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Identification of sex-specific DNA markers in betel vine ( Piper betle L

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The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in male and female plants of Piper betle L. Two bulks of DNA were made drawing one each from
  RESEARCH ARTICLE Identification of sex-specific DNA markers in betel vine(  Piper betle  L.) Sanghamitra Samantaray  • Arunkumar Phurailatpam  • Ashok Kumar Bishoyi  • K. A. Geetha  • Satyabrata Maiti Received: 18 November 2010/Accepted: 2 May 2011   Springer Science+Business Media B.V. 2011 Abstract  The Random Amplified PolymorphicDNA (RAPD) technique was used to amplify DNAsegments, with the objective of finding markerslinked to sex determination in male and femaleplants of   Piper betle  L. Two bulks of DNA weremade drawing one each from male and female, bypooling an equal volume of DNA samples from eachgroup of individual contributing to the bulk analysis.Fifty different random decamer primers werescreened with the two bulks to identify markersassociated with sex expression of which only fourprimers were found to be associated with sexexpression. These four primers were then tested withindividual plant DNA samples where sex-associatedRAPD markers were identified. A  * 1,400 and * 850 bp fragment from the primer OPA04 andOPN 02 respectively was found to be present in allthe male individuals and absent in all the femaleplants. In another primer, a  * 980 bp amplificationproduct from the primer OPC 06 was present only inthe female individuals. A common primer OPA 08showed both male and female specific markers of 650and 1,200 bp respectively. Thus, the three male-specific RAPD markers OPA04 1400 , OPA08 650  andOPN02 850  and two female-specific markersOPA08 1200  and OPC06 980  can reliably differentiatethe male and female plants of   P. betle  L. Ploidycomparison also showed the differences in male andfemale plants. Keywords  Dioecy    Piper betle  L.    Sexidentification Introduction Betel vine ( Piper betle  L.) belongs to the familyPiperaceae, is an important cash crop of India. Betelleaf is associated closely with cultural traditions of India and is considered as a holy plant. Fresh leavesare generally consumed along with betel nuts (  Arecacatechu  L.). It has many medicinal usages in Indiansystem of medicines to cure indigestion, stomach-ache, diarrhoea, flatulence and to heal wounds,bruises, swellings due to sprains, bruises, respiratorydisorders, constipations, boils and gum disorders(Chopra et al. 1956). Studies also revealed that theleaf improves immune system and inhibits cancergrowth (Amonkar et al. 1986). Moreover, oil of betelleaf has been used in the treatment of variousrespiratory catarrhs, and as a local application, eitherby gargle or by inhalation, in diphtheria (Anonymous1969). S. Samantaray ( & )Central Rice Research Institute (CRRI), Cuttack 753006,Orissa, Indiae-mail: smitralok@rediffmail.com; smitraray@gmail.comA. Phurailatpam    A. K. Bishoyi    K. A. Geetha    S. MaitiDirectorate of Medicinal and Aromatic Plants Research,Boriavi, Anand 387310, Gujarat, India  1 3 Genet Resour Crop EvolDOI 10.1007/s10722-011-9707-4  Betel vine is a native of Central and easternMalaysia and has spread through out tropical Asiaand Malayasia. It was also taken to Madagascar andEast Africa at a later date. It is widely cultivated inthe states of Uttar Pradesh, Bihar, Madhya Pradesh,Northeastern India, Maharasthra, Karnataka, WestBengal, Orissa, Andhra Pradesh, Tamil Nadu, Keralaand Andamans in India. Betel vine  (Piper betle  L.) isa perennial, evergreen creeper grown in shadyconditions having moderate temperature with highhumidity. It is cultivated also in Bangladesh, Srilanka, and to a limited extent in Pakistan, Malayasia,Vietnam and Papua New Guinea. This crop has greatmarket value both inside and outside India and leavesare exported from India to countries like Pakistan,Bangladesh, Indonesia, Burma and Thailand.Betel vine is a dioecious plant having male andfemale plants. Since sexual dimorphism is associatedwith economic traits (leaf length and width), greatimprovement in leaf yield and quality in the cropcould be achieved through heterosis breeding (Maitiet al. 1992) which has been already initiated todevelop new cultivars. Though phenotypic characterslike leaf shape are available to identify the male andfemale plants in the absence of flowering (Maiti andBiswas 1991), however, it does not reflect conspic-uously at early seedling stage. However, since theinability to identify gender at early ages can createproblems in advanced-generation breeding pro-grammes, particularly when all superior parentalselections or all progeny are unknowingly composedof one gender. Therefore, effective molecular markerbased sex identification would be very useful fordiscrimination of gender. Moreover, such test wouldallow a significant reduction in time for evaluatingthis trait, permit the rapid and separate evaluation of plants of both sexes during breeding processes, andconsiderably reduce the amount of labour and fieldspace. Besides, understanding the molecular basis of sex expression has immense importance both in basicand applied research. The evolution of sex in planthas been hypothesized variously as the plants displaya great variety of sexual phenotypes (Tanurdzic andBanks 2004).Betel vine cultivars in traditional farming systemsof India are mainly composed of two groups viz.,  Bangla  and  Kapoori . These cultivars are the selec-tions made by the farmers over a period of time basedon their personal preferences influenced by themarket trends or consumer trends from time to time.Till the discovery of male and female flowering andfruiting in India (Rao and Maiti 1989), it wasbelieved that only males were being cultivated. Later,Maiti and Biswas (1991) reported sexual dimorphismalso in the species wherein male plants have narrowlyovate leaves and females have cordate leaves.Fragmented work has so far been reported onchromosome numbers and ploidy status of the speciesby a few workers (Kumar and Subramaniam 1986)and different chromosome numbers were reported inthe species. Basic chromosome number of the speciesis x  =  13. Samuel and Bavappa (1981) reportedchromosome numbers as 2n  =  26 and 52; Sharmaand Bhattacharya (1959) and Dasgupta and Datta(1976) reported 2n  =  32; 2n  =  64 and Mathew(1958) and Jose and Sharma (1988) reported 2n  =  78 chromosomes. They also reported thatpolyploidy had played important role in the evolutionof   P. betle  L. The extensive vegetative propagation of the species has established these polyploidy varia-tions in the cultivars. All these cytological workswere carried out without considering the sex of theplant since rare flowering without fruit set wasreported at that time. Hence, no information isavailable on the mechanism of sex expression in thisspecies.Molecular marker systems based on direct analysisof genomic DNA have been used extensively forgenetic diversity, disease diagnostics and evolution-ary studies, and they can be also very useful in thestudy of sexual determination and identification indioecious plants. Genetic diversity amongst landracesof betel vine was studied earlier using RAPD markers(Ranade et al. 2002; Verma et al. 2004). However, an easy to score DNA marker to distinguish male andfemale plants has not been developed so far in P. betle  L. Recently, RAPD markers have shown itsreliability for determining sex in  Pistacia vera L. (Hormaza et al. 1994; Kafkas et al. 2001),  Atriplexgarrettii  Rydb. (Claudete et al. 1998),  Trichosanthesdiocia  Roxb. (Singh et al. 2002),  Salix viminalis L. (Alstrom-Rapaport et al. 1998),  Borassus flabel-lifer   L. (George et al. 2007),  Simmondsia chinensis (Link) C. K. Schneid (Agrawal et al. 2007),  Carica papaya  L. and  Cycas circinalis  L. (Gangopadhayaet al. 2007) and  Commiphora wightii  (Arn.) Bhandari(Samantaray et al. 2010). Here we report for the firsttime on the identification of RAPD markers and Genet Resour Crop Evol  1 3  ploidy levels associated with the sex determination inmale and female plants of   P. betle  L. Materials and methods Plant materialPlants from eleven accessions of male and eightaccessions of female individuals collected fromdifferent centers of the All India Co-ordinatedResearch Project (AICRP) on betel vine (Table 1)were used for determining sex-associated markers inDNA by RAPD analysis. Immature leaf materialscollected from sample of adult pistilate and staminateplants after complete observation of flower typeswere used for DNA extraction.Genomic DNA isolationTotal genomic DNA was isolated from 3 g of leaf tissues from 19 accessions (11 male: 8 female) withthe modified CTAB method (Doyle and Doyle 1990).DNA was treated with RNaseA (Qiagen, USA) foreradication of RNA followed by two washingswith chloroform:iso-amyl-alcohol (24:1; v/v). Subse-quently, quality and quantity were checked byrunning the dissolved DNA in 0.8% agarose gelalong side uncut  k  DNA (Bangalore Genei, Banga-lore, India) of known concentration. The DNA wasdiluted to 30 ng/  l l for RAPD analysis.Bulk analysisTwo bulk samples were prepared separately bypooling an equal amount of DNA from individualplants of 11 male and 8 female accessions andamplified with 60 decamer primers. A DNA markerpresent in the corresponding male and female bulksand absent in the alternate sex bulk was considered asa potential sex-linked markers. Bulked analysis wasused to screen each individual of known sexindependently to identify the sex specificity of themarker.RAPD marker analysisFifty 10-base primers (series OPA; 20, OPC; 20 andOPN;10 (Operon Technologies, Alameda, USA) wereused for Polymerase Chain Reaction (PCR) forscreening of known sex to ascertain their potentialof clear amplification in polymorphism and also thereproducibility. Amplification reactions were per-formed in volumes of 25  l l containing 2.5  l l of 10Xassay buffer (100 mM Tris–HCl; pH 8.3, 500 mMKCl, 15 mM MgCl 2 ), 100 mM of each dNTPs(dATP, dCTP, dGTP and dTTP) (MBI FermentInc., Maryland, USA) 5 pg of primer, 1.0 unit of Taq DNA polymerase (Bangalore Genei., Bangalore,India) and 30 ng of template DNA. The amplificationreaction was carried out in a DNA Thermal Cycler(Eppendorf AG, Hamburg, Germany) programmedfor 44 cycles as follows: 1st cycle of 5 min at 94  Cfollowed by 43 cycles each of 1 min at 92  C, 1 minat 37  C, 2 min at 72  C. The final step consisted of one cycle of 7 min at 72  C for complete polymeri-zation. After completion of the PCR, 2.5  l l of 6Xloading dye (MBI Ferment Inc., Maryland, USA) wasadded to the amplified products and were electroph-orized in a 1.5% (m/v) agarose (Bangalore Genei,Bangalore, India) gels with 1X TAE buffer, stainedwith ethidium bromide and documented by a geldocumentation system (Syngene, Cambridge, UK). Table 1  The betel vine accessions collected from differentcentres of AICRP on betel vine with identification of sexAccession name Place of collection GenderTellaku Ponnur Andhra Pradesh MaleKuljedu Cuddapah Andhra Pradesh MaleKapoori Chinacheppai Andhra Pradesh MaleKapoori Doddipatla Andhra Pradesh MaleKapoori Chilumuru Andhra Pradesh MaleKapoori Cuddapah Andhra Pradesh MaleKapoori Tamilnadu MalePachaikodi Tamilnadu MaleSwarna kapoori Andhra Pradesh MaleKapoori Chittikavata Andhra Pradesh MaleKapoori Arvi Andhra Pradesh MaleGangeri Andhra Pradesh FemaleVellaikodi Tamilnadu FemaleRamtek Bangla Maharashtra FemaleMeetha Pan West Bengal FemaleGhanaghette West Bengal FemaleGach Pan Assam FemaleBilhari Madhya Pradesh FemaleSimmurali Bhavna West Bengal FemaleGenet Resour Crop Evol  1 3  The size of amplification products was estimated bycomparing with standard DNA ladder (O’Gene Ruler1.0 kbp DNA ladder; MBI Ferment Inc., Maryland,USA). All the reactions were repeated three times.Eighteen primers were amplified on the basis of theclarity of the banding patterns.Ploidy analysisLeaf samples were used for flow cytometry studies.Relative fluorescence intensity of stained nuclei wasanalysed using a PA II flow cytometer (Partec,Germany). The reference standards used for theanalysis were Kapoori lines (Male) and Bangla lines(Female) for which different ploidy level wasconfirmed. Results In the present study, screening of the male and femaleDNA pools of the betel vine with a total of 50oligonucleotide primers, only 18 primers showedreproducible results and a total of 163 bands wereamplified; 76 and 87 bands were amplified in maleand female respectively. However, out of them, 4potential markers such as OPA 04, OPA 08, OPC 06and OPN 02 produced distinct polymorphismsbetween the sexes. The number of bands amplifiedby these four primers varied from 2 to 6 and thefragment size ranged between 400 and 2,200 bp inmale and female bulks.In Bulked analysis, among the four oligonucleotideprimers showing sex differences, two primers OPA 04and OPN 02 showed sex specificity only for the malesand OPC 06 was specific only for the females(Figs. 1a, b, 2a). Furthermore, one marker OPA 08 (5 0 GTGACGTAGG3 0 ) was found to be specific forboth sexes. Male plants resulted in a  * 650 bp andfemale plants in a * 1,200 bp respectively (Fig. 2b).The primers OPA 04 (5 0 AATCGGGCTG 3 0 ) and OPN02 (5 0 ACCAGGGGCA3 0 ) produced unique bands of  * 1,400 and  * 850 fragment respectively, in malebulk DNA. Similarly, a unique band of 980 bpspecific to female plants, was amplified by the OPC06 primer (5 0 GAACGGACTC3 0 ) and was absent inmale counterparts.For confirmation of this observation, the fourprimers which generated polymorphisms in thedifferent sex type were used to re-test the DNAsamplification with individual plants of each sex typeof male and female which produced consistent andreproducible amplification pattern with 11 male and 8female individual plants. The unique bands of  * 1,400 and * 850 bp were produced with the primerOPA 04 and OPN 02 in male individuals, which wasabsent in female (Fig. 1a, b). Similarly, anotherunique band * 980 bp specific for female individualsamplified by OPC 06 primer was only present infemale plants not in males (Fig. 2a). Thus, the RAPDmarkers OPA04 1400  and OPN02 850  could be con-firmed as putative sex-linked marker for male andOPC06 980  for female plants of   P. betle  L. As shownbefore, the marker amplified by OPA08 was specificfor both sexes and resulted in amplification productsof expected size * 650 and * 1,200 bp for male andfemale respectively (Fig. 2b.).Ploidy comparison showed that all the male andfemale plants collected from different regions couldbe grouped into two classes with all males beingtriploids (2n  =  3X  =  39) and females being tetra-ploids (2n  =  4X  =  52). Thus, the study revealed adistinct ploidy differences between the sexes of maleand female plants (Fig. 3). Discussion Molecular genetics can be integrated with traditionalmethods of selective breeding for desired phenotypesthrough the use of marker-assisted selection (MAS).The availability of suitable molecular markers canpermit breeding programmes from elite 10 cultivarbackgrounds to be completed in a shorter time and ina more cost-effective manner (Tanksley et al. 1989).For example, in hybrid production notable effortshave been made to overcome the problem of discriminating male and female plants using pheno-typic characters like leaf shape in the absence of flowering (Maiti and Biswas 1991), however, at earlyseedling stage these differences are not reflectedconspicuously. Similarly, Kumar et al. (2006) devel-oped chlorophyllase activity as a marker for distinc-tion of male and female landraces of   P. betle  L.which is environmental i.e. temperature dependant.Since the inability to identify gender at early ages cancreate problems in advanced-generation breedingprogrammes, particularly when all superior parental Genet Resour Crop Evol  1 3  selections or all progeny are unknowingly composedof one gender. On the other hand, manual selection of male and female during the flowering period con-sumes time, labour and money. Moreover, micro-propagation protocol already developed for  P. betle L. by Bhat et al. (1995) might be adopted forindividual propagation of male and female plants,however, sometimes, plant hormones in the mediummay change the sex of the plant reported earlier(Matsubara 1977).Our result showed two primers OPA 04 and OPN02 that produced unique bands of  * 1,400 and * 850fragment respectively, in male bulk DNA, and noproduct in female DNA. Bulked analyses involvingRAPD products have been successfully used toidentify molecular markers associated with sexdetermination in several dioecious plant speciesinstead of assessing amplification products fromDNA of individual plants (Mulcahy et al. 1992;Hormaza et al. 1994; Parrish et al. 2004). Subse- quently, Parrish et al. (2004) suggested a method of AFLP-BSA for development of more reproducibleresults for the detection of sex-specific markers;however this approach is more expensive than PCR-BSA.To confirm the results of the bulk analysis (BA)we performed the DNA analysis of individual plantsof males and females from which we noted poly-morphism for the sex type in four primers generatedwith individual plants of each sex type of male andfemale which produced consistent and reproducibleamplification pattern with male and female individ-uals. Similar findings were observed for male sex-associated genetic factors in other organisms forexample a Y-chromosome-specific restriction frag-ment has been reported in white campion ( Silenelatifolia  Poir.) (Dominson et al. 1996) and two RAPDbands, OPD05  961  and UBC354 151  have been foundin hemp ( Cannabis sativa  L.) (Torzek et al. 2002).Similarly, sex-linked markers OPA 08 945 , UBC345 560 , OPC 07 567  were amplified in female individ-uals of   Pistacia vera  L.,  Salix viminalis  L. and Trichosanthes diocia  Roxb. respectively reported byseveral groups (Hormaza et al. 1994; Alstrom-Rapa-port et al. 1998; Singh et al. 2002). Interestingly, a common marker specific to both sexes amplified by Fig. 1 a  RAPD bandingprofile of bulk andindividual male and femaleplants of   P. betle  L. usingOPA04.  Arrow  indicates theunique band of  * 1,400 bppresent in male bulk andmale individuals. M: 1 Kbpladder;  lane 1 : bulk male; lane 2 : bulk female;  lanes3 – 13 : male individuals; lane 14 – 21 : femaleindividuals.  b  RAPDbanding profile of bulk andindividual male and femaleplants of   P. betle  L. usingOPN 02.  Arrows  indicatethe unique band of  * 850 bp present in malebulk and male individuals.M: 1 Kbp ladder;  lane 1 :bulk male;  lane 2 : bulk female;  lanes 3 – 13 : maleindividuals;  lane 14 – 21 :female individualsGenet Resour Crop Evol  1 3
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