Identification and Validation of Quantitative Trait Loci of Mungbean Yellow Mosaic Virus Disease Resistance in Blackgram [Vigna mungo (L). Hepper]

Blackgram [Vigna mungo (L.) Hepper] is an important food legume crop of India. It is an important short duration crop and widely cultivated in India. It gives an excellent source of easily digestible good quality protein and ability to restore the fertility of soil through symbiotic nitrogen fixation.  The seeds are highly nutritious with protein (24-26%), carbohydrates (60%), fat (1.5%), minerals, essential amino acids and vitamins. The nutritional value improves greatly, when wheat or rice is combined with blackgram because of the complementary relationship of the essential amino acids such as arginine, leucine, lysine, isoleucine, valine and phenylalanine etc. (Mehra et al., 2016). In India, the cultivated area under blackgram is about 4.50 million hectares with the production of 2.83 million tonnes (AICRP on MULLaRP, 2018). Mungbean yellow mosaic virus (MYMV) disease is the major problem in blackgram. The disease is economically important, destructive, wide spread and inflicts heavy losses annually. The disease mainly occurs in South Asian countries India, Sri Lanka, Pakistan and Bangladesh (Malathi and John 2009). The disease can reduce seed yield upto 100% or even kill a plant infected at an early vegetative stage (Naimuddin, 2001). Mungbean yellow mosaic virus, a member of family Geminiviridae is transmitted by the polyphagous insect vector, whitefly (Bemisia tabaci).  It has a bipartite genome (DNA A and DNA B) (Mansoor et al., 2003; Jeske 2009). Begomo viruses include mungbean yellow mosaic virus (MYMV), mungbean yellow mosaic India virus (MYMIV), Dolichos yellow mosaic virus (DoYMV) and horsegram yellow mosaic virus (HgYMV), which are wide spread throughout South Asia (Haq et al., 2011). Both MYMV and MYMIV cause yellow mosaic virus (YMD) on blackgram. MYMV is prevalent in southern India where Tamil Nadu is situated (Nair et al., 2017). In blackgram, MYMV diseases cause irregular yellow green patches on older leaves and complete yellowing of young leaves of susceptible varieties. The characteristic yellow mosaic symptoms include two types, yellow mottle and necrotic mottle that can be distinguished visually (Nair and Nene, 1974). Moreover, the control of MYMV disease is often based on limiting the vector population by insecticides, which are ineffective under severe whitefly infestations. In highly susceptible plants, symptoms include shortening of internodes, severe stunting of plants with no yield or few flowers and deformed pods producing small, immature and shriveled seeds. The most effective way to prevent the occurrence of this disease is to develop genetically resistant cultivars of blackgram. Hence understanding the inheritance pattern and the nature of gene action involved in MYMV disease reaction is essential. There are some conflicting reports about the genetics of resistance to MYMV disease claiming both resistance and susceptibility as dominant. In blackgram, monogenic dominant nature of resistance was reported by Dahiya et al., (1977), Kaushal and Singh (1988) and Gupta et al., (2005) while it was reported to be digenic recessive by Singh (1980), Dwivedi and Singh (1985) and Verma and Singh (1986). Monogenic recessive control of yellow mosaic resistance was also reported by some authors (Pal et al., 1991). Improved resistance to MYMV is now the major goal for breeding programs in several blackgram and greengram production countries. However, a major difficulty in breeding MYMV disease resistant in greengram or blackgram is field screening for the virus disease.  Hence identification of QTL followed by Marker-assisted selection (MAS) is highly useful for genetic improvement of crops.  MAS can accelerate the development of new cultivars by reducing the number of generations and increasing precision in phenotypic evaluations (Collard and Mackill 2008). So far, there are few reports on DNA markers associated with mungbean yellow mosaic virus disease resistance in blackgram. With this background, a study was made for identification as well as validation of quantitative trait loci (QTL) for MYMV disease resistance in blackgram.

Plant material and development of mapping population
 
A total of 112 F_2:3 recombinant inbred lines (RILs) were developed from a cross between MDU 1 (female parent) and Mash 1008 (male parent). MDU 1 is a high yielding, popular blackgram variety grown in Tamil Nadu, but highly susceptible to MYMV disease. This variety was developed at Agriculture College and Research Institute, Tamil Nadu Agricultural University, Madurai and released in 2014 for commercial cultivation in October to November season of Tamil Nadu (MYMV disease free season). Mash 1008 is a variety released at Punjab Agricultural University, Ludhiana and is resistant to MYMV disease. Mash 1008 was used as a male parent and crossed with MDU 1 during July-September 2017 to produce F₁ seeds. An F₁ hybrid plant confirmed through SSR marker was selfed during October to November, 2017 to produce F₂ seeds. A total of 112 plants were selfed to obtain F₃ seeds and used to raise as F_2:3 recombinant inbred lines (RILs). 
 
Phenotypic data
 
A total of 112 F2:3 lines were evaluated for MYMV disease resistance along with parents at the National Pulses Research Center, Tamil Nadu Agricultural University, Vamban, Tamil Nadu (10.363505^o N, 78.902283^o W) during July-September 2018 under Augmented Design in the field.  Each line was sown in one row of 3 m in length with a spacing of 30 cm as between row and 10 cm as within row. Susceptible genotypes CO 5 and MDU 1 were sown as disease spreader rows after every eight rows and also around the plots. No insecticide was sprayed in order to maintain the natural white fly populations. The MYMV disease score was recorded on 60 DAS, by using phenotype rating scale from 1 (resistant) to 9 (highly susceptible) (Table 1), as suggested by Singh et al., (1995). The mean of each progeny was calculated and used as phenotypic data.


 
DNA isolation, SSRs and PCR condition
 
Total genomic DNA of each parental and F₂ plants were extracted from fresh young leaf tissue using the CTAB method (Lodhi et al., 1994). The collected samples were ground in pestle and mortar with preheated (65^oC) 500 μl of CTAB buffer. Extracted samples were taken into eppendorf tubes and incubated in the water bath for 30 min at 65^oC. After incubation, 300 μl of phenol: chloroform: iso-amyl alcohol (25:24:1) was added into tubes and inverted twice to mix and kept in centrifuge for10 min at 10000 rpm. The supernatant was collected in the fresh tubes. The tubes were added with 500 μl of chloroform: iso-amyl alcohol (24:1) and inverted twice to mix. The tubes were centrifuged for 5 minutes at 10,000 rpm.  The aqueous layer was transferred in to the new eppendorf tubes. An amount of 0.7 volume of isopropanol (stored at -20^oC) was added to each sample and inverted once to mix and kept overnight at 4^oC. The samples were centrifuged at 8000 rpm for 10 min on the next day. The supernatant was discarded from each sample and the pellets settled in the bottom were air dried for 30 min. A quantity of 50 μl of TE buffer was added into each sample and stored overnight at 4oC. RNAse (3 μl) was added into each sample and kept at 65^oC for 30 min. The DNA quality and quantity were checked on 0.8% agarose gel and DNA concentration was normalized to10 ng/μl.
 
The polymerase chain reaction (PCR) mixtures, containing2 μl of 10 ng template DNA, 1.0 μl of 10 X Taq buffer (M/s Genei, India) + MgCl₂ (1.5 mM), 1.0 μl of dNTPs (2 mM), 1.0 μl of forward and reverse SSR primers (0.5 μM), 0.3 μl of taq polymerase (3 IU) and 4.7 μl of sterile double distilled water. The DNA was amplified in a thermocycler (M/s Eppendorf, Germany, model AG 6325) under the following conditions: 94oC for 4 min followed by 30 cycles of 94^oC for 30 s, 55^oC for 45 s, 72^oC for 1 min, with a final extension step of 72^oC for 20 min. The PCR products were separated on 3% agarose gel and photographed using GELSTAIN 4x advanced gel documentation unit (M/s Medicare, India).
Linkage map and QTL analysis
 
Linkage analysis was carried out using QTL IciMapping (version 4.1.0.0) (Wang et al., 2016). A minimum LOD threshold of 3.0 and maximum distance of 50 cM were used for construction of linkage groups. Map distance in centimorgan (cM) values was calculated using Kosambi mapping function (Kosambi 1944). QTL analysis was performed using QGene 4.4.0 (Joehanes and Nelson 2008) following composite interval mapping (CIM) and automatic cofactor selection. LOD threshold significance for each QTL was calculated with1000 runs of a permutation test at P = 0.05. 
 
Validation of markers linked with MYMV disease resistance
 
Two mapping populations MDU 1 x Mash 114 and CO 5 x VBN6 in F_2:3 and F₂ generations respectively were used in this study to validate the identified QTL regions. The material was screened for the MYMV disease reaction in the field conditions at National Pulses Research Centre, Tamil Nadu Agricultural University, Vamban, Tamil Nadu during July - Sep 2018.  Normal cultural practices were followed, except the insecticide application. CO 5 and MDU 1 which are highly susceptible cultivars to MYMV disease were sown as disease spreader rows in every ninth row and around the perimeter of plots to increase sufficient disease pressure. The disease scores were recorded on 60 DAS as described by Singh et al., (1995).

Analysis of phenotypic variation
 
In the present study, mean and variability parameters were estimated for MYMV disease scores and presented in Table 2. Parameters, skewness and kurtosis help the breeder to understand the nature of distribution of individuals in the population. MYMV disease score at 60 DAS had non-significant skewness which indicates no skewness for this trait. MYMV disease score at 60 DAS had non-significant kurtosis. It indicates the mesokurtic nature of this trait. The results indicated the presence of normal distribution for the MYMV disease scores at 60 DAS (Fig 1).




 
Construction of linkage map
 
A total of 525 SSR primers were used to test polymorphism between parents MDU 1 and Mash 1008. Of these, 315 were from mungbean (Isemura et al., 2012; Gwag et al., 2006; Somta et al., 2008; Seehalak et al., 2009; Tangphatsornruang et al., 2009) and 210 from adzuki bean (Wang et al., 2004; Chankaew et al., 2014). Among 525 markers only 35 (14.1%) showed polymorphism between parents. Genotyping was carried out for 112 F_2:3 RILs of the cross MDU 1 x Mash 1008 with 35 polymorphic SSR markers. Linkage analysis was performed using QTL IciMapping (version 4.1.0.0) (Wang et al.,  2016).  Linkage groups were established using a minimum LOD score of 3.0, ordering by RECORD, rippled by SARF criterion with a window size of 5. Nine linkage groups were established with 29 SSR markers (Fig 2). Remaining six markers were found as unlinked. The total length of the map was 586.08 cM. 


 
QTLs of MYMV disease resistance
 
Composite Interval Mapping (CIM) was employed to locate QTL for MYMV disease resistance on the linkage map. The LOD threshold for MYMV disease resistance was determined by a permu­tation test with 1000 runs. Two QTLs for MYMV disease score at 60 DAS were located one each on LG2 and LG10 and designated as qmymv2_60 and qmymv10_60 (Fig 2 and Table 3) respectively. The qmymv2_60 was flanked by CEDAAG002, CEDG225 and GMES4236, showing a LOD score of 5.71 and significant at P = 0.05. This QTL had an additive effect of 9.1 and explained 20.90 % of variation for the MYMV disease score at 60 DAS.  The qmymv10_60 was flanked by cp05325, CEDG180 and GMES4431, showing LOD score of 6.98 and significant at P = 0.05. This QTL showed an ad­ditive effect of 9.0 and accounted for 24.90% of the vari­ation for the MYMV disease score at 60 DAS. In all cases, alleles from the resistant parent Mash 1008 contributed towards the reduction in disease score, i.e. increasing resistance. 


 
Validation of markers linked with MYMV disease resistance
 
A total of 80 SSR primers of LG 2 and LG 10 were used to test polymorphism between parents MDU 1, CO 5, Mash 114 and VBN 6. Of these, 55 and 25 primers were associated with LG 2 and LG 10 respectively. Among 55 markers of LG 2 only two showed polymorphism between parents in both crosses. Three and four primer showed polymorphism in LG 10 for the crosses MDU 1 x Mash 114 and CO 5 x VBN6 respectively. Genotyping was carried out for 39 F_2:3RILs and 68 F₂RILs of the crosses MDU 1 x Mash 114 and CO 5 x VBN6 respectively.
 
The LOD threshold for MYMV disease resistance was determined by a permutation test with 1000 runs. One QTL qmymv10_60 was detected by CIM for MYMV disease score at 60 DAS in both populations (Table 4). The qmymv10_60 was flanked by cp05325 and CEDG198 with LOD score of 5.12 and significant at P = 0.05. It had an additive effect of 9.67 and explained 45.40% of variation in the MYMV disease score at 60 DAS in the cross MDU 1 x Mash 114. In CO 5 x VBN 6 cross combination qmymv10_60 was flanked by CEDG198, CEDG180 and CEDG097 at LOD scores 9.16 on LG10. It had an additive effect of 2.63 and explained 46.00 % of variation for the MYMV disease score at 60 DAS.



Kang et al., (2005) reported that more than 80% of viral resistance in plants is controlled by single gene. In the present study, composite interval mapping (CIM) on F_2:3 RILs populations showed two major QTLs qmymv2_60 and qmymv10_60 for MYMV disease score at 60 DAS. These two QTLs on LG2 and LG 10 explained more than 20 per cent of variation on MYMV disease score at 60 DAS. Hence these QTLs can be considered as highly robust and can be used in marker-assisted selection programme for MYMV disease resistance. In blackgram, resistance gene analog markers YR4 and CYR1 were reported to be linked with resistance to MYMIV (Maiti et al., 2011). Marker CYR1 was also asso­ciated with resistance in mungbean. CYR1 is proposed as part of the candidate disease resistance (R) gene (Maiti et al., 2011). However, markers YR4 and CYR1 were found as monomorphic between the parents MDU 1 and Mash 1008. Gupta et al., (2013) reported that the SSR marker CEDG180 was associated with a major gene control­ling MYMV disease resistance in blackgram and amplified an allele of 136 and 163 bp in resistant and susceptible parents respectively. In the present study, qmymv10_60 has the marker CEDG180. But this marker amplified an allele of 136 and 163 bp for susceptible and resistant parents respectively. This type of deviation might be due to the influence of the third gene which may have inhibitory gene action.
 
In this study, identified QTLs were validated with two other mapping populations MDU 1 x Mash 114 and CO 5 x VBN 6 in F2:3 and F2 respectively. However, to validate the identified QTLs, SSR markers reported on LG 2 and LG 10 of Vigna group Azudiki bean (Han et al., 2005), Greengram (Isemura et al., 2012) and Blackgram (Chaitieng et al., 2006) were screened for polymorphism in the parental lines of MDU 1 x Mash 114 and CO 5 x VBN 6 crosses.  In the present study, composite interval mapping (CIM) detected one QTL qmymv10_60 for MYMV disease score at 60 DAS in both populations. The validation study revealed that the qmymv10_60 alone validated in both mapping population and explained very high phenotypic variation of 45.40 - 46.00%. Hence it may conclude that qmymv10_60 may be utilized for marker assisted selection/ marker assisted backcross in other mapping population with assured improvement on MYMV disease resistance lines in blackgram.

KV performed the field experiments, measurements, data analysis and KV and SR drafted the manuscript, NM and AM supervised the work, worked on the manuscript and AM supervised the work, worked on the manuscript and aided in interpreting the results, NM, AM, VKS and CV were involved in planning. All authors provided critical feedback on research, analysis and manuscript.
 

Authors are acknowledging the help rendered by Mr. Arul Doss, Agricultural Supervisor, NPRC, Vamban in the trial.

Not applicable

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.