Unclasping potential chickpea resources for biofortication of the antioxidant enzyme Superoxide Dismutase

Context: The Superoxide Dismutase enzyme plays a very decisive role in governing abiotic and biotic stresses infused the hypothesis for the study. Aims: The investigation was conducted to assess the diverseness and identify novel resources to be utilized in Superoxide Dismutase induced abiotic-biotic stress resistance breeding of chickpea. Methods: The plants were grown in triplicates under recommended agronomic practices using PUSA 256 as check in a randomized block design. Fresh leaves were collected for estimation of enzyme superoxide dismutase and DNA extraction. Number of pods was recorded on 20 individual plants from middle of the row for each of the 12 genotypes. Employing 32 STMS markers together with morpho-biochemical data, Jaccard’s similarity coecients along with dendrograms were generated to compare and assess the diversity. Key results: Amongst genotypes, the BGD-70 vs ICRISAT-3668 were identied as poorest vs best performers for superoxide dismutase activity. Out of 32 STMS primers, 80 alleles with 2.5 an average per loci were found. The marker TA-80 was identied as most polymorphic. The genotypes ICRISAT-3668 and SBD 377, distantly located on different molecular clusters, expressed higher SOD activity indicating genetic governance, probably by limited number of polygenes / OTLs and might be utilized as potential resources for abiotic-biotic stress resistance. Conclusions: The genotypes ICRISAT-3668, SBD 377 and polymorphic marker TA-80 were identied as novel potential genetic resources. Implications: The identied resources may be employed to widen the germplasm base, prepare maintainable catalogue, systematic blueprints and bifortication for future chickpea breeding strategies targeting abiotic-biotic stresses. of each during 2019-20in experimental eld randomized block set of three replications all agronomic using 256 as and 37.8 respectively. total annual ranged from to with a mean chickpea individual middle of each for each genotype for collection of fresh to estimating enzyme SOD and DNA extraction together with data for number of pods per of SOD trait specic development of mapping populations, construction of genetic maps, marker trait associations, localization of genes / QTLs in future marker assisted selection (MAS) and abiotic-biotic stresses resistance breeding programmes.


Introduction
Chickpea(Cicer arietinum L.), commonly known as Bengal gram or garbanzo beans belongs to Fabaceae family and on the basis of total production with 14.25million tons (MT), total harvested area of 13.72 million hectares (MHa), 1038.4 kg yield per hectare (Kg / Ha)(FAOSTAT 2019)andgrown over 40 countries representing all the continents, is the 2nd most important food legume globally. The 95% of the area, production and consumptions of chickpeas are represented by developing countries. During the span of last 30 years , the global chickpea area increased by 138.56%, yield by 143.29% and production by 198.53% (FAOSTAT 2019). Presently, it is cultivated in several countries with largest harvested area of 9.55 million hectares by India followed by Pakistan, Russian Federation, Turkey, Myanmar etc(FAOSTAT 2019). Currently, India represents as the largest producer of chickpeas accounting for around 69.76% of the global production followed by Turkey, Russian Federation, Myanmar and Pakistan are measured as the top ve major world producers(FAOSTAT 2019).
India expects 11.99 MT of total chickpea production for the year 2020-21 as per recently released 4th advance estimates (GOISTAT 2021). However, In India,during 2019-20, it was cultivated in 10.17 M Ha acreage with 11.35 MT productions and 1116 Kg / Ha productivity (GOISTAT 2020).Rajasthan ranked 1st with the highest acreage of 2.46MHa followed by Maharashtra, Madhya Pradesh, Karnataka and Uttar Pradesh. The highest production of 2.66 MT was produced by Rajasthan followed byMaharashtra, Madhya Pradesh and Uttar Pradesh. The highest yield of 1574 Kg / Ha was produced by Gujarat followed by Telengana(1532 Kg / Ha), Uttar Pradesh (1371 Kg / Ha) and Madhya Pradesh (1288 Kg / Ha)(GOISTAT 2020).
In recent years, utilization of DNA-based markers is increasing owing to their phenotypic appearance, elevated polymorphism, alleviated development and varied applications in plant improvement. Chickpea mapping is acutely obstructed by amazing little genetic polymorphisms in cultivated genotypes and previously utilized molecular oligos such as isozymes, RFLP, RAPD, AFLP etc being unsuccessful to disclose intra-speci c differentiations(Ghaffariet al.2014; Hajibarat et al.2015;Joshi and Reddy 2014). Amidst an array of DNA-based markers, Sequence Tagged Microsatellite Site (STMS) markers are often preferred in varied crop plants, inclusive of chickpea owing to their plethora, genomic compass, genetically co-dominant nature along with alleviated polymorphism (Bakshi et al. 2016;Bhardwaj et al. 2014;Harshavardhana et al. 2019;Katochet al. 2016;Kumar et al. 2017;Kumar et al. 2020;Singh et al. 2011;Singh et al. 2013) and their distribution on various linkage groups or chromosomes (Bharadwajet al. 2011;Gaur et al. 2011;Verma et al. 2015). Chickpea speci c STMS molecular markers were developed rst timeby Huttelet al.1999 that divulged polymorphism up to prudent degree. After some time, legume family indicated that some of the species peculiar STMS were recognized as very e cient in divulging polymorphism also in additional species, it indicates once evolved in chickpea they can also be employed in other chickpea linked species (Choumane et al.2000). Thus, DNA marker analysis will help the identi cation and differentiation of landraces with different genetic make-up. The present study was conducted to ascertain the pattern and extent of molecular characterization, relatedness and potential utility of STMS markers in analyzing molecular polymorphism in chickpea.
Plants have developed an e cient antioxidant system that can protect plants from any disaster injury(Joseph and Jini 2011). The toxic effects of reactive oxygen species (ROS) are counteracted by enzymatic as well as non-enzymatic antioxidative system such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), ascorbic acid (AsA), tocopherol, glutathione and phenolic compounds, etc. Normally, each cellular compartment contains more than one enzymatic activity that detoxi es a particular ROS. The presence of these enzymes in almost all cellular compartments gives their clear crucial role in ROS detoxi cation for the survival of the plant (Ahmad et al.2013;Mittler 2002). SODs are ubiquitous metalloenzymes that constitute the rst line of defense against ROS. SODs also constitute one of the major enzymatic components of detoxi cation of superoxide radicals generated in biological system by catalyzing its dismutation to H 2 O 2 and nally to H 2 O (Berwal and Ram 2018). SODs are metalloproteins working with Cu, Zn, Mn or Fe as cofactors and occur in the chloroplasts, mitochondria, cytosol, peroxisomes and the apoplast (Mittler 2002). Accordingly, three isoenzymes can be separated in plants having different structures and function as Cu/Zn-SOD, Mn-SOD and Fe-SOD (Alscher et al. 2002;Gill and Tuteja 2010). Mn-SOD is present in the mitochondria and peroxisomes, while Cu/Zn-SOD is mainly cytosolic, mitochondrial and plastidic.

Materials And Methods
B.1. Experimental plots -The experimental research and eld studies on chickpea were carried out during 2019-20 in the experimental plot "New Area A-Block" allocated to the Division of Genetics, IARI, New Delhi. This area is located between latitude 28.61 o N and longitude 77.23 o E and found at an altitude of 225 m above mean sea level. The topography of the experimental plot was uniform. The soil was sandy loam with physical and nutritional compositions as follows: The pH of soil was mild alkaline (7.5-8.5) with low EC (0.4-0.6 dS / m), low organic content (<0.5%), low nitrogen (<280kg/ha), high phosphorous (25-50kg/ha) and high potassium (>280kg/ha), medium sulphur (10-20mg/kg), adequate zinc (1-5mg/kg), de cient iron (5.8-10 mg/kg) adequate manganese (10-25mg/kg) and adequate copper (0.5-10mg/kg) respectively. B.2. Plant Genetic Material -12 Chickpea genotypes (Table 1) were selected for the morpho-biochemical and molecular characterization, based on visual observations recorded for biotic and abiotic stresses over a large pool of germplasm including core germplasm obtained from gene bank (ICRISAT 2021) and maintained at Division of Genetics, Indian Agricultural Research Institute, New Delhi, India. Healthy seeds of each genotype were sown during 2019-20in the experimental eld following a randomized block design with a set of three replications under all suitable agronomic practices using PUSA 256 as check. The mean daily minimum and maximum temperatures were 25 o C and 37.8 o C, respectively. The total annual rainfall during the season ranged from 0.2 mm to 66 mm with a mean of about 1.643 mm. The chickpea 20 individual plants from middle of each row for each genotype were used for collection of fresh leaves to estimating enzyme SOD and DNA extraction together with data recording for number of pods per plant. Separate controls (lacking enzymes) were used for total SOD and inhibition studies. The absorbance was recorded at 560 nm and 1 unit of enzyme activity was taken as that amount of enzyme, which reduces the absorbance reading to 50% in comparison with tubes lacking enzymes. was performed for 7 minutes at 72 o C and ampli cation products were analyzed using 3% Metaphor agarose (Resophor) gels stained with ethidium bromide. A 100 bp DNA ladder was run alongside the ampli ed products to determine the approximate size of the ampli cation products / bands.
The CCD camera (Sony XC-75 CE) attached to a gel documentation system with the Quantity One software (BIORAD) was used for photography of the gels. Each of the gel sections were scored done manually. The alleles were numbered as 'a 1 ', 'a 2 ' etc.
sequentially from the largest to the smallest size bands. The bands were designated as '1' for presence of a band and '0' for absence of a band in the data matrix. The polymorphic information content (PIC) for each STMS marker, was ascertained followed by standard procedure (Senior et al. 1998). PIC is an estimate of allele variation at a locus and comparable to 1-Σ (P 2 ij), where P ij is the frequency of the j th allele for i th locus summated including all alleles in the locus. The 0-1 data matrix was further used to calculate genetic similarity between genotypes following Jaccard'scoe cient (Jaccard 1908) using NTSYS software (Rohlf 2000). UPGMA (Sneath and Sokal 1973) on the similarity matrix was performed to identify genetic variation patterns among the chickpea genotypes using NTSYS pc version 2.11s.
B.2.4. Clusters analysis for measurement of distances -Software NTSYS-PC version 2.11s (Rohlf 2000) was employed to categorize genotypes into discrete conglomerations followed by dendrograms constructions using the UPGMA method (Sneath and Sokal 1973  C.1.3. Similarity Vs Dissimilarity Analysis -The integrated data for the enzyme SOD activity and yield trait number of pods was utilized for estimating pair wise genetic similarities among 12 genotypes of chickpea using Jaccard's coe cient method ( Table  3). The genetic similarity matrix was used for construction of dendrogram (Fig. 1)  Estimation of size range of alleles were approximately estimated, as the resolution power of agarose gel is relatively less, as compared to that of polyacrylamide gel, which can resolve nucleotide difference of even one base pair. Speci c STMS markers contained high differential capability for demarcating C. arietinum germplasm lines as the present study demonstrated that out of 80 STMS alleles, only 12 STMS alleles were found to be unique or rare; unique or rare allele is one with a frequency less than or equal to 0.10. The present ndings also indicated instances where the STMS pro les for some of the genotypes displayed deviation from the expected pattern. Chickpea germplasm / varieties are assumed to be highly homozygous and thereby should reveal only a single band (allele) per locus for a large majority of them if not all. However, double bands could clearly be seen in many of the lines.
C.2.2. Molecular Similitude Vs Dissimilitude Inspection -The STMS data was employed for inspecting pair wise genetic similarities among various genotypes using Jaccard's method (Table 3). The genetic similarity matrix was further explored following UPGMA clustering algorithm employing NTSYS pc version 2.11 software programme. The dendrogram extracted from the investigation as portrayed in Fig. 2 exhibited that the cluster I, II and III comprised of 5, 5 and 2 genotypes. The

Discussion
The investigation targeted for dissecting the chickpea genetic variability utilizing morpho-biochemical traits (SOD activity, number of pods) and STMS markers for determining the potential utility of these genotypes and molecular markers. Molecular markers are employed for variable purposes including construction of linkage maps, assessing genetic relationships and identi cation of crop cultivars. Microsatellite genotypic data generated from an array of loci have prospective for furnishing distinct allelic pro les or DNA diagnostics for displaying genotypic signatures. Because of compartmentation of different isoenzymes, SOD plays more effective role in stress resistance mechanism. Increased activity of SOD is often correlated with increased tolerance of plant against environmental stresses. It was suggested that SOD can be used as indirect selection criterion for screening stress resistant plant materials. Over production of SOD has been reported to result in enhanced oxidative stress tolerance in plants (Shukla and Verma 2019).
Expression pro ling of chickpea in response to biotic and abiotic stress has been reported (Mantri et al.2007), only based on customized microarray chip based on probe homologs. Metabolic pathways involved in cell wall synthesis, energy production, nitrogen metabolism, defense mechanism, regulation of transcription and signal transduction the key processes that were modulated by heavy metal stress also provide stress tolerance in chickpea. Many biotic stress associated genes were also reported to be differentially expressed during the exposure. The microarray data are available in the public domain and can be accessed to perform transcription factor based study. Insights from the transcriptome level expression pro ling provide clues for a future molecular breeding approach to developing stress tolerant chickpea varieties (Yadav and Mani, 2018).
The diversity analysis studies on 12 chickpea genotypes for the morpho-biochemical traits number of pods and SOD activity revealed that the close uniform similarity (0.991) values amongst BGD 1004 vs CSG 9505, ICRISAT-3668 vs Vijay followed by Pusa 256 vs GPF-2, ICRISAT 3688 vs GPF-2 with uniform similarity values (0.981) could be due to ancestral association. The largest distances observed between the genotypes ICRISAT-3673 vs ICRISAT-3155, ICRISAT-3673 vs SBD 377 with dissimilarity (0.899) followed by ICRISAT-3668 vs Pusa 362 with dissimilarity (0.893) and Pusa 256 vs BGD-1004 with dissimilarity (0.890) values respectively, could be due to lack of ancestral association.
Molecular dissimilarity examination, employing 32 STMS markers generated 80 alleles along with an average 2.5 alleles per loci, indicated the presence of remarkable polymorphism for the examined microsatellite loci and disclosed a moderate level of genetic variability in the chickpea genotypes, which is also supported by several others (Ghaffari et al. 2014;Khan et al. 2010).
The PIC values ranging from 0 to 0.705 for the markers disclosed enough heterogeneity amongst genotypes and potential utility of already mapped marker TA 80 on the linkage group-6 (Gaur et al. 2011). Cluster formations using morpho-biochemical traits together with STMS markers segregated all chickpea genotypes into four and three perceptible groups, respectively. The occurrence of heterozygosity in pure lines revealed through STMS analysis could possibly draw the attention of the chickpea breeders for effective maintenance breeding. Thus, molecular characterization can give very useful information to chickpea breeder (Stephens and Lombardi 2014).
Results from the present investigation disclosed potential utility of STMS in characterizing chickpea germplasm and is supported by several others (Flandez-galvez et al. 2003;Soi et al., 2014). The reasonably high rate of polymorphism shown by the marker TA 80 with four alleles indicated the potentiality of this marker for further employability. The occurrence of rare alleles or speci c STMS alleles gives an enormous chance for producing exhaustive ngerprint database. The noticed "null" alleles could be attained owing to mutations in the primer binding site causing to non-ampli cation. The PIC value is determined by the incidents of variants per locus along with relative dispersal of the alleles. The one to six extents of alleles per locus were found for example, CaSTMS 14 with single alleles with '0' PIC value compared to TA 45 with four alleles with higher PIC value (0.680) disclosed the signi cance of dispensation of alleles over the genome (Supplementary Table 2 Thus, the genotype ICRISAT-3668 located on molecular cluster II and the molecular marker TA-80 mapped on LG-6 need further investigation and validation to be utilized as potential resources for the SOD activity in chickpea. The results included identi cation of SSR marker TA-80 and high SOD content chickpea genotype ICRISAT-3668. The identi ed resources will allow exploration for their utilization as potential marker / donor resources for the bioforti cation of SOD trait speci c development of mapping populations, construction of genetic maps, marker trait associations, localization of genes / QTLs in future marker assisted selection (MAS) and abiotic-biotic stresses resistance breeding programmes.

Declarations
Author's contribution RK conceptualized and supervised the experiment. AS, RKS, PS and RY executed eld and laboratory investigations. NS, VS and NY analyzed the data and interpreted the results. RK and RA contributed to the original writing, reviewing and editing of the manuscript. All authors read and approved the manuscript.
Funding: The study was supported by ICAR-Indian Agricultural Research Institute, New Delhi.
Con ict of interest: The authors declare no con icts of interest.
Ethical approval: Not applicable.

Figure 1
Dendrogram depicting genetic relationship among selected genotypes of Chickpeas based on the enzyme SOD activity and yield trait number of pods data