Validation of Groundnut (Arachis hypogaea L.) Mini-core Genotypes for Phenotypic Extremities along with LLS Resistance Through Genetic and Molecular Intervention

Groundnut (Arachis hypogaea L.) construed as one of the most potent oilseed crop, delivering high protein besides meeting the larger share of country’s oil requirement. Globally, India ranks first in Groundnut area under cultivation and is the second largest producer in the world with 101 lakh tonnes with productivity of 1863 kg per hectare in 2021-22. Tamil Nadu ranks first in terms of productivity (2980 kg/ha), followed by Gujarat (2751 kg/ha) and Telangana (2391 kg/ha). Peanut can also be eaten directly because of its greater food value glorified by the fact that it contains more protein (24.35 per cent), carbohydrate (21.26 per cent) and oil content (45-56 per cent) besides several minerals, antioxidants, flavonoids and vitamins such as niacin (12.1 mg/100 g) and choline (52.5 mg/100 g) (Bonku and Yu, 2020). Groundnut is always been a fascinating crop to small and marginal farmers but its yield was greatly challenged by Late leaf spot which cause severe defoliation of diseased leaflets during 30-50 days after sowing and reduce pod and fodder yield by 50 per cent besides the kernel quality (Damicone et al., 2010 and Ndifon, 2022). Though LLS is normally managed by application of fungicides, yet the incorporation of host resistance gene in susceptible cultivar has always been an eco-friendly and sustainable approach in crop improvement (Wankhade et al., 2021). Plant breeders exploit the genetic diversity to choose superior genotypes with the estimation of heritability because of good indicator of transmission of traits from parents to progeny (Gangadhara et al., 2023).

Experimental sites and source of materials
 
The field trials were laid at Agricultural College and Research Institute, Killikulam, Tamil Nadu during Kharif and Rabi seasons of 2021-23 in ‘D’ block farm. The experimental materials consisted of 220 groundnut mini-core collections made from ICRISAT, Hyderabad besides the four check varieties availed from Genetics and Plant Breeding, Killikulam. Biometrical data on yield and its contributing traits viz., days to fifty per cent flowering, plant height, number of primary branches per plant, number of secondary branches per plant, kernel length, kernel breadth, number of seeds per plant, 100 seed weight, shelling per cent and pod yield per plant were recorded on five plants of each genotype. Oil content on the genotypes was assessed on Near Infra-red spectroscopy instrument adopting non-destructive method. The collected data were interpreted for genetic diversity utilizing the D2 statistical analysis (Mahalanobis, 1936) besides the genetic variability parameters such as PCV and GCV, heritability and GAM were also estimated according to Burton et al., (1953), Lush (1940) and Johnson et al., (1955) respectively. Further, ten plants were selected at random from each genotype and observation on late leaf spot disease was recorded based on modified 9 point scale (Subrahmanyam et al., 1995).
 
Molecular analysis-DNA extraction and PCR amplification
 
The extracted DNA pellets vide modified CTAB method as suggested by Kumari et al. (2020) were purified by ethanol wash and the purified DNA was treated with RNase enzyme to remove RNA contamination and the DNA was quantified using gel electrophoresis unit on a 0.8 per cent agarose gel and loading dye. DNA samples were amplified in a 10 μl reaction mixture consisting of 2.5 μl of template DNA, 0.75 μl of each forward primer and reverse primer, 3.5 μl of PCR master mix and 2.5 μl of sterile distilled water. PCR amplification was carried out using thermal cycler, programmed for an initial denaturation at 94°C for 4 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 1 minute and a final extension at 72°C for 20 minutes. PCR products were analyzed in 3% agarose gels, visualized and documented using BIORAD gel documentation system. The gel score as interpreted as ‘0’ for no bands and as ‘1’ for polymorphic bands were further fitted to the equation as given by Anderson et al. (1993) and the polymorphism information content (PIC) value was computed.
                   

 PIC = 1- ∑P_ij²
 

Where,
P_ij = Frequency of the jth allele for the ith marker.

Two hundred and twenty groundnut genotypes received from ICRISAT, Hyderabad were evaluated for their late leaf spot resistance along with yield superiority through phenotypic and genotypic intervention. The phenotypic screening for LLS resistance was carried out in groundnut genotypes using Percent Disease Index (PDI) score (Subrahmanyam et al., 1995 and Zanjare et al., 2023). The genetic diversity was assessed for two hundred and twenty four groundnut genotypes through the D2 analysis and Principal Component Analysis (PCA) besides clustering of genotypes was also performed based on molecular data for late leaf spot resistance.

The extent of genetic variability and the degree of its heritability available in the germplasm determines the effective superior genotype (Johnson et al., 1955).The present investigation had shown that the phenotypic coefficient of variation (PCV) value for all thirteen biometrical traits was higher than that of genotypic coefficient of variation (GCV) suggesting the role of environment on the phenotype of the crop and presented in Table 1. Similar results was also reported by Mubai et al., (2019) and Sab et al., (2018). In this study, the higher estimates of  PCV and GCV was expressed for plant height, primary branches per plant, secondary branches per plant and hundred seed weight. Patil et al., (2014) and Rao et al., (2014) have also reported high GCV and PCV for hundred seed weight. Further, Patil et al. (2014) reported high GCV and PCV for primary and secondary branches per plant. Higher heritability and genetic advance as a per cent of mean indicating greater non-additive gene action was observed for all traits  except kernel width and oil content  wherein the lower h2, GCV and GAM were reported suggesting more of additive gene action. The results were akin to the reports given by Sab et al., (2018) and Patil et al., (2014).



Clustering is also used to summarize information on relationships between objects by grouping similar units so that the relationship may be easily understood and communicated. All the two hundred and twenty four genotypes were evaluated based on Mahalanobis D2 method for thirteen traits and the analysis revealed the presence of wide genetic diversity as they formed seven different group constellations and cluster I comprised of maximum number of genotypes (79), followed by cluster III (68), cluster VI (67) and presented in Table 2. The maximum intra cluster distance was observed for cluster VII (22.171) which represented the presence of diverse nature of genotypes within the cluster followed by cluster III (21.533). The inter cluster distance between cluster VI and VII was maximum (31.795), followed by the clusters III and VII (31.174). The maximum inter cluster distance exists between the clusters having highly diverse genotypes.



In this study, 220 groundnut genotypes had also 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) and presented in Fig 1. On perusal of data, five groundnut genotypes viz., ICG 532, ICG 2381, ICG 14179, ICG 15233, ICG 6022 were registered to be resistant while forty five genotypes had shown moderately resistant reaction. Susceptible and highly susceptible reaction was exhibited by 126 and 48 genotypes. The groundnut mini-core germplasm were also evaluated for late leaf spot resistance under glasshouse condition adopting the inoculum spraying method. The results were pursued and found two genotypes as resistant namely ICG 15233 and ICG 6022. Further, fourteen genotypes were adjudged as moderately resistant while one hundred and eighteen as susceptible and ninety as highly susceptible to late leaf spot.



Among the twenty two markers deployed, five molecular markers had shown polymorphism for the resistant and susceptible checks (IPAHM524 at 280 bp; TC7H11 at 360 bp; Ah3TC23H10, Ah3TC24B05 at 160 bp and Ah3TC28B01 at 220 bp) and hence these markers were used for the detection of genetic background of 224 groundnut genotypes for late leaf spot resistance and presented in Table 3 and Fig 2. The assessment of level of association existing between the molecular markers used was made through the interpretation on Polymorphic Information Content (PIC) value and this study inferred that all markers had expressed significant level of polymorphism. The maximum allele size was observed for the genetic marker TC7H11 while the minimum allele size was observed for Ah3TC23H10. As such the PIC values for the molecular markers IPAHM 524, TC7H11, Ah3TC23H10, Ah3TC24B05 and Ah3TC28B01 were recorded as 0.57, 0.509, 0.501, 0.502 and 0.53 respectively (Fig 2 to 7) and the result akin with findings of (Khedikar et al., 2010).















The studies on genetic diversity made by assessment of phenotypic extremities of 220 genotypes along with checks validated banding pattern differences generated by five molecular markers and thereby further clustering of 220 genotypes were made following UPGMA pattern (Fig 2). Based on amplification pattern, the genotypes were investigated for genetic divergence with SSR markers. The dendrogram constructed using molecular data had grouped the groundnut genotypes into five clusters. The maximum number of genotypes were presented in cluster III (168), followed by cluster V (32), cluster IV (12), cluster I (9) and cluster II (3). The susceptible (TMV 2, TMV 7, TMV 13) and resistant (ICG 6022) checks had exhibited very low dissimilarity index and were assigned in cluster VII. The results highlighted the cluster III for inclusion of maximum genotypes for yield and LLS resistance.

Molecular intervention of LLS resistance among 220 groundnut germplasm had revealed that the genotypes viz., ICG 15233, ICG 8760, ICG 297, ICG 405, ICG 15234 had shown the exact band size as the resistant check attributing resistance to late leaf spot disease. Further, these genotypes also had proven their resistance to LLS under field and controlled condition.

In this investigation, two hundred and twenty groundnut germplasm were interpreted for the phenotypic extremities besides expression of late leaf spot tolerance through in vitro and in vivo protocols.  The results had exhibited the higher PCV and GCV estimates were recorded for plant height, primary branches per plant, secondary branches per plant, hundred seed weight. Higher heritability and genetic advance as a per cent of mean was observed for all traits included in this study except kernel width and oil content that suggested for effective selection for genetic improvement of such traits. Assessment of phenotypic variation quantified that the inter cluster between VI and VII was maximum the maximum, followed by the clusters III and VII. The crosses generated between parents from these clusters could deliver a greater level of heterosis in F1. Similarly, the germplasm were subjected for screening against late leaf spot both under in vitro and in vivo conditions and as such, 5 genotypes were found to be resistant, 45 genotypes were moderately resistant, 126 genotypes were susceptible and 48 genotypes were highly susceptible under natural conditions. Further, upon artificial screening, two genotypes found to be resistant, 14 were moderately resistant, 118 were susceptible and 90 were highly susceptible. Molecular analysis for LLS resistance among the groundnut germplasm also had shown the exact band size as the resistant check attributing resistance to late leaf spot disease among ICG 15233, ICG 8760, ICG 76, ICG 111, ICG 297, ICG 405, ICG 238, ICG 4598, ICG 5051, ICG 6057, ICG 8253, ICG 8285, ICG 10701, ICG 15234. Hence, use of such resistant genotypes as donor can help to tailor LLS resistance in locally adopted and superior groundnut varieties.

The research grant given by BRNS, BARC (GOI), Mumbai, has been greatfully acknowledged. 

The all authors declared that they have no conflict of interest.