Introduction

Cashew is native to Brazil, but India is the largest producer and exporter of cashew owing to its highly valued kernel and fruits. Its kernel is nutritionally equivalent to milk, egg and meat and it reduces level of cholesterol in blood. Major producer of cashew are India, Tanzania, Mozambique and Kenya. It is widely adapted to varying agro-climatic conditions and there exists a large variation in cashew genotypes. Cashew is a naturally cross pollinated crop and therefore, it maintains high level of genetic variability amenable for commercial exploitation. Genetic diversity can be studied using morphological, biochemical and molecular markers. However, molecular markers offer special advantage as they cover appreciably large area of the genome (Smith and Smith, 1989) and show detailed genetic differences in a faster way without any environmental influence (Souza et al., 2008). Besides, these are highly heritable and exhibit wide polymorphism to discriminate cultivars, hybrids, mutants and chimeras. Molecular data associated with phenotypic observations can increase the efficiency and quantum of selection in limited time span (Melo et al., 2002). Therefore, many workers attempted to associate molecular data with morphological information to analyze genetic diversity and detection of QTL for agro-economic traits of commercial importance (Cavalcanti et al., 2012). Among the molecular marker techniques, RAPD is simple, cheaper, rapid, and economical and requires very small amount of DNA with lower purity and eliminates the need for blotting, sequence information and radio-active detection. Therefore, a set of high yielding cashew hybrids along with their parents were subjected to molecular analysis for DNA profiling using RAPD primers. Besides, the probable association of DNA profile based clustering pattern in relation to specific characteristic agro-economic features have been investigated for genetic improvement in cashew nut.

Materials and Methods

Genomic DNA was isolated from 2 g tender young leaves of a set of 20 high yielding experimental cashew hybrids comprising ten cross combinations (e.g., Cross A – RP-1X Kalyanpur Bold Nut, Cross B - RP-1 x VTH -711/4, Cross C – RP-2 x Kankadi, Cross D – M-44/3 x VTH 711/4, Cross E – RP-1 x Kankadi, Cross F – RP-2 x VTH711/4, Cross G-RP-2 x Kalyanpur Bold Nut, Cross H-M-44/3 x Kalyanpur Bold Nut, Cross I-Vittol-44/3 x VTH 711/4 and Cross J-BPP-30/1 x Kalyanpur Bold Nut) along with their parents following Rout et. al. (2002). Each of the above 20 cashew hybrids was designated in terms of alphabetical letters followed by numerical numbers to refer cross combination and hybrid clone number. The plant materials were homogenized in liquid Nitrogen and extracted with extraction buffer (1 M Boric acid pH 8.0, 2 mM EDTA, 1.4 M NaCl, 1.5% Cetyltrimethyl ammonium bromide (CTAB), 0.2% β-mercaptoethanol) at 60^oC for one hour with occasional shaking and an equivalent volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture was added and centrifuged at 8,000 rpm for 15 min. at 4^oC. The supernatant was added with equal amount of ice cold absolute ethanol and kept for overnight to precipitate DNA. The intact genomic DNA was hooked out and washed with 70% ethanol and finally re-dissolved in TE buffer (10 mM Tris-HCl, pH-8.0 and 1mM EDTA). The DNA was purified by DNase free RNase-A (GeNei, Bangalore, India) @ 20 µg per ml of DNA extract to remove contaminating RNAs. Finally the quality of DNA was checked using the ratio of absorbance at 260nm and 280nm and also rechecked by running each sample in 0.8% agarose gel. The DNA was quantified through UV-VIS Nanodrop-2000 spectrophotometer (Thermo Electron Scientific Instruments LLC, USA) at 260 nm and diluted to a working concentration of 10 ng/µL for PCR analysis.

Genomic DNA sample of each genotype was individually primed and amplified using 25 random decamer RAPD primers (Chromos Biotech. Pvt. Ltd., Bangalore, India). The amplification was performed in a reaction volume of 25 µL containing 1X reaction buffer (10 mM Tris HCl, pH 9.0, 1.5 mM MgCl₂, 50 mM KCl, 0.01% gelatin), 2.5 mM each of dNTPs, 10 ng of single random primer, 20 ng of genomic DNA and 1 unit of Taq polymerase (Genei, Bangalore). DNA amplification was performed in the Gene-Pro Thermocycler (Bioer Tech. Co., Ltd, Japan), programmed for 4 min at 94^oC, 40 cycles of 1 min at 94^oC, 1 min at 37^oC and 2 min at 72^oC and final extension for 10 min at 72^oC followed by storing at 4^oC till loading to the agarose gel. The amplified products were loaded in 1.6% agarose gel containing 1 µg/mL ethidium bromide and electrophoresed in a constant voltage at 60V. The amplifications were checked for their reproducibility.

The gels were documented by gel doc system (Fire Reader-Uvtec, Cambridge, UK) for scoring the bands. The amplification products were checked for their reproducibility using each primer at least twice. Reproducible bands at about 0.5 mm apart or more were considered for scoring. The presence and absence of bands were scored as 1 and 0 respectively across all the genotypes. The size of amplicons was determined by comparing with the lambda DNA ladder (500 bp) with known size (bp) fragments. Polymorphism information content (PIC) and resolving power (Rp) of a primer were estimated as PIC=∑(1 –pi²)/n and Rp=Σ Ιb, where pi is the proportion of genotypes showing i^th band amplified by the primer, Ιb (band informativeness) =1–[2× (0.5–pi)] and n = total no. of bands produced by the primer (Prevost and Wilkinson, 1999).

The binary data matrix of RAPD score was analysed using the multivariate analysis program NTSYS-PC 2.1(2000-01) to estimate Jaccard’s similarity coefficient (Jaccard, 1908) values. The dendrogram was constructed using Unweighted Paired Group Method with Arithmetic averages (UPGMA) (Sneath and Sokal, 1973) employing Sequential Agglomerative Hierarchic and Non-overlapping clustering (SAHN).

Results and Discussion

Cashew nut is amenable for molecular analysis owing to its small genome size (1.11pg/2C) (Aliyu, 2012). DNA profiling using RAPD markers have been standardized and employed successfully by different workers (Samal, 2002; Croxford et al., 2005 and Thimmappaiah et al., 2009) to analyze samples of Anacardium species. The success in generating polymorphic loci depends on proper choice of primers for DNA amplification and extent of genetic variation among the test genotypes. Dhanaraj et al. (2002) used only five primers for DNA amplification of 90 cashew nut accessions, while only 2-3 primers and even a single primer  were sufficient to distinguish between cultivars of broccoli (Hu and Quaros, 1991) and jack fruit (Gopalsamy et al., 2012) . However, it is in vogue to use more number of primers to differentiate closely related cultivars in cashew nut. In the present investigation, initially twenty five random primers were examined out of which four RAPD primers gave smeared background without distinct fragments and a few of the primers did not produce any amplified products. As a result, fifteen RAPD primers were used for genomic DNA amplification of the test genotypes. The list of such informative primers used in this investigation is presented in Table 1. In the present study, Primer OU-1 amplified two monomorphic amplicons (1560 bp and 610 bp) while, Primer OU-3 and Primer OU-30 produced four monomorphic bands each and Primer OU-20, OU-32 and OU-33 each revealed a single monomorphic band of amplicon size 1950 bp, 1020 bp and 750 bp respectively. A wide array of reproducible PCR products (Figure 1a to Figure 1c) ranging from 210 to 3250 bp was amplified and it happens to be produced by the primer OU 34 (Figure 1c). Mneney et al. (1998) obtained stable and reproducible RAPD profiles of Tanzania cashew using optimal PCR reaction conditions. In the present study, the total number of bands ranged from 4-15 with an average of 7.66 bands per primer and the maximum number of bands being produced by primer OU-34. Neto Silva et al. (1995) observed a total of 27 amplification products with 0-4 bands per primer using six RAPD primers. In the present investigation, RAPD produced as a whole 94 polymorphic amplicons out of total 107 scorable amplified products resulting 87.85% polymorphism. Thimmappaiah et al. (2009) generated 60 bands in 100 cashew germplasm accessions, of which 51 bands were polymorphic (85%) and produced on an average of 5.45 polymorphic bands per primer. Similarly, Thimmappaiah (2011) assessed 60 accessions of cashew in another study using 20 RAPD markers that generated 138 bands, of which 8.1 bands were polymorphic per primer. In mango, RAPD primers yielded 14 monomorphic bands and 96 revealed polymorphism that resulted 87.3% polymorphism as compared to ISSR 79.38% using 46 cultivars (Bajpai et al., 2008), while Samant et al. (2010) obtained 100% polymorphism by all primers. In this context, 31 bands were reported to be polymorphic out of 120 primers tested in grape (Singh and Singh, 2011).

As a whole, the total number of bands produced by 15 RAPD primers varied widely among the genotypes. It ranged from 56 bands in I-12 and J-6 to as high as 72 bands in cashew hybrid E-16. Such differential response of cashew cultivars was also noted by Samal et al. (2003a). The data matrix in the present study resolved a total of 1,378 polymorphic amplicons out of 1,742 PCR products across the test genotypes using 15 RAPD primers. This reveals 79.1% polymorphism indicating higher genetic variation among the hybrids and parents involved. Nine out of 15 RAPD primers produced 100% polymorphism among which OU-34 revealed highest number of polymorphic bands (14) of fragment size 210-3250 bp (Figure 1c). Besides, another RAPD primer OU-3 (Figure 1a) amplified ten distinct polymorphic bands (680-2800 bp) followed by Primer OU-29, OU-32, OU-1 and OU-24 producing 8-9 conspicuous bands each with amplicon size variation 390-2850 bp, 270-1510 bp, 480-1830 bp and 660-2500 bp respectively. Thus, these primers could be considered highly informative.

GC content, melting temperature(Tm) and annealing temperature are considered most critical factors for amplification of DNA. In general, melting temperature of primers proportionately increase with the increase in GC content; and GC-rich primers are expected to yield more polymorphism in the agarose gel. In the present study, a RAPD primer OU 34 with comparatively high GC content (70%), responded well for production of more number of amplicons as well as higher level of polymorphism (Table 1). Annealing temperature more than 1-2^°C over the melting temperature gave more number of amplicons with good resolution irrespective of the primers used for amplification.

DNA banding pattern of each of the genotypes is expected to differ if they are genetically different. Even subtle difference at genotypic level which other-wise some time could not be possible to differentiate by phenotyping, can be confirmed by use of markers. The DNA markers could pave the way for success. Polymorphism information content (PIC) ranged from 0.13(Primer OU-30) to as high as 0.86 (Primer OU-5). Thimmappaiah (2011) reported a narrow range PIC from 0.147 to 0.354 with an average of 0.229 in 60 cashew nut accessions using 20 RAPD markers In the present pursuit, PIC value of four RAPD primers e.g., OU 5, OU 16, OU 28 and OU 34 were appreciably higher (>0.70) than the average PIC value of 0.568 over 15 primers and each such primer had also revealed 100% polymorphism over the test genotypes. Thus, these RAPD primers may be considered more informative in terms of the extent of polymorphism.

Rp (resolving power) value ranged from 3.64 (Primer OU-5) to as high as 15.36 (Primer OU-3) with a mean value of 8.27. It is worth to note that the RAPD primer OU-34 revealed highest number of polymorphic bands (14) with 100% polymorphism as well as very high PIC and Rp values. Such a RAPD primer alone could discriminate all 28 cashew genotypes. Thus, this informative and discriminative primer is of immense value for study of genetic diversity in a set of cashew genotypes. Similarly, Neto Silva et al. (1995) identified one of the 6 primers that distinguished each of the 4 cashew clone seedlings. However, Ye Chunjiang et al. (2005) proposed combination of RAPD with ISSR primers for detection of new genomic loci for genome mapping, finger printing and gene tagging.

A few of the RAPD markers were specific to test genotypes used. The cashew hybrids A 71 and E 16 revealed a specific 2640 bp band amplified by Primer OU 3 (Figure 1a). In this context, band 280 bp and 210 bp produced by Primer OU-34, were unique to D 19 and H 6 respectively (Figure 1c). Similarly, Primer OU 34 specifically revealed three specific bands of fragment size 3250 bp, 1960 bp and 880 bp in VTH 711/4 and E 16; RP-2 and Kalyanpur Bold Nut; and RP 1 and D 19 respectively. Further, it is worth to note that the polymorphic band of fragment size 675 bp amplified by Primer OU 34 was unique to RP 1, RP 2 and Kalyanpur Bold Nut , but an adjacent band of bit smaller amplicon size (530 bp) produced by the same primer was specifically absent in these three parent varieties of cashew nut. Primer OU-28 produced a specific band of amplicon size 1340 bp in A 62 and J 6; and another 485 bp amplicon in RP-1 and D 19 only. Neto Silva et al. (1995) also detected cashew cultivars using RAPD profiling. Tomar et al. (2014) scored 119 polymorphic amplicons and they reported presence of 34 unique bands specific to land races of mango.

The RAPD markers are dominant in nature and therefore, absence of a specific band could serve as valuable information for varietal discrimination and assessment of genetic relationship. The 2160 bp and 680 bp polymorphic bands produced by Primer OU-3 were specifically absent in J-20 and A 48; and E 16 and A 71 respectively. While 1320 bp band was amplified by RAPD primer OU-16 in all  cashew test genotypes except G 8, H 6 and D 19 respectively. Similarly, OU-28 failed to reveal 1600 bp amplicon in A 62 and RP 1; and the primer OU 34 also could not produce a 935 bp amplicon in cashew hybrid H 6 and D 19. Besides, the unique 1510 bp and 1120 bp band amplified by Primer OU 32 and Primer OU 34 were specifically absent in C 30 and H 6 respectively. The absence of characteristic bands in a few cashew test genotypes might be attributed to the fact that these markers might be linked to negative traits. Thus, presence or absence of bands revealed by specific primers could certainly serve as valuable markers for varietal identification (Samal, 2002) and more often could serve as valuable markers for elimination of duplicates (Virk et al., 1995). Castiglione et al. (1993) used RAPD markers to define all the commercial poplar clones including those which could not be distinguished with morphological traits.

Similarity index value (S.I) between each pair of genotypes is likely to give a clear picture of the extent of genomic homology in terms of gene content and nucleotide sequence. It ranged from 0.37 to as high as 0.71 between A 62 with J 6 with average S.I. value of 0.585. Thus, RAPD was found to be potent enough to sort out divergent genotype combinations with similarity index value as low as 0.37. In this context, Thimmappaiah et al. (2009) observed similarity co-efficient values varying from 0.43 to 0.94 between different pair of 100 cashew nut accessions. In the present investigation, the most divergent genotype pair exhibiting highest genetic distance between them was identified to be J 6 and H 6 (S.I = 0.37). Besides, high level of genetic distance was revealed also in case of genotype pair A62 and H 6 (S.I = 0.40); and A 62 and H 8 (S.I. = 0.41). Samal et al. (2004) also revealed genetic relatedness among cashew varieties using RAPD markers.

Similarity index values of parent varieties constituting cashew nut hybrids can be considered as a basis to reveal the extent of genetic variation among .the cashew hybrids. Cashew hybrids H 6 and D 19 maintained very high level of average genetic dissimilarity with rest of the test genotypes (0.49 and 0.50). The most divergent high yielding hybrids e.g., H 6 and D 19 were derived from the cross combination of M 44/3 with Kalyanpur Bold Nut and VTH 711/4 respectively. This could be attributed to the fact that the above two specific parental combinations had minimum inter se genomic homology (S.I. = 0.50-0.51) leading to produce heterotic hybrids (H 6 and D 19). However, Archak et al. (2003) reported no correlation between molecular data and the pedigree of the varieties. Besides, the cashew hybrids e.g., D19, H6, G8, B27, A71, H8 and C30 were shown to be genetically distant from their respective parents. These can be sorted out as genetically superior elite hybrids (Asolkar et al., 2011) at even seedling stage.

The whole range of fifteen RAPD primer-based UPGMA clustering of 28 cashew test genotypes revealed hierarchical separation of four distinct genetic clusters at 56.5% phenon level (Fig. 2). Among the test genotypes, seven cashew hybrids e.g., D 19, H 6, G 8, B27, A 71, H 8 and C 30 were initially separated as a broad genetic divergent group ‘Cluster I’ from rest of the genotypes at even 51% phenon level. Three dimensional scaling based on Principal Co-ordinate Analysis (PCA) (Fig. 3) also revealed similar clustering pattern. This corroborated with the findings of Malik et al., (2013).

Cluster I was further sub-divided into three sub-clusters (Cluster IA, Cluster IB and Cluster IC). Cashew hybrids D 19 and H 6 constituting Cluster IA may be considered most divergent followed by G 8 included in Cluster IB; and cashew hybrids B27, A 71, H 8 and C 30 forming Cluster IC. Rest 21 cashew test genotypes were grouped into three distinct clusters e.g., Cluster II, Cluster III and Cluster IV. Cluster II and Cluster IV were shown to be separated into two sub-groups each at 59.3% phenon level.

Cluster IIA contained cashew hybrids e.g., J 12, J 6 and A 62. Among the test genotypes, J 6 and A 62 maintained maximum inter se homology up to 71% beyond which these can be discriminated. Cluster IIB was shown to have three cashew hybrids (A 48, C 41 and G 9) and the parent genotype M 44/3. It is worth to note that Cluster IV is the bottom most cluster in the present set of hierarchical clustering pattern, which clubbed all parental test genotypes except M 44/3. This clearly suggests that the recombination breeding programme in the present pursuit has unequivocally resulted wide genetic diversity (genetic variation) exceeding the parental gene pool in terms of heterotic hybrids. Among the parents, M 44/3 was genetically far distant followed by RP 1 which constituted a mono-genotypic group ‘Cluster IVB’. Thus, RAPD markers were potent enough to discriminate all test genotypes beyond 71% phenon level. Neto Silva et al. (1995) used RAPD markers in cashew to distinguish four dwarf cashew seedlings in Brazil. Similarly, Thimmappaiah et al. (2009) reported clear distinction of 67 accessions out of which NRC-142 and NRC-12 emerged as highly divergent while, NRC-231 and NRC-232 were genetically similar among 100 accessions of cashew. In an another study, Thimmappaiah (2011) reported genotype combinations of NRC 335 with NRC 338; and NRC 362 with NRC 388 to be highly divergent. Mneney et al. (2001) used RAPD to determine the genetic diversity within and between populations of cashew (Anacardium occidentale L.).

RAPD technique together with study of morphological traits has proved useful for assessment of genetic diversity (Ramessur and Ranghoo-Sanmukhiya, 2011). Therefore, an effort was made to note agronomic performance of the test genotypes in relation to their distribution in the dendrogram clusters using RAPD markers. The order of occurrence of the genotypes in the above RAPD based clusters was used as a reference for arranging performance values of the accessions for 14 agro-morphological traits that were observed in the field over two consecutive seasons laid out in RBD. The average values of these traits are given in Table 2. For better clarity, the significantly lower/higher performance values for plant height and for rest of the morpho-economic traits significantly higher mean values compared to the experimental grand mean have been marked with an asterisk (*), are plant types of special consideration for cashew genetic improvement. In fact, genotypes with significantly higher values than the grand mean were identified to assess relation (if any) of genotypic performance for yield and yield attributing traits with RAPD marker based clustering. The table values arranged cluster-wise allow a simultaneous comparison of several quantitative traits in the test genotypes with reference to the position of each genotype in the dendrogram. It is observed that the genotypes in the same cluster based on DNA profiling have some common phenotypic performance and such clusters with unique phenotypic performance could be identified for genetic improvement in cashew nut.

Dwarf plant types are of special consideration to restructure plant ideotype. Cluster IVB and Cluster IIB had shown characteristically dwarf plant types. Among the genotypes under these genetic groups; M 44/3 in Cluster IIB and RP 1 comprising Clusters IVB, exhibited significantly dwarf plant height. Such dwarf type cashew genotypes have been purposefully used as parents for development of heterotic cashew hybrids. For instance, Cluster IC and Cluster III are characterized by tall plant types. B 27 included in Cluster IC and B- 5 in Cluster III had shown significantly tall plant type among the selected 20 hybrids under study. It is worth to note that the dwarf type parent RP 1 was one of the parents in the heterotic hybrids B-27 and B-5. Besides, cashew hybrids D-19 and H-6 constituting the most divergent heterotic genetic group ‘Cluster IA’ have M 44/3 (dwarf plant type) as one of the parent. Barros et al. (2002) reported that clones of early dwarf cashew have greatly impacted cashew cultivation as they are more productive, early, short, easily harvestable, and have uniform nuts and apples.

In cashew nut, trunk girth, canopy spread (N-S), number of flowering laterals/m², number of perfect flowers, sex ratio, nuts/panicle have considerable role in determining nut yield. Cluster IA was unique in significantly higher trunk girth while Cluster IC had highest mean values for canopy spread (N-S) and number of flowering laterals/m² which has direct bearing on productivity. In this context, Cluster IB and Cluster IIB had shown characteristically higher mean values for number of perfect flowers, sex ratio and nuts/panicle.

RAPD based clustering differentiated all parental genotypes except M 44/3 into the Cluster IV which revealed appreciably lower mean values for almost all agro-economic traits including nut yield. This is indicative of the fact that the 20 selected experimental hybrids developed from different cross combinations have expressed  heterotic performance for agro-economic traits leading to separate them from the above parental cluster.

The analysis of genetic relationships in cashew using morphological traits and RAPD banding data can be useful for plant improvement, description of new variety and also for assessing varietal purity in plant certification programmes (Samal et al., 2003b). Cashew hybrids comprising the most divergent broad genetic group ‘Cluster-I’ had shown significantly above average productivity. Phylogenetic tree based on RAPD markers which correlated with morphological characters was developed by Betal et al. (2004). Lavanya et al (2008) reported typical agronomic performance of genotypes that were included in each of the clusters based on RAPD.  Such a distribution of clusters based on agronomic performance and supported by RAPD profile are a very good starting point for further breeding efforts involving contrasting parental lines, and will further enable tagging of genes and identification of QTLs for these traits with molecular markers. Ghafoor et al. (2005) identified few QTLs for agro-economic traits based on significant correlation of polypeptide bands with the quantitative traits. It is worth to note that a RAPD band of amplicon size 1150 bp (amplified by primer OU 32) was revealed only in first four top ranking high yielding cashew hybrids e.g., D 19, H 6, B 27 and A 71. Such a DNA marker might be linked to a major QTL for nut yield in cashew nut and therefore, may serve as a molecular marker for identification of high yielding cashew genotypes. Shobha and Thimmappaiah (2011) identified RAPD markers linked to nut weight and plant stature in cashew.  Similarly, Ghallab et al. (2007) correlated superiority of two mungbean genotypes (L 2520 and L 1720) in seed yield with absence of two bands at around 94.6kd and a presence of a polypeptide band with molecular weight 12.1kd under drought stress condition. However, such an interpretation needs critical validation.

Thus, the RAPD markers can generate wide array of polymorphism for varietal identification and study of genetic diversity, and could be successfully used in conjunction with morpho-economic traits for genotype sorting to support cashew breeding programme. The high yielding divergent cashew hybrids H 6 and D 19 identified in this study, can be used in cashew breeding programme for further genetic improvement of nut quality and yield per se.

References

Aliyu O.M., 2012, Development of the flow cytometric protocol for ploidy analysis and determination of relative nuclear DNA content in cashew (A. occidentale L.), Amer. J. Biochem & Mol. Biol., 2(4): 200-215

http://dx.doi.org/10.3923/ajbmb.2012.200.215

Archak S., Gaikwad A.B., Gautam D., Rao E.V.V.B., Swamy K.R.M., and Karihaloo J.L., 2003, DNA fingerprinting of Indian cashew (Anacardium occidentale L.) varieties using RAPD and ISSR techniques, Euphytica, 230: 397-404

http://dx.doi.org/10.1023/A:1023074617348

Asolkar T., Desai A.R., and Singh N.P., 2011, Molecular analysis of cashew genotypes and their half-sib progeny using RAPD marker, Biotechnol. Bioinf. Bioeng., 1(4): 495-504

Bajpai A., Srivastava N., Rajan S., and Chandra R., 2008, Genetic diversity and discrimination of mango accessions using RAPD and ISSR markers, Indian J. Hort., 65(4): 377-382

Barros L. M., Paiva J. R., Cavalcanti J.J.V., and Araujo J.P.P., 2002, Cajueiro. In Bruckner CH (ed.) Melhoramento de Frueiras Tropicals, Editora UFV, Vicisa, p. 159-176

Betal S., Chowdhury P.R., Kundu S., and Raychaudhuri S.S., 2004, Estimation of genetic variability of Vigna radiata cultivars by RAPD analysis, Bilogia Plantarum, 48(2): 205-209

http://dx.doi.org/10.1023/B:BIOP.0000033446.43495.0c

Castiglione S., Wang G., Damiani G., Bandi C., Bisoffi S., and Sala F., 1993, RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp) clones, Theor. Appl. Genet., 87: 54-59

http://dx.doi.org/10.1007/BF00223744

Cavalcanti J.J.V., Costa dos Santos F.H., Pereira da Silva F., and Pinheiro C.R., 2012., QTL detection of yield-related traits of cashew, Crop Breed. and Appl. Biotechnol., 12: 60-66

http://dx.doi.org/10.1590/S1984-70332012000100008

Croxford A. E., Robson M., and Wilkinson J., 2005, Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew and their cross­-species utilization, Mol. Eco. Notes, 10 (11): 1-3

Dhanaraj, A.L., Bhaskara Rao E.V.V., Swamy K.R.M., Bhat M.G., Teertha Prasad D., and Sondur S.N., 2002, Using RAPDs to assess the diversity in Indian cashew (Anacardium occidentale L.) germplasm, J. Hortic. Sci. and Biotech., 77: 41-47

http://dx.doi.org/10.1080/14620316.2002.11511454

Ghallab K.H., Ekram A.M., Afiah S.A., and Ahmed S.M., 2007, Characterization of some superior mungbean genotypes on the agronomic and biochemical genetic levels, Egyptian J. Desert Res., 57(2): 1-11

Ghafoor A., Ahmed Z., and Afzal M., 2005, Use of SDS-PAGE markers for determining quantitative traits loci in blackgram (Vigna mungo (L.) Hepper). Germplasm, Pak. J. Bot., 37(2): 263-269

Gopalsamy J., Anburaj J., Sundaravadivelan C., Kuberan T., Kumar P., Starlin T., and Mariselvan M., 2012, Molecular marker (RAPD) based fingerprinting on jackfruit to estimate the genetic diversity, Int. J. Appl. Biores., 7: 1-7

Hu J., and Quaros C.F., 1991, Identification of broccoli and cauliflower cultivars with RAPD markers, Plant Cell Reports, 10: 505-511

http://dx.doi.org/10.1007/BF00234583

Jaccard P., 1908, Nouvelles recherches Sur la distribution florale, Bulletin Society Vaud Science National, 44: 223-270

Lavanya G.R., Srivastava J., and Ranade S.A., 2008, Molecular assessment of genetic diversity in mungbean germplasm, J. of Genet., 87(1): 65-74

http://dx.doi.org/10.1007/s12041-008-0009-3

Malik S.K., Kumar S., Choudhary R., Kole P.R., Choudhary R., and Bhat K.V., 2013, Assessment of genetic diversity in Khirni (Manilkara hexandra): an important underutilized fruit species of India using RAPD markers, Indian J. Hort., 70(1): 18-25

Melo L.C., Santos J.B., and Ferreira D.F., 2002, Mapping and stability of QTLs for seed weight in common beans under different environments, Crop Breed. and Appl. Biotechnol., 2: 227-236

http://dx.doi.org/10.12702/1984-7033.v02n02a09

Mneney E.E., Mantell S.H., Tsoktouridis G., Amin S., Bessa A.M.S., and Thangavelu M., 1998, RAPD profiling of Tanzania cashew.  Proc. of the Int. Cashew Coconut Conf. , Dares Salaam, Topper C.P., Caligari, P.D.S., Kullaya, A.K., Shomari, .S. H., Kasuga, L.J., Masawe, P.A.L. and Mpunami, A.A. Eds). Bihybrids Int. Ltd., Reading, UK, pp.108-116

Mneney E.E., Mantell S.H., and Mark B., 2001, Use of random amplified polymorphic DNA (RAPD) markers to reveal genetic diversity within and between populations of cashew (Anacardium occidentale L.), J. Hortic. Sci. Biotech., 76: 375-383

http://dx.doi.org/10.1080/14620316.2001.11511380

Neto Silva S. P., Maruta L., Takaiwa F., Oono K., and Matsumuto K., 1995, Identification of cashew (Anacardium occidentale L.) seedlings with RAPD markers, Acta Horticulturae, 370: 21-26

http://dx.doi.org/10.17660/ActaHortic.1995.370.3

Prevost A., and Wilkinson M.J., 1999, A new system of comparing PCR Primers applied to ISSR finger printing of potato cultivars, Theor. Appl. Genet., 98(1): 107-112

http://dx.doi.org/10.1007/s001220051046

Ramessur A.D., and Ranghoo-Sanmukhiya V.M., 2011, RAPD marker-assisted identification of genetic diversity among mango (Mangifera indica) varieties in Mauritius. Int. J. Agric. and Biol., 13:167-173

Rout G. R, Samal S., Nayak S., Rashmi M., Nanda R.M., Lenka P. C., and. Das P., 2002, An alternative method of plant DNA extraction of cashew for randomly amplified polymorphic DNA (RAPD) analysis, Gartenwissenschaft, 67 (3):1-4

Samal S., 2002, Evaluation of genetic relationship and variability in cashew (Anacardium occidentale L) through morphological, biochemical and molecular analysis. Ph. D. Thesis. Utkal University. India, pp.51-78

Samal S., Rout G.R., Nayak S., Nanda R.M., Lenka P.C., and Das P., 2003a, Primer screening and optimization for RAPD analysis of cashew, Biologia Plantarum, 46(2): 303-304

http://dx.doi.org/10.1023/A:1022827416677

Samal S., Rout G. R., and Lenka P. C., 2003b, Analysis of genetic relationships between populations of cashew (Anacardium occidentale L) by using morphological characterization and RAPD markers, Plant Soil Environ., 49 (4): 176-182

Samal S., Lenka P.C., Nanda R.M, Nayak S., Rout G.R., and Das P., 2004, Genetic relatedness in cashew (Anacardium occidentale L.) germplasm collections as determined by randomly amplified polymorphic DNA, Genet. Resour. and Crop Evol., 51: 161-166

http://dx.doi.org/10.1023/B:GRES.0000020856.77201.7d

Samant D., Singh A.K., Srivastav M., and Singh N.K., 2010, Assessment of genetic diversity in mango using inter simple sequence repeat markers, Indian J. Hort., 67 (special issue): 1-8

Shobha D., and Thimmappaiah W.G., 2011, Identification of RAPD markers linked to nut weight and plant stature in cashew. Scientia Horticulturae, 129(4): 637–641

http://dx.doi.org/10.1016/j.scienta.2011.05.006

Singh A.K., and Singh R., 2011, Analysis of genetic relationship of Indian grape genotypes using RAPD markers, Indian J. Hort., 70(1): 18-25

Smith J.S.C., and Smith O.S., 1989, The description and assessment of distances between inbred lines of maize. II. The utility of morphological, biochemical and genetic descriptors and a scheme for the testing of distinctiveness between inbred lines, Maydica, 34: 151-161

Sneath P. H.A., and Sokal R. R., 1973 Numerical taxonomy: The principles and practice of numerical classification, W.H.Freeman & Co Ltd., San Francisco. 573 p.

Souza S. G.H., Carpentieri- Pipolo V., Ruas C.F., and Carvalho V.P., 2008, Comparative analysis of genetic diversity among the maize inbreed lines (Zea mays L.) obtained by RAPD and SSR markers, Braz. Arch. Biotechnol, 51: 183-192

Thimmappaiah W.G., Santhosh D.S., and Melwyn G.S., 2009, Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers, Scientia Horticulturae, 120: 411-417

http://dx.doi.org/10.1016/j.scienta.2008.11.022

Thimmappaiah W.G., 2011, Molecular characterization of germplasm of cashew, Annual Report, Directorate of Cashew Research, pp.50-53

Tomar Rukam S., Gajera H.P., Viradiya R.R., Patel S.V., and Golakiya B.A., 2014, Characterization of mango genotypes of Gir regions based on ISSR markers, Indian J. Hort., 71(1): 1-5

Virk P.S., Ford-Lloyd B.Y., Jackson M.T., and Newbury H.J., 1995, Use of RAPD for the study of diversity within plant germplasm collection. Heredity, 74: 170-179

http://dx.doi.org/10.1038/hdy.1995.25

Ye Chunjiang Yu Z., Kong F., Wu S., and Wang B., 2005, R-ISSR as a new tool for genomic finger printing, mapping and gene tagging, Plant Mol. Bio. Reporter, 23:167-177

http://dx.doi.org/10.1007/BF02772707