Introgression of Vigna mungo Genome into Vigna radiata Towards the Broadening of Genetic Base for MYMV Resistance and Yield Attributes

Greengram [Vigna radiata (L.) Wilczek] is an important pulse crop of Asia, Africa and Latin America, where it is consumed as dry seeds and fresh green pods (Karuppanapandian et al., 2006). It is one of the important edible food legumes of Asia, widely cultivated and consumed throughout India. It is cultivated in tropical and sub-tropical zones of Asia including Bangladesh, India, Pakistan, Myanmar, Indonesia, Philippines, Srilanka, Nepal, China, Korea and Japan (Shanmugasundaram, 2001). India is the largest greengram producer with 55% of the total world acreage and 45% of total production (Rishi, 2009; Singh et al., 2013). In India, greengram is being cultivated in an area of 4.26 million hectares with an annual production and productivity of 2.01 million tones and 472 kg/ha respectively. In Tamil Nadu, next to blackgram, greengram is cultivated in an area on 1.8 lakh hectare with a production of 0.86 lakh tonnes and productivity of 487 kg/ha (Project Coordinator’s Report, 2018-19).
 
The main cause for low yield is susceptibility to pests and diseases, among which Mungbean Yellow Mosaic Virus (MYMV) is one of the most devastating diseases of greengram causing 85-100 per cent yield loss (Mahalingam et al., 2018). It is transmitted by white fly, Bemisia tabaci. The use of resistant varieties is the most desirable strategy to reduce the disease incidence in an economical and environment friendly way. Continuous breeding efforts towards the improvement of greengram [Vigna radiata (L.) Wilczek] and blackgram [V. mungo (L.) Hepper] has exhausted the available variability. Interspecific hybridization is a method for creation of genetic variability and widening of genetic base in a crop species. Greengram possess early maturity, erect growth habit and long pods with a greater number of seeds/pods whereas blackgram possess non-shattering pods with synchronous maturity, more clusters/plant and comparatively more durable resistance to yellow mosaic virus disease. These desirable traits can be transferred among them via wide hybridization (Singh, 1990). With these background, the present investigation was undertaken to study the interspecific derived progenies between greengram and blackgram for yield and related traits.

Crosses were made between greengram [Vigna radiata cv. VBN (Gg) 2] and blackgram (Vigna mungo cv. Mash 114) and forwarded up to F₄ generation. The F₄ population was evaluated at National Pulses Research Centre, Tamil Nadu Agricultural University, Vamban during July-Sep., 2017 for yield components and MYMV disease resistance. Each progeny was evaluated along with highly susceptible variety, CO5 of blackgram [Vigna mungo (L.) Hepper] as infector row to raise MYMV disease incidence. Individual plants with MYMV disease resistance and high yield were selected and evaluated in F₅ generation as progeny rows during Dec- Feb., 2017-18.
 
Genomic DNA of parents and selected MYMV disease resistance plants in F₄ generation were extracted using CTAB method suggested by Doyle and Doyle (1987) with modifications. DNA quality was assessed through 0.8% (w/v) agarose gel electrophoresis. Polymerase Chain Reaction (PCR) was carried out in total volume of 10 μl. PCR profile started with 94°C for 4 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds and extension at 72°C for 1 minute. A final extension at 72°C for 20 minutes was included. PCR was performed using thermocycler (Eppendorf, Germany). The final PCR product was electrophoresed using 3% (w/v) agarose gel with 100 bp ladder. Agarose gel was documented using GELSTAIN 4X advanced gel documentation unit (Medicare, India). Poly Acrylamide Gel Electrophoresis (PAGE) (6% w/v) was used for better separation of DNA fragments. The allelic pattern was marked as A, B and H for greengram homozygote, blackgram homozygote and heterozygote genome respectively. The allelic data was subjected to GGT (Graphical Genotyper version 2.0) to find out the genome contribution viz., number of recombinant segments and heterozygous segments from blackgram in greengram background. The proportion of blackgram genome was worked out as the per cent of (homozygote allele of blackgram + (0.5 x heterozygote allele) / total alleles.

Crosses between species of the same or different genera have contributed immensely in crop improvement program, gene and genome mapping, understanding of chromosome behavior and evolutionary aspects in many crops (Sharma, 1995). Stalker (1980) elaborated the gaps between hybridization and its utilization, along with approaches for the utilization of wild species in food legumes. However, it is well recognized that gene transfer through wide crosses is a long and tedious process, due to lack of homology between chromosomes of participating species in the cross, pre and post zygotic crossability barriers between wild and cultivated species. Chromosomal elimination is also seen in advanced generations, the unstable chromosomal composition of wide crosses tends to revert back to parental forms by gradual elimination of chromosomes (Gill et al., 1983; Yadav et al., 1986 and Pal et al., 1991).

Most reports on crossability among different Vigna species indicate that V. radiata produced more successful hybrids as seed parent with V. mungo, V. umbellata and V. angularis, although their reciprocal cross hybrids facing the loss of viability. However, by using sequential embryo rescue methods, the reciprocal hybrids between V. mungo and V. radiata could be successfully generated  (Gosal and Bajaj, 1983; Verma and Singh, 1986). V. mungo was also successfully crossed with V. delzelliana (Chavan et al., 1966), V. glabrescens (Dana, 1968; Krishnan and De, 1968) and V. trilobata (Dana, 1966).
 
In the present investigation, F₄ population obtained from the cross between Vigna radiata cv VBN (Gg) 2 and Vigna mungo cv Mash114 were evaluated. The female parent, Vigna radiata cv VBN(Gg)2 has lobed leaf shape and highly susceptible to MYMV. Whereas the male parent Vigna mungo cv Mash114 has ovate leaf shape and highly resistant to MYMV disease. A total of 23 single plants with MYMV resistance were selected in F₄ generation and evaluated in F₅ generation. In F₅ generation the variation in plant morphology and MYMV resistance proves the successful transfer of blackgram (Vigna mungo cv. Mash 114) genome into the interspecific derivatives. The delay in the attainment of homozygosity in the interspecific derivatives is common as reported by Singh and Dikshit (2002). Among the 23 F₅ progenies, six progenies viz. 3-1-11, 3-7-6, 3-7-8, 3-10-19, 3-12-13 and 3-13-3 recorded significant superior performances for seed yield per plant than check variety VBN(Gg)2 (Table 2). Among these superior progenies, four interspecific progenies viz. 3-1-11, 3-7-6, 3-10-19 and 3-12-13 had ovate leaf shape. which showed the introgression of Vigna mungo cv. Mash 114 into Vigna radiata cv. VBN (Gg) 2. Wider variation for morphology traits viz., seed colour, hypocotyl pigmentation and branching pattern was observed among progenies.
 

 
In the present study, background analysis at molecular level was performed to assess the level of genome introgressed from blackgram to greengram. DNA samples collected from all the 23 individuals in F₄ generation. Six progenies viz. 3-1-11, 3-7-6, 3-7-8, 3-10-19, 3-12-13 and 3-13-3 were studied for background analysis. A total of 33 polymorphic SSR primers were involved in the background analysis (Table 1). These markers were uniformly distributed among the 11 linkage groups of mungbean (Isemura et al., 2012). The background analysis revealed that 10.9 to 34.9 per cent of Vigna mungo cv. Mash 114 genome has been successfully introgressed in progenies. Among the progenies, the proportion of homozygote segments ranged from 5.4 to 23.8 per cent and heterozygotes ranged from 7.2 to 22.2 per cent for blackgram genome (Table 3). Among these progenies, a total of 5 to 11 recombinant segments and 1 to 3 heterozygotes segments were recorded. The progeny, 3-10-19 recorded maximum (34.9%) of blackgram genome (Fig 1) and it showed resistance towards MYMV disease incidence (Fig 2). The variation in the allelic pattern viz., homozygotes, heterozygotes and recombinant segments among the interspecific derivatives even in the F5 generation might be due to non-attainment of homozygosity in early generation. The superior performances of the progenies for number of branches per plant, pods per plant, seed yield per plant and MYMV disease resistance than the female parent VBN(Gg)2 might be due to the influence of introgressed blackgram genome. Further selection among the selected progenies may result in a high yielding greengram variety with MYMV disease resistance.
 

 

 

 

Our results demonstrate that wide hybridization is an important tool in crop improvement. In the present investigation, successful introgression of Vigna mungo genome into Vigna radiata genome has been achieved. The morphological variation observed for the traits such as leaf shape, seed color, hypocotyl pigmentation and branching pattern emphasize that greengram has been introgressed with blackgram genome. Further, superior performance for yield attributing traits and MYMV disease resistance in the interspecific progenies than the check, VBN (Gg)2 indicates the influence of introgressed blackgram genome into the progenies. Background analysis using SSR markers confirmed the successful introgression of blackgram genome into greengram genome. Selection among the progenies may help to obtain high yielding, MYMV disease resistant cultivars.