Molecular Screening For Resistance In Interspecific Hybrids Of Greengram Using Tobacco N Gene Primers

ABSTRACT

Inter specific hybridization was made between susceptible greengram variety CO 5 and wild Vigna sp. Crossed seeds were raised in field condition for testing the MYMV incidence. F1 showed dominant resistance reaction against MYMV disease incidence. The field growing F2 populations of C0 5 x wild Vigna sp cross produced a wide range of segregant viz., susceptible, parental and intermediate type. In order to understand whether there are R Gene sequences whose products contain NBS and LRR motifs in mungbean, the F2 progenies DNA subjected to PCR amplification using tobacco N gene primers designed based on the tobacco N-gene and Arabidopsis RPS gene sequences. The PCR reaction was carried out and expected product was amplified at annealing temperature at 40°C. In parental polymorphisms, a detectable single band of 500bp being present in MYMV resistant Wild Vigna species, which was absent in MYMV susceptible CO 5 greengram variety. All the F2 resistant individuals showed 500bp DNA band with use of the same tobacco N gene primers.

Introduction

Greengram, Vigna radiata (L) Wilczek is one of the important, ancient and well known grain legume in almost all states of India. It is an excellent source of easily digestable good quality protein for the growing population in many developing countries of south Asia and south-east Asia (Surashi et al., 2017). The seeds are favoured for their digestible protein with low flatulence, rich in phosphorus, provitamin-A and are relatively free from anti-nutritional factors (Adsule et al., 1986). Its high protein levels and high lysine or low methionine amino-acid profile compliment to cereals to form a balanced amino acid diet (Shahrajabian et al., 2019). Greengram is widely grown in India and other countries like Pakistan, USA and Australia etc. India accounts for more than two-third of total production of greengram. Annual production of greengram in Tamil Nadu is 1.21 lakhs tonnes cultivated in 1.89 lakhs ha with an average yield of 642 kg/ha (Mariyammal et al., 2019).

However, the national average grain yield of 461 kg/ha is very low compared to world average yield. There are several biotic and abiotic factors are responsible for low productivity in greengram crop. Among the biotic stresses, the mungbean yellow mosaic virus (MYMV) disease cause serious yield loss up to 85%. MYMV disease can be controlled by different methods (Paul et al., 2013). But, the host resistance alone is a best method to check the disease and then it is to be preferred over other method because it is the least expensive and has no adverse environmental effects.

Utilization of wild species for improving their cultivated counterparts is steadily increasing in various crops where genetic diversity in the primary gene pool is limited. The wild relatives of greengram such as V. umbellata, V. vexillata , V. trilobata and other wild V. spp. carrying desirable genes for many yield components coupled with resistance to MYMV. The use of wild Vigna species in greengram breeding programme has been difficult because of problems encountered in obtaining successful F1 hybrids due to crossability barriers. In spite of these difficulties, interspecific hybridization between V. radiata and its wild relatives of Vigna was successfully accomplished by many workers (Renganayaki, 1985, Pandae et al. 1990, Ganeshram 1993, Subramanian and Muthiah, 2000, Umamaheswari 200,. Sidhu and Satija. 2003, Pandiyan et al., 2010; Pandiyan et al., 2012; Sudha et al., 2013; Chitra et al., 2018; Basavaraja et al., 2019). Developing molecular markers or cloning genes of traits known only phenotypically is not an easy task; mungbean yellow mosaic disease is one such category. However, a handful of plant disease resistance (R) genes have been recently cloned by transposon tagging or map based cloning. These approaches are, on the other hand, time consuming and cumbersome. However, many laboratories followed Polymerase Chain Reaction (PCR)-based approach for isolating and using R genes and or Resistant Gene Analogous (RGA) for identification of MYMV resistance in greengram genotypes (Kabi et al., 2017; Sai et al., 2017; Patel et al., 2018; Sing et al., 2018). In the present study the occurrence of R gene (tobacco N gene) sequences was tested in the progenies derived from greengram with wild Vigna species for MYMV resistance.

Materials and Methods

The parental greengram (Var.CO 5) seed material was obtained from Department of Pulses, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, and Vigna wild species was collected from Top slip area of Tamil Nadu. The ovule parents Var.C0 5 and tester parent (wild Vigna sp.) was raised in rows to accommodate 10 plants per row with an intra row 30 cm. The female parent was crossed with pollen parent and the crossed seeds were collected carefully. The emasculation and crossing in greengram were exercised following the procedure developed by Boling et al. (1961). F1 seeds were raised in the field for assessing the MYMV resistance based on morphological characters. The infector row method was followed for assessing the MYMV reaction in both F1 and F2 populations. The susceptible greengram variety CO 5 was planted every 10th row of planting as the check (Singh, 1980).

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The present study was also carried out with an aim to tag the MYMV resistance gene by using tobacco N gene primers. The amino acid sequence homology between these two gene was 100% restricted to two short regions one in the kinase-1a or P-loof motif (GGVGKTT; Sarste et al, 1990) of the nucleotide-binding site (NBS) and the second (GLPLAL; Mindrinos et al., 1994) approximately 160 amino acids further downstream. The consensus sequences and possible primers are noted below.

Discussion

Expeditions and evaluation of wild Vigna sp.

The expeditions and collection of wild Vigna sp. in resistance to yellow mosaic virus disease was carried out at two different places viz., top slip area of Tamil Nadu and Kotaimaidan (Western Ghat) area of Kerala. Among the expeditions, one wild Vigna species was collected from Top slip area of Tamil Nadu. The collected wild Vigna species was raised in the field for assessing the yellow mosaic virus incidence using susceptible variety CO 5 as check. The collected wild Vigna species was phenotypically similar to cultivated Vigna species (Fig.1- P2). The flower buds, pod and seed colours were closely resembles the cultivated Vigna species. The collected wild Vigna species was phenotypically resistant to yellow mosaic viral disease.

Evaluation of F1 and F2 segregants

Interspecific hybrdization has been made between susceptible greengram variety CO 5 and a wild Vigna sp and F1 plants were produced (Fig 1). The hybridization was done with more than 500 flowers and obtained only few pods (12-15 seeds). The occurrence of low pod and seed set is mainly due occurrence of pre and post fertilization barriers. These results agree with the results of Gopinathan et al. (1986), Mendioro and Ramirez (1994), James et al. (1999), Uma Maheshwari (2002) and Pandiyan (2010) and Chitra et al., (2018). The filed raised F1 plants showed dominant resistance reaction against MYMV disease incidence. A similar finding was also reported by Pandiyan (2004) in V. radiata V. umbellata interspecific crosses. The selfed seeds of the F1 hybrid was collected and raised in the field for evaluating the MYMV incidence. The segregating population produced a wide range of segregant viz., susceptible, parental and intermediate type (Fig 2). The F2 population produced a wide range of segregants viz., susceptible, parental and intermediate types (Fig. 2). Similarly, Satija and Ravi (1996) reported that hybrids of the cross V. radiata x V. umbellata were either transgressive or intermediate compared to their parents. Earlier same findings also reported for the production of fertile hybrids with intermediate phenotypes compared to both the parents (Monika et al. 2001; Uma Maheswari, 2002, Sidhu and Satija, 2003; Chitra et al., 2018, Basavaraja et al. 2019). These results are also agree with the results of Parida et al. (1985), Subramanian and Muthiah, (2000), Premkumar et al. (2007), Pandiyan et al. (2010), Sudha et al. (2013) and Chitra et al. (2018) in interspecific hybridization of Vigna species..

Molecular screening F2 population by using Tobacco N gene primers

In order to understand whether there are R Gene sequences whose products contain NBS and LRR motifs in mungbean, the F2 progenies DNA subjected to PCR amplification using tobacco N gene primers designed based on the tobacco N-gene and Arabidopsis RPS gene sequences. The PCR reaction was carried out and expected product was amplified at annealing temperature at 40°C. In parental polymorphisms, a detectable single band of 500bp being present in MYMV resistant Wild Vigna species, which was absent in MYMV susceptible CO 5 greengram variety. All the F2 resistant individuals showed 500bp DNA band with use of the same tobacco N gene primers (Fig. 3). This band corresponds to the product size expected from the primers designed as evidenced in studies on potato (Leister et al., 1996) and soybean (Yu et al., 1996). Whitham et al. (1996) demonstrated that the tobacco N gene confers a hypersensitive responsive to tobacco mosaic virus (TMV) disease in transgenic tomato. He also found the expression of N gene is effectively inhibit and systemic movement of TMV in transgenic tomato.

Conclusion

In the present study, the expeditations and search for MYMV resistant wild Vigna species was undertaken and collected one wild Vigna species. The Vigna species was phenotypically resistant to MYMV. Interspecific hbridization was made between elite, MYMV susceptible greengram Var. CO 5 wild Vigna sp. and progenies followed up to F2 generation. In molecular analysis, all F2 Progenies, wild Vigna sp. amplified a 500bp DNA products and susceptible parent CO 5 didn’t yield any characteristic PCR products using tobacco N gene-primer sequences.

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