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Elucidation of Genetic Relationships in the Genus Cajanus Using Random Amplified Polymorphic DNA Marker Analysis | OMICS International
ISSN: 2329-9002
Journal of Phylogenetics & Evolutionary Biology
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Elucidation of Genetic Relationships in the Genus Cajanus Using Random Amplified Polymorphic DNA Marker Analysis

Ramya Ranjan Mishra1, Bharati Behera2 and Jogeswar Panigrahi1*
1Plant Biotechnology Laboratory, School of Life Sciences, Sambalpur University, Jyoti Vihar, Burla, Sambalpur-768019, Odisha, India
2Parija PG Department of Botany, Ravenshaw University, Cuttack-753003, Odisha, India
Corresponding Author : Dr. Jogeswar Panigrahi
Reader, Plant Biotechnology Laboratory
School of Life Sciences
Sambalpur University, Jyoti Vihar
Burla, Sambalpur-768019, Odisha, India
Tel: +91-9437130857
E-mail: [email protected]
Received January 15, 2013; Accepted February 06, 2013; Published March 04, 2013
Citation: Ramya Ranjan M, Bharati B, Jogeswar P (2013) Elucidation of Genetic Relationships in the Genus Cajanus Using Random Amplified Polymorphic DNA Marker Analysis. J Phylogen Evolution Biol 1:102. doi:10.4172/2329-9002.1000102
Copyright: © 2013 Ramya Ranjan M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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 In this present investigation, a total of 27 RAPD primers were used to elucidate the genetic relationships between cultivated Cajanus cajan cultivars and 10 allied species of primary, secondary and tertiary gene pool, including C. cajanifolius, C. scarabaeoides, C. platycarpus, C. albicans, C. volubilis, C. sericeus, C. acutifolius, C. lineatus, C. lanceolatus and C. reticulates. All primers showed polymorphism at species level and produced 215 unequivocal polymorphic bands, with an average of 7.96 bands per primer. These polymorphic primers exhibited variation with regard to average band informativeness, resolving power, and showed high polymorphism information content value. No single primer was able to distinguish between all the two cultivars of C. cajan and ten allied species of Cajanus, but several species specific amplified fragments were observed. The pair wise Jaccard’s similarity coefficient values revealed high level of inter-specific genetic variation in the genus Cajanus. Cluster analysis exhibited the grouping of two C. cajan accessions with C. cajanifolius in one cluster, while except C. platycarpus, all the nine wild Cajanus species belonging to the secondary and tertiary gene pool form another cluster. The present analysis more or less agreed with the sectional classification of the genus Cajanus, and it has been hypothesized that cultivated pigeonpea has evolved through multi-genomic interaction through C. cajanifolius, and it has experienced minor genomic reorganization during its divergence. Again, identification of species specific amplification pattern substantiated the utility of RAPD markers on selection of suitable species to transfer the desirable trait into cultivated C. cajan, through marker aided breeding for its genetic augmentation, and also for the effective management of genetic resources of C. cajan.

Cajanus; Phylogenetic analysis; RAPD
RFLP: Restriction Fragment Length Polymorphism; RAPD: Random Amplified Polymorphic DNA; AFLP: Amplified Fragment Length Polymorphism; SSR: Simple Sequence Repeat; PIC: Polymorphism Information Content; Ib: Band informativeness; AvIb: Average Band informativeness; Rp: Resolving power; UPGMA: Unweighted Pair group Method with Arithmetic mean
Pigeonpea, botanically known as Cajanus cajan (Linn.) Millsp, is an important grain legume crop of the semi-arid tropics, with somatic chromosome complement 2n=2x=22. This legume crop refers as SKIPPER of pulse world. It is widely cultivated in Asia and Africa, in addition to some parts of Australia and Latin America [1]. The major centre of world production is undoubtedly India, where pigeonpea is the second most important pulse crop next to chickpea. Among the countries growing this crop, India has the largest area (3.53 million hectares) under pigeonpea cultivation, and also contributes a major share (2.51 million tons), about 75% of world production, with an average yield of 0.78 tons/hectare [2]. Being a monotypic crop with poor cultivation practice in marginal and submarginal land and heterozygous genome structure, the genetic improvement depends upon the alien gene transfer from its secondary and tertiary gene pool [1,3,4], and to selection and perpetuation of useful kind of gene action through conventional recombination breeding [5]. Hence, there is a need for the assessment of genetic relationship between C. cajan and its wild allies, the donor source for desired agro-economic traits. Studies on genetic origin of pigeonpea are still unsettled and reports were available in favour of both monophyletic and polyphyletic origin. Studies on morphology [3,4,6], cytology and crossability [7,8], isozymes [9] and nuclear RFLPs [10], suggested a monophyletic origin from C.cajanifolius. On the other hand, studies on seed protein profiling [11-13] and nuclear DNA amounts [14], suggested a polyphyletic origin of the C. cajan.
In the last couple of decades, the DNA based markers are potentially used for elucidation of phylogenetic relationships across the taxa, in addition to its exploitation in marker aided breeding, molecular tagging and mapping of genes [15]. Among the DNA markers, RAPD based marker system was simple, and are equally reliable for genetic studies because RAPDs are indefinite in number, ubiquitously distributed throughout the genome, and capable of high level polymorphism. RAPD markers have also been widely used in the grain legumes for the depiction of genetic relationships among cultivars, among wild allies, or between cultivars and wild allies [16-25]. In Cajanus species, RAPD markers have been employed for genetic fingerprinting, genetic diversity assessment and gene tagging [26-28]. We report here on the utilization of RAPD markers to elucidate and validate the genetic relationships between the cultivated C. cajan and ten allied species of Cajanus, being the potential donor source of genes during inter-specific breeding programmes.
Materials and Methods
Plant materials
Seeds of two cultivars of pigeonpea(C. cajan (L.) Millsp.), BDN- 2 and DSLR-17 and 10 wild species (Table 1) were obtained from ICRISAT, Patancheru, Andhra Pradesh. The species are maintained in the experimental garden of School of Life Sciences, Jyoti Vihar, Odisha.
DNA extraction and purification
Fresh and young leaf samples of equal quantity (~ 1.2g) were collected for isolation of genomic DNA. Genomic DNA was isolated by using SDS method [29], with few modifications [30]. DNA was dissolved in 10 mM Tris/1 mM EDTA (T10E1) buffer. DNA concentration and purity was measured by using UV-Vis spectrophotometer (UV 1601, Shimadzu, Japan), with T10E1 buffer (pH 8.0). For further confirmation, the quantification of DNA was accomplished by analyzing the purified DNA on 0.8% agarose gel, along with diluted uncut λ DNA as standard. DNA from two cultivars of C. cajan and 10 wild species were equilibrated to concentration of 10 ng/μl, using T10E1 buffer.
RAPD marker generation
For RAPD analysis, PCR amplification of 20 ng of genomic DNA, obtained from two cultivars of C. cajan and 10 wild species, were carried out using 27 standard decamer oligonucleotide primers (OPA and OPB series; Operon Tech., Alameda, CA, USA), individually [31]. Each amplification reaction mix of 25 μl contained the 30 ng template DNA, 2.5 μl of 10X assay buffer (100 mM Tris.Cl, pH 8.3; 0.5 M KCl; 0.1% gelatin), 2 mM MgCl2, 200 μM each of the dNTPs, 20 ng primers, 1.0 U Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Bangalore, India). The amplification was carried out in a thermal cycler (Veriti-96, Applied Biosystem, USA), programmed for initial denaturation of 5 min at 94°C; 45 cycles of 2 min denaturation at 94°C, 1 min annealing at 37°C and 2 min elongation at 72°C, and final elongation step of 5 min at 72°C. The PCR products were separated on 1.4% agarose gel for electrophoresis in TAE (40 mM Tris acetate; 2 mM EDTA) buffer at 50 V for 4 h, stained with ethidium bromide and visualized by UV transilluminator (M-15, UVP, Upland, CA 91786, USA). The size of the amplified fragments were determined using 250 bp ladder (B. Genei, Merck Bioscience, India), as molecular weight marker and TOTAL LAB SOLUTIONS-V 2003.02 software. The presence and absence of amplified fragments in each case was scored as ‘1’ and ‘0’, respectively.
Statistical analysis
The information content of RAPD marker system was calculated for each marker and locus using the polymorphism information content {PIC=1-Σ f2(1-n), where ‘f ’ is the frequency of allele (1-n) } [32], band informativeness {Ib=1- (2×[0.5-p]), where, ‘p’ is the proportion of the total genotypes having the band}, average band informativeness {Av Ib=Σ Ib/n, where, Ib–Band informativeness and ‘n’ is the number of markers loci analysed}, and resolving power {Rp=Σ Ib} of the primer [33]. The RAPD marker data was arranged in a binary matrix, with rows corresponding the RAPD banding pattern, and column to the taxa in question (OTUs). The scores were ‘1’ for presence and ‘0’ for absence of a RAPD band. NTSYS-PC 2.02e was used to estimate the similarity matrices among the taxa using Jaccard’s coefficient indices. The similarity matrix thus generated was used to construct a dendrogram by Unweighted Pair Group Method with Arithmetic mean (UPGMA), using Sequential, Agglomerative, Hierarchical and Nested (SAHN) method [34], and the confidence limits were measured by squared elucidation distance interval. Again, the Principal Coordinate Analysis (PCoA), based on Jaccard’s similarity coefficient was done using eigen vector of NTSYS-PC 2.02e.
Generation of RAPD markers and genotypic characterization
For this purpose, two cultivars of C. cajan and ten different species of the genus Cajanus were considered. A total of 27 decamer primers (Table 2) were used for RAPD analysis. Amplification of all 27 primers generated 215 unequivocal scorable bands, which are polymorphic at species level (Figures 1a and b). The size of amplification products ranges from 140 bp to 4,030 bp. The maximum of 13 loci were amplified with primer OPA 15, whereas only one loci was observed with primer OPB 04. These 27 polymorphic primers exhibited variation, with regard to average band informativeness (AvIb) and resolving power (Rp). The primer OPA 16 showed highest AvIb (0.675), while OPB 03 and OPB 08 showed lowest AvIb of 0.142. The primer OPA 16 showed highest Rp (7.426) and the primer OPA 10 showed lowest Rp (0.432) values. All the 27 primers exhibited high PIC values. But among them, OPB 03 and OPB 08 showed high PIC (0.994) and OPA 16 showed low PIC (0.806) values. Although all 27 primers were found to be polymorphic, no single primer was able to differentiate the 11 species of Cajanus. But, amplification by different primers was informative for the identification of two cultivars of C. cajan and eight species of Cajanus (Table 3).
Genetic relationships in the genus Cajanus
The similarity matrix indices were estimated among 11 species of Cajanus using 215 RAPD markers, to quantify the level of polymorphism for inter-specific studies. The pair wise Jaccard’s similarity indices values ranged from 0.416 to 0.954 (Table 4), which evidenced large amount of genetic variation exist between the species of Cajanus at the genome level. Among all the allied species, C. cajanifolius is pretty close to C. cajan genotypes, with similarity indices (0.796 and 0.833). The Jaccard’s similarity matrix data was utilized to construct a dendrogram. Dendrogram analysis exhibited the clustering of C. cajan accessions with C. cajanifolius (Section-Cajanus) in one cluster, while rest of the wild Cajanus species except C. platycarpus, belonging to the tertiary gene pool, formed another cluster respectively (Figure 2). However, two species (C. lanceolatus and C. reticulates), belonging to section Fruticosa were clubbed together with similarity indices 0.928. Similarly, species belonging to Atylia (C. lineatus and C. sericeus), Cantharospermum (C. albicans and C. scarabaeoides) and Fruticosa (C. acutifolius, C. lanceolatus and C. reticulates) also form close clusters. C. sericeus (sec. Atylia) shows greater similarity with C. volubilis (sec. Volubilis) than with C. lineatus of the same section. C. platycarpus (sec. Rhynchosoides) is found to be out grouped. These clustering patterns were further supported by two and three dimensional principal coordinate analysis (Figures 3a and b).
C. cajan is the only domesticated species in the subtribe Cajaninae (genus-Cajanus), and is predominantly self-pollinated. So, its genetic augmentation has been restricted to selection and hybridization within cultivars. With gradual realization that wild relatives of C. cajan are potential sources for its genetic improvement, attempts were made for wide hybridization, to introduce some desirable traits into C. cajan. However, this requires the knowledge of species affinities and the phylogenetic relationships inter se. RAPD based DNA markers has been extensively utilized to deduce the genetic relatedness of plant cultivars and plant populations, as well as the inter- and intra-specific genetic relationships between plant species [15-25]. This is due to the simplicity of this technique, as only very small amounts of DNA are required, and information on template DNA sequence is not needed [31].
In the present investigation, the RAPD marker used not only for the elucidation of genetic relationship in the genus Cajanus, but also for the identification of species specific RAPD markers in the genus Cajanus, at least for the 11 species used. All the responded 27 primers were found to be polymorphic at species level and generated 215 unequivocal scorable polymorphic bands. The size of amplification products ranges from 140 bp to 4,030 bp. Maximum 13 loci were amplified with one primer OPA 15, whereas minimum one amplicon was observed with the primer OPB 04. The PIC, AvIb and Rp values were varied from one primer to another. The RAPD markers OPA 02725, OPA 05290, OPA 081450, OPA 051020, OPA 091160, OPB 06530,OPA 03700, OPA 04955, OPA 031183, OPA 131500, OPA 15865, OPA 051450, OPA 061490, OPA 08860, OPA 181665, OPA 191725, OPB 011710, OPB 0603530,OPA 031585, OPA 05690, OPA 071315, OPA 181525, OPA 071420, OPA 161090, OPB 023055 and OPA 10640,555 were unique to different species of Cajanus, while OPA 02725, OPA 05290 and OPA 081450 were unique to the C. cajan cultivars used in the present study. Several RAPD markers have detected for the identification of pigeonpea cultivars, as well as the allied species of Cajanus [25]. However, the primer set, cultivar set and allied species are different in both studies. Recently, species specific RAPD and ISSR markers have detected to differentiate C. cajan and C. scarabaeoides [35], and SSR markers to differentiate C. cajan and C. cajanifolius [36]. No other information was available on the identification of pigeonpea cultivars and wild species at DNA level. As a result, pigeonpea breeding relies heavily on a phenotypic selection method. Secondly, pigeonpea is one of the exceptions among grain legumes, in that it has tendency towards out crossing. As result of frequent outcrossing, existing standard cultivars have become heterogeneous for several important agronomic characters, such as pest and disease resistance. For instance, the cultivar ‘Bahar’ lost its resistance to sterility mosaic disease, perhaps as a result of out crossing with susceptible cultivars [37]. The maintenance of germplasm in pigeonpea is very tedious, and problems of contamination have been enormous. The identification of DNA markers for specific traits in pigeonpea will, therefore, be helpful in assessing the purity and stability of the genotypes entering into the breeding programme. Similarly, the species could clearly be distinguished, with as few as one selected primer, or with polymorphic amplicons. These species specific markers may also be utilized to track the introgressive wide hybridization programme for the genetic augmentation in pigeonpea.
The phylogenetic analysis in the genus Cajanus is equivocal. Studies based on morphology [3-4,6], cytology and crossability [7], isozymes [9], nuclear RFLPs [10] and r-DNA analysis [38], suggest a monophyletic origin from C. cajanifolius. On the other hand, the seed storage protein profiles [11-13] and nuclear DNA amounts [14] suggest a polyphyletic origin of the cultigens. In the present study, eleven species representing the genus Cajanus (including cultigen C. cajan) showed high genetic variation, ranging from 0.416 to 0.954. The low similarity indices might be due to non-response of four species, C. volubilis, C. platycarpus, C. reticulates and C. lanceolatus to five primers for amplification. However, earlier studies reported the similarity indices value of 0.22 to 0.801 among eight wild species of Cajanus, including C. acutifolius, C. albicans, C. goensis, C. grandifolius, C. sericeus, C. volubilis, C. lineatus, C. reticulates, and five species of three genera Dunbaria, Rhynchosia and Flemingia [25]. This incongruence in the result might be due to the different set of primers, different species, and different genotypes taken into consideration. However, the variation between two cultivated accessions is much less than their differences with C. cajanifolius pattern. The allied species showed varying degrees of similarity with the cultigens, indicating their relative genetic closeness to C. cajan. This substantiates the gene introgression from allied species (which is still effective) has also taken place during the course of evolution of pigeonpea in the genus Cajanus [13]. Further diversification of cultivated species might be due to structural alterations of the genome and environment specific expression of house keeping genes.
On the basis of RAPD banding pattern and Jaccards similarity indices, a dendrogram was constructed (SAHN), which exhibited the clustering of C. cajan accessions with C. cajanifolius (Section-Cajanus) in one cluster, while species belonging to the section Cantharospermum (C. albicans and C. scarabaeoides), Atylia (C. lineatus and C. sericeus), Fructicosa (C. acutifolius, C. lanceolatus, C. reticulates), and Volubilis (C. volubilis) form separate cluster. The species C. platycarpus belongs to section Rhynchosoides, out grouped by maintaining equidistant from species of both clusters. The clustering pattern was also supported by two dimensional and three dimensional principal coordinate analyses. RAPD banding pattern indicated C. reticulates and C. lanceolatus are close to each other than to C. acutifolius and, C. acutifolius, showed equal sharing of RAPD bands with the species of the section Fructicosa, Atylia and Cajanus, including C. cajan. Likewise, a study based on esterase zymogram [9], also showed a close relationship between C. albicans and C. scarabaeoides and close homology between C. cajan and C. cajanifolius, while the pattern of C. platycarpus was found to be distinctly different from other species. However, the clustering of C. lineatus and C. sericeus (belongs to section Atylia) into a single cluster was also reported earlier [25]. This variation might be due to different set of primers used in both studies. The overall cluster pattern based on RAPD banding pattern partially supports the sectional classification of the genus [3]. These results also agreed with nuclear RFLPs based parsimony analysis [10], and seed albumin and globulin profiling [13]. Again in the present study, C. platycarpus is out grouped and justified its status in the tertiary gene pool [3]. However, the results of present study were at variance with crossability relationships and DNA content studies [8,14], and it might be due to the genetic divergence during the course of evolution under selection pressure. The evidences obtained from different studies till now, including the present study, unequivocally support C. cajanifolius to be the closest relative of cultivated pigeonpea [3,4,6,7,9-11,13]. RAPD banding pattern shared several homology not only with C. cajanifolius, but also with several allied species, including C. scarabaeoides, C. lineatus, C. albicans, C. volubilis and C. sericeus. These results based RAPD pattern, similarity indices and clustering pattern substantiated that C. cajan and C. cajanifolius are a product of multigenomic interaction, at least involving C. scarabaeoides, and both had experienced minor genomic reorganization during its divergence, due to existing gene flow within the genus.
Again this study presented the suitability of RAPD marker as potential tool on elucidation of genetic relationship and species differentiation. Future studies on genetic association and validation of identified species specific RAPD markers led to a platform for transfer of the desirable agronomic traits into cultigen C. cajan for its genetic augmentation, at least by involving two species C. cajanifolius and C. scarabaeoides.
The authors are highly grateful to University Grant Commission (F. No. 37- 331/2009 (SR)) and Department of Biotechnology (BT/PR13468/AGR/02/702/ 2010), Govt. of India for financial assistance through R & D project to Jogeswar Panigrahi. The authors are also grateful to Dr. H.D. Upadhyaya, Principal Scientist, ICRISAT, for providing the germplasm of the plant material required for the study.


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