Development of Mapping Population and Validation of Molecular Markers Associated with MYMV Resistance in Mungbean

Among the major diseases causing signiûcant yield loss in mungbean, yellow mosaic disease (YMD) is the most devastating, especially in South Asian countries. YMD can cause yield loss up to 100 per cent (Alam et al., 2014). Developing a resistant variety for MYMV through conventional breeding methods takes a longer period due to rapid development of new isolates and also the complexity of mechanism in controlling MYMV resistance as it involves both the dynamic virus and the vector. The most economic and effective method to control the disease is the breeding of resistant cultivars against MYMV through marker assisted breeding. F₂ population is the simplest type of mapping population developed for self-pollinating species and can be developed in relatively short time (Alam et al., 2014). In the present study, efforts were made to develop new mapping population with a resistance source and to validate molecular markers associated with MYMV resistance.

Development of mapping population
 
DGGV-2 is agronomically superior, high yielding and popular variety developed at UAS, Dharwad, but highly susceptible to MYMV. IPM-2-14 is a field resistant genotype to MYMV. The present investigation was carried out with 260 F₂ individuals. These were derived from crossing DGGV-2 and IPM 2-14 during Kharif-2017 at Main Agril Research Station, UAS, Dharwad. A modification of the mungbean hybridization technique (Boling et al., 1961) was followed to carry out crossing. Hybrid seeds of this cross were harvested individually and sown during rabi 2017 along with the two parents, as checks for distinguishing the true hybrids (Plate 7). Hybridity of F₁s was conûrmed through molecular marker analysis and the true F₁s were selfed to raise the F₂ generation.
 

 
DNA extraction and Simple sequence repeats analysis
 
Total genomic DNA was isolated from young leaves of 15 days old plants using cetyltrymethyl ammonium bromide (CTAB) method described by Agbagwa et al., (2012) with certain modifications. Here the genomic DNA extraction was done by using liquid nitrogen and lyophilisation which is avoided in the Agbagwa et al., protocol. A set of 24 microsatellites or simple sequence repeat (SSR) primers (Table 1) which were found to be associated with MYMV resistance in previous reports of legume species viz., black gram (Gupta et al., 2013) and mungbean (Alam et al., 2014, Wang et al., 2004 and Han et al., 2005) were used to screen the parents for identification of polymorphic markers.
 
PCR reaction mixtures were prepared in the volume of 20 µl containing, 1 µl of the extracted genomic DNA (50 ng/µl), 2 µl of Taq buffer (10X) with MgCl₂, 0.8 µl each of forward and reverse primer (0.2 μM), 0.8 µl of dNTPs (2.5 mM), 0.5 µl of Taq polymerase (3 U/µl) and 14.1 µl of sterile distilled H₂O.
 
The PCR reactions with the specific primers were carried out in a DNA thermal cycler as per the following stated cycle regime except for the annealing temperatures, which were standardized based on temperature gradient PCR. Initial denaturation at 95ºC for 5 minutes, then 35 cycles consisting each of a denaturation at 94ºC for 1 minute, annealing for 1.10 minutes and extension at 72ºC for 1 minute. Final extension was performed at 72ºC for 10 minutes and finally, the reaction mixtures were held at 4ºC. Amplicons were mixed with DNA loading dye and 10 µl of product was loaded onto 4 per cent agarose gels and electrophorised at a constant voltage of 80 V for 2 hours, then the ethidium bromide stained gels were visualised in a gel documentation system.
 
Evaluation of F₂ population for MYMV infection
 
The F₂ population along with the parents were screened for mungbean yellow mosaic virus disease reaction under field conditions during summer 2018 following the method of Singh and Singh (2000). Each entry was sown in a single row of four meter length with the spacing of 30´10 cm. All the recommended agronomic practices were followed. No insecticidal spray was given in order to allow the whitefly population to spread the disease. To ensure enough availability of the inoculum, susceptible check (DGGV-2) was planted in each block and around the experimental layout.
 
The test entries were scored for MYMV disease after 80 % of the plants in infector rows showed MYMV incidence. Based on the disease severity, symptom severity grades (Plate 6) designated with numerical values of (0-4) and a scale of response value (0-1) corresponding to such grade, the coefficient of infection (CI) was calculated by multiplying the Per cent Disease Incidence (PDI) to the response value assigned for each grade (Singh and Singh, 2000).
 

 
Genotyping of F₂ population
 
Each individual in the F₂ population was screened with polymorphic primers. The PCR products were separated on 4 per cent agarose gel electrophoresis and the bands were visualized and documented in gel documentation system (Alpha ImagerTM1200, Alpha Innotech Corp., CA, USA). The amplicons were scored as “A” for the female parent (homozygous dominant), “B” for the male parent (homozygous recessive) and “H” for the heterozygotes.
 
Analysis for marker-trait association
 
Single marker analysis was performed to find the contribution of the markers towards the MYMV resistance by win QTL cartographer version 2.5. The coefficient of determination (R²) which explains the per cent of phenotypic variance by the polymorphic marker was derived.

Identification of polymorphic markers between MYMV resistant and susceptible parent
 
Of the 24 SSR markers (Table 1) used to screen the MYMV susceptible (DGGV-2) and resistant (IPM-2-14) variety to know the polymorphism, two markers viz., CEDG305 and CEDG115 were found to be polymorphic (Plate 1). The approximate size of the amplicons and their primer annealing temperature for all the primers are presented in Table 1.
 

 

 
Molecular confirmation of F₁
 
One hundred and twenty F₁ seeds were sown in separate pots along with their respective parents. They were genotyped using two polymorphic markers CEDG305 and CEDG115. Of the 120 F₁ plants, 27 were confirmed as true F₁s (Plate 2 and 3). These F₁s were self pollinated to produce F₂.
 

 

 
Phenotyping of F₂ mapping population
 
 A total of 260 F₂ plants along with parents were evaluated for MYMV under field conditions .Of these 260 plants, 11 plants showed resistant reaction, 47 plants recorded Moderate resistance and 202 plants showed susceptible reaction, thus exhibiting phenotypic segregation in the ratio of 3 S:1 R (202 susceptible: 58 resistant) implying that MYMV resistance is governed by a single recessive gene .
 
The coefficient of infection in F₂ ranged from 7.66 to 53.51 per cent with the mean value of 31.63 per cent. Coefficient of infection of DGGV-2 was 53.51 per cent and categorized as susceptible whereas coefficient of infection of IPM-2-14 was 7.66 per cent and categorized as resistant (Table 2).
 

 

 
Genotyping of F₂ population using polymorphic markers
 
In the present study MYMV resistant (IPM-2-14) and susceptible (DGGV2) parents were screened using a set of 24 SSR markers for identification of polymorphic SSR markers. Of these 24 SSR markers, some were reported to be linked with yellow mosaic disease resistance in green gram (Alam et al., 2014) and some of them were found to be polymorphic for MYMV resistance in black gram (Gupta et al., 2013) and mungbean (Wang et al., 2004; Han et al., 2005). Genotyping of two hundred and sixty F₂ individuals was done using CEDG305 (Plate 4) and CEDG115 (Plate 5) which had shown polymorphism between DGGV2 and IPM-2-14. The markers CEDG305 and CEDG115 segregated as 69:125:66 and 74:132:54 (Homozygous dominant: Heterozygotes: Homozygous recessive), respectively in the F₂ population. Chi-square test indicated the good fitness for 1:2:1 ratio (Table 4). Similar inheritance pattern of MYMV resistance was studied by Sai et al., (2017) using F₂ as a mapping population. Single marker analysis revealed that the molecular markers CEDG305 and CEDG115 were associated with MYMV resistance with a phenotypic variance of 24.47 and 10.31 per cent respectively at probability of 0.05 and 0.01 (Table 5). The SSR marker, CEDG 305 showed polymorphism between the two parents and amplified an allele of 110 bp and 120 bp in the resistant and susceptible parents. Alam et al., (2014) also have reported the association of the marker CEDG305 with yellow mosaic resistance (27.13% of phenotypic variance). Wang et al., (2004), Han et al., (2005) and Gupta et al., (2013) reported that the marker CEDG115 was found to be polymorphic for MYMV resistance in mungbean and urdbean. However, the composite interval mapping using high number of polymorphic markers to know the fine association of molecular markers with MYMV resistance should be carried out to facilitate marker-assisted selection of resistant genotypes/lines in developing MYMV resistant varieties. The monogenic recessive inheritance of YMV resistance observed in the present study is consistent with the results reported by Jain et al., (2013) and Anusha (2014). There are, however, a few contrasting published reports on the inheritance of resistance to MYMV in mungbean. Involvement of two recessive genes for the control of YMV resistance in mungbean by Dhole and Reddy (2013) and complementary recessive genes reported by Shukla and Pandya (1985). Single dominant gene controlling resistance to MYMV was reported by Gupta et al., (2005) and Gupta et al., (2013). These contradictory reports on inheritance of mungbean yellow mosaic virus disease resistance are probably due to the differences in the genetic background of the parents used in their study and the different strains of virus prevalent at different locations and interaction between them.
 

 

 

 

 
Performance of superior genotypes
F₂ poulation was evaluated for yield and yield attributes. Segregant number 183 has recorded lowest coefficient of infection (4.34) and moderate yield of 28.19 grams per plant this genotype can be used as donor for breeding agronomically superior genotype with MYMV resistance. The other segregants, such as number 88 and 161 have recorded moderately resistant reaction to MYMV under natural field conditions with yield of 18.28 and 20.25 grams per plant. These genotypes can be stringently screened for reaction to MYMV under artificial epiphytotic conditions and on confirmation of their resistance reaction, these genotypes can be  utilized in downstream breeding programmes aimed at releasing high yielding mungbean variety with MYMV resistance.

In the current investigation, the SSR marker, CEDG 305 showed polymorphism between the two parents and amplified an allele of 110 bp and 120 bp in the resistant and susceptible parents. Single marker analysis revealed that CEDG 305 marker is associated with MYMV resistance (24.47% phenotypic variance.) This will be useful in future for the development of high yielding mungbean yellow mosaic disease resistant cultivars of mungbean through marker assisted selection. Further the material produced can be forwarded by single seed-descent method to develop RILS.