences
also have homology with other NB-ARC domains (results not shown). Majority
of these accessions were either plant R genes or R gene
homologues. Both of these sequences also have high sequence similarity
with the MYMIV-resistance linked marker VMYR1 of V. mungo (Accession
No. AY297425, Basak et al., 2004). Similarities between VMYR1 and
VMYR6 and between VMYR1 and VMYS1 represented by E-value (expected frequency)
= 0 and 4e-90, respectively. The conserved domain search revealed
that VMYR6 sequence is a part of the NB-ARC domain containing conserved
reported motifs (Pal et al., 2007). In silico translated
amino acid sequence obtained from the nucleotide sequence of VMYS1 revealed
the presence of stop codons within the ORF (Fig. 2B).
Agro-Morphological Traits of MYMIV-Susceptible and -Tolerant Genotype
The MYMIV-tolerant lines produced more seeds/pod than those produced
by the T9. The results also showed that the variation was due to year
(environment) and the year X genotype interactions were insignificant
(Table 3). These results clearly show that in terms
of agro-morphology and yield traits the MYMIV-tolerant lines were statistically
nearly identical to the MYMIV-susceptible T9 cultivar.
Inheritance of MYMIV-Resistance Trait
Assuming Mendelian inheritance, the almost perfect fit to a ratio
of 3:1 (susceptible: resistant) for segregating progenies under natural
condition suggest the monogenic recessive control of MYMIV-resistance
in V. mungo mutant line, VM6 (Table 4). Inheritance
of resistance to MYMIV was studied in crosses of mungbean, blackgram and
their interspecific crosses with V. sublobata (Singh, 1980, 1988).
Resistance to MYMV was recessive in the three Vigna species. The
segregation ratios in F2 and back crosses indicated that the
resistance was digenic recessive in the crosses of mungbean and in interspecific
crosses of mungbean with blackgram and V. sublobata but MYMV-resistance
was monogenic recessive in blackgram crosses. Frisch and Melchinger (2001)
reported that several important genes in breeding for resistance and quality
traits are inherited recessively. Especially, resistance traits for plant
viruses has been reported to be recessive in crop plants (Park and Tu,
1991; Pal et al., 1991; Miklas et al., 2000; Diaz-Pendon
et al., 2004; Hayes et al., 2004; Ritzenthaler, 2005). The
significant p-value from the expected 3:1 segregation for MYMIV-reaction
in the F2-population under forced feeding condition (Table
4) was probably due to the following reasons:
||Experimental error due to the non-viruliferous nature of the white
||Vectors may not feed on the plant during the feeding period
||Vectors were weak and incapable of transmit the virus
Monogenic recessive nature of the genetic control for MYMIV-tolerance
was also reported earlier and that was re-confirmed by phenotypic segregating-F2
progenies of a third cross in the present investigation. By understanding
the genetic basis of the MYMIV-reaction trait and the allelic variation
at the locus, the breeder would be able to design superior genotypes of
Probable Cause of Gain-in-Function Mutants from the MYMIV-Susceptible
The findings of the present study corroborate with our contention
that the MYMIV-tolerant plant arose due to the natural mutation/s in the
susceptible T9 genome. Firstly, the collection and cultivation history
of the T9 genotype was in favour of the natural mutation hypothesis (Basak
et al., 2004). The combined analysis of variance over two years
presented have shown that the tolerant and susceptible genotypes are homogeneous
with respect to most of the yield related traits. Secondly, except two
RGA primer pairs, all the rest primers tried so far produced monomorphic
banding profiles from the genomes of the MYMIV-tolerant lines and the
susceptible cv. T9. It indicates that most probably the tolerant lines
arose through a natural mutation in the genome of the susceptible cultivar
T9 and have high genomic homology except for a small portion of the genome
including R gene/s; which is evident from the differential MYMIV
It is further evident from the analysis of marker fragment (VMYS1), generated
from within the NB-ARC domain (which is a novel signaling-motif shared
by plant R gene products and regulators of cell death in animals)
of the susceptible genotype, that it is a pseudo-ORF and such no transcripts
was found even after challenging with the virus (result not shown). Whereas,
perhaps due to the spontaneous mutation, the tolerant genotypes salvaged
the function; which is also evident from the presence of the transcripts
of VMYR6 after challenging with the virus. Therefore, it is assumed that
the tolerant genotypes are gain-in-function mutants.
Chemical pesticides and insecticides are commonly applied in the farmer`s
field to protect crop plants from the attack of pathogens. The extensive
use of these toxic chemicals not only forced the insects to build up resistance
and new biotypes, but also adversely affects the ecological balance and
natural pest controlling agents. Cultivation of biotic stress tolerant
varieties endowed with favoured allele, like R-gene are globally preferred
to keep the environment free from chemical and toxic pollutants and to
sustain ecological balance. The V. mungo cultivar T9 is
a superior genotype with high agronomic yield and cultivated at different
states of India. MYMIV-tolerant lines derived from the T9 genotype would
prove useful for farming and also for further improvement of V. mungo.
We thank the Department of Biotechnology, India, for financial assistance
(BT/PRO 0689/AGR/07/27/97 and BT/01/COE/06/03) and research fellowships
to J.B, SK and A.K. We are thankful to Dr. Aparajita Mitra for the identification
of the mutant lines from the MEF, Bose Institute.
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