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The NucleusAn International Journal of Cytologyand Allied Topics ISSN 0029-568XVolume 55Number 1 Nucleus (2012) 55:27-35DOI 10.1007/s13237-012-0050-8

Cyto-morphological and molecularcharacterization of Cajanus cajan × C.scarabaeoides F1 hybrid

Ramya Ranjan Mishra, Alok RanjanSahu, Sobha Chandra Rath, SatyendraPrasad Mishra & Jogeswar Panigrahi

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ORIGINAL ARTICLE

Cyto-morphological and molecular characterizationof Cajanus cajan×C. scarabaeoides F1 hybrid

Ramya Ranjan Mishra & Alok Ranjan Sahu &

Sobha Chandra Rath & Satyendra Prasad Mishra &

Jogeswar Panigrahi

Received: 16 October 2011 /Published online: 8 May 2012# Archana Sharma Foundation of Calcutta 2012

Abstract Cajanus scarabaeoides has many desirable agro-nomic traits that deserve attention for introgression intopigeonpea cultivars. An inter-specific F1 hybrid was raisedusing C. cajan (ICP-26) as seed parent and C. scara-baeoides (ICPW-94) as pollen parent. Hybridity of the F1plant was affirmed by analyzing the morphological attrib-utes, such as seed coat color, leaf shape, brown striations inthe petal, pod features, and intermediate branching type etc.Most of the pollen mother cells (PMC) in the F1 hybrid haveshown regular meiosis, but in some PMCs chromosomeheteromorphism was observed for two pairs of bivalentsduring diakinesis, leading to precocious separation of biva-lents (62.81 %) at metaphase-I, and bridge formation(7.44 %) and laggards (2.48 %) at anaphase-I. SDS-PAGEof two major seed protein fractions (albumin and globulin)detected five polypeptide bands (Mol wt. 40.7, 39.8, 32.4,23.5 and 19.5 KDa) unique to C. scarabaeoides, the maleparent, in the F1 hybrids that evidenced the hybridity of F1plants. Further, RAPD and ISSR marker analysis revealedthe hybrid nature of the putative F1 plant. These analysesshowed the inheritance of 28 RAPD and 12 ISSR markers,unique to C. scarabaeoides - the pollen parent, to F1hybrids. This complementary use of cyto-morphologicalanalysis, and molecular marker based genotyping allowedprecise evaluation of the C. cajan×C. scarabaeoides inter-specific hybrid.

Keywords Cajanus . Hybridity . Heteromorphic bivalent .

Seed protein profile . RAPD and ISSR

Introduction

Cajanus cajan (Linn.) Millsp. is an important grain legumecrop of the semi-arid tropics, and is the second most impor-tant pulse crop next to chickpea. India has 3.53 millionhectares land under Pigeonpea cultivation. India contributesabout 2.51 million tons (75 % of world production) with anaverage yield of 0.78 tons/hectare [41] among the countriesgrowing this crop. However, the yield ceiling was con-strained with several biotic and abiotic stresses. Limitedgenetic variation, long life cycle of this monotypic crop,and heterozygous genome structure [35] make its geneticimprovement only to selection and perpetuation of usefulkind of gene action. However, the wild Cajanus constitutethe germplasm of the cultivated taxon C. cajan, and possesgenes for annuality/perenniality, earliness, photoperiod in-sensitivity, high protein content, biotic and abiotic stressresistance [36]. Among the species present in the wild genepool, C. scarabaeoides accessions possessed with gene(s)for resistance to pod borer, pod fly, early flowering, andphotoperiod insensitivity [34, 36]. These agro-economictraits could be incorporated into the cultivated C. cajanthrough wide introgressive hybridization assisted by thetools of molecular breeding to improve Pigeonpea cultivars,and to evolve novel genotypes.

The inter-specific F1 hybrids could be useful as startingmaterial for the production of advanced breeding lines,widening the genetic base of this crop, and allowing thestudy of the interaction between both the genomes in asingle cytoplasmic background. Some attempts were alsomade to generate inter-specific hybrid between C. cajan and

R. R. Mishra :A. R. Sahu : S. C. Rath : S. P. Mishra :J. Panigrahi (*)Plant Biotechnology Laboratory, School of Life Sciences,Sambalpur University,Jyoti Vihar, Burla-768019,Sambalpur, Orissa, Indiae-mail: drjpanigrahi@gmail.com

J. Panigrahie-mail: drjogeswar_panigrahi@ymail.com

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C. scarabaeoides [1, 14, 27, 32, 36, 42], and success wasachieved to certain extent. Identification of true hybrids atan early stage is mandatory in the studies involving inter-specific hybridization. Morphological and cytological anal-ysis has been used to ascertain hybridity in many cropspecies, including the genus Cajanus. Few reports are alsoavailable on cyto-morphological analysis of C. cajan×C.scarabaeoides hybrids, which evidenced heteromorphicbivalents [32], followed by meiotic abnormalities [28].The reproducibility of the cyto-morphological markers toenvironmental and developmental variations limits their ap-plicability; but molecular markers can be utilized to screenand identify true hybrids at an early stage. However, noevidence has yet been made for identification of C. cajan×C. scarabaeoides hybrid using markers based upon eitherthe gene or gene products. Uses of these markers unique toC. scarabaeoides, being the donor parent, could beexploited to ascertain the hybridity as well as marker assis-ted selection of germplasm during breeding programmes.

Seed protein markers have been effectively employed forcultivar characterization, genetic diversity assessment, andverification of hybridity in several crop plants [2, 12, 20,25]. In the genus Cajanus, seed protein markers were used

for generating seed protein profiles [15], identification ofhybridity during wide hybridization [6, 26], and elucidationof phylogenetic relationship [11, 25, 28].

DNA markers based on the PCR technique, such asrandom amplified polymorphic DNA (RAPD), and intersimple sequence repeat (ISSR) markers [43, 44], have beenused as handy molecular tools for plant breeders. RAPDmarker analysis were used in the genus Cajanus for geno-type characterization [29–31], elucidation of genetic rela-tionship [29], hybrid identification [18, 19], and genetagging [5, 13, 33]. But, ISSR markers were used only forgenetic diversity assessment, and genotype characterization[9, 22]. Of course, ISSR markers have also been used forhybrid identification in several species [3, 8, 17–19], inaddition to genetic fingerprinting, gene mapping and eluci-dation of phylogeny.

In this study, we evaluated the morphological char-acters, and analysed the meiotic behaviour of the F1hybrid involving C. cajan (ICP-26) and C. scara-baeoides (ICPW-94; source for resistance to pod borercomplex). We have also characterized the F1 hybridwith respect to their parents using seed protein, RAPDand ISSR markers.

Table 1 Morphological featuresof C. cajan (cv ICP-26), C.scarabaeoides (ICPW-94) andtheir F1 hybrids

Morphological traits C. cajan(♀ Parent)

ICP-26 x ICPW-94 F1hybrid plant

C. scarabaeoides(♂ Parent)

Shape of primary leaves Lanceolate Ovate lanceolate Ovate

Shape of side leaflets Lanceolate with cuneatebase

Ovate lanceolatewith oblique base

Ovate withoblique base

Growth habit Erect Erect Twining

Branching Racemose with wideangled branches

Racemose withintermediateangled branches

Racemose with basalbranches

Branch tip Straight Drooping Drooping

Days to first flush offlowering

112±1.4 100.6±1.6 94.0±2.3

Colour of petioles Greenish white Brownish yellow Brownish yellow

Colour of standard petal Uniformly yellow Pale yellow withred striations

Pale yellow with redstriations

Young pod colour Green with brownpatches on thepericarp

Uniformly reddishbrown

Greenish

Young seed Green with no aril Green withintermediate aril

Green withprominentaril

Pod surface Sparse, Glabrous Sparse, Pubescent Dense, Pubescent

Pod beak Prominent Intermediate Minute

Pod shattering Non shattering Shattering Shattering

Locules per pod 3.8±0.44 4.2±0.83 5.6±0.55

Seed colour Brownish yellow Mottled black Mottled black

Seed aril Non-arrilate Arrilate Arrilate

100 seed mass(in gm.; Mean±SD)

8.7±0.32 4.8±0.1 3.3±0.13

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Materials and methods

Plant materials 32 genotypes of 10 species of the genusCajanus were obtained from ICRISAT, Patancheru, Hyder-abad, and maintained at the experimental garden, School ofLife Sciences, Sambalpur University, Orissa, India. Basedon parental screening, two potential genotypes- C. scara-baeoides (ICPW-94), and C. cajan (ICP-26) were identified.The inter-specific F1 hybrids were raised by involving selfedC. cajan genotype as seed parent, and that of C. scara-baeoides as pollen parent.

Morphological analysis The following morphological fea-tures were studied for three F1 plants along with theirparents: shape of primary leaf, shape of leaflet, growth habit,branching, leaf colour, nature of branch tip, colour of stan-dard petal and calyx, pod characters, seed characters, and100 seed weight. The pollen parent specific morphologicalcharacters were used as marker to characterize the F1 hybrid.

Cytological analysis Meiotic analysis of F1 hybrids and itsparents were carried out to study the chromosome homolo-gy. Anthers containing PMCs previously fixed in ethanol :acetic acid (3:1) were used for the preparation of meioticchromosome spread by aceto-carmine (2 %) squash tech-nique, and appropriate stages like diakinesis, metaphase-Iand anaphase-I were observed. A total of 121 PMCs wereanalysed from each parent, and F1 hybrid plants. The pollenfertility was also accessed by staining the pollen grains in 1glycerin : 1 aceto-carmine mixture, and an average fiveslides were scored for the purpose.

Seed protein electrophoresis Extraction of the albumin andglobulin fraction from seed flour, and its denaturation weredone following the procedures as described by Panigrahi etal. [25]. Protein samples (approximately 25.0 μg) wereelectrophoresed in the modified discontinuous sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [16] using 10 % resolving gel (0.375 M Tris–HCl,pH 8.8) and 4 % stacking gel (0.125 M Tris–HCl, pH 6.8).The running buffer was Tris–Glycine (0.1 % SDS, 25 mMTris–Glycine, pH 8.3), and the sample was electrophoresedat 1.5 mA per well constant current until the tracking dyeentered the separating gel, and then the current was in-creased to 2 mA per well until the tracking dye entered thebottom of the gel. The sizes of the polypeptides were deter-mined using molecular weight marker-PMWM (BangaloreGenei Pvt. Ltd.) as a standard.

RAPD and ISSR marker analysis Genomic DNA from threeF1 hybrids, and their parents were isolated using the modi-fied CTAB method [37]. The DNA was dissolved in 2.0 mlTris-EDTA (TE) buffer (10 mM Tris; 1 mM EDTA, pH 8.0),

and stored at −20 °C. DNA concentrations and purity weremeasured using a UV–Vis spectrophotometer (UV 1601;Shimadzu, Kyoto, Japan) with TE buffer as the blank. Forconfirmation, quantification of the DNA was accomplishedby analyzng the purified DNA on 0.8 % (w/v) agarose gelsalong with diluted, uncut phage lambda DNA as standard.All DNA samples were diluted to 10 ng μl−1 in TE buffer.

For RAPD and ISSR analysis, PCR amplification of25 ng of genomic DNA was carried out separately usingeach of 20 random decamer oligonucleotide primers, OPM-01-20 (Operon Technologies, Alameda, CA, USA) and nineISSR primers from the set 100/9 (University of BritishColumbia, Vancouver, Canada) respectively. Each amplifi-cation reaction (25 μl) contained the 25 ng template DNA,2.5 μl of 10X assay buffer [100 mM Tris-Cl, pH 8.3; 0.5 MKCl; 0.1 % (w/v) gelatin], 1.5 mM MgCl2, 200 μM of eachdNTP, 0.25 μM primer, 1.0 units Taq DNA polymerase

Fig. 1 Morphological characteristics of in the C. cajan (ICP-26)×C.scarabaeoides (ICPW-94) interspecific hybrid and its parents a Leafmorphology (1× magnification), b Striation on petals and floral attrib-utes (2×), c Pod characters (1×), d Mature seed (2×) showing colourand aril (Left to right C. cajan, hybrid, and C. scarabaeoides)

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(Bangalore Genei Pvt. Ltd., Bangalore, India). Amplifica-tion conditions for RAPD analysis include an initial dena-turation step of 94 °C for 5 min, followed by 45 cycles ofdenaturation at 94 °C for 60 s, a primer annealing step at37 °C for 60 s, and an extension at 72 °C for 2 min; then afinal extension was carried out at 72 °C for 5 min. For ISSRamplification following conditions were used—an initialdenaturation step of 94 °C for 5 min, followed by 40 cyclesof denaturation at 94 °C for 30 s, a primer annealing step at40–60 °C for 45 s, and an extension at 72 °C for 2 min; thena final extension was carried out at 72 °C for 5 min.

Both RAPD and ISSR amplification was carried out in athermal cycler (GENEAMP-9700; Applied Biosystems, FosterCity, USA), and PCR products were separated in 1.4 % (w/v)agarose gel containing 0.5 μg ml−1 ethidium bromide in TAEbuffer (40 mMTris acetate, pH 8.0; 2 mMEDTA) at a constant50 V. A gel loading buffer [20 % (w/v) sucrose; 0.1 M EDTA,1.0% (w/v) SDS; 0.25% (w/v) bromo-phenol blue; 0.25% (w/v) xylene cyanol] was used as a tracking dye. Amplified DNAfragments were visualised on the gel documentation system(Geldoc XR system, Biorad, USA) and photographed. Thesizes of the amplified products were determined using 250 bpstep up ladder (Bangalore Genei Pvt. Ltd.) as standard, and TL-120 software (Non-linear Dynamics, Total Lab Ltd., NewcastleUpon Tyne, UK). Each amplification products (fragments) wasconsidered as one RAPD or ISSR marker, and each reactionwas repeated twice. The parental polymorphism of each primerwas computed as the ratio of number of polymorphic bands tothe total number of bands amplified.

Results and discussion

Ascertaining the hybridity of the inter-specific F1 hybrids atan early stage is mandatory in the studies involving widehybridization, being the starting material for all breedingendeavours [17]. In this study, the C. cajan×C. scara-baeoides inter-specific F1 hybrid was characterized using

Fig. 2 Chromosome pairing behaviour of C. cajan (ICP-26)×C. scar-abaeoides (ICPW-94) inter-specific hybrid ( ) at 1000× magnifica-tion. a–b Heteromorphic bivalents at diakinesis, c Precocious meioticseparation of bivalents at metaphase-I, d Anaphase bridge, and eLaggards at anaphase-I

Table 2 Chromosome behaviour of C. cajan (ICP-26), C. scarabaeoides (ICPW-94), and their inter-specific F1 hybrid in diakinesis, metaphase-Iand anaphase-I

Plant Diakinesis Metaphase-I Anaphase-I Pollen viability(%) Mean±SD

Ring bivalents Rod bivalents Precociousseparation (%)

Laggards(%)

Anaphasebridge (%)

Range Mean±SD Range Mean±SD

C. cajan cv. ICP-26 10–11 10.87±0.35 0–1 0.14±0.35 – – – 80.32±11.63

C. scarabaeoides (ICPW-094)

10–11 10.93±0.26 0–1 0.07±0.26 – – – 83.36±10.8

C. cajan cv. ICP-26 × C.scarabaeoides (ICPW-094)

8–11 9.41±0.93 0–3 1.59±0.93 62.81 2.48 7.44 64.96±7.72

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cyto-morphological analysis, seed albumin and globulinprofiling, and DNA marker analysis.

Morphological characterization Morphological characterslike plant habit, seed coat color, presence of seed aril, brownstriations on the petal, pod shape, and pod trichomes etc.serve as morphological marker to distinguish C. scara-baeoides (ICPW-94) from C. cajan (ICP-26). In many suchmorphological traits the F1 hybrid was intermediate betweenthe parents with predominance of the characters of C. scar-abaeoides, such as seed coat colour, presence of seed aril,striation on the petal and pod trichomes, though this acted aspollen parent (Table 1; Fig. 1a–d), and their appearance inthe intermediate form was very vivid for the identification ofthe F1 hybrid. These findings support the earlier observationmade by Kumar et al. [14]. Characters those proved to bedominant were drooping branch tip over straight branch tip,brownish color of the petiole over greenish white, pubescentand shattering nature of pod over glabrous and non shatter-ing, mottled black colour of seed over orange yellow, andarillate nature of seed over nonarillate seed. In most of thequantitative traits such as leaf size, size of the flower and100 seed weight the hybrid showed mid parental value asobserved in the inter-specific hybrid of C. cajan and C.cajanifolious [21].

Meiotic analysis Meiotic analysis of both the parentsrevealed 11 bivalents (II) at diakinesis, and metaphase-I ineach of them, being the somatic chromosome number 2n02x022. No abnormality was noticed at anaphase-I, and 11:11 separation to two poles was fairly regular in both theparents. A total of 121 PMCs of the F1 plants were studied,and the chromosomal behaviour at various stages of meiosisrevealed clear inter-genomic homeology between the twospecies (Table 2). In most of the hybrid PMCs 11 bivalentswere observed at diakinesis and metaphase-I, and regular11: 11 separation noticed at anaphase-I. But, in certaincases, the F1 hybrid PMC showed loose pairing between

the two pairs of heteromorphic chromosomes at diakinesis,which led to precocious separation (62.82 %) of chromo-somes at metaphase-I, and laggard formation (2.48 %) inanaphase-I (Fig. 2a, b, c and e). Similar features were alsonoticed in the inter-specific hybrids of the genus Cajanus[21, 28, 32, 40], which is indicative of partial non-homologybetween certain homologues or the development of de-synaptic genes in course of evolution. In most PMCs,precociously separated chromosomes were found to beregularly distributed between the poles. Despite regular11 :11 separation in most PMCs of F1 hybrid atanaphase-I, certain frequency (7.44 %) of bridge forma-tion was encountered involving two pairs of chromo-somes. This might be due to cryptic structural alterationsbecause of crossing over with paracentric inversion [40].The pairing behavior of heteromorphic chromosomes atdiakinesis, precocious separation of chromosomes atmetaphase-I, and chromosome bridge formation atanaphase-I confirmed the hybrid nature of the putativeF1. However, this result contradicts with the number ofheteromorphic bivalents [32] in C. cajan×C. scara-baeoides inter-specific F1 hybrid, and this might be dueto different genotypes used in both the studies. The pollenfertility of F1 hybrid was found to be low (64.96 %) incomparison to the parents. This might have been causedby the meiotic irregularities since pollen fertility has beenfound to be directly related to the chromosome associa-tions [39]. These cyto-morphological observations couldbe utilized for the confirmation of hybridity in the breed-ing programmes aiming at the allien gene transfer to thecultivated genetic background of Pigeonpea.

Seed protein marker analysis Seed protein expression isusually controlled by homologous multigene familiesexhibiting monogenic segregation with co-dominancefor molecular weight variants and presence of polypep-tide bands being completely dominant over absence[24]. Deletion or mutation of structural genes coding

Table 3 Details of seed albuminand seed globulin markersinherited in the interspecific (C.cajan-ICP-26×C.scara-baeoides-ICPW-94) F1 hybrid

Cc, C. cajan specific band; Cs,C. scarabaeoides specific band

Marker Totalno. ofbands

Range ofMolecularweight(KDa)

No. ofPolymorphicbands(Percentage)

No. ofpolymorphicbands

No. ofpoly-morphicbandsinheritedto F1

C.scarabaeoidesspecificpolypeptidesinherited to F1plants (Mol.wt. in KDa)

Cc Cs Cc Cs

Seed albumin 24 14.5–95.5 06 (25.0) 02 04 02 01 40.7

Seed globulin 18 16.6–55.0 06 (33.33) 01 05 01 04 39.8, 32.4,23.5 & 19.5

Total 42 14.5–95.5 12 (28.57) 03 09 03 05 40.7, 39.8,32.4,23.5 & 19.5

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for the polypeptides or their regulatory loci results ininhibition of transcription or translation of polypeptides[25] leading to the lack of expression of the concernedpolypeptides. This kind of variations, revealed in elec-trophoretic banding patterns, could lead to the detectionof genotype or species specific bands. Therefore, thepolypeptides varying for their presence could be usedas reliable molecular markers for verification of hybrid-ity of inter-varietal [2], and inter-specific [12, 26]crosses. In the present study SDS-PAGE of seed albu-mins of three genotypes including C. cajan (ICP-26), C.scarabaeoides (ICPW-94), and their F1 hybrids led tothe detection of 24 polypeptide bands with molecularweight 16.6 to 55.1 KDa (Table 3; Fig. 3a). Out ofthese, 18 polypeptides were monomorphic. F1 hybridrevealed the summation of five albumin/globulin poly-peptides (Mw 40.7, 39.8, 32.4, 23.5, and 19.5 KDa)specific to pollen parent, C. scarabaeoides (ICPW-94),and three unique polypeptides (Mw 34.7, 26.3 and20.9 KDa) of seed parent C. cajan (ICP-26) in additionto the common monomorphic polypeptides. Since C.scarabaeoides is used as pollen parent, five polypepti-des specific to C. scarabaeoides appeared in the F1hybrid can be potentially used as markers for identifi-cation of hybrids involving at least C. scarabaeoides-ICPW-94 as the donor parent for resistance to pod borercomplex.

RAPD and ISSR marker analysis RAPD and ISSR baseddetection of genetic polymorphism relies on differentialenzymatic amplification of small DNA fragments usingpolymerase chain reaction (PCR) with random decamerprimers [43], and simple sequence repeat (SSR) motifs[44], respectively. Both, RAPD and ISSR, markers areindefinite in number, ubiquitously distributed throughoutthe genome, and capable of detecting high level of poly-morphism. Hence, these markers have been successfullyutilized in molecular breeding endeavours in several cropspecies. In the present pursuit, both the DNA markersshowed polymorphism at par, among the parents used(Table 4). All the 20 RAPD primers and nine ISSR primersshowed parental polymorphism, and this might be due tothe genetic divergence between the parents at species level.There are some contradictory reports on detection of poly-morphism by RAPD and ISSR markers; ISSR markers aremore polymorphic than RAPD markers [7, 17, 23, 44] andvice versa [22]. This contradiction might be due to the useof different decamer oligonucleotides or SSR motifs asprimers, and varied primer-annealing site in the genomes.Again, ISSR polymorphism depends on the frequency ofSSRs, which changes with species or even varieties, andthe SSR motifs that are targeted [4]. Identification of inter-and intra-specific hybrids has been carried out in several

species using either RAPD or ISSR markers individually,or in combination [3, 8, 12, 17]. In this study, both RAPDand ISSR primers were equally efficient for identificationof the F1 hybrid (Fig. 3b and c). The total number ofamplified fragments, number of polymorphic fragments, per-centage of polymorphism, number of polymorphic parentalfragments appeared in the F1 hybrid, and C. scarabaeoides(pollen parent) specific fragments inherited to the hybrid pereach RAPD and ISSR primer were shown in Table 4. Twenty

Fig. 3 a Seed protein profile of C. cajan (Cc), C. scarabaeoides (Cs)and their interspecific hybrid (F1); Lane ‘M’ represents mol. weightmarker (PMW-M, GENEI, India), and arrow indicates the C. scara-baeoides specific polypeptides inherited to the F1 hybrid. b RAPDbanding pattern generated by five primers showing inheritance of C.scarabaeoides specific polymorphic bands to the F1 plants (whitearrow), and amplification of non parental bands (black arrow). c ISSRbanding pattern generated by four primers showing inheritance of C.scarabaeoides specific polymorphic bands to the F1 plants (whitearrow), and amplification of non parental bands (black arrow)

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eight RAPD and 12 ISSRmarkers, unique to the pollen parentC. scarabaeoides, were appeared in the F1 hybrids used in thepresent study. The primers (Table 4), generating thesemarkers, were useful to select true hybrids based on theamplification of male parent specific markers in the F1 hybrid.However, in the F1 hybrids, 18 of the parental amplified frag-ments were missing and four non-parental amplified frag-ments appeared. The appearance of non-parental amplifiedfragment and the disappearance of parental amplified frag-ments might be attributed to DNA recombination followed byminor genomic reorganization [10], and to loss of priming sitesdue to chromosomal crossing over during meiosis [38]. Sincethe objectives here pertain to identification of hybrids, the

confirmation of the hybrid nature of putative seedlings at thejuvenile stage by screening with either RAPD or ISSR markerswould be practical, and of economic significance to this crop.

Previous studies have reported analysis of inter-specifichybrids, belonging to the genus Cajanus, using either mor-phological or cytological approaches. These cyto-morphological attributes are constrained with the environ-mental fluctuations and level of polymorphism. Hence, thecomplementary use of cyto-morphological, seed proteinprofiling, and DNA marker based characterization allowmore precise evaluation of F1 hybrids,. This could be usefulfor further studies in gene mapping, marker assisted breed-ing involving wide hybridization.

Table 4 Inheritance of RAPD and ISSR markers in the inter-specific (C. cajan, ICP-26×C.scarabaeoides, ICPW-94) F1 hybrid

Primer Ref. Primersequence(5′→3′)

No. offragmentsamplified

Amplifiedfragmentsize (bp)

Parentalpolymor-phism (%)

No. of Non-parental fragmentsa

in F1

C. scarabaeoides(ICPW-94) specificinherited fragmenta

OPM-01 5′-GTTGGTGGCT-3′ 10 195–1500 40 – 470, 375

OPM-02 5′-ACAACGCCTC-3′ 11 395–1885 54.5 – 1885

OPM-03 5′-GGGGGATGAG-3′ 09 165–2000 55.5 – 275

OPM-04 5′-GGCGGTTGTC-3′ 11 525–2500 70 1 (1330) 2500, 1175, 4940, 525

OPM-05 5′-GGGAACGTGT-3′ 11 370–2855 30 1 (450) 1480

OPM-06 5′-CTGGGCAACT-3′ 07 450–1500 28.5 – None

OPM-07 5′-CCGTGACTCA-3′ 09 475–2125 44.4 – 1165, 580

OPM-08 5′-TCTGTTCCCC-3′ 04 640–1195 50 – 710

OPM-09 5′-GTCTTGCGGA-3′ 07 760–3125 100 – 3125

OPM-10 5′-TCTGGCGCAC-3′ 08 565–2710 25 – None

OPM-11 5′-GTCCACTGTG-3′ 11 550–2890 45.4 – 1000, 550

OPM-12 5′-GGGACGTTGG-3′ 12 510–2430 75 – 2320, 1645, 1130, 510

OPM-13 5′-GGTGGTCAAG-3′ 05 395–865 20 – None

OPM-14 5′-AGGGTCGTTC-3′ 10 500–1855 60 – None

OPM-15 5′-GACCTACCAC-3′ 06 535–2395 66.6 – 2395

OPM-16 5′-GTAACCAGCC-3′ 06 540–1250 16.6 – None

OPM-17 5′-TCAGTCCGGG-3′ 06 320–1500 83.3 – 1270, 1100, 320

OPM-18 5′-CACCATCCGT-3′ 08 625–2000 62.5 – None

OPM-19 5′-CCTTCAGGCA-3′ 05 915–2750 40 – 2250

OPM-20 5′-AGGTCTTGGG-3′ 09 540–2785 77.7 – 2785, 1670, 1305, 1140

Total 165 165–3125 52.72 02 28

UBC-809 5′-(AG)8G-3′ 08 775–2375 50 – None

UBC-810 5′-(GA)8T-3′ 08 450–1580 50 – 1350, 1230, 825

UBC-861 5′-(ACC)6-3′ 07 695–1525 42.8 – None

UBC-865 5′-(CCG)6-3′ 05 410–1290 20 – None

UBC-868 5′-(GAA)6-3′ 13 320–2540 83.3 1 (1050) 1615, 1420, 1165

UBC-873 5′-(GACA)4-3′ 09 525–2040 55.5 – 1615, 1270, 965

UBC-872 5′-(GATA)4-3′ 06 525–2000 100 1 (525) 2000, 1250, 1105

UBC-808 5′-(AG)8C-3′ 07 515–2375 14.3 – None

UBC-807 5′-(AG)8T-3′ 08 315–2125 50 – None

Total 71 315–2540 52.11 02 12

a Fragment size in bp

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Acknowledgments The authors are highly grateful to IFS, Sweden(4584/2), and University Grant Commission [F. No. 37-331/2009(SR)], Govt. of India, New Delhi for financial assistance though R&D project to JP. The authors are also grateful to Dr. H. D. Upadhyaya,Senior Scientist, ICRISAT for providing the germplasm of the plantmaterial required for the study.

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