Journal Pre-proof

Phenotypic and molecular evaluation of Arachis hypogaea L. against foliar fungal

diseases

Sunil Yadav, Sushma Tiwari, Manoj Kumar Tripathi, Neha Gupta, Sangeeta Singh,
Niraj Tripathi

PII: S2772-8994(23)00014-9
DOI: https://doi.org/10.1016/j.cropd.2023.100036
Reference: CROPD 100036

To appear in: Crop Design

Received Date: 1 May 2023

Revised Date: 6 June 2023
Accepted Date: 7 June 2023

Please cite this article as: S. Yadav, S. Tiwari, M.K. Tripathi, N. Gupta, S. Singh, N. Tripathi, Phenotypic
and molecular evaluation of Arachis hypogaea L. against foliar fungal diseases, Crop Design (2023), doi:
https://doi.org/10.1016/j.cropd.2023.100036.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2023 The Authors. Published by Elsevier B.V. on behalf of Zhejiang University and Zhejiang University
Press Co. Ltd.
 1 Phenotypic and Molecular Evaluation of Arachis hypogaea L. against Foliar Fungal
2 Diseases

3 Sunil Yadav1, Sushma Tiwari1*, Manoj Kumar Tripathi1, Neha Gupta2, Sangeeta Singh3 and
4 Niraj Tripathi4
1
5 Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Rajmata
6 Vijayaraje Scindia Agricultural University, Gwalior-474002, India,
2
7 Biotechnology Centre, Jawaharlal Nehru Agricultural University, Jabalpur 482004, India,
3
8 National Institute of Plant Genome Research, New Delhi, India,
4
9 Directorate of Research Services, Jawaharlal Nehru Agricultural University, Jabalpur 482004,
10 India

of
11 *Correspondence: sushma2540@gmail.com

ro
12 ABSTRACT
13
-p
Groundnut improvement task are generally engrossed to breed foliar fungal diseases resistant
re
14 varieties. Early and late leaf spot diseases of groundnut cause significant loss in yield and quality
15 of groundnut seeds. Simple Sequence Repeat (SSR) markers represents high polymorphic
lP

16 amplification, while Inter-simple sequence (ISSR) is being used widely for diversity assessment
na

17 due to their large size and reproducible results. Current investigation was carried out with 96
18 groundnut genotypes for morpho-physiological characterization, disease indexing for early and
ur

19 late leaf spot diseases under field conditions and by employing SSR and ISSR markers for
Jo

20 diversity assessment. In correlation analysis, kernel weight was found positively and highly
21 significantly correlated with harvest index and pod weight (r = 0.906) at 1% level of
22 significance. While, pod weight is negatively correlated with early leaf spot at 30 days (r=-0.401)
23 and at 45 days (r=-0.410), late leaf spot at 70 days (r=-0.342) and at 85 days (r=-0.309) at 1%
24 significant level. With the use of SSR markers, total 16 alleles were recognized with a mean of
25 3.20 alleles per locus. The gene multiplicity differed from 0.6074 to 0.6491 for the markers
26 Seq5D05 and PM137 respectively with an average of 0.6289. Total 18 alleles were produced by
27 ISSR markers with an average of 3.6 alleles per locus. In our study, the gene diversity varied
28 from 0.5877 to 0.6625 for the markers ISSR3 and ISSR21 respectively with an average of
29 0.6164. Diverse group of groundnut germplasm has been identified on the basis of SSR and
30 ISSR markers along with morphological characterization.
31 Keywords: Groundnut, molecular markers, cluster analysis, disease, stress
32 1. INTRODUCTION

33 Groundnut (Arachis hypogaea L.) is a main oilseed and food crop being cultivated in more than
34 100 countries on 315 lakh hectares area with worldwide production 536 lakh tonnes with the
35 productivity of 1701 kg per hectare [1,2]. India positions first with occupying 55.71 lakh
36 hectares of land area and second in terms of pod production with 102 lakh tonnes with
37 productivity of 1831 kg per hectare in 2020-21[1]. The crop is utilized largely as confection,
38 cooking oil and in innumerable food products in the Indian subcontinent. Its kernels are occupied
39 with alimentary components and comprises in the range of 40–55% fat, 20-30% protein and 10-
40 20% carbohydrates together with an array of nutritional components including niacin, vitamin E,

of
41 iron, zinc, magnesium, calcium, potassium, phosphorus, riboflavin and thiamine.

ro
42 An array of factors is responsible for reduction in yield and quality of produce. Among
43
-p
these some of the biotic and abiotic factors play major role. In groundnut, various fungal diseases
re
44 have been reported. However, the crop is generally infected with two common fungal diseases
45 viz., early leaf spot (ELS) and late leaf spot (LLS) [3-6]. Early leaf spot is caused by Cercospora
lP

46 arachidicola while late leaf spot by Cercosporidium personatum. Among different biotic
na

47 constraints, both foliar fungal diseases are responsible for about 70% yield loss in groundnut.
48 Apart from yield reduction, these diseases also reduce quality of seeds and their grade [7-10].
ur

49 These fungal diseases also deteriorate quality and quantity of haulm which is used as animal
Jo

50 fodder [2,3,9-11]. Applications of chemical fungicides are the common practices to counter these
51 problems, however these approaches are labour-intensive, expensive and are not eco-friendly.
52 One of the most successful methods for developing cultivars resistant to leaf spot is breeding for
53 host plant tolerance. So, it is important to identify resistant cultivars against these diseases and
54 exploit them for development of new resistant varieties.

55 Limitations of traditional breeding approaches make it essential to apply molecular tools

56 and techniques in oilseed crop improvement programmes. Molecular tools are being utilized in
57 groundnut improvement programmes [12]. In this investigation, we evaluated the efficacy of two
58 different markers, Inter-simple sequence (ISSR) and Simple Sequence Repeat (SSR), in
59 groundnut genotype diversity analysis, using local germplasm lines collected from different parts
60 of Madhya Pradesh, India, and some were procured from DGR, Junagarh, Gujrat, India. The SSR
61 marker has highly polymorphic information content (PIC) with their high reproducibility rate,
62 multiallelic nature, they have been useful for diversity assessment in groundnut widely and have
63 successfully applied in breeding as an efficient tool to link phenotypic and genotypic variation
64 [3]. However, in the last decade, ISSR markers have been successfully applied for diversity
65 studies in many crop species including groundnut, as one of the most frequently employed
66 markers in intra-specific diversity analysis [13]. Reproducibility of ISSR is better than RAPD
67 owing to their longer size and higher annealing temperatures [13, 14]. In this sequence, Shoba et
68 al. [15] screened nine simple sequence repeat (SSR) primers linked with LLS severity score.
69 Various other reports are also available on identification and validation of LLS and rust resistant
70 gene linked markers in groundnut [16, 1718-20]. Besides, various advantages of molecular

of
71 markers in diversity assessment. Conventional method of screening is one of the powerful tools
72 to see the expression at field level and it could not be ignored for successful breeding

ro
73 programme. The present investigation was performed to analyze variability among groundnut
74
-p
genotypes based on morphological traits, foliar fungal disease scoring and SSR as along with
re
75 ISSR markers.
lP

76 2. MATERIALS AND METHODS

77 2.1. Plant Material
na

78 In current study, 96 groundnut germplasm lines including 23 local germplasm lines collected
79 from Shivpuri, Madhya Pradesh, India, 66 germplasm lines belonging to ICGV series received
ur

80 from Directorate of Groundnut Research, Junagadh, Gujrat, India and 7 check varieties including
Jo

81 TG-26, GPBD4, KDG128, JGN3, Sunolic 95-R, Girnar-2 and Gangapuri were chosen for
82 evaluation of different morpho-physiological traits under field conditions and characterization
83 for ELS and LLS (Table 1). Among these genotypes, GPBD4 and KDG128 are known for foliar
84 fungal diseases resistant, and others as sensitive [21].
85 2.2. Morpho-physiological characterization

86 The field and laboratory experiments were conducted at Department of Plant Molecular Biology
87 & Biotechnology, Rajmata Vijayaraje Scindia Agricultural University (RVSKVV), Gwalior,
88 Madhya Pradesh, India during Kharif 2019-20 and 2020-21. The seed material was sown in three
89 replications for each of the 96 germplasm lines with row spacing of 30 cm and plant to plant10
90 cm in augmented design. The seeds were treated with disinfectant fungicide (Dithane M-45 @2g
 91 kg-1 seed + Bavistin @ 1gkg-1 seed) before sowing. The crop was grown succeeding the endorsed
92 agronomical practices of NPK with ratio 20:60:20 and gypsum in essential dose.

93 A set of 96 uncharacterized groundnut germplasm lines including check varieties, were

94 evaluated for plant height (average height of five plants of each plot taken at physiological
95 maturity from ground to tip of the shoot), canopy temperature, days to 50% flowering (the day
96 after sowing when 50% plants bear flowers), days to maturity (the day after sowing when plants
97 of a plot reached 90% physiological maturity), total number(s) of pods/plant (average of real
98 count of pods of five randomly selected plants), 100-pod weight (g) (average weight of 100 pods
99 in three replications), 100-kernel weight (g) (average weight of 100 kernels in three replications),

of
100 sound mature kernel %, shelling percentage (about 200 g mature pods were used for the

ro
101 estimation), biological yield (shoot dry weight) and harvest index (ratio of kernel yield per plant
102 and shoot dry weight). Morphological data was recorded as per method suggested by Pramanik
103 et al. [3].
-p
re
104 2.3. Disease scoring
lP

105 Groundnut genotypes were evaluated for resistance to ELS and LLS in the scale of 1 to 9 (with 1
106 means no disease and 9 shows 81 to100 percent severity) in two repetitions at 35 and 45 days
na

107 after sowing for ELS and at 75 and 85 days after sowing for LLS [22]. Disease scoring was
108 based on 1-9 scale visual score indicated as 1= highly resistance 0%, 1-3= resistance 1- 20%, 4-
ur

109 5= moderate resistance, 21-50%, 6-7= susceptible 51-70% and 8-9= highly susceptible 70-100%
Jo

110 [22].
111 2.4. Genomic DNA extraction
112 Twenty days old young leaves were sampled from each genotype. The genomic DNA was
113 isolated using cetyl tri-methyl ammonium bromide (CTAB) method [23] with slight changes as
114 suggested by Tiwari et al. [24]. The DNA was checked for quality on 0.8% agarose gel and
115 quantity was assessed using nano-drop spectrophotometer (Thermo-fisher). The final DNA
116 concentration (20 ng/µl) was maintained after dilution of each DNA sample for further
117 polymerase chain reaction (PCR) process.
118 2.5. Marker validation
119 DNA templates were amplified in 10 µl reaction volume with 5µl master mix (Fermentas, USA),
120 1µl of each primer (forward and reverse), 1 µl of genomic DNA and required volume of nuclease
121 free water. Total 16 reported SSR markers as suggested by Varma et al. [16], Mace et al. [25],
122 Shoba et al. [15], Sujay et al. [17], Sukruth et al. [19] for resistance against foliar fungal diseases
123 were considered for screening. For SSR markers the PCR protocol of following steps with initial
124 denaturation at 94°C for 3 min, 35 cycles at 94°C for 1 min, annealing at 55°C for 30 sec,
125 elongation at 72°C for 1 min and final extension at 72°C for 10 min was employed. The PCR
126 products were separated on 3% agarose gel and visualized under gel documentation system
127 (Syngene, USA). Among them only 5 primers i.e., PM137, Seq 5D05, GM2079, GM2301 and
128 Seq13A07 showed polymorphism in four groundnut genotypes including Gangapuri, KDG 128,
129 GPBD 4 and JGN 3, hence used for further amplification of all the 96 genotypes.
130 Furthermore, a set of 5 ISSR markers recommended by Mondal et al. [26] for evaluation

of
131 of resistant and susceptible groundnut genotypes were also applied. For ISSR markers the PCR
132 programming included first step of denaturation at 94°C for 5 min tracked by 45 cycles at 94°C

ro
133 for 1 min, annealing at 50°C for 1 min, elongation at 72°C for 2 min. The final extension was at
134
-p
72°C for 7 min. The amplified PCR products were subsequently resolved on 1.4 % agarose gel
re
135 through electrophoresis at 65 volts for 2 hours (Bio-Rad, USA).
136 2.6. Statistical analysis
lP

137 Morphological and physiological traits were analyzed for variance (ANOVA), standard error
na

138 (S.E.), critical difference (CD) and coefficient of variation (CV). using the mean values of
139 two years data. At maturity of the crop the coefficient of correlation among all morphological
ur

140 and physiological traits was calculated using software SPSS ver19.0. Heat map and
Jo

141 dendrogram analysis was done using “heatmap” function of ‘R’ package [27]. During
142 molecular analysis the gel scoring for both types of markers was done according to the
143 differences in band/allele sizes. Allele number, frequency, gene diversity and polymorphism
144 information content (PIC) were analyzed using software Power Marker v3.25 [28]. The
145 dendrogram based on unweighted pair group method for arithmetic average (UPGMA) and
146 bootstrap value of 1000 permutations was constructed using MEGA 6.0 software [29].
147 3. RESULTS
148 3.1. Morpho-physiological characterization and disease scoring
149 Significance of correlation of different morpho-physiological traits along with disease
150 scoring against ELS and LLS is presented in Table 2. The highly significant and positive
151 correlation was documented between pod weight and kernel weight (r=0.906) at 1%
152 significant level. Similarly, Early leaf spot after 30 days showed highly significant and
153 positive correlation with early leaf spot trait after 45 days (r=0.969), and late leaf spot after
154 70 days (r=0.843) and 85 days (r=0.831) at 1% level of significance. Subsequently, highly
155 significant, and positive correlation was detected between early leaf spot after 45 days and
156 late leaf spot at 70 days (r=0.816) and late leaf spot after 85 days (r=0.801) at 1% level of
157 significance. Highly significant and positive correlation was detected between late leaf spot
158 at 70 days and late leaf spot after 85 days also (r=0.983) at 1% level of significance. Some of
159 the other characters were also found positively and significantly correlated with each other.
160 Pod weight is negatively correlated with ELS at 30 days (r=-0.401), ELS at 45 days (r=-
161 0.0.410), LLS at 70 days (r=-0.342) and LLS at 85 days (r=-0.309) at 1% significant level.

of
162 Likewise, kernel weight is also negatively correlated with ELS at 30 days (r=-0.392), ELS at
163 45 days (r=-0.394), LLS at 70 days (r=-0.350) and LLS at 85 days (r=-0.332) at 1%

ro
164 significant level. Diversity assessment based on heatmaps for representing expression levels
165
-p
of different morphological traits (Fig. 1) showing variations among studied genotypes. In
re
166 Fig. 1, Green color represents a lower and red color signifies a higher value. The increasing
167 intensity of color from green to black and then to red embodies increasing values
lP

168 accordingly. Dendrograms representing diversity among groundnut genotypes on the basis of
na

169 field observations (Fig 1).

170 Groundnut genotypes were categorized based on disease indexing data od ELS and LLS
ur

171 (Fig 2). Total 21 groundnut germplasm lines were considered highly resistant against ELS
Jo

172 and LLS diseases as they represented (Fig. 2) disease score in range of 1-3. Phylogenetic tree
173 based on expression level of ELS and LLS diseases at field represented diversity and
174 different level of disease intensity (Fig 3). Based on disease scoring data Shivpuri Local 74,
175 ICGV-5660, ICGV-13573, Shivpuri Local 93, Shivpuri Local 73, ICGV-8171, Shivpuri
176 Local 88, Shivpuri Local 92, ICGV-13214, Shivpuri Local 80, Shivpuri Local 83, Shivpuri
177 Local 87, Shivpuri Local 91, Girnar-2, ICGV-9934, Shivpuri Local 84, Shivpuri Local 81,
178 Shivpuri Local 89, SUNOLIC 95-R and Shivpuri Local 82 were found to be resistant for ELS
179 and moderately resistant for LLS. Shivpuri Local 93, ICGV-8171, ICGV-13214, ICGV-
180 13573, Shivpuri Local 87 and Shivpuri Local 81 were considered to be resistant for LLS and
181 moderately resistant for ELS.
182 3.2. SSR markers-based diversity
183 With the use of SSR markers in the present investigation, total 16 alleles were recognized
184 with a mean of 3.20 alleles per locus. The gene multiplicity differed from 0.6074 to 0.6491
185 for the markers Seq5D05 and PM137 respectively with an average of 0.6289. However, in
186 our study, PIC values were ranged between 0.5328 to 0.6019 for Seq5D05 and PM137
187 markers correspondingly, with a mean value of 0.5612 (Table 3). In the current investigation,
188 PM137 was recognized highly efficient marker according to the gene diversity and PIC
189 values. The lowest value for major allele frequency was investigated 0.4375 for marker GM
190 2079 whilst highest (0.5208) for PM137 marker. The mean value for major allele frequency
191 was evidenced 0.4938 (Table 3).

of
192 The DNA based affiliation among the studied groundnut genotypes grouped them into 8
193 clusters. Third cluster is a group of foliar diseases resistant genotypes whereas first cluster

ro
194 has the genotypes with high fatty acids and biochemical composition (Fig.4). Cluster1 had
195
-p
four genotypes viz., ICGV13523, ICGV4278, SL77 and SL85. Genotype SL85 showed
re
196 moderate resistance against ELS and LLS under disease indexing under field condition. They
197 may have resistance genes for ELS and LLS which is also supported by morphological and
lP

198 molecular data gathered during the present investigation. Cluster 2 has eleven genotypes
na

199 including SL92, ICGV13214, ICGV8171, SL75, ICGV13554, SL76, KHNRCG13574,

200 ICGV13297, ICGV-9931, ICGV-13549 and SL-83. Most of the Shivpuri-local collection is
ur

201 fallen in cluster 2 and represented resistance or moderately resistance under field condition.
Jo

202 Cluster 4 included 34 genotypes which is the largest group among 8 clusters signified most
203 of the DGR- Junagadh collection in cluster 4 including two check verities viz., KDG128 and
204 TG26 (both resistant check varieties for ELS and LLS). These genotypes epitomized
205 resistance or moderately resistance under field condition. While cluster7 included 22
206 genotypes, which is the second largest group among all clusters, represented most of the
207 DGR- Junagadh collection.
208 3.3. Diversity assessment using ISSR markers
209 Total 18 alleles were produced by ISSR markers with an average of 3.6 alleles per locus. In our
210 investigation, the gene diversity varied from 0.5877 to 0.6625 for the markers ISSR3 and
211 ISSR21 respectively with a mean value of 0.6164. An average worth of major allele frequency
212 was 0.4854 (Table 3) with a range of 0.3854 (ISSR21) to 0.5833 (ISSR8).
213 The obtained data grouped the investigated genotypes into 13 small clusters (Fig. 5). According
214 to the dendrogram, most of the genotypes collected from farmers field of Shivpuri district
215 Madhya Pradesh, India i.e., Shivpuri local 90, Shivpuri local 88, Shivpuri local 84, Shivpuri
216 local 91, Shivpuri local 87, Shivpuri local 77, Shivpuri local 86, Shivpuri local 72 and Shivpuri
217 local 71 were grouped together in C IVIX cluster. One more cluster, CIX also had genotypes of
218 Shivpuri viz., Shivpuri local 85, Shivpuri local 78, Shivpuri local 79, Shivpuri local 80, Shivpuri
219 local 85, Shivpuri local 73 and Shivpuri local 92. Cluster XIII contains only one genotype
220 ICGV13208. Grouping of the locally collected genotypes along with other germplasms in
221 different clusters C1 to CXIII indicates existence of variability among them. These findings

of
222 suggest huge diversity among them which could be further utilized them for the improvement of
223 groundnut crop. Like this, most of the ICGV genotypes collected from Directorate of Groundnut

ro
224 Research, Junagadh, Gujrat, India shown genetic similarity with each other and formed separate
225 sub clusters.
-p
re
226 4. DISCUSSION
227 4.1. Morpho-physiological characterization and disease scoring
lP

228 The measurement of morphological traits has been played essential role to analyze the
na

229 differences in crop genotypes in the expression of these traits. Like the findings of the present
230 study, Pramanik et al. [3] also observed significant and positive correlation between hundred pod
ur

231 weight (r=0.769) and kernel yield (r=0.899); mature kernel and pod weight with kernel yield and
Jo

232 weight of kernels and harvest index. Recently, Mubai et al. [30] documented that grain yield had
233 significant (P < 0.001) and positive correlation with one of the important yield attributing
234 characters i.e., number of pods per plant (r = 0.87). Previous investigations conducted by Zaman
235 et al. [31] and Rao et al. [32] addressed similar relationships. These favorable connections show
236 that direct or indirect selection for these traits may boost yield. During the current study a
237 substantial positive relationship was identified between numbers of pods per plant and grain
238 yield. This positive relation indicates genetic relationship between both traits [33, 34] and their
239 expression are governed by interdependent genes. Like the current study's findings, the numbers
240 of pods per plant and per hundred seeds have repeatedly been found to have a significant direct
241 effect to production of groundnut [35-37]. In our investigation we found highly significant and
242 positive correlation between pod weight and kernel weight (r=906) at 1% significant level.
243 Similar correlation was also found in a recent study conducted by Killi and Beycioglu [38].
244 According to these experts, these features had a good and considerable direct impact on grain
245 yield, so, these features should be given preference in selection for improvement of economic
246 yield in groundnut.
247 In a recent investigation, Mubai et al. [30] reported negative relationship between disease
248 incidences (%) (P>0.05, r=−0.09) and pod weight. In our study, also pod weight is negatively
249 correlated with ELS at 30 days (r=-0.401), ELS at 45 days (r=-0.0.410), LLS at 70 days (r=-
250 0.342) and LLS at 85 days (r=-0.309) at 1% significant level. Likewise, kernel weight is also
251 negatively correlated with ELS at 30 days (r=-0.392), ELS at 45 days (r=-0.0.394), LLS at 70
252 days (r=-0.350) and LLS at 85 days (r=-0.332) at 1% significant level. The incidence of ELS and

of
253 LLS diseases was shown to have a substantial negative connection with the traits responsible for
254 grain yield, supporting previous claims that the diseases have a disastrous effect on groundnut

ro
255 output [36, 37]. These findings also support the disease's severe impact on grain yield, with
256
-p
complete yield losses possible. The loss in yield varies according to the growth stage of crop at
re
257 which pathogen causes severe infection. Infection at early growth stage may cause heavier loss
258 than infection at later stages. Grouping of genotypes based on morphological characteristics is
lP

259 beneficial for identifying and selecting the top performers and ethnically varied parents that
na

260 could be used in breeding programs [39, 40]. The study revealed that the groundnut variants
261 studied had a wide range of variability. Groundnut genotypes from separate clusters might be
ur

262 tested for their capacity to combine to form a reservoir of superior individuals. According to the
Jo

263 findings of Siddiquey et al. [41] and Banerjee et al. [42], there was presence of considerable
264 genetic diversity in groundnut genotypes taken under their studies and these reports back up the
265 findings of the present investigation. High diversity among groundnut genotypes based on
266 morphological and qualitative traits has also been reported earlier by Gokidi [43] and Upadhyaya
267 et al. [44]. High variations in groundnut genotypes alongside ELS response have been formerly
268 reported [45, 46]. Sometimes the expression of traits depends upon genetic makeup of the
269 genotypes considered for the study or the experimental environment in which the study
270 conducted [45].
271 4.2. SSR based diversity
272 Earlier, very less information was available on linkage of SSR markers with targeted genes in
273 peanut but, recent research made it possible. Now, many reports are available on linked SSR
274 markers with these diseases [46,9,10]. Like this, Mandloi et al. [10] applied four gene based SSR
275 markers for identification of LLS and rust resistant groundnut lines. During this study, PIC
276 values were ranged between 0.397-0.579 (an average 0.47). In a previous investigation,
277 conducted by Pandey et al. [47] the PIC value was ranged between 0.06 to 0.86 which
278 categorized the used markers in more efficient and less efficient categories. According to some
279 former studies based on SSR markers, higher PIC values are indication of superiority of that
280 marker [48, 49]. In some earlier experiments, efficiency of the molecular markers for LLS and
281 rust resistance gene transfer with the applications of marker assisted selection were authenticated
282 and employed through MABC in groundnut varieties viz., TAG24, ICGV91114 and JL24 [50,
283 51]. Three popular varieties of Gujarat viz., GJG9, GG20 and GJG-HPS1 have been developed

of
284 for foliar disease resistance recently [2]. Although the number of markers is less for the diversity
285 but morpho-physiological data interpretations, along with ISSR diversity provided useful

ro
286 information for the study.
287 4.3. ISSR based diversity
-p
re
288 A set of 5 polymorphic ISSR markers recommended by Mondal et al. [26] for evaluation of ELS
289 and LLS resistant and susceptible groundnut genotypes were chosen. Recently, Abbas et al. [52]
lP

290 used five ISSR markers for the evaluation of groundnut genotypes and reported a total of 36
na

291 amplified fragments with an average 7.2 alleles per locus. PIC values for ISSR markers were
292 ranged between 0.5137 to 0.5886 with the mean value of 0.5462. However, Abbas et al. [52]
ur

293 reported the average PIC value only 0.19 for ISSR markers employed for diversity assessment in
Jo

294 groundnut genotypes. These results indicated the efficiency of ISSR21 due to highest gene
295 diversity and PIC values. Earlier, PIC values were used to assess the edifying prospective of
296 markers in diverse genotypes, accessions, and germplasm lines [53-55]. As, molecular markers
297 with PIC values >0.5 are considered more informative. So, all ISSR markers applied in the
298 current study may be considered as more informative markers for DNA polymorphism and
299 genetic variation investigation in groundnut germplasm lines as well as varieties. According to
300 the field data obtained after evaluation of these genotypes against ELS and LLS diseases and
301 ISSR molecular markers-based clustering dissimilar genotypes may be selected as parents for
302 further breeding programmes. In some of the earlier studies, ISSR markers were used for
303 diversity assessment in groundnut genotypes [56] and these markers were found appropriate.
304 Abbas et al. [52] used ISSR markers for diversity assessment among groundnut genotypes and
305 reported variability in association with biochemical profiling. Recently, Khan et al. [57] used
306 ISSR markers for DNA fingerprinting of Bambara groundnut genotypes collected from different
307 regions of Malaysia. They reported the effectiveness of ISSR markers in diversity assessment
308 and tagging of potential genotypes.

309 5. Conclusions

310 Due to high demand of groundnut for the purpose of human consumption and animal feed, it is
311 important to develop superior cultivars to fulfill the requirement. Among different biotic factors
312 responsible of yield reduction in groundnut two foliar fungal diseases viz., ELS and LLS are
313 major causes of yield reduction in groundnut. During the present investigation, total 96
314 groundnut genotypes were evaluated based on different morpho-physiological as well as

of
315 molecular markers. In correlation analysis, kernel weight was found positively and significantly

ro
316 correlated with harvest index, pod/plant, pod weight, shelling% and biological yield. SSR and

-p
317 ISSR markers are highly polymorphic and appropriate for diversity analysis in groundnut.
318 Genotype Sun Oleic 95R was identified as resistant against foliar fungal diseases based on
re
319 morpho-physiological and molecular markers data. More studies are needed to establish
lP

320 molecular basis of disease resistance in groundnut. This genotype may be employed as parents in
321 crossing programme to breed varieties resistant against these major fungal diseases in future.
na

322 REFERENCES
ur

323 1. Agricultural Statistics at a Glance (2021) Available online

Jo

324 at:https://eands.dacnet.nic.in/PDF/Agricultural%20Statistics%20at%20a%20Glance%20-
325 %202021%20(English%20version).pdf.

326 2. Shasidhar, Y.,Variath, M.T., Vishwakarma, M.K., Manohar, S.S.,Gangurde, S.S.,Sriswathi,

327 M., et al. (2020). Improvement of three Indian popular groundnut varieties for foliar disease
328 resistance and high oleic acid using SSR markers and SNP array in marker-assisted
329 backcrossing. Crop J. 8, 1-15. doi: 10.1016/j.cj.2019.07.001.

330 3. Pramanik A, Tiwari S, Tomar RS, Tripathi MK and Singh AK (2019) Molecular
331 characterization of groundnut (Arachis hypogaea L.) germplasm lines for yield attributed
332 traits. Indian J. Genet. 79 (1): 56-65

333 4. Bhawar, P.C., Tiwari, S., Tripathi, M.K., Tomar, R.S. and Sikarwar, R.S. (2020).
334 Screening of groundnut germplasm for foliar fungal diseases and population structure
335 analysis using gene based SSR markers. Current Journal of Applied Science and Technology.
336 39(2): 75-84.

337 5. Adlak T., Tiwari S, Gupta N, Tripathi M K, Sikarwar R S, Sastya R and Gupta V.
338 Assessment for yield and nutritional profiling of groundnut with the help of allele specific
339 markers for desirable fatty acids. Int.J.Curr.Microbiol.App.Sci. 2021. 10(02): 1625-1637.
340 doi: https://doi.org/10.20546/ijcmas.2021.1002.193

341 6. Rathore, M.S., Tiwari, S., Tripathi, M.K., Gupta, N., Yadav, S., Singh, S. and Tomar,
342 R.S. 2022. Genetic diversity analysis of groundnut germplasm lines in respect to early and
343 late leaf spot diseases and biochemical traits. Legume Research; DOI: 10.18805/LR-4833.

of
344 7. Tomar, Y.S., Tiwari, S., Tripathi, M.K., Singh, S. and Gupta, N. (2022). Genetic

ro
345 diversity, population structure and biochemical parameters estimations driving variations in
346
-p
groundnut germplasm. Legume Research. DOI: 10.18805/LR-4965.
re
347 8. Yadav, S., Tiwari, S., Tripathi, M. K., Tripathi, N., Gupta, N. and Tiwari, S. (2023).
lP

348 Evaluation of high oleic acid content in a set of 96 genotypes of Arachis hypogaea L.
349 Scientist, 2, 132-143.
na

350 9. Pramanik, A., Tiwari, S., Tripathi, M.K., Mandloi, S., Tomar, R.S. (2021). Identification of
351 groundnut germplasm lines for foliar disease resistance and high oleic traits using SNP and
ur

352 gene-based markers and their morphological characterization. Legume Research, DOI:
Jo

353 10.18805/LR-4666.

354 10. Mandloi S, Tripathi MK, Tiwari, Tripathi, N. (2022). Genetic diversity analysis among late
355 leaf spot and rust resistant and susceptible germplasm in groundnut (Arachis hypogea L.).
356 Israel Journal of Plant Sciences. doi:https://doi.org/10.1163/22238980-bja10058

357 11. Varshney, R.K., Thudi, M., Pandey, M.K., Tardieu, F., Ojiewo, Vadez, V, Whitbread,
358 K.H.M. et al., (2018). Accelerating genetic gains in legumes for the development of
359 prosperous smallholder agriculture: integrating genomics, phenotyping, systems modelling
360 and agronomy. J. Exp. Bot., 69, 3293-3312

361 12. Deshmukh, D.B., Marathi, B., Sudini, H.K., Variath, M.T., Chaudhari, S., Manohar, S.S. et
362 al. (2020). Combining High Oleic Acid Trait and Resistance to Late Leaf Spot and Rust
363 Diseases in Groundnut (Arachis hypogaea L.). Front. Genet.11, 514. doi:
364 10.3389/fgene.2020.00514

365 13. Verma KS, Ul Haq S, Kachhwaha S, Kothari SL. RAPD and ISSR marker assessment of
366 genetic diversity in Citrullus colocynthis (L.) Schrad: a unique source of germplasm highly
367 adapted to drought and high-temperature stress. 3 Biotech. 2017 7(5):288.

368 14. Raina SN, Rani V, Kojima T, Ogihara Y, Singh KP, Devarumath RM. (2001) RAPD and
369 ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal
370 identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and
371 wild species. Genome,44(5):763-72. PMID: 11681599.

of
372 15. Shoba, D., Manivannan, N., Vindhiyavarman, P., Nigam, S.N. (2012). SSR markers

ro
373 associated for late leaf spot disease resistance by bulked segregant analysis in groundnut
374
-p
(Arachis hypogaea L.). Euphytica, 188, 265-272.
re
375 16. Varma, T.S.N., Dwivedi, S.L., Pande, S., Gowda, M.V.C. (2005). SSR markers associated
lP

376 with resistance to rust (Puccinia arachidis Speg.) in groundnut (Arachis hypogaea L.).
377 SABRO J. 37, 107-119.
na

378 17. Sujay, V., Gowda, M.V.C., Pandey, M.K. (2012). Quantitative trait locus analysis and
379 construction of consensus genetic map for foliar disease resistance based on two recombinant
ur

380 inbred line populations in cultivated groundnut (Arachis hypogaea L.). Mol. Breed. 30, 773-
Jo

381 788.

382 18. Shirasawa, K., Bertioli, D.J., Varshney, R.K., Moretzsohn, M.C., Leal-Bertioli, S.C.M.,
383 Thudi, M., et al. (2013). Integrated consensus map of cultivated peanut and wild relatives
384 reveals structures of the A and B genomes of Arachis and divergence of the legume genomes.
385 DNA Res. 20, 173-184.

386 19. Sukruth, M., Paratwagh, S.A., Sujay, V, Kumari, V., Gowda, M.V.C., Nadaf, H.L. (2015).
387 Validation of markers linked to late leaf spot and rust resistance, and selection of superior
388 genotypes among diverse recombinant inbred lines and backcross lines in peanut (Arachis
389 hypogaea L.). Euphytica, 204,343–351.

390 20. Pandey, M.K., Khan, A.W., Singh, V.K., Vishwakarma, M.K., Shasidhar, Y., Kumar, V
391 (2017). QTL-seq approach identified genomic regions and diagnostic markers for rust and
392 late leaf spot resistance in groundnut (Arachis hypogaea L.). Plant Biotechnol. J. 15, 927–
393 941. doi: 10.1111/pbi.12686

394 21. Mahatma, M.K., Thawait, L.K., Jadon, K.S., Thirumalaisamy, P.P., Bishi, S.K., Rathod, K.J.,
395 Verma, A., Kumar, N., Golakiya, B.A. (2021) Metabolic profiling for dissection of late leaf
396 spot disease resistance mechanism in groundnut. Physiol Mol Biol Plants, 27 (5): 1027-
397 1041. doi: 10.1007/s12298-021-00985-5.

398 22. Subrahmanyam, P., McDonald, D., Waliyar, F., Reddy, L.J., Nigam, S.N., Gibbons, R.W.
399 (1995). Screening methods and sources of resistance to rust and late leaf spot of groundnut.
400 Information Bulletin no. 47. Patancheru 502324, Andhra Pradesh, India: International

of
401 Crops Research Institute for the Semi-Arid Tropics., 24 pp.

ro
402 23. Murray, M.G., Thompson, W.F. (1980). Rapid isolation of high molecular weight plant
403 DNA. Nul. Acids Res., 8, 4321-4325.
-p
re
404 24. Tiwari, S., Tomar, R.S., Tripathi, M.K., Ahuja, A. (2017) Modified protocol for plant
lP

405 genomic DNA isolation. Indian Res J Genet Biotechnol, 9 (4): 478–485.

406 25. Mace, E.S., Phong, D.T., Upadhaya, H.D., Chandra, S., Crouch, J.H. (2006). SSR analysis of
na

407 cultivated groundnut (Arachis hypogaea L.) germplasm resistant to rust and late leaf spot
408 diseases. Euphytica, 152, 317-330.
ur

409 26. Mondal, S., Sutar, S.R., Badigannavar, A.M. (2008). Comparison of RAPD and ISSR marker
Jo

410 profiles of cultivated peanut genotypes susceptible or resistant to foliar diseases. Food Agric
411 Environ 6:181–187.

412 27. Zhao, S., Guo, Y., Sheng, Q., Shyr, Y. (2014) Advanced heat map and clustering analysis
413 using heatmap3. Biomed Res Int. 986048. doi: 10.1155/2014/986048.

414 28. Liu, K., Muse, S. (2004). Power marker: New genetic data analysis software, version 27.

415 29. Tamura, K., Dudley, J., Nei, M., Kumar, S. (2007) MEGA4: Molecular evolutionary genetics
416 analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24:1596-1599

417 30. Mubai, N., Sibiya, J., Mwololo, J., Musvosvi, C., Charlie, H., Munthali, W., et al., (2020)
418 Phenotypic correlation, path coefficient and multivariate analysis for yield and yield-
419 associated traits in groundnut accessions. Cogent Food Agri. 6, 1, 1823591, DOI:
420 10.1080/23311932.2020.1823591.

421 31. Zaman, M., Tuhina-Khatun, M., Ullah, M., Moniruzzamn, M., Alam, K. (2011). Genetic
422 variability and path analysis of groundnut (Arachis hypogaea L.). The Agricult., 9 (1–2), 29–
423 36. https://doi. org/10.3329/agric. v9i1–2.9476.

424 32. Rao, V.T., Venkanna, V., Bhadru, D., Bharathi, D. (2014). Studies on variability, character
425 association and path analysis on groundnut (Arachis hypogaea L.). Int. J. Pure Appl. Biosci.
426 2 (2), 194-197. http://www.ijpab.com/vol2-iss2.php.

of
427 33. Almeida, W.S.D., Fernandes, F.R.B., Teófilo, E.M., Bertini, H.C.D.M. Bertini. (2014).
428 Correlation and path analysis in components of grain yield of cowpea genotypes.

ro
429 Rev.Ciên.Agron., 45(4), 726–736. https://doi.org/10.1590/S1806-66902014000400010.

430
-p
34. Kozak, M.; Azevedo, R.A. (2014). Sequential path analysis: What does “sequential” mean?
re
431 Scientia Agricola, 71 (6), 525–527. https://doi.org/10.1590/ 0103-9016-2014-0186.
lP

432 35. Reddy, A.T., Sekhar, M.R., Vijayabharathi, A., Pathy, T.L., Reddy, G.L., Jayalakshmi, V.
433 (2017). Correlation and path analysis of kernel yield and its components in groundnut
na

434 (Arachis hypogaea L.). Int. J. Curr. Microbiol. Appl. Sci. 6 (12), 10–16.
435 https://doi.org/10.20546/ijcmas.2017. 612.002.
ur

436 36. Mohammed, K.E., Afutu, E., Odong, T.L., Okello, D.K., Nuwamanya, E., Grigon, O.,
Jo

437 (2018). Assessment of groundnut (Arachis hypogaea L.) genotypes for yield and resistance to
438 late leaf spot and rosette diseases. J. Exp. Agri. Int. 21(5), 1–13. https://doi.org/
439 10.9734/JEAI/2018/39912.

440 37. Muitia, A. (2011). Farmer perceptions and genetic studies of rosette disease in groundnut
441 (Arachis hypogaea L.) in northern Mozambique. PhD Thesis, University of KwaZulu-Natal,

442 38. Killi, F., Beycioglu, T. (2022). Genetic and environmental variability, heritability and genetic
443 advance in pod yield, yield components, oil and protein content of peanut varieties. Turk. J.
444 Field Crops, 27 (1), 71-77.

445 39. Govindaraj, M.M. Vetriventhan, Srinivasan M. (2015). Importance of genetic diversity
446 assessment in crop plants and its recent advances: An overview of its analytical perspectives.
447 Genet. Res. Int.,1–14. https://doi.org/10.1155/2015/431487.
448 40. Niveditha, P.D., Sudharani, M., Rajesh, A.P., Nirmala, P.J. (2016). Genetic diversity based
449 on cluster and principal component analysis for yield, yield components and quality traits in
450 peanut stem necrosis tolerant groundnut (Arachis hypogaea L.) genotypes. J. Res. ANGRAU,
451 44, 6–12. https://angrau.ac.in/ angrau/pdf/44(3&4) %202016%20volume.pdf.

452 41. Siddiquey, M.N.H., Haque, M.M., Ara, M.J.F., Ahmed, M.R., Roknuzzaman, M. (2006).
453 Correlation and path analysis of groundnut (Arachis hypogaea L.). Int. J. Sust. Agri. Technol.
454 2 (7), 6–10.

455 42. Banerjee, P., Maity, S., Maiti, S.S., Banerjee, N. (2007). Influence of genotype on in vitro
456 multiplication potential of Arachis hypogaea L. Acta Botany Croat, 66 (1), 15–23.

of
457 43. Gokidi, Y. (2005). Evaluation of a mini core set of germplasm in groundnut (Arachis

ro
458 hypogea L.). MSc Thesis, Dharwad University of Agricultural Sciences.

459
-p
44. Upadhyaya, H.D., Bramel, P.J., Ortiz, R., Singh, S., (2002). Developing a mini core of
re
460 peanut for utilization of genetic resources. Crop Sci., 42 (6), 2150-2156.
lP

461 45. Shaibu, A.S., Miko, Z.L., Ajeigbe, H.A., Mohammed, S.G (2020). Genetic diversity and
462 stability of groundnut mini-core collections for early and late leaf spot resistance in Nigeria.
na

463 Afr. Crop Sci. J., 28, 23-32.

ur

464 46. Zanjare, S.R., Suryawanshi, A.V., Zanjare, S.S., Shelar, V.R., Balgude, Y.S. (2020).
465 Screening of groundnut (Arachis hypogaea L.) genotypes for identification of sources of
Jo

466 resistance against leaf spot disease. Leg. Res., DOI: 10.18805/LR-4370

467 47. Mondal, S., Badigannavar, A.M. (2010). Molecular diversity and association of SSR markers
468 to rust and late leaf spot resistance in cultivated groundnut (Arachis hypogaea L.). Plant
469 Breed., 129, 68–71.

470 48. Pandey, M.K., Monyo, E., Ozias-Akins, P., Liang, X., Guimarães, P., Nigam, S.N. (2012).
471 Advances in Arachis genomics for peanut improvement. Biotechnol. Adv., 30, 631–51.
472 http://dx.doi.org/10.1016/j.biotechadv.2011.11.001.

473 49. Sahu, P., Khare, D., Tripathi, N., Saini, N. (2012). Molecular screening for disease resistance
474 as strategic and tactical gene pool in soybean. J. Food Leg., 25, 200-205.
475 50. Tiwari, S.,Tripathi, N., Tsuji, K., Tantwai, K. (2019). Genetic diversity and population
476 structure of Indian soybean (Glycine max (L.) Merr.) as revealed by microsatellite markers.
477 Physiol. Mol. Biol. Plants, 25, 953-964

478 51. Varshney, R.K., Bertioli, D.J., Moretzsohn, M.C., Vadez, V., Krishnamurthy, L., Aruna, R.
479 et al., (2009). The first SSR-based genetic linkage map for cultivated groundnut (Arachis
480 hypogaea L.). Theor Appl Genet. 118, 729–739

481 52. Yeri, S.B., Bhat, R.S. (2016). Development of late leaf spot and rust resistant backcross lines
482 in Jl 24 variety of groundnut (Arachis hypogaea L.). Electron J. Plant Breed.7,37–41.
483 10.5958/0975-928X.2016.00005.3.

of
484 53. Abbas, M., El-Shabrawi, H., Hamza, M., Wahba, H., Shaba, M. (2020). Association between

ro
485 Productivity, Fatty Acid Profiles, Oil Bodies’ Ultrastructure and Molecular Markers in
486 Peanut (Arachis hypogaea L.)
-pCultivars. Agronomy, 10, 1401;
re
487 doi:10.3390/agronomy10091401.
lP

488 54. Bera, S.K., Kamdar, J.H., Kasundra, S.V., Patel, S.V., Jasani, M.D., Maurya, A.K. (2019).
489 Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty
na

490 acid desaturase mutant alleles and its effect on seed germination along with other seedling
491 traits. PloS One, 14, e0226252. doi: 10.1371/journal.pone.0226252.
ur

492 55. Hammond, E.G., Duvick, D., Wang, T., Dodo, H., Pittman, R.N (1997). Survey of the fatty
Jo

493 acid composition of peanut (Arachis hypogaea) germplasm and characterization of their
494 epoxy and eicosenoic acids. J Am. Oil Chem. Soc., 74,1235–1239

495 56. Yol, E., Furat, S., Upadhyaya, H.D., Uzun, B. (2018). Characterization of groundnut
496 (Arachis hypogaeaL.) collection using quantitative and qualitative traits in the Mediterranean
497 Basin. J. Integ. Agri. 17, 63-75.

498 57. Khan, M.M.H., Rafii, M.Y., Ramlee, S.I., Jusoh, M., Mamun, M.A., Halidu, J. (2021). DNA
499 fingerprinting, fixation-index (Fst), and admixture mapping of selected Bambara groundnut
500 (Vigna subterranean [L.] Verdc.) accessions using ISSR markers system. Sci Rep. 11, 14527.
501 https://doi.org/10.1038/s41598-021-93867-5

502
503
 507
506
505
504

Jo
ur
na
lP
re
-p
ro
of
508 Table 1 List of groundnut genotypes with their parentage, origin, habitate and cluster pattern of
509 SSR and ISSR markers

SSR ISSR
S No. Genotype Parentage ICG Origin HBT
cluster cluster

1. ICGV 13282 AH 7808 41103 - VUL V III

2. ICGV 13569 SALOUM 44.3 SEN HYR V I

3. ICGV 13208 AH5 4119 - HYR V XIII

4. ICGV 13536 CS 888 1376 IND VUL V I

5. ICGV 13220 MD 351 4335 - HYR V IV

of
IPM. SP. II VII
6. ICGV 13271 145 ARG VUL

ro
PEANUT

7. ICGV 13212 AH 675 4172 - - II VII

8. ICGV 13213 AH7245 4201-p - HYR II VII

re
9. ICGV 8003 S 7-2-26 2691 SDN HYB II XII
lP

KANYOMA II XI
10. ICGV 13240 2637 - HYR
BULK
na

11. ICGV 13295 U 2-24-6 4688 - VUL III XII

ur

12. NCC NO. 75 - - - - V II

Jo

COTE DE V VI
13. ICGV 1936 2709 AUS HYR
IVORIE

14. ICGV 13562 R-7-47-9 4386 SDN HYR VI I

SPANISH II III
15. ICGV 13224 2837 - HYR
PEANUT

16. ICGV 13270 U 4-7-24 1808 SDN VUL V VII

17. ICGV 9912 V 32 4567 ARG FST V VII

18. ICGV 8110 28-13 2879 SEN HYR II XII

19. ICGV 13543 BANSAI NO.1 4611 TZA HYB II VI

20. ICGV 13575 SOLAM A 4404 SEN HYR

21. ICGV 13574 KONGWA 4329 TZA HYR V VII

 RUNNER

22. ICGV 2850 V 53 630 UNK HYB IV II

23. ICGV 13283 AH 7999 4111 - VUL V XII

24. ICGV 13574 AH 7599 4452 SDN HYB V VII

SOURTHERN II V
25. ICGV 9930 4643 USA VUL
CROSS

26. ICGV 3 RS 101 30 IND FT VI V

27. ICGV 9955 III V

of
28. ICGV 2521 10-1 4734 UNK VUL II XI

ro
29. ICGV 4276 NC 1 4478 YSA HYB VIII XII

30. ICGV 7123 RCM 593 1924 UNK FST II XII

31. ICGV 13519 U 4-47-16 4713

-p ZAF VUL V VIII
re
32. ICGV 13228 A6 4542 - VUL V IX
lP

33. ICGV 13281 U 4-47-11 1867 SDN VUL V VII

na

34. ICGV 9934 TIPO 4662 ARG VUL III VII

35. ICGV 13549 TESO 4527 UGA HYB I VII

ur

VIRGINIA V IX
36. ICGV 8115 2885 USA HYB
Jo

RUNNER

37. ICGV 9895 R 7-4-9 4368 TZA HYR II V

38. ICGV 9525 CS 110 11386 IND VUL V X

39. ICGV 13269 EC 24395 1804 BOL VUL VI V

40. ICGV 13554 41-98 4401 SEN HYR IV V

41. ICGV 13218 AH 7267 1271 SDN FST V IV

42. ICGV 8171 AH 6712 2985 MYA HYB IV VII

43. ICGV 13518 AH 1720 2016 USA VUL V III

RUSSAIN V II
44. ICGV 9112 1127 SUN FST
INTERNA
45. ICGV 13274 AH 7522 2487 CHN HYB V V

46. ICGV 5672 MK 371 602 NGA HYB VI II

47. ICGV 13535 AH 7311 4546 CHN VUL V I

48. ICGV 7349 B 28 5641 UNK HYB V XII

49. ICGV 13264 U 1-2-1 4528 - HYB II XI

50. ICGV 6300 U 2-1-1 1374 ZAR VUL II XI

COLORADO II V
51. ICGV 13293 4569 - VUL
MANFRE

of
52. ICGV 9931 TAINAN #1 4650 TWN VUL IV XI

ro
53. ICGV 9224 AH 7276 1579 TZA VUL II IX

54. ICGV 13544 ZM 2617-1 11292 ZMB HYB II VI

TENNESSEE
-p II VIII
re
55. ICGV 9932 4657 USA FST
RED
lP

56. ICGV 13523, 948/DLEO/32 4737 ARG VUL VIII VII

57. ICGV 13297 U 4-7-17 4692 - VUL IV VIII

na

58. G13547, AH 7599 4230 SDN HYB V VII

ur

59. ICGV 6360 U 4-7-3 1447 NGA VUL II VII

Jo

60. ICGV 13214 AH 7295 4207 - HYR VII VII

61. ICGV 9249 AH 7276 1579 TZA VUL V X

62. ICGV 5660 28-206- RR 589 SEN HYB V VII

63. ICGV 8171 AH 6712 2985 MYA HYB IV VIII

64. ICGV 7988 S 7-1-9 2668 NGA HYR IV VII

65. ICGV 13238 A 477-1 2465 - HYR IV VII

66. ICGV 13573 AC 17213 4305 USA HYR V IX

Farmers field of II VII

67. Shivpuri Local 71 Shivpuri, MP, IND HYR
India

68. Shivpuri Local 72 Farmers field of IND HYR IV VII

Shivpuri, MP,
 India

Farmers field of IV X
69. Shivpuri Local 73 IND HYR
Shivpuri

Farmers field of III IX

70. Shivpuri Local 74 IND HYR
Shivpuri

Farmers field of IV IX
71. Shivpuri Local 75 IND HYR
Shivpuri

Farmers field of IV IX
72. Shivpuri Local 76 IND HYR
Shivpuri

Farmers field of VIII VIII

of
73. Shivpuri Local 77 IND HYR
Shivpuri

ro
Farmers field of IV IX
74. Shivpuri Local 78 IND HYR
Shivpuri

75. Shivpuri Local 79

Farmers field of
-p
IND HYR
II IX
re
Shivpuri
lP

Farmers field of III IX

76. Shivpuri Local 80 IND HYR
Shivpuri
na

Farmers field of III VII

77. Shivpuri Local 81 IND HYR
Shivpuri
ur

Farmers field of III IX

78. Shivpuri Local 82 IND HYR
Shivpuri
Jo

Farmers field of I IX
79. Shivpuri Local 83 IND HYR
Shivpuri

Farmers field of VI VII

80. Shivpuri Local 84 IND HYR
Shivpuri

Farmers field of VII IX

81. Shivpuri Local 85 IND HYR
Shivpuri

Farmers field of I VII

82. Shivpuri Local 86 IND HYR
Shivpuri

Farmers field of I VII

83. Shivpuri Local 87 IND HYR
Shivpuri

Farmers field of VI VII

84. Shivpuri Local 88 IND HYR
Shivpuri
 Farmers field of V IX
85. Shivpuri Local 89 IND HYR
Shivpuri

Farmers field of VI VII

86. Shivpuri Local 90 IND HYR
Shivpuri

Farmers field of VI VII

87. Shivpuri Local 91 IND HYR
Shivpuri

Farmers field of VII IX

88. Shivpuri Local 92 IND HYR
Shivpuri

Farmers field of VI X
89. Shivpuri Local 93 IND HYR
Shivpuri

of
Virginia III VII
90. Girnar-2 M 13 x R 33-1 IND
bunch

ro
(ICGV 87121 X V IX

-p
ICGV 87853) X
91. KDG-128 IND HYR
ICGV 92023) X
re
ICGV 98300)
lP

TG-23 [TGS-2 X V V
92. TG-26 TG-1 (X-ray mut)] IND HYR
na

Virginia V VIII
93. GANGAPURI Local Check IND
bunch
ur

Check variety with Virginia V IX

Jo

94. SUNOLIC 95-R IND

high oleic contents bunch

Local check foliar Virginia V VIII

95. JGN-3 IND
disease sensitive bunch

KRG 1 x ICGV V VII

86855
Virginia
96. GPBD-4 Check Variety IND
bunch
Foliar disease
resistant
510 SEN=Senegal; IND=India; ARG= Argentina; SDN= Sudan; AUS=Australia; TZA= Tanzania; UNK=Kosovo;
511 USA= United states; YSA; yuvaslabia; UNK= United nation; ZAF= South Africa; UGA= Uganda; BOL=Bolovia;
512 MYA= Myanmar; CHN= China; NGA= Nigeria; ZAR= South Africa; TWN=Taiwan; TZA=Tanzania; ZMB=
513 Zambia

514
515

516 Table 2 Correlation coefficient among morpho-physiological traits of groundnut genotypes.

Correlations

DF DM PHT PPP WP WK SP BYD HI ELS30 ELS45 LLS70 LLS85

DF 1 0.028 -0.246* -0.170 0.039 0.012 0.017 0.171 -0.063 0.068 0.029 -0.007 -0.034

DM 1 -0.094 -0.154 -0.198 -0.265** -0.185 0.063 -0.239* 0.141 0.128 0.182 0.209*

PHT 1 0.119 0.214* 0.184 0.003 -0.065 -.013 -0.177 -0.141 -0.083 -0.063

PPP 1 0.346** 0.296** -0.009 0.377** 0.348** -0.210* -0.223* -0.204* -0.159

WP 1 0.906** 0.034 0.321** 0.302** -0.401** -0.410** -0.342** -0.309**

of
WK 1 0.439** 0.265** 0.205* -0.392** -0.394** -0.350** -0.332**

ro
SP 1 -0.036 -0.137 -0.056 -0.046 -0.056 -0.082

-p
BYD re 1 -0.119 -0.145 -0.180 -0.207* -0.193

HI 1 -.086 -0.051 -0.005 0.005

ELS30 1 0.969** 0.843** 0.831**

lP

ELS45 1 0.816** 0.801**

na

LLS70 1 0.983**

LLS85 1
ur

517 *. Correlation is significant at the 0.05 level (2-tailed).

518
Jo

**. Correlation is significant at the 0.01 level (2-tailed).

519 DF= Days to 50% flowering, DM = Days to maturity, PHT = Plant height, PPP = Pod per plant, WP = Pod weight, WK = Kernel weight, Sp=
520 Shelling percentage, BYD = Biological yield, HI = Harvesting index, ELS30 = Early leaf spot at 30 days, ELS45 = Early leaf spot at 45 days,
521 LLS70= Late leaf spot at 45 days, LLS85= Late leaf spot at 85 days.

522

523
524

525 Table 3 Molecular data analysis of groundnut for SSR and ISSR markers

Marker MAF Allele No Gene Diversity PIC

SSR

PM 137 0.5208 4.00 0.6491 0.6019

GM 2079 0.4375 3.00 0.6398 0.5642

Seq 5D05 0.5104 3.00 0.6074 0.5328

Seq 13A07 0.4896 3.00 0.6287 0.5573

of
GM 2301 0.5104 3.00 0.6196 0.5499

ro
Mean 0.4938 3.20 0.6289 0.5612

ISSR
-p
re
ISSR 21 0.3854 4.00 0.6625 0.5886
lP

ISSR 7 0.4479 3.00 0.6131 0.5313

ISSR 20 0.4688 3.00 0.6196 0.5416

na

ISSR8 0.5833 4.00 0.5992 0.5557

ur

ISSR 3 0.5417 4.00 0.5877 0.5137

Jo

Mean 0.4854 3.6 0.6164 0.5462

526 MAF-Major allele frequency

527
528
529
530
 of
ro
-p
re
lP
na
ur
Jo

531
532
533 Fig.1. Diversity assessment and heat map of groundnut genotypes based on morphological data.

534
 of
ro
535

536
-p
Fig.2 Disease scores of early and late leaf spots and their symptoms on groundnut leaves
re
537
lP

538
na
ur
Jo
 of
ro
-p
re
lP
na
ur
Jo

539

540 Fig. 3 Heat map of groundnut genotypes based on disease scoring of early and late leaf spot in different
541 groundnut genotypes.

542
543

544

of
ro
-p
re
lP
na
ur
Jo

545

546 Fig. 4 Dendrogram of groundnut genotypes based on SSR markers data

547

548
549

550

551

of
ro
-p
re
lP
na
ur
Jo

552

553 Fig.5 Dendrogram showing relationship among groundnut genotypes based on ISSR markers.

554
On behalf of all authors, the corresponding author states that there is no conflict of
interest.

f
r oo
-p
re
lP
na
ur
Jo