Background
Blackgram (Vigna mungo (L.) Hepper), popularly known as urdbean or mash in India, is one of the important a grain legumes. Yellow Mosaic Disease (YMD) is a significant biotic stress causing profound yield loss in blackgram. Yellow Mosaic Virus (YMV) belongs to the genus Begomovirus and transmitted by the vector whitefly, Bemisia tabaci. Yield loss due to this disease varies from 5 to 100 per cent depending upon disease severity, susceptibility of cultivars and population of whitefly (Nene, 1972; Singh, 1980; Rathi, 2002). It has been confirmed that two virus species causing YMD are prevalent in Indian sub continent viz., mungbean yellow mosaic India virus (MYMIV) is commonly occurring in northern part of India and mungbean yellow mosaic virus (MYMV) is confined to Southern India. Existence of different species of yellow mosaic viruses changes the crop behavior on nature of resistance exhibited by blackgram genotypes from southern to northern parts of India. Thus assessing the varietal behavior of blackgram with respect to the YMV predominant in the regions through effective screening is a considerable criterion in varietal development programme. The development of YMD resistant varieties along with disease screening is curtailed by the complexities on the viral strains, vector population, crop biology and environmental factors (Malathi and John, 2008). Screening may be either on natural field conditions or through forced inoculation employing viruliferous vectors. In both the cases, vector population remains specific for the disease spread. Practical difficulties include climatic conditions that greatly affect the building up of the vector population. If optimal conditions occur throughout the year, vector population will be present continuously. Marker assisted indirect selection of resistant genotypes using linked markers has been reported as an effective breeding approach for developing YMD resistant cultivars in blackgram. It is assuming increased importance due to lack of uniform field screening procedure as well as difficulty in direct selection due to complex virus, vector, host and environmental interaction (Souframanien and Gopalakrishna, 2006). Few molecular markers linked with YMD resistance were identified (Basak et al., 2004; Souframanien and Gopalakrishna, 2006; Maiti et al., 2011). Yet, validation of such markers becomes an essential criterion with regard to the intricate disease nature. Thus the present study was undertaken to validate available RGA and SCAR markers linked with YMD resistance in blackgram. The present study was carried out in Tamil Nadu where, MYMV is prevalent.

1 Results and Discussion
Molecular markers are now widely used to tag important genes and genomic regions in may important crops. Marker assisted selection for those traits which are difficult to screen phenotypically has become an important tool for breeding programs. Screening for YMD in blackgram is also difficult owing to practical difficulties in creation of artificial epiphytotic conditions and dependency on several factors such as vector population and climatic conditions. The aim of the present investigation is to validate markers associated with YMD resistance in blackgram.

In the present study, amplicons were obtained with all the four markers and produced an allele of expected size to that of original papers. Among the four markers screened, the marker VMYR1 produced an approximate allele size of 445 bp in all the 14 genotypes evaluated and not differentiated the resistant and susceptible genotypes (Table 1). The VMYR1 marker profile of 14 blackgram genotypes is shown in Figure 1. Hence, it is concluded that it is not linked with YMV resistance genes. Similar results were also reported by Souframanien and Gopalakrishna (2006) for the marker VMYR1 in Blackgram, while evaluating different set of blackgram genotypes.

Table 1 Validation of molecular markers linked with YMD resistance

Figure 1 VMYR 1 marker profile for 14 blackgram genotypes

 
The markers YR4, CYR1 and SCARISSR 811 produced an allele in certain genotypes and were absent in remaining genotypes (Table 1). The above markers behaved as dominant, either with presence or absence of allele. By comparing the marker data and disease response, the following conclusions were drawn. Those genotypes which were resistant to YMD had null allele or absent whereas, susceptible varieties produced an allele of respective size. But, in the original papers an allele was present in resistant lines and absent in susceptible genotypes which was just the opposite of the result of the present study.

However in the present study, few exceptions were also noticed in all the three markers where, the respective allele was absent in susceptible genotypes and present in resistant genotypes. Among the three markers, YR4 had relatively few such exceptions than CYR1 and SCARISSR 811. As mentioned, the exceptional results for YR4 marker were recorded for five genotypes. The genotypes TU 94-2, IPU 02-033 and VBN(Bg) 4 were observed as resistant genotypes and had a respective allele. The genotype VBN(Bg) 5 recorded as moderately resistant variety with no allele and TNAU Blackgram Co 6 recorded as moderately resistant but the allele was present (Figure 2).

Figure 2 YR4 marker profile for 14 blackgram genotypes

 
The disparity in the behaviour of VBN(Bg) 5 and TNAU Blackgram Co 6 could be reasoned as they were categorized as moderately resistant varieties from the standard procedure of Mayee and Datar (1986). Since YR4 is a dominant marker, the moderately resistant variety can behave on either way viz., presence or absence of allele. The other exception was with TU 94-2, IPU 02-033 and VBN(Bg) 4 where no correlation was observed with phenotypic and genotypic data. These genotypes were classified as resistant genotypes from results of disease screening. But, they exhibited presence of allele. This showed that YR4 is partially linked with YMV resistance genes.

Similar results were observed for other two markers viz., CYR1 and SCARISSR 811 with more number of exceptions (Table 1). The exceptions with respect to the disease reaction and CYR 1 marker were with TU 94-2, IPU 02-033, VBN(Bg) 5, VBN(Bg) 4, TNAU Blackgram Co 6, VBN(Bg) 6 and Vigna mungo var. silvestris 22/2 (Figure 3). The variety VBN(Bg) 6 was a resistant genotype whereas, it produced an allele of respective size. Similarly, the genotype Vigna mungo var. silvestris 22/2 showed null allele but it was found to be a susceptible variety in both the locations.

Figure 3 CYR1 marker profile for 14 blackgram genotypes

 
The SCAR_{ISSR 811} marker produced allele with an approximate size of 1357 bp and the marker profile of 14 blackgram genotypes is shown in Figure 4. The exceptional genotypes for this marker was observed to be many viz., TU 17-4, Vigna mungo var. silvestris 22/10, IPU 02-033, VBN(Bg) 5 and VBN(Bg) 4. The above results clearly showed that the markers are partially linked or not very close to the resistance locus and showed several discrepancies. The results obtained on validation of YR4, CYR1 and SCAR_{ISSR 811} markers agreed with the existence of two separate strains of yellow mosaic virus as stated by Malathi et al. (2004), based on marker and phenotype interaction. This was also justified from the behaviour of Co 5 variety with SCAR_{ISSR 811} marker. The variety Co 5 produced an allele of 1357 bp and found to be resistant in Maharashtra, where the screening was carried out (Souframanien and Gopalakrishna, 2006). Correspondingly in the present study, the marker produced the same size of allele in Co 5 but it was found to be susceptible to YMV disease in two locations viz., Coimbatore and Vamban of Tamil Nadu. The above results clearly indicated that viral strains present in North/Western and South India may be different. Usharani et al. (2004) and Malathi et al. (2004) described that while the species mungbean yellow mosaic virus (MYMV) is prevalent in southern India, the species Mungbean Yellow Mosaic India Virus is predominant in northern India. It is possible that the markers validated in the present study viz., SCAR_{ISSR 811} marker was developed in Western India and YR4, CYR1 and YMVR1 in Eastern India. From the discrepancies seen in the markers profile, it can be inferred that markers have been developed targeting a specific virus, which may not be predominant in southern India. The varieties found to be resistant in North India could get infected by the virus strain in South India. Markers used in the present study were originally developed at Institutes from eastern and western parts of India where, genotypes with an allele were found to be resistant and absent in susceptible genotypes. However, the results were opposite in the present study where, specific allele were found to be susceptible and resistant varieties had no allele.

Figure 4 SCAR ISSR 811 marker profile for 14 blackgram genotypes

 
To conclude, among the four markers validated, VMYR1 was found to be not linked with YMV resistance genes whereas, the markers viz., YR4, CYR1 and SCAR_{ISSR 811} were found to be partially linked with YMV resistance genes. While comparing the marker and phenotypic data, few deviations were noticed for certain genotypes in all the three markers. The existence of two different YMV viral strains got confirmed through the present study since the results were opposite in the present study where, the genotypes with specific allele were found to be susceptible and resistant varieties had no allele. These markers could be used in the marker assisted selection only for the genotypes whose marker expression coincides with the disease reaction. However identification makers associated with YMD resistance genes caused by south Indian viral species viz., MYMV is highly essential and to use MAS for the development of YMD resistant varieties in Blackgram for South Indian condition.

2 Materials and Methods
Fourteen blackgram genotypes viz., VBN(Bg) 6, TU 17-4, TU 94-2, IPU 02-033, IPU 02-43, LBG 17, VBN(Bg) 5, VBN(Bg) 4, Co 5, TNAU Blackgram Co 6, Vigna mungo var. silvestris 8/2, T 9, Vigna mungo var. silvestris 22/10 and Vigna mungo var. silvestris 22/2 were selected for the present study. It comprises of lines having resistance or susceptible response for YMV disease. Field screening for YMV disease was carried out in the hot spot areas (more prone to disease susceptibility) of Tamil Nadu during summer, 2012 using infector row technique. Two locations namely Coimbatore and Vamban were selected for screening. Fourteen genotypes were sown in the order of two rows test entries and one row of infector variety. The blackgram variety Co 5 was chosen as the infector variety as it is highly susceptible to YMV disease. Disease incidence was scored according to the extent of symptom expression calculated from per cent disease incidence.

Percent disease incidence = [(Number of plants infected in a row) / (Total number of plants in a row)] × 100

The genotypes were later grouped into different categories based on 0 to 9 scale (Mayee and Datar, 1986) (Table 2).

Table 2 Grouping of genotypes into different categories based on 0 to 9 scale

 
Correspondingly, the DNA of 14 blackgram genotypes was isolated using CTAB mini-prep method. Leaf samples for DNA isolation were collected from the YMD resistant / susceptible plants of respective genotypes during phenotypic scoring.

A set of three RGA markers (Basak et al., 2004; Maiti et al., 2011) and one SCAR marker (Souframanien and Gopalakrishna, 2006) were validated using above 14 genotypes. PCR reaction was carried out in a volume of 15 μL containing 50 ng of genomic DNA, 1 × PCR buffer, 10 mM dNTP’s, 25 mM MgCl₂, 5 μM of (forward and reverse) primer and 1 unit of Taq DNA polymerase. Amplification was performed in master cycler gradient PCR (eppendorf). Amplification conditions were, initial denaturation at 94? for 3 minutes

followed by 35 cycles of denaturation at 94? for 45 seconds, annealing at 45 - 58? for 1 minute and extension 72? for 1 minute and a final extension at 72? for 10 minutes. The PCR amplified products were subjected to gel electrophoresis in 3% agarose gel in 1 × TBE at 100 V for 5 hours using gel electrophoresis unit. The ethidium bromide stained gels were documented using BIO-RAD gel documentation system. The three RGA markers got amplified at the same annealing temperature as mentioned in the original publications (Basak et al. 2004; Maiti et al. 2011). The annealing temperature for SCAR marker was fixed through gradient PCR and the amplification was performed. The details of the markers used in this study are mentioned in Table 3. The results obtained for the four markers were verified through independent PCR.

Table 3 Molecular markers linked with YMD resistance in blackgram

 
Authors’ contributions
SK carried out the overall experiment and preparation of the manuscript. JP supervised the experiment as chairman of the advisory committee for the master degree thesis work.

Acknowledgement
We thank the Department of Bio-Technology, a division under the Department of Science and Technology, India, for the funding and supporting the research programme (BT/PR13464/AGR/02/698/2010 dt.01.12.2010).

References
Basak J., Kundargrami S., Ghose T.K., and Pal A., 2004, Development of yellow mosaic virus (YMV) resistance linked DNA marker in Vigna mungo from populations segregating for YMV- reaction, Molecular Breeding, 14: 375-383
http://dx.doi.org/10.1007/s11032-004-0238-y

Maiti S., Basak J., Kundagrami S., Kundu A., and Pal A., 2011, Molecular marker-assisted genotyping of mungbean yellow mosaic India virus resistant germplasms of mungbean and urdbean, Molecular Biotechnology, 47: 95-104
http://dx.doi.org/10.1007/s12033-010-9314-1

Malathi V.G., Usharani K.S., Sivalingam P.N., Rouhibaksh A., Latha K.V.P., and Periasamy M., 2004, Diversity and complexity of begomoviruses, Annual Reviews of Plant Pathology, 3: 225-270

Malathi V.G., and John P., 2008, Geminiviruses infecting legumes, In G.P. Rao, P. Lava Kumar and R.J. Holguin-Pena (Eds.). Characterization,Diagnosis and Management of Plant Viruses: Vegetables and Pulses Crops (pp. 97–123). Houston, TX: Stadium Press, LLC

Mayee C.D., and Datar V.V., 1986, Phytopathometry, Technical Bulletin No. 1, Marathawada Agricultural University, Parbhani, pp.145-146

Nene Y.L., 1972, A survey of the viral diseases of pulse crops in Uttar Pradesh, G.B. Pant University of Agriculture and Technology, Pantnagar, Res. Bull., 4: 191

Rathi Y.P.S., 2002, Epidemology, Yield losses and management of major diseases of Kharif pulses in India, Plant Pathology and Asian congress of Mycology and Plant Pathology, University of Mysore, Mysore

Singh J.P., 1980, Effect of virus diseases on growth component and yield of mungbean and urdbean, Indian phyto- pathology, 8: 405-408

Souframanien J., and Gopalakrishna T., 2006, ISSR and SCAR markers linked to the mungbean yellow mosaic virus (MYMV) resistance gene in blackgram (Vigna mungo (L.) Hepper), Plant Breeding, 125: 619-622
http://dx.doi.org/10.1111/j.1439-0523.2006.01260.x

Usharani K.S., Surendranath B., Haq Q.M.R., and Malathi V.G., 2004, Yellow mosaic virus infecting soybean in Northern India is distinct from the species infecting soybean in southern and western India, Current Science, 86: 845-850