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 F
2 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 F
1s was conûrmed through molecular marker analysis and the true F
1s were selfed to raise the F
2 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 MgCl
2, 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 H
2O.
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 F
2 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 F
2 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 (R
2) which explains the per cent of phenotypic variance by the polymorphic marker was derived.