Introduction

Blackgram [Vigna mungo (L.) Hepper] is an important food legume crop of Asian countries. It is referred to as the ‘king of the pulses’ due to its delicious taste and numerous other nutritional qualities. In India, it occupies 4.50 million hectares with a production of 2.83 million tonnes (MULLaRP 2019). Both biotic and abiotic constraints limit grain production across southern Asia and most parts of the world. Viral diseases are the major biotic causes responsible for production losses (Ilyas et al. 2009).

This disease has seriously increasing because of lack of genetic resistance in most cultivated high-yielding varieties. The transmission of the virus has been carried by whitefly (Bemisia tabaci). The symptoms appear in tiny irregular yellow specs and spots along the veins, which enlarge later until leaves completely yellowed under severe infestation. Diseased plants also have very few flowers and pods; pods were curled and reduced in size, and the percentage of shrivelled seeds increased (Shukla et al. 1978). MYMV disease infection may cause yield losses up to 85–100% in blackgram. Since vector whitefly attributes the virus transmission, controlling MYMV disease limits the vector population by using insecticides. However, it is ineffective under severe whitefly infestations and also not an eco-friendly approach. The most effective way to prevent this disease is to develop genetically superior resistant cultivars for blackgram.

Improved resistance to MYMV disease is now the primary goal of blackgram breeding programmes. However, breeding for MYMV disease resistance in blackgram is difficult with field screening. It is hampered by nonuniform development of the disease due to the fluctuation of the whitefly population in different locations and at different seasons. Hence, understanding the genetic resistance mechanisms and discovering genes or QTLs are essential for developing MYMV disease resistance in blackgram. SSRs as DNA markers have many advantages over other markers and have been used to assess genetic variation (Morgante and Olivieri 1993). Much work has not been done towards the development of genomic resources in this crop, but by using genomic resources from adzuki bean, common bean and mungbean, genetic maps were constructed (Chaitieng et al. 2006; Gupta et al. 2008; Isemura et al. 2012). By using SSR primers, a low level of polymorphism has been observed in cultivated blackgram (Chaitieng et al. 2006).

Previously, an attempt was made in the same mapping population to identify the QTL responsible for bruchine resistance and got two major QTL regions with a high PVE % of 17.01 on LG 5 and LG 8, respectively (Subramaniyan et al. 2021). In the present study, an attempt was made to impart multiple resistances in the MDU1 × TU 68 mapping population in addition to pest resistance by identifying the genomic regions responsible for MYMV resistance. Hence, the objectives of the present study were (i) to assess the genetic variation for MYMV disease resistance among the mapping population. (ii) To understand the inheritance pattern of the MYMV disease resistance among the mapping population. (iii) To identify the QTL region responsible for the MYMV disease resistance.

Materials and methods

The present field experiment was carried out at National Pulses Research Centre (NPRC), Tamil Nadu Agricultural University, Vamban, India, located at the latitude of 10.363505oN longitude of 78.902283oW. The NPRC, Vamban, is the best hotspot to assess MYMV disease incidence during June–July. Hence, the MYMV disease screening was performed following the infector row method. The molecular analysis was performed at the Pulses Marker Assisted Selection Laboratory, NPRC, Vamban.

Plant material and development of mapping population

MDU 1 (female parent) and TU 68 (male parent) were used to develop an F2 mapping population. MDU 1 is a high yielding popular blackgram variety of Tamil Nadu which was developed at Agriculture College and Research Institute, Tamil Nadu Agricultural University, Madurai and released in 2014. It is recommended for commercial cultivation in MYMV disease free period, namely October to November season of Tamil Nadu. This variety (MDU 1) is highly susceptible to MYMV disease. TU 68 is a genotype developed from Bhabha Atomic Research Centre, Trombay. It is a wild cross derivative obtained from the cross TU 94-2 × Vigna mungo var. silvestris and resistant to MYMV disease. TU 68 was selected as a male parent and crossed with female parent MDU 1 during January–March 2019 to produce F1 seeds. Polymorphic SSR markers between the parents were employed to find true F1s. Further, selected true F1s were selfed during April–June 2019. The F2 generation was evaluated during July–September, 2019 season.

Phenotyping of MYMV disease resistance

All the 108 single plants of the mapping population and the parents were screened for MYMV disease resistance under the infector row method at NPRC, Vamban, during July–September 2019. Susceptible genotypes CO 5 and MDU 1 were sown as disease spreader rows after every eight rows and around the plots. No insecticide was sprayed to maintain the natural whitefly populations. The MYMV disease incidence was recorded on all plants of F2 population and parents based on the visual scores on the 60th day. The MYMV disease score was recorded using the phenotype rating scale as suggested by Singh et al. (1995) (table 1). Based on the disease grade, two phenotypic classes were formed among F2 plants with the scales of 1 to 3 as resistant and 4 to 9 as susceptible phenotype. The goodness of fit for MYMV disease resistance in the segregating population was tested by the chi-square test (Stansfield 1991).

Table 1 Phenotypic rating of MYMV disease score.
Full size table

Genotyping

The genetic linkage map of MDU 1 × TU 68 mapping population was constructed using polymorphic SSR markers between the parents. The linkage map construction, QTL analysis and other standard methods involved are described below.

DNA isolation and SSR-based genotyping

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 polymerase chain reaction (PCR) mixtures contain 2 μL of 10 ng template DNA, 1.0 μL of 10 × Taq buffer + MgCl2 (15 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 thermo cycler (M/s Eppendorf, Germany, model AG 6325) under the following conditions: 94°C for 4 min followed by 30 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 1 min, with a final extension step of 72°C for 20 min. The PCR products were separated on 3% agarose gel and photographed using GELSTAIN 4× advanced gel documentation unit (M/s Medicare, India).

Linkage map and QTL analysis

Linkage analysis was carried out using QTL IciMapping (v. 4.1.0.0) software (Wang et al. 2016). Linkage groups were constructed using a minimum LOD threshold of 3.0 and a maximum distance of 50 cM. Map distance in centimorgan (cM) values were calculated using the Kosambi mapping function (Kosambi 1944). Further, QTL analysis was employed using QGene4.4.0 software (Joehanes and Nelson 2008). Composite interval mapping (CIM) was carried out with automatic cofactor selection. LOD threshold significance for each QTL was calculated with 1000 runs with a permutation test.The study performed is true with novelty in the interpretations.

Results and discussion

Parents and mapping population were evaluated for MYMV disease resistance. Resistant (TU 68) and susceptible (MDU 1) parents showed resistant (score 1) and highly susceptible (score 8) reactions for the MYMV disease at 60 DAS, respectively (table 2). The parents were more variable for MYMV disease resistance. In the present study, mean and variability parameters were estimated for MYMV disease scores and are presented in table 2. The mean and variability calculated for MYMV disease reaction show much variation among the population. The MYMV disease score at 60 DAS had positive skewness and leptokurtic nature (figure 1). Kang et al. (2005) reported that monogenic host resistance factors control more than 80% of viral resistance in plants. The digenic recessive nature of resistance was also stated by some authors (Singh 1980; Verma and Singh 1980; Vadivel et al. 2021). The inheritance pattern of MYMV disease resistance in F2 generation was studied, and the results are presented in table 3. The nonsignificant chi-square value (0.034) indicates digenic inhibitory gene action with an 80–90% probability at 5% for MYMV disease.

Table 2 Phenotypic distribution of the MYMV disease resistance trait at 60 DAS in MDU 1 × TU 68 mapping population.
Full size table
Figure 1
figure 1

Frequency distribution for MYMV disease resistance trait at 60 DAS.

Full size image
Table 3 Chi-square test for the inheritance of MYMV disease resistance trait at 60 DAS in MDU 1 × TU 68 mapping population.
Full size table

Further, this study involved 625 SSR primers in identifying the polymorphism between parents, i.e. MDU 1 and TU 68. The SSR primers and its sequence information were utilized from the maps of related Vigna species, namely mungbean (Gwag et al. 2006; Somta et al. 2008; Seehalak et al. 2009; Tangphatsornruang et al. 2009; Isemura et al. 2012; Chotechung et al. 2016) and adzuki bean (Wang et al. 2004; Chankaew et al. 2014). These SSR markers were cross-species amplification in nature. Among the 625 SSR markers, 49 showed polymorphism between parents. Genotyping was carried out with 49 polymorphic SSR markers for the F2 mapping population of the cross between MDU 1 × TU 68. A linkage map was constructed using QTL IciMapping (v. 4.1.0.0) (Wang et al. 2016). Among the polymorphic markers obtained, 46 SSR markers were found to be linked with 10 linkage groups, and three primers were left unlinked. These markers covered a total map length of 454.7 cM.

QTL analysis 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 (figure 2). In the present study, a QTL was found for MYMV disease score at 60 DAS on LG 10 and designated as qMYMVD_60 (figure 3; table 4), respectively. Markers CEDG180 and CEDG116 flanked the qMYMVD_60 with a LOD score of 4.56 and significant at P = 0.01. This QTL explained 21% of variation for the MYMV disease score at 60 DAS. Alleles from the resistant parent of TU 68 contributed towards an increase in the resistance against MYMV disease. It was supported by the previous reports of Vadivel et al. (2021).

Figure 2
figure 2

Permutation analysis on the MYMV disease resistance trait to identify QTL regions based on LOD threshold in MDU 1 × TU 68 mapping population.

Full size image
Figure 3
figure 3

QTL regions linked with the MYMV disease resistance trait on the linkage group 10 in MDU 1 × TU 68 mapping population.

Full size image
Table 4 QTL regions associated with MYMV disease resistance trait at 60 DAS in MDU 1 × TU 68 mapping population.
Full size table

However, the presence of another gene that alters the gene expression is still unknown. Hence, the obtained information can be improved further through fine mapping in the near future. The large PVE explains the major gene linked with the MYMV disease resistance on the LG 10. Further, SSR marker CEDG180 was frequently reported as associated with a major gene controlling MYMV disease resistance in blackgram (Gupta et al. 2013; Vadivel et al. 2021). Markers flanking the QTL can be used in marker-assisted selection for MYMV disease resistance in blackgram. Based on the previous research, the mapping population possessed bruchine resistance QTL regions on the LG 5 and LG 8. In addition, the present study identified the major QTL region linked with MYMV disease resistance on LG 10. Chen et al. (2013) reported QTL regions for bruchine pest (LG 7 and LG 9) and mungbean yellow mosaic India virus (MYMIV) disease resistance (LG 7, LG 8 and LG 9) in blackgram. Hence, the resistance QTLs can be involved in the marker-assisted selection procedure to impart multiple resistances into the desired cultivar. Further, it is planned to perform additional research to reduce mapping distance and to be more precise in locating the genes through fine mapping. The cloning of the QTL region is also intended in the near future to identify the gene involved in the MYMV disease resistance.

In conclusion, based on the preceding discussion, a robust QTL, i.e. qMYMVD_60 was identified on LG 10, which accounted for a PVE of 21%. The goodness of fit ratio indicates inhibitory gene action in the expression of MYMV disease resistance. The flanking markers of the qMYMVD_60, namely CEDG180 and CEDG116 could be used in the MYMV disease resistance breeding programme. Further, the information on both the QTL regions responsible for storage pest bruchine and MYMV disease resistance can be utilized to impart multiple resistances into the cultivar through marker-assisted breeding and marker-assisted backcross breeding procedures.