Assessment of Molecular Diversity and Mapping of Bruchid Tolerance Loci in Green Gram [Vigna radiata (L.) Wilczek]

Greengram, scientifically known as Vigna radiata (L.) Wilczek, holds significant importance as a pulse crop in Asia, with a consistent increase in its production. This crop is renowned for its high protein content and serves as an abundant source of folic acid and iron. Due to its small genome size of 578 Mb, close genetic relationship to other legumes, short life cycle and self-pollinating nature, it has become a preferred model organism for molecular studies among all legume crops. The versatility and genetic traits of greengram make it an important candidate for further research and development in the field of agriculture.
       
Bruchid beetles pose a major challenge to greengram production, particularly in stored conditions. These destructive pests, including species like Callasobruchus chinensis and Callasobruchus. maculatus, significantly impact both the quantity and quality of greengram seeds. Infestation starts modestly in the field but escalates during storage as eggs hatch into larvae, leading to rapid population growth. Within a span of two to three months, bruchid beetles can cause complete seed loss, resulting in financial setbacks for farmers who also lose valuable seed for subsequent planting. Effective management strategies are crucial to mitigate the damage caused by these pests and safeguard greengram yields.
       
The level of resistance towards bruchids in pulses relies on morphological barriers preventing colonization of the seed by bruchid larvae, or on secondary metabolites and other toxic compounds interfering with bruchid growth, development or reproduction. Genetic diversity studies either at phenotypic level or by combining molecular studies employing varied genotypes is pre-requisite for any crop improvement through trait breeding and molecular mapping of targeted traits. Previous reports suggest that some wild mungbean accessions (TC1966, ACC41, V2709, V2802 etc.) and cultivated (RS4, RMG11, IC325770, IC333175, IC329039, Sonamung, Dantan, etc.) genotypes showed resistance to bruchids (Sarkar et al., 2011; Chen et al., 2013 and War et al., 2017).
       
Identification of potent molecular markers that associated with bruchid resistance would help overcome the menace caused by bruchids both at field level and at storage level. However, the available bruchid tolerant loci and associated markers till now, haven’t been validated precisely, to use in the breeding program. Further, in-depth study on reported bruchid tolerance loci is needed to develop tightly linked markers to use in marker assisted breeding for bruchid-resistant greengram varieties. Hence, the current study was aimed to identify the molecular diversity at reported bruchid tolerant loci along with the markers linked with the trait, by screening of diverse greengram genotypes.

Field experiment was carried out with a panel of 47 diverse greengram genotypes comprising of popularly cultivated varieties, other released varieties and advanced breeding lines procured from different AICRP (All India Co-Ordinate Research Project) Center at Dryland farm, Regional Agricultural Research Station (RARS), ANGRAU, Tirupati during summer, 2019 in a randomized block design with three replications and molecular analysis was carried at Biotechnology Lab, Institute of Frontier Technology (IFT), RARS, Tirupati.
 
Per cent seed infestation (PSI -%)
 
The artificial study of the genotype’s response to bruchid pest tolerance/susceptibility was conducted using the following procedure. The pulse beetle, Callasobruchus chinensis (C. chinensis), which is a major storage pest of greengram and causes damage in both field and storage conditions, was used as the test insect. The culture of C. chinensis was obtained from the Department of Entomology, S.V. Agriculture College, Tirupati and was multiplied on locally available greengram seeds. To mass culture the insects, approximately 25 pairs of adult beetles were released into plastic boxes containing 500 g of disinfested greengram seed. The containers were covered with muslin cloth and secured with rubber bands. These containers were left undisturbed under ambient conditions until the progeny emerged. The newly emerged adult beetles (1-2 days old) from the culture after 25-30 days of release were used for the experimental studies. The adult beetles were identified as per the key traits given by (Southgate et al., 1958).
       
One hundred healthy, sound and disinfested seeds of each greengram genotype were placed in Petri plate. Those Petri plates were arranged in trays with the free choice test insect i.e., the Petri plates were not closed, mouth of the tray was covered with mosquito net and fastened with rubber bands. Ten pairs (10 male + 10 female) of freshly emerged adult pulse beetles were released into each tray for oviposition prior the trays were secured with mosquitoes’ net. Each genotype was replicated three times (Supplementary Fig 1). Control treatment was maintained similarly, without releasing bruchids. Seven days after infestation (DAI), all adults were removed and each seed was checked to make sure each had more than two eggs. The samples were examined at 40 DAI to record the number of bruchid holes that had emerged and percentage of seed damage was estimated.
 

       
The seed with hole is considered as damaged seed. The percentage of damaged seed was estimated Raghuwanshi et al., (2016) using the formula:
 
Genotypes were classified into different response groups based on per cent damaged seeds (Chen et al., 2007) viz., Highly resistance (0-10.0%), Resistance (10.1-35.0%), Moderately resistance (35.1-65.0%), Susceptible (65.1-90.0%) and highly susceptible (>90.0%) groups.
 
Statistical analysis
 
The data on pod pubescence, pod wall thickness, test weight, seed length, seed width, seed length to seed weight ratio, seed coat texture and percent seed infestation was subjected for statistical analysis viz., mean, range, ANOVA, standard deviation, standard error mean and critical difference using data analysis package of Windows-Microsoft/ Excel v. 2010.
 
Molecular analysis  
      
Molecular analysis was carried out by using reported markers for bruchid tolerance along with designed candidate gene markers for the reported bruchid tolerant greengram gene, Vr05G03950 (Supplementary Table 1). The genomic DNA of Vr05G03950 was downloaded from LIS (legume information system) database (https://legumeinfo.org/) and gene specific primers were designed using primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/) keeping the default parameters.
 

       
Association between marker alleles and bruchid tolerance was performed with TASSEL (Bradbury et al., 2007). The association between the marker and phenotypic traits was done using both GLM (General linear model) and MLM (Multiple linear model) methods, where markers tested and subpopulation data (Q matrix) was considered as fixed-effect factors, whereas the kinship matrix was considered as a random-effect factor.

The data on pod pubescence, pod wall thickness, test weight, seed length, seed width, seed length to seed weight ratio, seed coat texture and percent seed infestation was subjected for statistical analysis viz., mean, range, ANOVA, standard deviation, standard error mean and critical difference using data analysis package of Windows-Microsoft/ Excel v. 2010 (Table 1).
 

       
Analysis of Variance (ANOVA) study revealed existence of highly significant variation (p = <0.0005) for the entire pod and seed related traits along with the percent bruchid infestation. Hence, the genotype panel can be employed for diversity studies and for gene mapping approaches apart from development of mapping populations.
 
Per cent seed infestation (PSI-%)
 
40 days after treatment (artificial release of bruchids), seed damage per cent due to bruchid attack i.e., per cent incidence (%) was recorded among the genotypes and were classified into different response groups based on PSI (Chen et al., 2007).
       
The PSI among greengram genotypes used in control treatment (without bruchid release) was ranged from 0 to 5% with the mean of 0.85% where as under the artificial conditions per cent bruchid incidence was ranged from 19.67% to 99.33% with the mean of 59.38% (Table 2). The highest PSI was recorded in the genotype NRI-AMULYA (99.33%), followed by NRI-SAKTHI (POLISH) (93.33%), LGG-577 (92.33%) and hence, these are grouped into highly susceptible class. The genotypes VGG-15-030 (33.33%), NVL-722 (31.67%), COGG-13-19 (30.67%), OBGG-58 (26.42%), MGG-387 (23%) and OBGG-57 (19.67%) were grouped into resistant class (10.1-35.0%). A total of 16 genotypes were sustained under moderately resistant class (35.1- 65.0%) after 40 DAI (days after infestation).
 

       
The genotypes that sustained under resistant (10.1-35.0%) and moderately resistant classes were maintained under the treatment condition for another two complete life cycles of bruchid then the PSI was again recorded at 150 days after treatment (Table 3, Fig 1). Among the resistant genotypes, VGG-15-030 (37.67%), NVL-722 (41%), COGG-13-19 (36.33%), OBGG-58 (53%) and MGG-387 (39%) found with an increase of 4.34%, 9.33%, 5.66%, 26% and 16% bruchid incidence, respectively and thus, fall in moderately resistant class after four months of treatment. However, the genotype OBGG-57 showed 28.67% with an increase of 9% incidence and remained in resistant class after three complete life cycles of bruchid i.e., after 150 days after treatment. Hence, ‘OBGG-57’ can be used as bruchid resistant genotype either directly for release or as donor parent in trait-oriented breeding programmes.
 

 

 
Molecular analysis for bruchid resistance
 
Greengram genotypes were screened at molecular level by employing the reported bruchid tolerant primers along with designed candidate gene specific primers as detailed in material and methods section. Out of 19 primers amplified (Table 4), only one primer namely DMBSSR 125 showed polymorphic alleles viz., 240 bp and 310 bp (Fig 2) among the genotypes under study with 5.3% of polymorphism, which denotes the existence of very low polymorphism at the targeted bruchid tolerant loci.
 

 

Association study of DMBSSR 125
 
Association analysis employing MLM approach revealed that the marker DMBSSR 125 was linked to the greengram bruchid tolerance at p-value of 0.0378 and with R² value of 8.60 (Table 5). However, GLM model couldn’t reveal significant association. The marker-trait association can be confirmed either by employing larger and diverse population or through linkage mapping approaches. The primer that is having an R² value of about 15 can be termed as strongly associated marker to the trait. Hence, DMBSSR 125 with a moderate R² value of 8.60 needs to be re-validated employing large populations either through linkage or association mapping approaches.
 

       
The marker DMBSSR 125 is an EST-SSR marker. BLAST analysis of the marker sequence against greengram genome (https://legumeinfo.org/) aligns to a functional gene that belongs to Metallothionein (MTs) group of proteins on Chromosome 9 (vigra. Vradi09g07610) and to a scaffold with ID, Vradi0111s00070 (Table 6).
 

       
Metallothionein (MTs) proteins are reported to be as small cysteine-rich, heavy metal-binding proteins that participate in many of protective stress responses (Nedecky et al., 2013). Interestingly, a cysteine rich protein, ‘VrCRP’ that confers bruchid tolerance has been isolated from seed coat of wild greengram (Vigna radiata var. sublobata) genotype TC1966. The genotype showed resistance to Callasobruchus chinensis through VrCRP’s insecticidal activity. Greengram seeds that contain 0.2% VrCRP completely hinder the bruchid larval development (Chen et al., 2022).
       
The DMBSSR 125 marker is an ESTSSR that corresponds to a cystine-rich protein which reported to confer bruchid resistance can be further validated either through mapping or by introgression approaches to confirm its trait association and thereby to use in bruchid resistant breeding programmes.
       
Host plant resistance involving antibiosis, antixenosis and tolerance mechanisms are preferred in controlling bruchids. It is reported that in the cultivated greengram accessions V2709 and V2802, resistance to Callasobruchus chinensis is caused by secondary metabolites which leads to antibiosis in the cotyledons (Talekar and Lin, 1992).
       
Vignatic acid A, an alkaloid has been isolated from bruchid (C. chinensis) resistant wild accession of greengram viz., TC1966 (Sugawara et al., 1996). In addition, a peptide compound ‘GIF-5’ toxic to the bruchids has been identified from the source, TC1966 (Kaga et al., 2000). Accumulation of cyanogenic glycosides and phytic acids, in greengram seeds during seed maturation to drying plays an important role in defence against bruchids (Lattanzio et al., 2005). Bruchid resistance in the genotypes TC1966, VC6089A and a RIL 59 (recombinant inbred line) has been assigned to the presence of a resistant-speciûc protein, gag/pol polyprotein and aspartic proteinase (Lin et al., 2016). Recently, it has been fine mapped by Chen et al., (2022) to a candidate gene on chr 5, namely Vradi05g03810 (encoding a probable resistance-speciûc protein) that possess a 69-bp sequence deletion in the coding region of bruchid- susceptible lines (VC2778A, VC 1973A) when compared to the resistant lines, TC1966, VC6089A.
       
In the era of omics, identification of probable/potential candidate genes responsible for bruchid tolerance in the genome of a crop spp. can be precisely done. Thus, by employing the knowledge of known/orthologous genes, identification of functional variants that govern targeted trait(s) through re-sequencing/mapping is an immense need especially in the orphan crops like pulses, wherein it can overcome the problem of low molecular variability aroused while using random markers.

From the study it can be concluded that gene-based markers are helpful in unraveling the marker-trait association and landing onto a candidate gene than genome wide spanned SSR markers. The DMBSSR 125 marker loci can be used in development of bruchid tolerant varieties with prior validation as described. Further, the two candidate genes vigra. Vradi09g07610 and Vradi0111s00070 can be targeted to reveal the useful variations.

This research was supported by the Department of Biotechnology, Institute of Frontier Technology, Regional Agricultural Research Station, ANGRAU, Tirupati, A.P.
 
Author contribution
 
SVA, DK and PL planned the experiments. SY executed the experiments and recorded the phenotypic data and genotypic data. SVA and PL analyzed the data. PL, DK and SVA provided the required material and facility for laboratory screening. SY, SB and SVA wrote the manuscript. All authors read and approved the final manuscript.
 
Ethical approval
 
This article does not contain any studies with human participants or animals performed by any of the authors.

The authors declare that they have no conflict of interest.