Plant material
In present investigation, five check varieties including JGN 3, GPBG4, SunOleic 95 R, KDG-128 and Gangapuri and fifty-one groundnut germplasm lines collected from Junagarh, Gujarat (24), Shivpuri, Madhya Pradesh (11), Dhar, Madhya Pradesh (8), Badwani, Madhya Pradesh (5) and Jhabua, Madhya Pradesh (04) were taken. Temperature between 25°C-30°C suits best for the crop with an average rainfall ranging between 50-75 cm. The experimental field of College of Agriculture, Gwalior is located at 26 13” N latitude, 78 14” E longitude and at an altitude of 211.5 m above the sea level in gird belt. It has hot weather conditions and during summers the temperature rises beyond 45°C.
Methodology
Biochemical estimation for nutritional and anti-nutritional parameters
Ten different biochemical parameters including chlorophyll content, total sugar, total amino acid, proline, phenol, DPPH, phytic acid, flavonoid, lipid peroxidation and H
2O
2 peroxidation were estimated from the leaves at 35 Days after sowing for investigate the nutritional and antinutritional values of the groundnut genotypes. Based on the estimated values grouping of genotypes was done along with heat map to study the effect of level of expression in genotypes. Total chlorophyll was calculated as per method suggested by
Arnon et al., (1949), total sugars as per protocol described by
Dubois et al., (1956), proline as per method of
Bates et al., (1973), total amino acid by the method described by
Moore and Stein (1948), DPPH as per
Sultana et al., (2007), phenol as per method employed by
Swain and Hills (1959), Phytic acid as per the method given by
Wilcox et al., (2000), Flavonoid given by
Zhu et al., (2010), Lipid peroxidation by the protocol proposed by
Hodges et al., (1999). Assaying for superoxide dismutase activity (SOD) was calculated by the method of
Beyer et al., (1987).
Molecular characterization
Genomic DNA was isolated from 20-30 days young leaves of groundnut germplasms by modified CTAB method
(Murray and Thompson, 1980;
Tiwari et al., 2017). Twenty-six SSR primers unveiling polymorphism between two contrasting genotypes were employed for analysis of genetic diversity among 56 genotypes included germplasm lines and check varieties
(Pramanik et al., 2019). Out of 26 only 11 primers were found to be polymorphic. The SSR primers were synthesized by Eurofins Genomics India Pvt Ltd. Polymerase chain reaction was accomplished in 10μl reaction mixture encompassing of 1X PCR buffer, 0.1 U Taq DNA polymerase (Fermentas), 1 μl dNTP (1 mM), 0.5 μl of forward and reverse primers each (10 pM) and 20 ng/μl of genomic DNA in a thermocycler (Bio-Rad, USA). The PCR protocol comprised of initial denaturation step of 94°C for 3 min tracked by 35 cycles of 94°C for 1 min, annealing at 55°C for 30 sec, elongation at 72°C for 1 min with final extension at 72°C for 10 min. The PCR products were resolved on 3% agarose gel at 120V for 2-3 hrs and documented using Syngene, Gel Documentation System (USA).
Genetic diversity and population structure assessment
The genetic profile of groundnut genotypes was scored on the basis of difference in allele size using 11 highly polymorphic SSR molecular markers. The major allele frequency, number of alleles per locus, polymorphism information content (PIC) and gene diversity was analyzed using Power Marker v3.25 software
(Liu and Muse, 2005). The dendrogram based on unweighted pair group method for arithmetic average (UPGMA) and bootstrap value of 1000 permutations was constructed using MEGA 6.0 software
(Tamura et al., 2007). Groundnut germplasms were evaluated for resistance to early and late leaf spot on a 1 to 9 scale (no disease symptoms= 1 and 81 to 100% diseases severity= 9) at 35 and 45 days after sowing for ELS and at 75 and 85 days after sowing for LLS
(Subrahmanyam et al., 1995). The population structure for groundnut genotypes comprising both germplasm lines and released check varieties was inferred using Structure 2.3.4 software (
Pritchard et al., 2000). The structure outputs were visualized using Structure Harvester from which Evanno plots were constructed (
Earl and Von Holdt, 2012). An assumed admixed model with independent allele frequency and a uniform prior probability of the number of populations, K was used in structure. All the runs were conducted for K= 1 to 10 with 50,000 Markov Chain Monte Carlov (MCMC) replicates after a burn-in of 50,000 replicates. For each value of K, 3 independent runs were done to generate an estimate of the true number of sub-populations.
