Groundnut (Arachis hypogaea L.) construed as one of the most dynamic oilseed crop, delivering high protein besides significantly meet country’s major oil requirement. Globally, groundnut is grown on 327 million hectares recording a production potential of 539 million tonnes and a productivity of 1648 kg/ha (FAOSTAT, 2021). India stands second largest producer of Peanut after China, followed by Nigeria and USA. In India, 10.1 million tonnes of groundnut are produced over an area of around 5.7 million hectares at a productivity of 1777 kg/ha (INDIASTAT, 2022). Groundnuts are majorly grown in Gujarat state followed by Rajasthan, Tamil Nadu, Andhra Pradesh and Karnataka during Kharif and Rabi seasons.
       
Groundnut is always been a fascinating crop to small and marginal farmers but its yield was greatly challenged by biotic and abiotic stresses. The most important and widely distributed diseases of groundnut are foliar fungal diseases such as early leaf spot (Cercospora arachidicola Hori), late leaf spot (Phaeoisariopsis personata) and rust (Puccinia arachidis Speg.). Among these, late leaf spot is a major foliar fungal disease which cause severe defoliation of diseased leaflets and reduce pod and fodder yield by 50 per cent besides the kernel quality (Gujar et al., 2025; Ndifon, 2022). When both rust and late leaf spot occur simultaneously, exhaustive damage results to pod yield and kernel quality to a prediction level of 70 per cent (Adorada et al., 2025; Elmhirst, 2024). Groundnut exhibit an extensive genetic diversity and major share of groundnut germplasm have been maintained by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in India. Therefore, the diverse groundnut germplasm collections can be sourced from ICRISAT for late leaf spot resistance and yield related traits to tailor desirable progenies for advanced breeding (Abadya et al., 2021).
               
The genetic or molecular marker is referred to fragment of a gene or DNA sequence with a known chromosome location associated with particular trait. Phenotypic marker proclaimed as an initiative for trait based varietal assortment over greater decades as pointed by Sax (1923) who had proposed the idea of indirect selection using genetic markers. But now, new technologies have been developed that ensure the precise selection based on genetic variation at DNA level by crop breeders (Rathava et al., 2025). During recent times, the molecular biologist are quenched on adoption of simple sequence repeats (microsatellites) markers to screen, identify and evaluate the crop genetic diversity. Microsatellites representing the simple repeated motifs of 1-6bp are usually co-dominant, reproducible and exhibit the higher level of polymorphism (Yadav et al., 2023). 

Crop experiment
 
Field experiment was carried out at Department of Genetics and Plant Breeding, Agricultural College and Research Institute, Killikulam, Tamil Nadu during 2022 to 2024. Two hundred and twenty groundnut mini-core germplasm collections collected from ICRISAT, Hyderabad along with four check genotypes were evaluated for LLS resistance. The details of mini-core collections was given in Table 1.


 
Natural screening of mini-core collections for LLS resistance
 
During Rabi season of 2022-23, the screening was carried out on the groundnut field at two stages viz., 70^th and 90^th days after sowing (DAS) under natural field condition. Ten plants were selected at random from each genotype and observation on late leaf spot disease was recorded based on PDI scoring scale (Table 2). Disease reaction was assessed based on the disease severity level  as such 0 per cent as highly resistant, 1-20 as resistant, 21-50 moderately resistant, 51-70 as susceptible and 71 -100 as highly susceptible with the respective scale of 1, 2-3, 4-5, 6-7 and 8-9 (Pooniya et al., 2020).


 
Extraction of genomic DNA and PCR amplification
 
Young leaves were collected from the mini-core germplasm lines and DNA was extracted adopting CTAB method developed by Saghai-Maroof et al. (1984). PCR reaction was performed with a total of five gene specific markers. The germplasm were surveyed utilizing the list of SSR markers as furnished in Table 3. PCR reaction was performed with a total volume of 10 μl. The thermal cycler programme as follows, step 1-Initial denaturation (94^oC for 5 minutes), step 2-Denaturation (94^oC for 30 seconds), step 3-Annealing (55^oC for 30 seconds), step 4-Extension (72^oC for 1 minute), steps 2-4 (40 cycles), step 5-final extension (72^oC for 10 minutes) and step 6 (Final Hold-4^oC until sample retrieval).


 
Polymorphism information content (PIC)
 
For critical estimation of genetic variation at DNA level among the groundnut germplasm, the gene specific markers were used to identify the polymorphism. The amplified DNA products were assessed as bands with clearly resolved, unambiguous polymorphic were scored using the standard 100 bp molecular weight ladder. The banding pattern was scored in binary digits as absence of band was indicated as ‘0’ and presence of band was indicated as ‘1’.  The polymorphic Information Content (PIC) was evaluated vide the equation 1-Σ (Pi)², whereas Pi  hint the extent of samples carrying the i^th allele.
 
Assessment of genetic diversity through molecular intervention

Molecular markers were employed to validate the polymor-phism between groundnut germplasm at the DNA level. After the gel electrophoresis run, the gel was visualized to access the differences in the banding pattern of the amplified DNA products for each primer linked with disease resistance. Visually distinguishable polymorphic bands were assessed by comparing them to the standard 100bp ladder. A score of ‘0’ indicated that no bands were seen in the respective lane, while the score of ‘1’ indicated the presence of polymorphic bands. A two-way matrix was constructed by entering the genotype scores and allele sizes for a specific marker in rows and columns, respectively.
       
The amplified bands of the different groundnut genotypes in each lane were compared with bands of corresponding susceptible (TMV2, TMV7 and TMV 13) and resistant (ICG 6022) checks. A total of 224 genotypes were clustered using the software PAST version 4.03 by UPGMA method based on the dissimilarity matrix. Binary data generated during polymorphic survey has been taken as input and based on which a phylogenetic tree was  constructed.

Phenotypic Screening of groundnut genotypes for validation of late leaf spot resistance
 
Natural screening for LLS resistance
 
During Rabi 2022, 220 groundnut genotypes had been tested for their reaction to late leaf spot resistance along with the resistant check (ICG 6022) and susceptible checks (TMV 2, TMV 7 and TMV 13) at Agricultural College and Research institute, Killikulam. Scoring for the leaf spot disease was made at two stages viz., 75^th and 90^th days after sowing (DAS) which then interpreted using Percent Disease Index (PDI) of corresponding genotypes. On perusal of data, five groundnut genotypes viz., ICG 532, ICG 2381, ICG 14179, ICG 15233 and ICG 6022 registered to be resistant while forty five genotypes had shown moderately resistant reaction (Table 4).


 
Controlled screening for LLS resistance
 
The groundnut germplasm along with the resistant check (ICG 6022) and susceptible checks (TMV 2, TMV 7 and TMV 13) were evaluated for late leaf spot resistance under controlled (glasshouse) condition adopting the inoculum spraying method. The results were pursued and found two genotypes as resistant namely ICG 15233, ICG 6022 with the PDI values at 75 DAS and 95 DAS as 16.33 and 19.37, 13.44 and 18.56 respectively.
       
Further, fourteen genotypes were adjudged as moderately resistant while 118 as susceptible and ninety as highly susceptible genotypes to late leaf spot disease (Table 5).


 
Molecular intervention for LLS resistance

Polymorphic information content (PIC) value assessment
 
Among the forty markers deployed, five molecular markers had shown polymorphism for the resistant and susceptible checks and hence these markers were used for the detection of genetic background of 220 groundnut genotypes along with the resistant check (ICG 6022) and susceptible checks (TMV 2, TMV 7 and TMV 13) for late leaf spot resistance. The assessment of association existing between the molecular markers used was made through the interpretation on Polymorphic Information Content (PIC) value. Upon perusal of the PIC values, it was inferred that the markers had a significant level of polymorphism with PIC value >0.5 (Table 6). The maximum allele size was observed for the genetic marker TC7H11 while  minimum was observed for Ah3TC23H10. As such the PIC values for the molecular markers IPAHM 524 (Plate 1), TC7H11 (Plate 2), Ah3TC23H10 (Plate 3), Ah3TC24B05 (Plate 4) and Ah3TC28B01 (Plate 5) were recorded as 0.570, 0.509, 0.501, 0.502 and 0.530 respectively. The results are akin with the reports made by Mace et al., (2006) and Khedikar et al. (2010).













Molecular assessment of genetic diversity on groundnut germplasm
 
The studies on genetic diversity made by assessment of phenotypic extremities of 220 genotypes along with checks employing the validation of banding pattern differences generated by five molecular markers. The molecular data were elucidated in PAST 4.03 software following UPGMA (unweighted pair group method with arithmetic mean) pattern and thereby further clustering of 224 groundnut germplasm were made (Table 7; Fig 1). Henceforth, the groundnut genotypes were grouped into five clusters namely I, II, III, IV and V accommodating 9, 3, 168, 12 and 32 genotypes respectively. The susceptible (TMV 2, TMV 7, TMV 13) and Resistant (ICG 6022) checks were assigned in cluster III. The principal co-ordinate analysis was also performed using GenA1Ex 6.5 software. Similar studies have been reported by Sathees et al. (2019). The result had shown that the total variation in the genotypes was 12.78 per cent, 11.21 per cent and 10.12 per cent for PC1, PC2 and PC3 respectively and had given in Fig 2.






 
Molecular validation of groundnut germplasm for late leaf spot resistance
 
Five polymorphic molecular markers such as IPAHM 524, TC7H11, Ah3TC23H10, Ah3TC24B05 and Ah3TC28B01 were deployed to identify the late leaf spot resistant genotype among groundnut germplasm. The results had revealed that 35 genotype had shown that the same allele size (bp) as the resistant check at 280 bp for IPAHM 524 marker and 36 genotype out of 220 showed allele size (bp) as the resistant check at 360 bp for the marker TC7H11.  Also, the genetic markers Ah3TC23H10, Ah3TC24B05, Ah3TC28B01 had identified 44, 24, 67 genotypes with similar allele size as resistant check at 160 bp, 160 bp, 220 bp, respectively (Table 8). The genotype, ICG 15419 had shown the exact band size as the resistant check for late leaf spot disease and was confirmed by all five polymorphic markers whereas ICG 2511 had exhibited band size similar as the resistant check which was confirmed by four SSR polymorphic markers.  The results were akin with the findings of Saravanan et al., (2024). Further, confirmation of groundnut genotypes viz., ICG 405, ICG 721, ICG 14118, ICG 11144 for LLS resistance was made with record of similar band size using three SSR polymorphic markers as the resistant check for late leaf spot resistance.

Groundnut germplasm viz., ICG 978, ICG 1649, ICG 1703 and ICG 2280 were categorized as resistant genotypes for late leaf spot through molecular validation while ICG 8760, ICG 4598, ICG 5051, ICG 5891 and ICG 6057 had shown moderately resistant to LLS disease besides exhibited higher mean yield performance among the test genotypes. Hence, the groundnut germplasm viz., ICG 8760, ICG 4598, ICG 5051, ICG 5891 and ICG 6057 may be utilized for developing ideal recombinants for LLS resistance breeding in Groundnut.

The present study was supported by BRNS, BARC (GOI), Mumbai with Sanction No: BRNS GOI 55/14/05/2-2022.

Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.