Fenugreek (Trigonella foenum-graecum L.) or methi, is a crop with significant economic and medicinal value (Mehrafarin et al., 2011; Choudhary et al., 2024; Yadav et al., 2024). It belongs to the Fabaceae family. It is a multi-purpose crop, where leaves are consumed as vegetable and as fodder for livestock (Singh et al., 2025; Parveen and Samyuktha, 2026). For medicinal purposes, the leaves, seeds and sometimes the whole plant is used (Sinskaja, 1961). These applications have contributed to its popularity, leading to its cultivation in nearly every region of the world for centuries (Sinskaja, 1961). However, India is the largest producer of fenugreek in the world with the states of Madhya Pradesh, Haryana, Rajasthan, Gujarat, Maharashtra, Uttar Pradesh, Punjab, Bihar and Andhra Pradesh, as the major fenugreek producers (Meena et al., 2018). In India, it is one of the oldest used spices, seeds (vernacularly called “methi dana”) of which are rampantly used as a condiment or flavouring agent in many Indian cuisines (Srinivasan, 2014). Despite its economic importance, fenugreek remains largely underutilized and neglected as compared to other crops, and its potential in medicine remains underexplored. The germplasm of a species serves as a valuable resource by providing plant breeders unique opportunities to develop new crop varieties. The successful application of germplasm collections for enhancing crop productivity depends on comprehending the extant genetic diversity (Mondal et al., 2023). India being the primary or main fenugreek producer, has a rich diversity in fenugreek germplasm for many morphological traits like plant height, growth pattern, growth habit, yield related traits, etc. Many of these traits have been investigated to gauge the genetic diversity within fenugreek germplasm (Maloo et al., 2020; Roba and Mohammed, 2024). A cultivar demonstrates significant variation in growth performance when grown in different environments, a phenomenon referred to as genotype- environment interaction (GEI) (Dos et al., 2003).               

The phenotype is the result of the interplay between genetic factors, environmental conditions, and their interaction. In order to estimate GEI, it is therefore important to grow cultivars across various agro-climatic zones which allows selection of superior genotypes which can be subsequently utilized for varietal development.
       
The cultivated legumes acquired the determinate growth habit where the vegetative growth terminates into a cluster of flowers, during domestication as a component of the “domestication syndrome”. This type of growth habit is associated with several advantages like early and synchronous maturity, a compact plant type, and potentially higher per-plant yield (Avtar and Jhorar, 2002). Plants with determinate growth habit are compact and more amenable to mechanical harvesting and therefore are more desirable. Owing to these advantages, breeding for determinate growth habit remains a priority in crop improvement. The genetic control of the determinate growth habit has been deciphered in many legumes with the trait being under monogenic (pigeonpea) or digenic (chickpea) inheritance in most species (Kapoor and Gupta, 1991). The performance of RILs (recombinant inbred lines) derived from DT (determinate) x IDT (indeterminate) crosses of Indian bean depicted segregation for various traits like flowering time, plant height, number of racemes produced, and several important yield-related traits, likely due to co-localization of the genes governing the growth habit and these traits (Megha et al., 2023). Identifying the genetic loci governing the determinate growth habit would therefore help in developing desirable plant types by allowing improvement of associated traits. Typically like most legumes, fenugreek has an indeterminate growth habit where the plants grow continuously at the shoot apex and they keep producing new shoots, flowers and seed pods (Avtar and Jhorar, 2002). However, a few determinate type of fenugreek varieties have also been identified or developed (Chaudhary and Singh, 2001; Avtar and Jhorar, 2002; Choudhary, 2003). Notable amongst these is RMt-305 which is a determinate variety that was developed from RMt-1 using ethyl methane sulphonate (EMS) mutagenesis (Chaudhary and Singh, 2001). Identification of new determinate genotypes would allow broadening of the genetic base for genetic improvement for the trait.                
               
Keeping in view, the importance of morphological characterization for trait discovery, the present study was undertaken to morphologically characterize a set of 30 different fenugreek germplasm lines across four different locations. Through this analysis, we identified a novel determinate genotype. The genotype identified has not been previously reported as a determinate genotype. The novelty of this genotype as a genotype with determinate growth habit was established using DNA-based molecular markers. Based on the profiles obtained, it could be concluded that the identified genotype is different from the previously reported determinate genotype. The identification of this genotype would facilitate breeding for determinate plant type in fenugreek. 

Plant material and experimental design
 
A total of 300 accessions of fenugreek were grown in randomized block design (RBD) during Rabi 2023 for germplasm characterization at ICAR-National Bureau of Plant Genetic Resources, Pusa Farm, New Delhi (28^o34' North and 76^o50' East) (unpublished data). A genotype UM-370 was identified as a novel genotype with determinate growth habit. This genotype was shorter in height and the shoot apex terminated into inflorescence, typical of the determinate growth habit. In the following year (Rabi 2024) a set of thirty accessions, including UM-370 were evaluated in augmented block design (ABD) under natural field conditions, at four different locations, namely ICAR-NBPGR, Pusa Farm, New Delhi (28^o34' North and 76^o50' East); ICAR-NBPGR, Issapur Farm, New Delhi (28^o63' North and 77^o16' East); ICAR-NBPGR Regional Station, Jodhpur (26^o182  North and 73^o002  East) and ICAR-NBPGR Regional Station, Akola (20^o43' North and 77^o04' East). These accessions were selected based on the diversity for various traits recorded and few of these were cultivated varieties (Table 1). Each accession was grown in two rows of 2 m length each. The row-to-row distance was maintained at 45 cm and the plant to plant spacing was maintained at 15 cm. Standard agronomic practices were followed throughout the crop growth period (Bhutia et al., 2018). The check varieties included the varieties Hisar Sonali and RMt-1. 


 
Phenotypic evaluation
 
The germplasm was characterized for various qualitative and quantitative traits following the DUS (Distinctiveness, Uniformity and Stability) guidelines (https://plantauthority. gov.in). The data was recorded on five randomly selected plants of each accession (excluding the border plants). The quantitative traits included days to 50% flowering (DTF); plant height (PH); number of primary branches per plant (PB); number of pods per plant (PP); pod length (PL) and 1000 seed weight (TSW). The qualitative characters included shape of leaf blade, plant growth pattern, pod curvature and plant growth habit.
 
Statistical analysis
 
The phenotypic data collected was used for further analysis. XLSTAT (Addinsoft, Paris) software (2020) was utilized to construct boxplots, correlation coefficient and principal component analysis (PCA). To perform hierarchical clustering using the UPGMA method, we used the ‘dendextend’ and ‘factoextra’ packages using the R programming language. ANOVA (Analysis of Variance) analysis was carried out for the trait data across the four different locations using https://www.statskingdom.com.
 
Molecular marker based validation of UM-370
 
The genotype, UM-370 was identified as a new determinate type of fenugreek. As molecular markers are the ultimate line of evidence to establish the distinctness of different genotypes, we used SSR markers to establish the distinct-ness of UM-370 from the earlier identified determinate variety, RMt-305. PCR amplification was performed for these two genotypes along with the indeterminate varieties Hisar Sonali and RMt-1 as check samples. The PCR reactions were performed in a total volume of 20 µl, with the reaction mixture consisting of 1X PCR buffer, 2.5 mM MgCl₂, 1 µM primer, 0.2 mM of each dNTPs, 1 U Taq DNA polymerase (NEB) and 15 ng template DNA. The standard PCR amplification conditions were used (Gaikwad et al., 2025). The amplified products were resolved and visualized through EtBr staining of 3% metaphor agarose gels.

A large variability was observed for the traits studied, with highest variability for the trait PB (CV of 25%). The PB values ranged from 3.9 to 8.4 for the pooled data. This is in agreement with previous report where the PB was reported to range from 2.3 to 7.5 across 245 different fenugreek genotypes (Sharma and Sastry, 2008). The trait DTF was least variable (CV of 6.5%). The DTF ranged from a minimum of 45.6 to 58. Highest variation between the four locations was observed for the trait PP with the genotypes at Jodhpur with a substantially lesser number of average pods/plant compared to the average PP for genotypes grown at other locations (Fig 1a, Table 2). ANOVA analysis depicted a significant (p<0.05) difference in means for the traits of DTF, PP, PL and TSW depicting higher trait variability across the four different locations (Table 3).






       
Correlation analysis revealed a significant (p≤0.05) negative correlation (-0.45) between TSW and DTF; and a significant (p≤0.05) positive correlation between PB and PP (0.40) (Fig 1b). The PCA analysis indicated that the first two principal components explained a cumulative variance of more than 50% (Table 4, Fig 2a). The PC1 possessed the highest eigenvalue and % variance suggesting that it accounts for the largest variation in the dataset examined. The traits DTF and PP possessed the highest positive eigenvalues for the PC1 indicating their significant influence in governing the overall variability among the fenugreek genotypes. In the correlation circle for PCA, the varied contribution of each of the traits towards the PC1 and 2 is depicted in the form of variation in vector lengths (Fig 2b). The traits PH and PL possessed the least vector lengths. In the scatter plot, the proximity of the vectors depicts presence of a strong correlation between the variables. Such vectors include PP and PB. Overall, the results of this analysis, concurred well with the correlation analysis.




       
Cluster analysis revealed the presence of two distinct clusters with cluster I containing twelve genotypes and cluster II possessing eighteen genotypes (Fig 3). Cluster I was further divided into two sub-clusters and cluster II was also similarly divided into two sub-clusters with only one genotype, CO 1 in one of the subclusters.


       
The genotypes were analysed for the qualitative characters (Table 5). This includes the plant growth habit, shape of leaf blade (basal and apex) at first primary branch axis and first pod axis, shape of leaf blade for fully grown terminal leaf, plant growth pattern and curvature of the pods. There was little variation for these traits across the four locations, as qualitative traits show little environmental variation. This is because they are oligogenic in nature being controlled by a single gene or a small number of genes. Notably, for a few categories no observation was recorded. For instance, none of the genotypes were found to possess rounded shape of the apex of leaf blade on first pod axis. For pod curvature, only Hisar Sonali was observed to possess strongly curved pods with the remaining genotypes having pods with moderate curvature. For the trait of plant growth habit, only two genotypes were observed with the determinate growth habit, namely RMt-305 and UM-370.


       
Amongst the genotypes, a determinate genotype, UM-370, was confirmed through phenotypic observations, at all the four locations. This genotype was earlier maturing and shorter than the other determinate variety, RMt-305 (Fig 4a and b). It is a local collection from village Manpura, in Nagaur, Rajasthan. The molecular profiling of this genotype with SSR (Simple Sequence Repeat) markers confirmed its distinctness from RMt-305 (Fig 4c). SSR markers have the advantage of identifying both genic and intergenic variation by virtue of their presence in either regions of the genome and they are widely used for genetic diversity analysis and gene mapping or tagging (Jethra et al., 2020; Maloo et al., 2023). The SSR markers which are identified to be polymorphic between the determinate and indeterminate genotypes in the present study could potentially indicate allelic variation for the genomic region governing the plant growth habit in fenugreek. Based on the obtained SSR profiles, UM-370 was identified to be a genotype different from the previously known determinate variety RMt-305. The identification of a novel determinate genotype would provide unique opportunities for breeding for determinate growth habit in fenugreek. In the absence of trait variability for plant growth habit in fenugreek, identification of a novel determinate genotype would broaden the genetic base for development of determinate varieties in fenugreek. Since the determinate genotypes are earlier maturing, they are capable of escaping the peak heat or drought periods, thereby ensuring stable yields under the rapidly changing environmental conditions.

In this study, we characterized the fenugreek germplasm for various quantitative and qualitative traits, at four different locations. Substantial variation existed for the quantitative traits while not much variation was observed for the qualitative traits. Highest variation was observed for the number of primary branches per plant while least variation was observed for the days to 50% flowering. A determinate genotype, UM-370 was identified and its distinctness from the previously known determinate genotype, RMt-305 was established using SSR markers. This genotype can be used for breeding fenugreek for the determinacy trait. The genotype identified would broaden the genetic base for fenugreek improvement in the current scenario of rapidly changing climate as the determinate genotypes are higher yielding and with synchronous maturity. 

The authors acknowledge the facilities provided at ICAR-National Bureau of Plant Genetic Resources.
 
Funding
 
The work was supported by the funding provided by ICAR-Consortium Research Platform on Genomics (Project number 1007341).
 
Author contributions
 
ABG. conceptualized the study, procured the grants, drafted and edited the manuscript. Sheel Yadav, Neelam Shekhawat, Sunil Gomashe, Ratna Kumari, Vinod Kumar Sharma. carried out the experiments, Sheel Yadav drafted the manuscript, Dhirendra Singh and D.K. Gothwal provided the material, Narendra Kumar Gupta edited the manuscript. All authors have read and approved the final manuscript.

All authors declared that there is no conflict of interest.