Ten pran genotypes were evaluated using RAPD markers to determine the genetic relationships among genotypes showing morphological variations. Fourty three decamer primers were used in this study. Genetic diversity in the genotypes studied was analyzed using several variability parameters. Genotypes were grouped into two main clusters based on jaccard similarity coefficient. A dendrogram based on UPGMA analysis grouped the ten genotypes into two main clusters. Genotypes representing cluster-I are superior over genotypes in cluster-II with respect to yield attributes. Some genotypes did not show any similarity with either cluster, depicting their diverse nature. The grouping that was obtained with the RAPD analysis was at par with the morphological grouping based on yield attributes. The results of Principal Co-ordinate (PCO) analysis were comparable to the cluster analysis. Finally, the investigation of the genetic variation of pran with RAPD indicated that this marker is a suitable tool for assessing polymorphism and estimating the genetic similarity.
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K A Mir, M A Sheikh, M H Khan,, J I Mir, N Ahmed, S Wajida, R Rizwan,

U Noor, Z F Shafia,

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194IntroductionThe genus Allium L. (Alliaceae) exhibits greatdiversity in various morphological characters,particularly in life form (bulb) and ecologicalhabitat. It is of major economic importance asa vegetable and herbal crop and ornamentalplant (Ricroch et al. 2005). This genus consistsmostly of perennial and bulbous plant and it iswidely distributed over Holarctic regions fromthe dry subtropics to the boreal zone (Stearn1992). This species was used in traditionalpharmacopoeia for its expectorant properties(Marcucci & Tornadore 1997). Contrary to thecultivated garlic (Allium sativum L.) which isvery well studied in terms of its morphological,agronomic and molecular aspects (Baghalianet al. 2006; Singh et al. 2011), studies about rosygarlic (A. roseum L.) are few and limited tomorphophenology (Jendoubi et al. 2001) andcytocaryology (Marcucci & Tornadore 1997).In the genetic improvement process, it isdesirable to use molecular markers forevaluating genetic diversity of accessions,choosing of parents and selection of progeny.Diversity analyses of different garlic accessionshave been carried out using RAPD, AFLP andMicrosatellite markers for detection of putativeduplicates in germplasm collections (Ipek et al.2003) or grouping with respect tophotoperiodism (Maass 1997; Singh et al. 2011).Genetic diversity of pran (Allium cepa var. prolliferum) in KashmirJ I Mir*, N Ahmed, S Wajida, R Rizwan, K A Mir, M A Sheikh, M H Khan, Z F Shafia & U NoorCentral Institute of Temperate Horticulture,Rangreth, Srinagar-190 007, J & K, India.*E-mail: javidiqbal123@rediffmail.comReceived 23 May 2012; Revised 19 December 2012; Accepted 19 December 2012AbstractTen pran genotypes were evaluated using RAPD markers to determine the genetic relationshipsamong genotypes showing morphological variations. Fourty three decamer primers were usedin this study. Genetic diversity in the genotypes studied was analyzed using several variabilityparameters. Genotypes were grouped into two main clusters based on jaccard similarity coefficient.A dendrogram based on UPGMA analysis grouped the ten genotypes into two main clusters.Genotypes representing cluster-I are superior over genotypes in cluster-II with respect to yieldattributes. Some genotypes did not show any similarity with either cluster, depicting their diversenature. The grouping that was obtained with the RAPD analysis was at par with themorphological grouping based on yield attributes. The results of Principal Co-ordinate (PCO)analysis were comparable to the cluster analysis. Finally, the investigation of the genetic variationof pran with RAPD indicated that this marker is a suitable tool for assessing polymorphism andestimating the genetic similarity.Keywords: bunching onion, molecular markers, pran, RAPDJournal of Spices and Aromatic CropsVol. 22 (2) : 194–199 (2013)         Indian Society for Spiceswww.indianspicesociety.in/josac/index.php/josac195Pran, also known as bunching or Egyptianonion, is one of the high value spice of Kashmirused for culinary seasoning and to flavor foods.The range of foods that have been spiced withpran is wide, including cheese, chicken and meatand different Kashmiri cuisine favours the useof pran. While morphological differencesbetween pran cultivars can be observed, onlya few morphological traits are used forgrouping pran, and these can be affected byenvironmental conditions. Even with theintroduction of new molecular marker systemsduring the last years, reports of these markersin pran have been scarce (Tsukazaki et al. 2008).But further molecular characterization of pranis necessary to associate the desirable traits withthe molecular markers which can be furtherused in pran improvement programmes. In thepresent study, diverse pran accessions havebeen evaluated with respect to yield parametersand characterized at molecular level foridentifying the most divergent accessionswhich can further be used for crossing anddevelopment of superior cultivars.Material and methodsThe present experiment was carried out during2010–11 in which ten pran selections (CITH-Pran-1, CITH-Pran-2, CITH-Pran-3, CITH-Pran4, CITH-Pran-5, CITH-Pran-6, CITH-Pran-7, CITH-Pran-8, CITH-Pran-9, CITH-Pran-10) maintained at the Central Institute ofTemperate Horticulture, Srinagar (J&K) underopen field conditions were used. Thesegenotypes were used with respect to differenttraits like plant height, bulb length, bulbthickness, neck thickness, average bulb weight,number of bulbs/plant and plant yield. GenomicDNA was extracted from freshly emerged leavesof pran genotypes using the CTAB method(Murray & Thompson 1980). Approximately 0.5g tissue samples from each plant species weresnap-frozen in liquid nitrogen. DNA waspurified and quantified spectrophotometricallyusing uncut lambda DNA fragment as control.Final concentration of 20 to 40 ng μL–1 was usedfor PCR. Samples were screened for RAPDvariation using standard 10-base primerssupplied by Operon. DNA from an individualplant of each pran accession was screened with45 RAPD primers. The PCR reaction (25 μL)contained the following: 1x reaction buffer (20mM Tris-Cl pH 8.4, 50 mM KCl), 0.2 mMdNTPs, 2 mM MgCl2, 10 pM primer, 1.0 Unit ofTaq DNA polymerase and 25 to 50 ng genomicDNA. The DNA was amplified in a thermalcycler (Takara Thermal Cycler) that wasprogrammed as follows: initial DNAdenaturation for 5 min at 94°C; 45 cycles of 60sec at 94°C (denaturation), 60 sec at 37°C(annealing) and 120 sec at 72°C (extension) anda final extension for 7 min at 72°C. All primerstested using all cultivars and markers werechecked three times for reproducibility. TheRAPD amplified-DNA was analyzed byelectrophoresis on 2% agarose gel in a 0.5 TBEbuffer. The gels were stained with ethidiumbromide (0.5 μg L–1) and visualized under UVlight.In order to determine the utility of thesemarkers, polymorphic information content(PIC), effective multiplex ratio (EMR)/resolvingpower (Rp) and marker index (MI) werecomputed. PIC for genetic markers wascalculated from the sum of the squares of allelefrequencies: Din=1–∑ pi2 (where pi is the allelefrequency of the ith allele). The arithmetic meanheterozygosity was calculated for each markerclass: Diav=∑ Din/n, (where n is the numberof markers (loci) analyzed). The DI forpolymorphic markers is: (Diav) p=∑ Din/np(where np is the number of polymorphic lociand n is the total number of loci). EMR (E) isthe product of the fraction of polymorphic lociand the number of polymorphic loci for anindividual assay; EMR (E)=np (np/n). MI isdefined as the product of the average diversityindex for polymorphic bands in any assay andthe EMR for that assay, MI=Diavp * E.Prominent DNA bands that were amplified bya given primer were scored as present (1) orabsent (0). The PIC values of individual primerswere calculated based on the formula PIC=2 ×F (1–F) (Anderson et al. 1993). The Jaccard’ssimilarity index was calculated using NTSYS-pc version 2.02e (Applied Biostatistics, Inc.,Setauket, NY, USA) package to compute pair-wise Jaccard’s similarity coefficients (Jaccard1908) and this similarity matrix was used inPran genetic diversity196cluster analysis using an unweighted pair-group method with arithmetic averages(UPGMA) and sequential, agglomerative,hierarchical and nested (SAHN) clusteringalgorithm to obtain a dendrogram. To judgethe confidence of the group revealed in thedendogram, bootstrap analysis was performedusing the WINBOOT program (Yap & Nelson1996) with 1000 replications.Results and discussionEvaluation of pran genotypesThe performance of ten pran/bunching oniongenotypes during the year 2010–2011 withrespect to traits like plant height, bulb length,bulb thickness, neck thickness, average bulbweight, number of bulbs plant-1 and yield aregiven in Table 1. Average bulb weight variedfrom 22.97 g in CITH-Pran-1 to 49.93 g inCITH-Pran-10. Yield plant-1 varied from 214 gplant-1 in CITH-Pran-6 to 404 g plant-1 in CITH-Pran-2. Number of bulbs was maximum (15plant -1) in CITH-pran-2 and minimum (5 plant-1)in CITH-Pran-10.RAPD analysis and genetic diversityAll the chosen primers amplified with thenumber of amplified fragments varying in sizefrom 200–1200 bp. Out of 64 amplified bands,46 were found polymorphic, with an averagenumber of bands/polymorphic bands perprimer as 1.42. The respective values for overallgenetic variability for PIC, Rp and MI acrossall the 10 genotypes are given in Table 2. PIC isa feature of a primer and therefore the PICvalues were calculated for all primers. HighestPIC value (0.24) was observed for the primerOPP-10 and lowest PIC value (0.05) wasrecorded for the primer OPJ-5 (Table 1). AveragePIC value was 0.18.  The RP is a feature of theprimer that indicates the discriminatorypotential of the primer. RP ranged from 0.05 to0.93 with an average of 0.29 per primer. MI is afeature of a marker and therefore the MI valueswere calculated for all markers. MI valuesranged from 0.23 (OPJ-5) to 1.07 (OPP-10, OPJ-11, OPP-4, OPB-20, OPA-15, OPB-2, OPB-18,OPV-15 and OPP-5) with an average value of0.83 across all the primers. Similar results havebeen obtained earlier in Allium sativum usingRAPD markers (Abdoli et al. 2009; Singh et al.2011). Jaccard’s coefficient showed that thehighest similarity was observed between cv.CITH-Pran-10 and CITH-Pran-9 (0.90),implying that these are genetically closer thanthe other cultivars. Lowest similarity wasTable 1. Evaluation of pran genotypesVariety Plant Bulb Neck No. of Plant Totalheight length Bulb dia. thickness Avg. bulb bulbs yield yield(cm) (cm) (cm) (cm) wt. (g) plant-1 (g plant-1)  (q ha-1)CITH-Pran-1 46.50 8.5 1.88 0.76 22.97 14 321.60 536.00CITH-Pran-2 53.00 8.9 1.72 0.72 26.96 15 404.40 674.00CITH-Pran-3 63.00 9.6 2.42 0.70 24.02 13 312.30 520.50CITH-Pran-4 58.50 9.9 2.13 0.88 28.15 11 309.60 516.00CITH-Pran-5 58.50 11.5 2.49 1.26 49.03 7 343.20 572.00CITH-Pran-6 57.00 10.2 2.05 1.03 35.70 6 214.20 357.00CITH-Pran-7 54.50 8.8 2.20 0.86 34.07 10 340.66 567.77CITH-Pran-8 56.70 8.7 2.70 0.87 40.04 8 320.33 533.88CITH-Pran-9 60.10 8.0 2.80 0.99 39.11 9 352.00 586.66CITH-Pran10 52.8 10.4 2.80 1.30 49.93 5 249.66 416.10CD @ 5% 3.626 1.388 N.S. 0.209 3.076 2.505 5.765 6.393SE(d) 1.713 0.655 3.244 0.099 1.453 1.183 2.723 3.020SE(m) 1.211 0.463 2.294 0.070 1.027 0.837 1.925 2.135CV(%) 3.746 8.465 131.772 12.876 5.143 14.787 1.054 0.727Mir et al.197observed between cv CITH-Pran-7 and CITH-Pran-3 with Jaccard’s similarity coefficient of0.56. The average genetic similarity of 0.75among the cultivars clearly showed thatsignificant genetic diversity exists among thepran cultivars. The low level of genetic diversitypresent among these cultivars clearly suggestedthat they must have originated from geneticallyless divergent parents or have a short historyof adaptation to their respective microclimaticregions. Earlier studies using RAPD(Maniruzzaman et al. 2010) techniques alsoshowed lack of genetic variations amongdifferent onion cultivars. But similar studiesusing RAPD (Abdoli et al. 2009; Singh et al.2011) and AFLP (Ipek et al. 2003) techniques ingarlic showed large genetic variation amongcultivars from different countries. Adendrogram based on UPGMA analysisgrouped the 10 Pran genotypes into two mainclusters (I to II; Fig 1). Cluster I was representedby CITH-Pran-1, CITH-Pran-2, CITH-Pran-4and CITH-Pran-5 with an average similarityco-efficient of 0.84. Cluster II was representedby CITH-Pran-8, CITH-Pran-9 and CITH-Pran-10 with an average similarity co-efficientof 0.86. CITH-Pran-3, CITH-Pran-6 and CITH-Pran-7 form separate sub-clusters. Genotypesrepresenting cluster-I are superior overgenotypes in cluster-II with respect to yieldattributes (Table 1). Average number of bulbsof cluster-I genotypes was higher (12 plant-1)than in cluster-II genotypes (7 plant-1). Alsoaverage yield was 344 g plant-1 in cluster-Igenotypes which is much higher than averageyield per plant of 307 g plant-1 in cluster-IIgenotypes. Average similarity between cluster-I and cluster-II was 0.82, which is very similarTable 2. Polymorphic profile of 43 RAPD primers across 10 genotypes of pranPrimer PIC Rp MI Primer PIC Rp MIOPJ-1 0.17 0.59 0.86 OPP-5 0.21 0.73 1.07OPJ-4 0.16 0.17 0.78 OPV-3 0.21 0.49 0.64OPJ-5 0.05 005 0.23 OPV-6 0.18 0.61 0.91OPJ-7 0.17 0.39 0.52 OPV-15 0.21 0.24 1.07OPJ-9 0.20 0.22 0.97 OPV-14 0.20 0.22 0.97OPJ-3 0.20 0.93 0.81 OPV-10 0.18 0.20 0.88OPZ-J-6 0.21 0.71 1.04 OPZ-3 0.19 0.41 0.55OPJ-9 0.07 0.07 0.352 OPZ-4 0.16 0.17 0.78OPJ-11 0.21 0.24 1.07 OPA-15 0.21 0.24 1.07OPP-15 0.20 0.22 0.97 OPA-16 0.18 0.20 0.88OPP-16 0.18 0.20 0.88 OPA-17 0.20 0.44 0.58OPP-6 0.21 0.24 1.07 OPA-19 0.18 0.20 0.88OPP-13 0.18 0.20 0.88 OPA-8 0.20 0.22 0.97OPP-1 0.20 0.22 0.97 OPB-13 0.14 0.15 0.67OPP-2 0.16 0.17 0.78 OPB-1 0.18 0.20 0.88OPP-3 0.20 0.22 0.97 OPB-2 0.21 0.21 1.07OPP-4 0.21 0.24 1.07 OPB-5 0.14 0.29 0.40OPP-7 0.16 0.17 0.78 OPB-6 0.20 0.22 0.97OPP-9 0.18 0.20 0.88 OPB-10 0.09 0.10 0.46OPP-10 0.24 0.24 1.07 OPB-11 0.11 0.12 0.57OPB-19 0.20 0.22 0.97 OPB-18 0.21 0.24 1.07OPB-20 0.21 0.73 1.07 OPO-19 0.16 0.17 0.78OPO-15 0.21 0.49 0.64Pran genetic diversity198to average similarity within cluster-I (0.84) andwithin cluster-II (0.86). Although there is littledivergence between cluster-I and cluster-II,genotypes CITH-Pran-3, CITH-Pran-6 andCITH-Pran-7 showed least similarities with restof the genotypes, average similarity coefficientsbetween CITH-Pran-3, CITH-Pran-6 andCITH-Pran-7 with rest of the genotypes were0.66, 0.68 and 0.68, respectively.The genetic relationship between the accessionswas clearly depicted in the dendrogram whichwas constructed from the DNA profile and theconfidence of the cluster was further confirmedby bootstrap analysis. The grouping that wasobtained with the RAPD analysis was at parwith the morphological grouping based onyield attributes. The results of PCO analysiswere comparable to the cluster analysis (Fig 2).Overall 89% of the variability observed wasexplained by the first three coordinates, PCO1,PCO2 and PCO3 accounted for 78.63%, 5.82%and 3.99% of total variability, respectively.Molecular markers have been applied inclassification, identification and mapping inbunching onion (Tsukazaki et al. 2008). Thepresent study clearly identified the genotypeshaving better fruit quality. Improvement ofthese genotypes and inclusion of thesegenotypes in breeding programs for furtherquality enhancement can be done with the aidof molecular markers. First step in breeding isthe identification of closely related genotypesFig 2. Three dimensional PCO (principal co-ordinateanalysis) scaling of 10 pran genotypes using43 RAPD markersMir et al.Fig 1. UPGMA dendrogram showing clustering pattern of pran genotypes. The bootstrap values are given onthe nodes199and the study of genetic relationships. To assessorigin and genetic relationships among prancultivars by RAPD markers, a wider surveyincluding additional genotypes belonging to allestablished eco-geographical groups andadditional markers would be more desirable.Results from this study showed that RAPDmarkers are a useful tool for pran diversitystudies, which provided the basic knowledgefor improvement of pran quality throughfurther breeding programmes involvingdiverse genotypes.ReferencesAbdoli M, Habibi-Khaniani B, Baghalian K,Shahnazi S, Rassouli H & Naghdi Badi H2009 Classification of Iranian garlic (Alliumsativum L.) ecotypes using RAPD marker.J. Med. Plants 8: 1–7.Anderson J A, Churchill G A, Autrique J E, TanksleyS D & Sorrells M E 1993 Optimizingparental selection for genetic linkagemaps. Genome, 36: 181–188.Baghalian K, Sanei M R, Naghavi M R, Khalighi A&  Naghdi Badi H A 2006 Post-cultureevaluation of morphological divergencein Iranian garlic ecotypes. Sci. 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Nucleic Acid Res. 8: 4321–4325.Ricroch A, Yockteng R, Brown S C & Nadot S 2005Evolution of genome size across somecultivated Allium species. Genome, 48:511–520.Singh S R, Mir J I, Ahmed N, Rashid R, Wani S H,Sheikh M A, Mohi ud din H, Shafi W, JanN & Mir H 2011 RAPD profile basedgrouping of garlic Allium sativumgermplasm with respect tophotoperiodism, J. Trop. Agri. 49: 114–117.Stearn W T 1992 How many species of Allium areknown? Kew Mag. 9: 180–181.Tsukazaki H, Yamashita K, Yaguchi S, MasuzakiS, Fukuoka H, Yonemaru J, KanamoriH, Kono I, Hang T T H, Shigho M, Kojima A& Wako T 2008 Construction of SSR-basedchromosome map in bunching onion(Allium fistulosum). Theor. Appl. Genet. 17:1213–1223.Yap I P & Nelson R J 1996 Winboot: A program forperforming bootstrap analysis of binarydata to determine the confidence level.IRRI Discuss Paper Ser. 14 IRRI, Manila,Philippines.Pran genetic diversity

Genetic diversity of pran (Allium cepa var. prolliferum) in Kashmir

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Genetic diversity of pran (Allium cepa var. prolliferum) in Kashmir

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