SSR based Molecular Diversity and Marker-trait Association in Mungbean (Vigna radiata L.) under Deficit Moisture Stress

Mungbean [Vigna radiata (L.) Wilczek, 2n= 22] is the most important short duration pulse crop of India. It seems to be originated from Vigna sublobata in South Asia and belongs to the family Fabaceae. Nutritionally, mungbean is often preferred owing to its higher level of easily digestible protein (22.63-25.84%) (Idris et al., 2025), iron (5.9-7.6 mg/100g) (Dahiya et al., 2015), crude fiber (4.4%) (Offia and Madubuike, 2014) and low levels of oligosaccharides (Ihsan et al., 2013). It also harbors higher amount of vitamin A, vitamin B and phosphorous as compared to other grain legumes. Besides, sprouted mung bean is rich in vitamin C and bears traces of antinutritional factors without any health hazards.
       
Among a variety of abiotic stresses, deficit moisture stress alone affects more than 20% of agricultural lands world-wide (Rojas, 2020). It drastically affects plant growth (Jincya et al., 2019) by limiting the nutrient uptake (nitrogen, phosphorous, potassium and sulphur) and disrupts nitrate assimilation leading to alter amino acid concentrations and decrease in protein content in seeds. The tolerant cultivars show increased expression of transporters and aquaporins to maintain growth and metabolism under drought stress (Barzana et al., 2021). However, the progress of breeding for drought tolerance is indeed limited owing to its highly complex mechanism, time consumable selection of tolerant plants and expensive cost involved (Athar and Ashraf, 2009). Mungbean is considered  sensitive to deficit moisture stress among the pulses (Dutta and Bera, 2008) in the order of lathyrus >horsegram> cowpea >pigeonpea> chickpea > lentil >mungbean>urdbean> fieldpea> rajmah (Pratap et al., 2019). It is reported to have narrow range of optimum osmotic adjustment (0.3-0.4MPa) as compared to other legumes (urdbean: 0-0.5, chickpea: 0-1.3, pigeonpea: 0.1-1.3 and ground nut: 0.2 - 1.6 MPa) and it may not be possible to revive the mungbean plants once it reaches lethal leaf water potential nearly to “1.9MPa (Pratap et al., 2019). Elucidation of the molecular basis for moisture stress tolerance is still not clear as compared to other important crops. A few candidate genes regulating production of osmolytes like proline, starch content, soluble sugars etc. have been reported for stress tolerance in legumes (Dar et al., 2016). Molecular finger prints, more specifically SSR (Simple Sequence Repeat) markers (Panigrahi et al., 2013) those linked to deficit moisture stress are worth trying for genotyping and marker-trait association. Therefore, we carried out molecular profiling based on gene specific SSR primers to explore allelic variation, genetic diversity and marker-trait association (if any) in a set of selected deficient moisture stress tolerant mungbean genotypes.

In the present study, ten diverse mungbean core genotypes including susceptible checks (Dhauli and T34-1-5) for deficient moisture stress were selected from a set of 200 germplasm accessions comprising standard ruling varieties, important pre-released cultures and popularly adapted local land races (Ravada, 2023). These were rigorously screened twice based on germination percentage, drought tolerance score and seedling traits (shoot and root length and their fresh and dry weight) (Supplementary Table 1) under deficient moisture stress (-1.5 bar or - 0.15 MPa corresponding to LD50) in growth chamber (25+1^oC, RH: 68%, photoperiod 16/8h, light intensity 2000 lux) using PEG6000 (Ravada, 2023). The amount of PEG (in gram) required to impose deficit moisture stress at -1.5 bar was calculated as per Michel and Kaufmann, (1973). Besides, the selected genotypes were replicated thrice in Randomized Block Design (RBD) and studied for ten agronomic traits (Supplementary Table 1) including seed yield over two years under deficit moisture stress (65% of field capacity) (in poly house) during late winter season.  


       
Genomic DNA of each of the above genotypes was isolated following standard CTAB method (Doyle and Doyle, 1990). The crude genomic DNA was purified by DNase free RNase-A (GeNei), quantified using agarose electrophoresis followed by UV-VIS Nanodrop-2000 spectrophotometer at 260 nm and finally diluted to a working concentration of 10 ng/µl for PCR analysis.
       
Each individual RNA-free gDNA sample was primed and amplified using ten gene specific SSR primers (Table 1) in a reaction volume 20 µl. Amplifications were set up in a Gene Amp PCR (Applied Biosystems), programmed for 5 min at 94^oC, 40 cycles of 1 min at 92^oC, 1 min at varying annealing temperature specific to primer (53-58^oC, Table 1) and 2 min at 72^oC and final extension for 10 min at 72^oC. The amplified DNA was electrophoresed in an agarose gel at a constant voltage of 50V and scanned by gel doc system (Fire Reader-Uvtec, Cambridge, UK) for detection of gene specific SSR alleles. The amplifications were checked at least twice for their reproducibility and the size of the amplicons was determined by comparing with the lambda DNA ladder (100 bp) with known size (bp) fragments.


       
Gels were scored for the presence (1) or absence (0) of bands. Polymorphism information content (PIC) and resolving power (Rp) of each primer was estimated as per Prevost and Wilkinson, (1999). Besides, the binary data matrix of the unweighted paired 1/0 score was analyzed for estimation of similarity coefficients and construction of Dendrogram as per Jaccard, (1908) using computer program for Numerical Taxonomy and Multivariate analysis system (NTSYS-PC), Version 2.02 (Rohlf, 2002). Similarity matrix was also used for principal coordinate analysis (PCA) comprising first three Eigen vectors (PCA 1, PCA 2 and PCA 3) to reveal spatial distribution of test genotypes in three dimensional spaces.
               
Besides, the Marker-trait  Association (MTA) of different molecular markers was established using regression analysis (Elakhdar et al., 2016) as per SPSS (Statistical Package for Social Sciences) Software, version 16. The Marker-Trait Association with probability value at least less than 0.05 is considered a statistically significant relationship between a marker with the trait concerned. 

Deficit moisture stress is indeed a global problem for crop production. Ample genetic and genomic resources are now available in mungbean and related Vigna crops, which can be exploited for the development of climate smart mungbean cultivars. There is a need to explore suitable diverse genotype (s) with assured level of deficit moisture stress tolerance. In this context, study of genetic variation based on molecular markers (Tripathy et al., 2015; Baisakh et al., 2021) and follow-up tagging of appropriate marker(s) with stress tolerant trait (s) using marker-trait association (Dash et al., 2022) can be a way forward for identification of elite genotypes.
 
SSR based genotyping
 
Simple sequence repeat (SSR) markers are excellent for genotyping and assessment of stress tolerance (Younis et al., 2020) with the advantages of being codominant, abundant, highly reproducible, highly polymorphic and easy to assay. In the present set of materials, only ten primers out of 50 SSR primers (Supplementary Table 2), amplified 36 amplicons (Supplementary Table 3) with an average of 3.6 alleles per primer. Chen et al., (2015) got amplified products only in 58 among 500 SSR primers while evaluation of genetic diversity in a panel of 157 cultivated and wild mungbean accessions. In contrast, 2-3 primers considered to be sufficient to distinguish between cultivars of broccoli (Hu and Quaros, 1991).




       
CodA revealed maximum number of polymorphic bands (9) with the widest size variation of amplicons (380-2800 bp) followed by the Primer ICCM-0249 (Fig 1, Table 1), while HSP-20, AP-2 and Vr UBC1 produced only one fragment each. Higher polymorphism of the above marker may be attributed to appreciable variation in number of tandem repeats within the core sequence in the genome across the panel of genotypes under study. Overall, 19 polymorphic bands were recorded out of total 36 bands resulting 52.8% polymorphism indicating subtle response of some primers towards genetic variation. While, Mathivathana et al., (2018) reported polymorphism only in primers: VrSSR_6_746 and VrSSR_1_31 out of ten primers used across six mungbean genotypes. 


       
Presence or absence of specific band is the inherent feature of each genotype. Primer GmDREB 2 and ICCM-0249 produced 820 bp and 740 bp amplified products which were specific to Dhauli and Phulbani local respectively (Supple. Table 3). Vigna glabrescens produced unique molecular finger print by a number of primers e.g., ICCM-0249, TAA-170, MA-VF, RD 22, GmDREB-2 and Cod-A which amplified 600 bp, 320 bp, 1180 bp, 460 bp, 1180 bp, 460 bp, 1140 bp and 810 bp amplicons respectively. In contrast, all test genotypes except Vigna glabrescens amplified 250 bp and 520 bp allele by primer TAA-170 and MA-VF respectively. These results suggest that SSR markers can be used as an accurate and efficient tool for identification of mungbean genotypes (Liu et al., 2013).
       
Polymorphism information content (PIC) is a measure of allelic diversity (Table 1). The primers ICCM-0249 and GmDREB 2 may be considered highly informative as they revealed 100% polymorphism and PIC value more than 0.5 and the maximum value (0.99) being recorded by the later.  Major allele frequency (MAF) being  the  distinct feature of a primer, ranged from 0.1 for GmDREB-2 to 1.0 for the SSR primer TAA-170,TM-VF,RD-20-A, HSP-20, AP-2 and VrUBC1 (Table 1). While, resolving power (Rp) was estimated to be as high as  8.8  in  TM-VF  indicating its immense discriminative power for varietal identification.
 
Molecular diversity
 
Available literature revealed use of SSR markers for study of genetic diversity in germplasm (Liu et al., 2013; Ganguly and Bhat, 2012). Vigna glabrescens maintained high genetic dissimilarity with TARM-1(0.32), OUM-99-4(0.34), Phulbani local (0.35) and RCM-14(0.36) (Table 2) indicating better scope of using them in hybridization to achieve transgressive segregants. Similar molecular diversity has been also reported in many legume crops e.g., mungbean (Saini et al., 2010), common bean (Galvan et al., 2003) and chickpea (Rakshit et al., 2003).


       
The grouping of genotypes using UPGMA analysis (Fig  2) was found to be more or less consistent with that of three dimensional scaling based on PCA values (Fig 3). The erstwhile mentioned divergent genotypes (Vigna glabrescens with TARM-1, OUM-99-4 and RCM-14) which were initially separated from rest of the test genotypes in the UPGMA clustering  were also shown to be sorted out to diverse extreme positions in case of three- dimensional scaling with vectors (Fig 3). This supports the findings of Tripathy et al., (2015) in mungbean and Baisakh et al., (2021) in urdbean. However, the deficit moisture stress tolerant genotypes e.g., SML-668 and PUSA-9672 with inherent high yield potential (> 3.0 g/plant) (Ravada, 2023) formed a distinct cluster (Fig 3). Therefore, the above divergent and high yielding test genotypes may serve as valuable materials for further genetic improvement in mungbean using recombination breeding.




 
Marker-trait association (MTA) under deficit moisture stress
 
Deficient moisture stress tolerance is a highly complex trait having involvement of a number of genetic factors. Breeding for the trait in mungbean can be accomplished by using  MTA (Elakhdar et al., 2016), which enables indirect selection on markers avoiding the phenotypic assessment of traits. But, untill now, little information is available on MTA for abiotic stresses except cold tolerance for 230 germplasm lines in mungbean (Dash, 2021) and frost tolerance for 672 worldwide pea (Pisum sativum L.) collections (Liu et al., 2017). The MTA can be performed either using ANOVA based on marker genotypes or a regression testing for a linear trend of marker alleles. In addition, because of the lack of distinct trait similarities, MANOVA seems to be not an ideal option to test for marker-trait associations (Bodah et al., 2017). Therefore, MTA was established based on regression analysis which is considered as more reliable approach. In the present study, linear regression analysis of each SSR allele on ten agronomic traits including seed yield, seedling parameters (germination % and seedling growth traits) and deficient moisture stress tolerance score (under stress using PEG) was carried out  to explore significant MTA. Out of several possible combinations, we revealed thirteen significant MTAs (Table 3) under deficit moisture stress. 


               
CodA amplified nine amplicons within the range of 2800 bp to 380 bp position in the electrophoregram. Out of these, the 1610 bp marker designated as CodA_3 was shown to be strongly associated (p<0.001) with deficient moisture stress tolerance score (1-9 scale) and germination percentage in a panel of 10 selected mungbean core germplasm lines comprising tolerant, moderately tolerant and sensitive genotypes with 87.3% and 81.2% of phenotypic variation respectively (Table 3). Similarly, Dash et al., (2022) tagged few molecular markers with shoot and fruit borer resistance in brinjal. In our study,the markers ICCMO249_6 and RD-22_1 also revealed significant association with deficient moisture stress tolerance score and germination percentage. Besides, root trait is an important parameter which was affected significantly under deficit moisture stress using PEG (Ravada, 2023). Primer GmDREB2_2 revealed a significant marker-trait association with root length explaining 53.7% phenotypic variation for the trait. Hence, such above molecular fingerprints seem to be useful for validation and screening of mungbean germplasm under deficit moisture stress. The amplicon designated as CodA_1 produced by the primer CodA had shown significant marker-trait association with  root length as well as number of seeds/pod and explained about 55% of the phenotypic variation for the traits. Available literature for MTA with regard to seed traits in mungbean is indeed scanty. Lucas et al., (2013) revealed haplotypes determining seed size in Vigna unguiculata using association studies and legume synteny. The marker CodA_4 had shown significant association with cluster/plant. Such component trait being an important determining factor for seed yield, the said marker (CodA_4) eventually also maintained significant association with the trait. Besides, the markers TAA170_2 and RD-22_2 also had shown significant association with seed yield and explained around 75% of the phenotypic variation. MTA Analysis of seed traits in accessions of common bean (Phaseolus vulgaris L.) has been investigated using genome- wide association studies (GWAS) (Lei et al., 2020), They identified 21 significantly associated markers for four seed traits including 100-seed dry weight. 

The present investigation displayed genotype-specific polymorphism based on SSR based genotyping. Vigna glabrescens, Dhauli and Phulbani local emerged as highly divergent from high yielding cultivars cv. SML-668 and PUSA-9672. A few molecular markers strongly associated with seed yield as well as seedling traits related to moisture deficit stress tolerance. Thus, the MTA established in the present study would serve as immense value for marker assisted back cross breeding in mungbean to screen high yielding plants with deficit moisture stress tolerance.

We acknowledge the contributions of all researchers which have been included as references in this pursuit.
 
Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided but do not accept any liability for any direct or indirect losses resulting from the use of this content.

All authors declare that they have no conflicts of interest.