Indian Journal of Biotechnology Vol 16, July 2017, pp 346-356

Molecular markers in assessing genetic variation of Indian ( medica L.) collected from different parts of India

Ajit Uchoi1, S K Malik2*, Ravish Choudhary1, Susheel Kumar2, Digvender Pal1, M R Rohini1 & Rekha Chaudhury2 1Indian Agricultural Research Institute, Pusa, New Delhi-110012 India 2Tissue Culture and Cryopreservation Unit, National Bureau of Genetic Resources (NBPGR), Pusa Campus, New Delhi-110 012, India

Received 2 June 2014; revised 28 August 2015; accepted 28 August 2015

Genetic diversity of forty six representative accessions of Citrus medica L. () was estimated using 17 (RAPD) random amplification of polymorphic DNA and 11 inter simple sequence repeat (ISSR) markers. A total of 213 RAPD and 105 ISSR bands were generated, out of which, 175 (RAPD) and 77 (ISSR) bands were polymorphic. Polymorphism percentage of RAPD (82%) was comparatively higher than ISSR (73%). Genetic similarity value was found in the range of 0.49 to 0.96 (average 0.73), 0.38 to 0.99 (average 0.69) and 0.47 to 0.96 (average 0.72) in RAPD, ISSR and combined data analyses, respectively. Moderately high levels of polymorphism and genetic similarity within C. medica suggested that citron cultivars have a significant level of genetic diversity. A dendrogram generated based on unweighted pair group method of arithmetic- average (UPGMA) separated all the accessions into two main clusters. The placement of C. medica accessions in various sub-clusters and groups in the dendrogram was based on molecular differentiation of individual accessions rather than their geographical origin. Both, RAPD and ISSR analyses were able to identify elite genotypes of C. medica with unique accession specific DNA fragments. Thus, both the markers can be utilized for inferring the extent and distribution of genetic diversity in C. medica.

Keywords: Citrus medica, genetic diversity, ISSR, RAPD, UPGMA

Introduction these true or closely related genera followed, mainly, The genus Citrus belongs to the sub-tribe Citrineae by natural mutations. Recently, this idea has gained within the sub-family Aurantioideae of the family support from various biochemical and molecular Rutaceae and has a pre-dominantly tropical to semi- studies8-11. tropical distributions. It is one of the most important There exists a high level of genetic variations among fruit crops in the world with over 122 million tons of the cultivated Citrus species due to frequent bud international production1. mutations, widely sexual compatibility between Citrus Currently, there are two widely used classification genus and related genera, apomixes and the long systems in Citrus: Swingle2-3 and Tanaka4. The history of cultivation and the worldwide dispersion7. Swingle system included 16 species under two sub- Elucidating relationships, , and diversity genera - Citrus and , while the Tanaka system is important for developing breeding strategies, recognized 162 species under the sub-genera conserving biodiversity, and improving breeding ArchiCitrus and MetaCitrus. However, Scora5 and efficiency. Also understanding of genetic variability in Barrett & Rhodes6 suggested that there are only three Citrus is critical for characterizing germplasm, ‘basic’ true species of Citrus within the sub-genus controlling genetic erosion and the registration of new Citrus i.e., Citron (C. medica L.), Mandarin cultivars10,12. (C. reticulata Blanco) and Pummelo (C. maxima L. Citrus medica (commonly known as citron) one of Osbeck). Later, Scora7 added C. halimi as another the important Citrus fruits of the world, is considered a true species. Other cultivated species within native of India5,13 and is found to grow in a wild and Citrus were derived from hybridization between semi-wild state in primary as well as secondary forests along the foothills of the Eastern Himalayas and —————— North-East India4,14,15. The true citron is called bira- *Author for correspondence [email protected] jora or bakol-khowa-tenga (bakol means rind, Tel: +91-011-25848955 khowa to eat and tenga stands for sourness) in Assam UCHOI et al.: MOLECULAR MARKERS IN ASSESSING GENETIC VARIATION OF CITRUS MEDICA 347

valley plains, jaara-jamir in Sylhet and Cachar, extensive sampling from its natural habitat, is the first Soh-manong in the Khasi Hills, Sutrung in Bengal14 etc attempt to assess the RAPD and ISSR polymorphism of India. Citron is monoembryonic in nature and is and genetic variability in Indian citron cultivars considered as one of the basic species of Citrus. The collected from different parts of India particularly from citron is used mainly for medicinal purposes: to combat the North-East India. sea sickness, pulmonary troubles, intestinal ailments, and other disorders. Materials and Methods

Natural populations of C. medica are vanishing Plant Materials at alarming rate in the North-East and other parts Specific exploration and collection missions were of India due to lack of proper conservation efforts, undertaken to different parts of India particularly the hence necessitating need for systematic management North-Eastern region of India viz., Assam, Arunachal of its genetic resources. Preliminary morphological Pradesh, Meghalaya, Nagaland, Sikkim, North- characterization of Indian citron cultivars growing in Western states of Himachal Pradesh, Uttarakhand and North-East India has been carried out by Ray and Punjab for the survey and collection of C. medica Deka16, indicating significant variability within the germplasm. A total of forty six representative species. However, extensive studies using molecular accessions of citron cultivars were collected from wild, markers on the existing genetic diversity in different semi-wild and cultivated conditions for RAPD and parts of India has not been studied till now. The ISSR analysis (Table 1). Collections were mostly importance of C. medica in the ancestry of Citrus has made following selective sampling strategy, where been put into question mark till date. Due to this samples collected from single plant was given an confusion of the role of C. medica, there is an urgent indigenous collection (IC) number and treated as need for studying the extent of diversity occurring in individual accession. Leaf and fruit samples of citron from different parts of India. each accession were taken for confirmation of Use of molecular markers has more advantages than taxonomic identity, characterization and DNA that of morphology based phenotypic characterization, extraction. Detailed information of each accession because molecular markers are generally unaffected by was recorded in National Bureau of Plant Genetic external impact. It is possible to compare accessions Resources (NBPGR) database. of a collection at any time of year using molecular markers, while phenotypic characteristics can be DNA Isolation influenced by environmental or climatic affects. Many Total genomic DNA was isolated from all the forty six accessions through cetyl trimethyl ammonium studies have utilized molecular markers to examine the 39 genetic diversity and phylogenetic relationships among bromide (CTAB) method with some modifications . Citrus and its related genera, including isozymes12, Quantification of isolated DNA was done RFLP8, ISSR17-19, RAPD8-9, 20, DAMD21, cpDNA spectrophotometrically. DNA quality was determined sequence22-23, SSR10 and AFLP24. through electrophoresis on 0.8% agarose gel.

The random amplified polymorphic DNA (RAPD) RAPD-PCR Amplification and the ISSR (inter simple sequence repeats) markers The RAPD primers of Operon Technologies are widely used in different studies. For example, (Alameda, CA, USA) were used for molecular RAPD markers have been used for analysis of genetic analysis. A total of 50 primers were screened with C. diversity in Citrus20,25,27, for characterization of Citrus medica, of which 17 primers were selected for final hybrids in breeding programs28, for identification of profiling based on banding patterns and reproducibility C. lemon29 and for phylogenetic analysis9,19,30. ISSR (Table 2). The basic protocol of RAPD-PCR reported markers have been used for assessing genetic diversity by William et al.40 with minor modifications was in clementine31 and in lemon32, identification followed for PCR amplification in a total reaction in Citrus and Poncirus33-34, constructing genetic volume of 25 μl, containing 10 mM Tris-HCl, 50 mM 35 linkage map in C. grandis and P. trifoliata , in KCl, 2.5 mM MgCl2, 1.0 U Taq DNA polymerase management of Citrus germplasm resources36, (G-Biosciences, India), 0.2 mM of dNTP each, 0.2 μM discrimination of clones of sour oranges37, and of RAPD primer and 20-25 ng of DNA template. molecular differentiation in Citrus germplasm DNA amplification was carried out in a BioeR resources of Iran38. The present study, based on an Xp thermocycler and the thermal cycler conditions for 348 INDIAN J BIOTECHNOL, JULY 2017

Table 1 — Details of 46 accessions of Citrus medica collected from different parts of India, used for RAPD and ISSR analysis. Sl No. Collector Cultivar name Source Latitude Longitude IC number Biological status number 1 MD-17 Bemberia Sakhu, East Sikkim 27.13 88.30 586997 Semi-wild 2 MD-22 Bemberia Lingzo, South Sikkim 27.03 88.25 587002 Semi-wild 3 MD-30 Bemberia Darjeeling, West Bengal 27.04 88.39 587010 Semi-wild 4 MS-15 Themachi East Garo Hills, Meghalaya 25.29 90.19 558139 Wild 5 MS-21 Themachi East Garo Hills, Meghalaya 25.29 90.19 558140 Wild 6 MS-41 Themachi East Garo Hills, Meghalaya 25.29 90.19 587029 Wild 7 MS-42 Themachi East Garo Hills, Meghalaya 25.35 90.16 558147 Wild 8 MS-58 Themachi East Garo Hills, Meghalaya 25.30 90.20 - Wild 9 MD-154 Themachi East Garo Hills, Meghalaya 25.30 90.20 417222 Wild 10 MD-176 Themachi South Garo Hills, Meghalaya 25.44 90.82 417243 Wild 11 IC-344926 Themachi East Garo Hills, Meghalaya 25.30 90.20 344926 Wild 12 MD-11/48 Pati jora Tinsukia, Assam 27.31 95.21 591406 Cultivated 13 MD-11/49 Bira jora Tinsukia, Assam 27.31 95.21 591407 Cultivated 14 MD-11/52 Soh- mondong Tinsukia, Assam 27.31 95.21 591410 Cultivated 15 MD-11/63 Citron Tinsukia, Assam 27.31 95.21 591421 Cultivated 16 MD-11/64 Gondharaj Tinsukia, Assam 27.31 95.21 591422 Cultivated 17 MD-11/65 Pongam-Citron Tinsukia, Assam 27.31 95.21 591424 Cultivated 18 MD-11/66 Holong Tenga Tinsukia, Assam 27.31 95.21 591425 Cultivated 19 MD-11/73 Bore Tenga Tinsukia, Assam 27.31 95.21 591433 Cultivated 20 MD-11/83 Jora Tenga Joygukhawa, Assam 27.32 97.23 591442 Cultivated 21 MD-11/84 Citron Dibru Saikhowa, Assam 27.35 95.21 591443 Natural wild 22 MD-11/99 Chonchuno Tesophenyu, Nagaland 25.58 94.13 591458 Wild 23 MD-202 Upper Subansiri, 27.56 94.20 417268 Wild Arunachal Pradesh 24 CITRON-AP Tayum Upper Subansiri, 27.56 94.20 583259 Wild Arunachal Pradesh 25 A-I Tayum West Siang, Arunachal Pradesh 28.05 94.42 - Wild 26 CITRON-I Tayum West Siang, Arunachal Pradesh 28.05 94.42 583270 Wild 27 MSA-11/18 Etrog Hamirpur, Himachal Pradesh 31.50 76.30 - Cultivated 28 MBS-11 Citron Abohar Field Gene Bank, Punjab 30.21 74.22 - Cultivated 29 MD-61 Citron Abohar field Gene Bank, Punjab 30.21 74.22 311379 Cultivated 30 MDY-71 Citron Abohar field Gene Bank, Punjab 30.21 74.22 395400 Cultivated 31 MD-467 Citron Abohar field Gene Bank, Punjab 30.21 74.22 311349 Cultivated 32 N/SKV/1418 Citron Bahraich, Uttar Pradesh 28.35 79.42 219067 Cultivated 33 P/N/SKV-1418 Citron Bahraich, Uttar Pradesh 28.35 79.42 319067 Cultivated 34 MAU-01 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 35 MAU-02 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 36 MAU-03 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 37 MAU-04 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 38 MAU-05 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 39 MAU-06 Citron Bhowali Research Station, 29.20 79.30 - Cultivated Uttarakhand 40 IC-318908 Citron Bhowali, Uttarakhand 29.20 79.30 318908 Cultivated 41 IC-319049 Citron Bhowali, Uttarakhand 29.20 79.30 319049 Cultivated 42 IC-320210 Citron Bhowali, Uttarakhand 29.20 79.30 320210 Cultivated 43 IPS/PNB/319 Citron Bhowali, Uttarakhand 29.20 79.30 326648 Cultivated 44 IC-202113 Citron Bhowali, Uttarakhand 29.20 79.30 202113 Cultivated 45 IC-285406 Citron Bhowali, Uttarakhand 29.20 79.30 285406 Cultivated 46 A1N1 Citron Bhowali, Uttarakhand 29.20 79.30 - Cultivated

UCHOI et al.: MOLECULAR MARKERS IN ASSESSING GENETIC VARIATION OF CITRUS MEDICA 349

Table 2 — Details of amplified bands generated by 17 RAPD and 11 ISSR primers in 46 accessions of C. medica Sl. No. Primer code Sequence 5’-3’ Total no. of bands PBa PPBb PICc Amplified fragment length RAPD 1. OPA-04 AATCGGGCTG 15 13 87 0.3433 355-995 2. OPB-12 CCTTGACGCA 14 13 93 0.3220 220-936 3. OPC-08 TGGACCGGTG 9 7 78 0.3337 296-905 4. OPD-02 GGACCCAACC 18 14 78 0.2741 328-942 5. OPD-03 GTCGCCGTCA 15 12 80 0.2986 300-1005 6. OPD-11 AGCGCCATTG 12 12 100 0.3057 242-820 7. OPD-15 CATCCGTGCT 10 8 80 0.3205 275-882 8. OPD-20 ACCCGGTCAC 18 15 83 0.3068 295-880 9. OPF-01 ACGGATCCTG 5 3 60 0.2043 496-715 10. OPF-02 GAGGATCCCT 11 8 73 0.3303 290-770 11. OPF-03 CCTGATCACC 10 8 80 0.1630 320-700 12. OPF- 16 GGAGTACTGG 15 12 80 0.3170 225-895 13. OPU-01 ACGGACGTCA 11 9 82 0.2445 298-1200 14. OPU-02 CTGAGGTCTC 15 12 80 0.3191 332-1498 15. OPU-05 TTGGCGGCCT 14 11 79 0.3793 364-958 16. OPU-08 GGCGAAGGTT 10 8 80 0.3202 355-2000 17. OPU-14 TGGGTCCCTC 11 10 91 0.2590 580-1350

Total 213 175 82 % 0.2965 ISSR 1. UBC-808 (AG)8C 14 11 79 0.4069 310-565 2. UBC-809 (AG)8G 10 8 80 0.4021 280-982 3. UBC-810 (GA)8T 12 9 75 0.1398 300-746 4. UBC-811 (GA)8C 11 8 73 0.3781 250-824 5. UBC-812 (GA)8A 10 7 70 0.2632 275-750 6. UBC-834 (AG)8YT 11 8 73 0.3395 332-735 7. UBC-836 (AG)8YA 9 7 78 0.3503 360-1350 8. UBC-842 (GA)8YG 8 6 75 0.3641 140-325 9. UBC-868 (GAA)6 7 5 72 0.3215 385-642 10. UBC-880 (GGGTG)3 5 3 60 0.3829 745-962 11. UBC-889 DBD (AC)7 8 5 63 0.3081 298-805

Total 105 77 73 % 0.3324

PCR reactions were an initial denaturation cycle for conditions programmed as initial pre-denaturation at 2 min at 94°C, followed by 40 cycles comprising 94°C for 4 min, followed by 35 cycles of denaturation 1 min at 94°C, 1 min at 35°C and 2 min at 72°C. at 94°C for 1 min, annealing at optimized temperature An additional cycle of 5 min at 72°C was used for for 1 min, and extension at 72°C for 2 min. A final 7 min final extension. extension at 72°C followed the completion of 35 cycles.

ISSR-PCR Amplification Gel Electrophoresis For ISSR-PCR optimization trials, a total of 50 The amplification products were separated by primers of University of British Columbia (UBC # 9) electrophoresis on 1.8% agarose gels stained with were custom synthesized from Bioscript, India Gel view stain (G-Biosciences, India) at 120 V for 3 h and screened in citron accessions. Out of 50, only and bands were visualized and documented under 11 primers, gave the best amplification results with UV Gel Doc System (Mega Biosystematica, U.K). the entire DNA samples, were selected for the final Data Analysis ISSR-PCR analysis (Table 2). PCR-amplification was The amplification products were scored as 1 and 0 carried out in 25 ml reaction volume containing for present and absent bands or respectively. 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.0- 2.5 mM Molecular weight of the amplified bands was estimated MgCl2, 0.2 mM dNTP each, 1.0 U Taq DNA by using 1kb DNA ladder (G-Biosciences, India) as polymerase (G-Biosciences, India), 0.2 mM primer and standard. The data matrix was analyzed using the 20-25 ng genomic DNA. The DNA amplification was Numerical Taxonomy and Multivariate Analysis carried out in a BioeR Xp thermocycler with reaction System (NTSYS) ver. 2.10e software41. 350 INDIAN J BIOTECHNOL, JULY 2017

Polymorphism information content (PIC) value, Results which is an index of the usefulness of each primer for RAPD Analysis diversity analysis, was determined as described by A total of 213 bands were amplified with 17 RAPD Smith et al.42. primers in 46 accessions of C. medica, of which PIC = 1 -∑fi2 Where, fi is the frequency of the ith allele. 175 bands (82%) were polymorphic (Table 2). The

total number of amplified bands per primer varied A pair wise similarity matrix of all the accessions from 5 to 18 (average 12.5), while number of were estimated based on Jaccard’s coefficient43 and a polymorphic bands ranged from 3 to 15 (average dendrogram was generated based on the unweighted 10.29) bands per primer. A representative gel pair group method for Arithmetic average (UPGMA) profile generated using the primer OPD-02 is given in using the same software. Two-way Mantel test for Figure 1a The approximate size of amplified goodness of fit for UPGMA cluster to the binary data fragments ranged from 220-2000 bp. The maximum and principal coordinate analysis (PCOA) were also number of bands (18) was generated by OPD-02 and performed using the NTSYS ver. 2.01e software41. OPD-20 primers, while OPF-01 primer amplified The binary data were also used to calculate genetic the minimum number of bands (5). The maximum diversity parameters like observed number of polymorphic bands (15) were obtained with the primer alleles per locus (na), effective number of alleles per OPD-20 and minimum bands (3) were generated with locus (ne), total heterozygosity (Ht) and Shannon’s primer OPF-01. The PIC value of each primer varied information index (I) using the software POPGENE from 0.163 (OPF-03) to 0.379 (OPU-05) with an 1.3144. Considering the uneven distribution of the average of 0.297. Some of the RAPD primers were target taxon in the study sites, and the asymmetry in able to generate accession specific unique fragment the number of sampled genotypes from different in ten different accessions (Table 3). localities, the genetic diversity parameters were A pair wise similarity value among all the calculated using the single population approach under 46 accessions of C. medica ranged from 0.49 to 0.96 the assumption of Hardy Weinberg equilibrium. Table 3 — Unique bands for RAPD and ISSR markers identified in the present study. Sl. Primers Number of Band size Accession no. Nos. bands (bp) RAPD 1. OPA-04 1 980 MD-11/49 2. OPC-08 2 450 IC-219067 900 MAU-06 3. OPD-11 1 670 CITRON-AP 4. OPD-20 3 820 MD-22 590 MD-17 420 MD-22 5. OPF-03 3 700 IC-319049 470 MD-22 450 MD-22 6. OPF-16 3 750 MSA-18 270 MD-22 240 MD-17 7. OPU-01 1 1150 MD-17 8. OPU-02 1 750 IC-202113 9. OPU-05 1 700 MD-154 10. OPU-08 1 1800 IC-202113 11. OPU-14 1 1330 IC-202113 Total 18 ISSR 1. UBC-808 1 MS-42 310 2. UBC-809 1 MD-11/65 700

3. UBC-810 1 MD-22 440 Fig 1 a) — RAPD gel profile of primer OPD-02, b) ISSR gel profile 4. UBC-834 1 MD-22 350 of primer UBC-808 Total 4 UCHOI et al.: MOLECULAR MARKERS IN ASSESSING GENETIC VARIATION OF CITRUS MEDICA 351

with an average of 0.73. Clustering pattern of A 2D plot generated from principle co ordinate C. medica revealed that all the accessions from analysis (PCOA) of RAPD data also supported the different localities grouped on the basis of genetic clustering pattern of the UPGMA dendrogram. relationship despite their geographical distribution. First and second principal components accounted for All accessions were separated into two main clusters 31.03 and 9.71 %, respectively, of the total variation. in the UPGMA dendrogram (Fig. 2). The first cluster The values of mean observed number of alleles comprised of 23 accessions in which two accessions (na), mean effective number of alleles (ne), total namely MD-17 and MD-22 clustered distinctly heterozygosity (Ht), and Shannon’s information index with other accessions. The second cluster also (I) were 1.991, 1.418, 0.257 and 0.400, respectively. consisted of 23 accessions where accessions nos. A1N1 and IC-285406 was found to be closely related ISSR Analysis with a similarity value of 0.96. A total of 105 bands were scored by 11 primers Mantel test revealed high and significant co- among the 46 accessions of C. medica, of which phenetic correlation coefficient (r = 0.96), which shows 77 bands were polymorphic (73%) with an average of a very good fit of UPGMA dendrogram to the data. 7 polymorphic bands per primer (Table 2). The

Fig 2 — UPGMA dendrogram of 46 Indian citron accessions based on RAPD marker

Fig 3 — UPGMA dendrogram of 46 Indian citron accessions based on ISSR marker 352 INDIAN J BIOTECHNOL, JULY 2017

fragment size ranged from 140 bp to 1350 bp. such as observed number of alleles (na), effective A representative ISSR gel profile is shown in Figure number of alleles (ne), total heterozygosity (Ht), and 1(b). Each of the 11 primers amplified a range of 5 to Shannon’s information index (I) were recorded as 14 bands with an average of 9.5 bands per primer. The 1.981, 1.519, 0.312 and 0.475, respectively. primer UBC-808 generated the maximum number of bands (14), whereas UBC-880 amplified the minimum Cumulative Analysis of RAPD and ISSR Data number of bands (5). Primer UBC-808 generated the The binary data of 46 accessions of C. medica, highest number of 11 polymorphic bands. PIC value derived from RAPD and ISSR analyses were pooled to of each primer varied from 0.139 to 0.407 (average form a single data set. Similarity value ranged from 0.332). Some of the primers were able to generate 0.47 to 0.96 with an average of 0.72. Similarity value unique marker bands in certain accessions (Table 3). was found minimum (0.47) between IC-202113 and A pairwise similarity value among all the accessions of MD-11/65; A1N1 and MD-11/84 and maximum (0.96) C. medica ranged from 0.38 to 0.99 with an average between A1N1 and IC-285406 accessions. The value of 0.69. The UPGMA dendrogram placed all the UPGMA-based dendrogram from the combined data 46 accessions in two main separate clusters (Fig. 4). set separated all the accessions in two main clusters The first cluster consisted of eight accessions. The (Fig. 4). Cluster I contained 23 accessions represented second cluster consisted of 38 accessions which were from Northeastern states i.e., Assam, Meghalaya, further sub-divided into three sub-clusters. Within this Nagaland and Sikkim. Accession MD-17 (Bemberia) cluster, MSA-17 and MBS-11 accessions were closely and MD-22 (Bemberia) formed a separate sub-cluster related to each other with a similarity value of 0.99. within the main cluster. The second cluster also Correlation value (r) was calculated as 0.99 by comprised 23 accessions and represented accessions Mantel test, which showed a very good fit of the from Arunachal Pradesh, Punjab, Himachal Pradesh, clustering pattern to the data. A 2D plot generated from Uttarakhand and Uttar Pradesh. In Cluster II, Tayum principle co-ordinate analysis (PCOA) of ISSR data cultivars (Citron I, A-I and Citron-AP) from Arunachal also supported the clustering pattern of the UPGMA Pradesh was found to be distinct as it grouped into a dendrogram. First and second principal components separate sub-cluster within the main cluster. accounted for 23.48 and 15.44 %, respectively, of the A 2D plot generated from PCA of RAPD and ISSR total variation. Other genetic diversity parameters, data (Fig. 5) also supported the clustering pattern of

Fig 4 — UPGMA dendrogram of 46 Indian citro accessions based on combine data of RAPD & ISSRmaker UCHOI et al.: MOLECULAR MARKERS IN ASSESSING GENETIC VARIATION OF CITRUS MEDICA 353

UPGMA dendrogram. The first ten PCs contributed matrices were used for co-phenetic analysis, which 65.16 % of the total variability of the analyzed showed very significant correlation (r = 0.96) between accessions. The first five PCs accounted for 47.33 % of the two separate data sets. the total variability and the first three accounted for The values of genetic structure data like observed 37.16 % of the variance, in which maximum variability number of alleles (na), effective number of alleles (ne), was contributed by first component (22.45 %) followed total heterozygosity (Ht) and Shannon’s information by second component and third component (8.97 and index (I) were recorded to be 1.986, 1.468, 0.284 and 5.74 %, respectively). Both ISSR and RAPD similarity 0.437, respectively. The performance of two markers was evaluated using various parameters such as percentage of polymorphism, average bands and polymorphic bands per primer, polymorphic information content (PIC), average similarity values, observed number of alleles (na), effective number of alleles (ne), total heterozygosity (Ht), Shannon’s information index (I) (Table 6).

Discussion Several powerful marker techniques are currently available for genetic analysis of both plant and animal species. The choice of the most appropriate markers and technique principally depends on the objective of research, and the biology and genetic structure of the species. Therefore, comparisons are desirable in order to fix on the most appropriate technique for the issue being examined45. In this study, genetic diversity in 46 accessions of C. medica collected from different parts of India was assessed using RAPD and ISSR

Fig 5 — 2 D Plot of combined RAPD & ISSR data markers. This study has demonstrated the potential of RAPD and ISSR markers as a rapid, reproducible and Table 4 — Eigen values, differences, percentage of proportions useful method for distinguishing different accessions and cumulative for 10 principal co ordinate axes derived from of C. medica and for clustering of the accessions combined RAPD and ISSR data of C. medica into different groups. RAPD (17 primers) and Axis Eigenvalue Percent Cumulative ISSR (11 primers), were sufficient to provide 1 10.10518 22.4559 22.4559 significant information on the genetic diversity 2 4.036004 8.9689 31.4248 within C. medica. 3 2.580493 5.7344 37.1593 RAPD and ISSR data generated from 46 accessions 4 2.429374 5.3986 42.5579 of Citron with 17 and 11 primers, respectively, 5 2.14733 4.7718 47.3297 were sufficient to provide inferences on genetic 6 1.813052 4.029 51.3587 differentiation and relationships among the accessions. 7 1.682165 3.7381 55.0969 In the 46 accessions of C. medica, moderate level of 8 1.633686 3.6304 58.7273 polymorphism (73%) was estimated by ISSR markers, 9 1.56895 3.4866 62.2138 while RAPD markers revealed moderately high level 10 1.326139 2.947 65.1608 of polymorphism (82%). The variations in the level of

Table 5 — Comparison of polymorphism and population genetic data generated by RAPD and ISSR primers in 46 accessions of C. medica Marker system PPB Avg no. of Avg no. of Mean Range of na ne I Ht (Total primers used) bands/ PB/ primer PIC similarity Primer value (Avg.) RAPD (17) 82 12.5 10 0.2965 0.73 1.9906 1.4180 0.2567 0.4001 ISSR (11) 73 9.5 7 0.3324 0.68 1.9810 1.5178 0.3116 0.4742 Combined (28) 78 11 8.5 0.3146 0.72 1.9858 1.4679 0.2842 0.4372 354 INDIAN J BIOTECHNOL, JULY 2017

polymorphism generated by these two markers are due A search for unique bands was made for all the to different target regions in the genome. RAPD primer accessions tested, in which a total of eighteen unique targets entire genome including coding and non-coding bands were generated in ten accessions by 11 RAPD regions as well as repeated sequences40, while ISSR primers and four unique bands in three accessions by 4 primer targets only region between two microsatellite ISSR primers. In accession no. MD-22 (Bemberia), a loci. Similar results were found in several earlier maximum of two unique bands was given by primer studies based on RAPD and ISSR markers system in OPD-20 (820 bp, 420 bp) and OPF-03 (470 bp, Vigna umbellata46, Melocanna baccifera47, Melilotus48 450 bp). Similarly, unique bands were generated and Cymbopogon winterianus49, which revealed that in accession MD-17 (Bemberia), MD-11/49 (Pati RAPD marker is more efficient than ISSR for Jora), MD-154 (Themachi), MD-11/65 (Pongam detecting polymorphism at intra-specific level. Basha citron), MS-42 (Themachi), MAU-06, MSA-18 and Sujatha50 also reported higher level of (Etrog), CITRON-AP (Tayum), IC-202113 and RAPD polymorphism (42%) than ISSR (33.5%) in IC-219067. These unique fragments can be used as a Jatropha curcas. In the present study, PIC value marker for identification of these cultivars, which will (0.332) of ISSR markers were observed higher in be useful for future conservation, maintenance and comparison to RAPD markers (PIC = 0.297). Similar breeding programmes. These accessions can also be results were also reported by various other workers used for developing the core collection of C. medica on different crops32, 51-53. However, ISSR marker could germplasm. detect greater total heterozygosity (Ht = 0.312) and Shannon’s information index (I = 0.475) than RAPD, Conclusions i.e. Ht = 0.257 and I= 0.400, respectively. Jaccard’s Each marker has advantage and disadvantage, thus genetic similarity value was found in the range of combining different marker system were greatly better 0.38 to 0.99 (average 0.69), 0.49 to 0.49 to 0.96 for diversity study of Citrus and its relatives. The (average 0.73) and 0.47 to 0.96 (average 0.72) in ISSR, present study confirms that RAPD and ISSR markers, RAPD and combined data analyses, respectively. as a fast and simple technique, can detect enough Genetic similarity values analyzed among all the 46 polymorphism within C. medica germplasm and aid in accessions of C. medica through RAPD and ISSR understanding the inter-relationships. Both marker markers, individually as well as in combination, analyses were also able to generate some accessions showed significant level of genetic variation within specific unique bands in a few cases. Thus, both the C. medica (RAPD, ISSR and Combined). This markers can be utilized for inferring the extent and inference is correspondingly congruent with the values distribution of genetic diversity and were equally of total heterozygosity and Shannon’s information important for genetic diversity analysis in C. medica. index of C. medica generated through RAPD, However, if a finer molecular analysis of Indian citron ISSR markers individually as well as in combination. accessions is required, the use of other molecular marker techniques, e.g. amplification fragment length The resolution of combined dendrogram was 54 comparatively higher than the separate RAPD and polymorphisms (AFLPs) or simple sequence repeat ISSR dendrogram in term of genetic relationship. markers (SSRs) may be considered.

UPGMA dendrogram drawn from RAPD data also Acknowledgement supports the clustering pattern of ISSR data; in both the Authors are grateful to Director, NBPGR and Head, cases no geographic isolation of C. medica accessions Exploration and Collection Division, NBPGR, is shown. Grouping of 46 accessions of C. medica New Delhi for encouragement and support. Thanks was almost similar in all the three dendrograms are also due to officers and staff of Forest Departments (RAPD, ISSR and Combined) except few transitions and ICAR Institutes/ Centres in Northeast India like MD-17 (Bemberia) grouped along with MD-22 for assistance and support during exploration and (Bemberia) in Cluster I in ISSR dendrogram, while in survey trips. RAPD it formed a separate group within Cluster I. One accession from Dibru-saikhowa, Assam (MD-11/83) References: 1 FAO, Food and Agricultural Organization of the United formed a single cluster II in the ISSR dendrogram, but Nations, 2009. http://faostat.fao.org it segregated into a separate group within Cluster I in 2 Swingle W T, The botany of Citrus and its relatives of the the RAPD dendrogram. subfamily Aurantioideae of the family Rutaceae, in UCHOI et al.: MOLECULAR MARKERS IN ASSESSING GENETIC VARIATION OF CITRUS MEDICA 355

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