Genetic relationships among ginger accessions based on AFLP ...

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Jurnal Bioteknologi Pertanian, Vol. 8, No. 2, 2003, pp. 60-68 ABSTRAK Jahe merupakan salah satu tanaman penting dari jenis temu- temuan. Di Indonesia dikenal tiga tipe jahe, yaitu jahe merah, jahe besar, dan jahe kecil. Ketiga tipe jahe tersebut mempunyai bentuk, warna, aroma, dan komposisi kimia rimpang yang berbeda. Untuk mengetahui kekerabatan antartipe dan dalam tipe pada jahe, 28 nomor aksesi yang terdiri atas 22 aksesi jahe Indonesia dan masing-masing 3 aksesi jahe asal Afrika dan Jepang digunakan dalam penelitian ini. Total DNA diekstrak dari rimpang dengan metode cetyltrimethyl ammo- nium bromide (CTAB) kemudian dimurnikan menggunakan serbuk magnet. Amplified fragment length polymorphism (AFLP) dilakukan mengikuti prosedur pada AFLP TM plant mapping kit (PE Applied Biosystem) dan hasil akhir polymerase chain reaction (PCR) dipisahkan pada 5% gel poliakrilamid dalam ABI 373 sequencer . Jumlah fragmen yang teramplifikasi pada setiap kombinasi primer AFLP rata- rata mencapai 96 dengan kisaran 47-137 fragmen. Pengamat- an dengan menggunakan 21 kombinasi primer menghasilkan 221 pita polimorfis. Dendrogram berdasar unweighted pair group methods of arithmetic average (UPGMA) dari semua nomor aksesi yang digunakan dapat diklasifikasikan menjadi tiga kelompok utama. Jahe merah secara genetik jauh dari jahe besar, tetapi mempunyai kekerabatan yang dekat dengan beberapa aksesi jahe kecil. Keragaman genetik dari jahe kecil (Ht = 0,25) lebih tinggi dari jahe besar (Ht = 0,08). [ Kata kunci : Zingiber officinale, jahe, AFLP, keragaman ge- netik] ABSTRACT Ginger ( Zingiber officinale Rosc.) is a rhizomatous perennial herb and one of the important crop of the genus Zingiber. There are three types of ginger in Indonesia, i.e. big ginger, small ginger, and red ginger. Their rhizomes differ in shape, color, aroma, and chemical composition. To understand the genetic relationships among the three types of ginger, 28 accessions consisted of 22 Indonesian cultivated ginger and 3 accessions each of African and Japanese commercial ginger were analyzed. Total DNA was extracted from rhizome using cetyltrimethyl ammonium bromide (CTAB) method then purified by magnetic beads. Amplified fragment length polymorphism (AFLP) was carried out according to the protocol described in AFLP TM plant mapping kit (PE Applied Biosystem) and the final polymerase chain reaction (PCR) products were separated on 5% denatured polyacrylamide gel on an ABI 373 sequencer. The number of fragments produced by a primer combination of AFLP ranged from 47 to 137 with an average of 96. A total of 221 polymorphic bands were ob- served by using 21 selective primer combinations. Dendrogram based on unweighted pair group methods of arithmetic average (UPGMA) revealed that the gingers could be classified into three major clusters. The red ginger was genetically far from the big ginger, but close to some accessions of small ginger. There was no clear genetic differentiation between the small and big types of ginger. The genetic diversity of small ginger (Ht = 0.25) is higher than that of the big ginger (Ht = 0.08). [ Keywords : Zingiber officinale, ginger, AFLP, genetic varia- tion] INTRODUCTION Ginger ( Zingiber officinale Rosc.) is a perennial rhizomatous herb of the family Zingiberaceae. Its origin is unknown, probably in tropical Asia and China (Purseglove et al. 1981). In Indonesia, gingers are grown in 13 provinces, but the main producing areas are Aceh, East Java, North Sumatra, West Java, Lampung, and Central Java. Ginger cultivation in these areas is considered to be beneficial. Ginger can be planted at the altitude up to 1,000 m asl, but the optimum condition for high yield is at 300-500 asl. Ginger is used worldwide as a cooking spice, condiment, and herbal remedy. It is the underground roots or rhizomes that are used for culinary and medicinal purposes. The Chinese have used ginger for at least 2,500 years as digestive aid, antinausea and rheumatism. In Malaysia and Indonesia, ginger is widely used as beverage such ginger tea to warm body. In Arabian medicine, ginger is considered as an aphrodisiac, while some African believe that eating ginger regularly will help repel mosquito. Nowadays, ginger is extensively cultivated in Asia and Africa, but Genetic relationships among ginger accessions based on AFLP marker Kekerabatan antar nomor-nomor aksesi jahe berdasarkan marka AFLP S. Wahyuni 1 , D.H. Xu 2 , N. Bermawie 1 , H. Tsunematsu 2 , and T. Ban 2 1 Indonesian Spices and Medicinal Crops Research Institute, Jalan Tentara Pelajar No. 3, Bogor 16111, Indonesia 2 Japan International Centre for Agrobiological Sciences (JIRCAS)

Transcript of Genetic relationships among ginger accessions based on AFLP ...

Page 1: Genetic relationships among ginger accessions based on AFLP ...

60 S. Wahyuni et al.Jurnal Bioteknologi Pertanian, Vol. 8, No. 2, 2003, pp. 60-68

ABSTRAK

Jahe merupakan salah satu tanaman penting dari jenis temu-temuan. Di Indonesia dikenal tiga tipe jahe, yaitu jahe merah,jahe besar, dan jahe kecil. Ketiga tipe jahe tersebut mempunyaibentuk, warna, aroma, dan komposisi kimia rimpang yangberbeda. Untuk mengetahui kekerabatan antartipe dan dalamtipe pada jahe, 28 nomor aksesi yang terdiri atas 22 aksesijahe Indonesia dan masing-masing 3 aksesi jahe asal Afrikadan Jepang digunakan dalam penelitian ini. Total DNAdiekstrak dari rimpang dengan metode cetyltrimethyl ammo-nium bromide (CTAB) kemudian dimurnikan menggunakanserbuk magnet. Amplified fragment length polymorphism(AFLP) dilakukan mengikuti prosedur pada AFLPTM plantmapping kit (PE Applied Biosystem) dan hasil akhirpolymerase chain reaction (PCR) dipisahkan pada 5% gelpoliakrilamid dalam ABI 373 sequencer. Jumlah fragmenyang teramplifikasi pada setiap kombinasi primer AFLP rata-rata mencapai 96 dengan kisaran 47-137 fragmen. Pengamat-an dengan menggunakan 21 kombinasi primer menghasilkan221 pita polimorfis. Dendrogram berdasar unweighted pairgroup methods of arithmetic average (UPGMA) dari semuanomor aksesi yang digunakan dapat diklasifikasikan menjaditiga kelompok utama. Jahe merah secara genetik jauh darijahe besar, tetapi mempunyai kekerabatan yang dekat denganbeberapa aksesi jahe kecil. Keragaman genetik dari jahe kecil(Ht = 0,25) lebih tinggi dari jahe besar (Ht = 0,08).

[Kata kunci: Zingiber officinale, jahe, AFLP, keragaman ge-netik]

ABSTRACT

Ginger (Zingiber officinale Rosc.) is a rhizomatous perennialherb and one of the important crop of the genus Zingiber.There are three types of ginger in Indonesia, i.e. big ginger,small ginger, and red ginger. Their rhizomes differ in shape,color, aroma, and chemical composition. To understand thegenetic relationships among the three types of ginger, 28accessions consisted of 22 Indonesian cultivated ginger and3 accessions each of African and Japanese commercial gingerwere analyzed. Total DNA was extracted from rhizome usingcetyltrimethyl ammonium bromide (CTAB) method thenpurified by magnetic beads. Amplified fragment lengthpolymorphism (AFLP) was carried out according to the

protocol described in AFLPTM plant mapping kit (PE AppliedBiosystem) and the final polymerase chain reaction (PCR)products were separated on 5% denatured polyacrylamide gelon an ABI 373 sequencer. The number of fragments producedby a primer combination of AFLP ranged from 47 to 137 withan average of 96. A total of 221 polymorphic bands were ob-served by using 21 selective primer combinations. Dendrogrambased on unweighted pair group methods of arithmetic average(UPGMA) revealed that the gingers could be classified intothree major clusters. The red ginger was genetically far fromthe big ginger, but close to some accessions of small ginger.There was no clear genetic differentiation between the smalland big types of ginger. The genetic diversity of small ginger(Ht = 0.25) is higher than that of the big ginger (Ht = 0.08).

[Keywords: Zingiber officinale, ginger, AFLP, genetic varia-t ion]

INTRODUCTION

Ginger (Zingiber officinale Rosc.) is a perennialrhizomatous herb of the family Zingiberaceae. Itsorigin is unknown, probably in tropical Asia and China(Purseglove et al. 1981). In Indonesia, gingers aregrown in 13 provinces, but the main producing areasare Aceh, East Java, North Sumatra, West Java,Lampung, and Central Java. Ginger cultivation in theseareas is considered to be beneficial. Ginger can beplanted at the altitude up to 1,000 m asl, but theoptimum condition for high yield is at 300-500 asl.

Ginger is used worldwide as a cooking spice,condiment, and herbal remedy. It is the undergroundroots or rhizomes that are used for culinary andmedicinal purposes. The Chinese have used gingerfor at least 2,500 years as digestive aid, antinausea andrheumatism. In Malaysia and Indonesia, ginger iswidely used as beverage such ginger tea to warmbody. In Arabian medicine, ginger is considered as anaphrodisiac, while some African believe that eatingginger regularly will help repel mosquito. Nowadays,ginger is extensively cultivated in Asia and Africa, but

Genetic relationships among ginger accessionsbased on AFLP marker

Kekerabatan antar nomor-nomor aksesi jahe berdasarkan marka AFLP

S. Wahyuni1, D.H. Xu2, N. Bermawie1, H. Tsunematsu2, and T. Ban2

1Indonesian Spices and Medicinal Crops Research Institute, Jalan Tentara Pelajar No. 3, Bogor 16111, Indonesia2Japan International Centre for Agrobiological Sciences (JIRCAS)

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in commerce the main ginger exporting countries areChina and India, which supplied almost 75% of theworld annual requirement of over 18,000 tons of ginger.The major ginger importing countries are USA, Japan,Europe, and Middle East countries.

Indonesian ginger has been described as twovariants, i.e. white ginger (Zingiber officinale var.officinale) and red ginger (var. sunti) (Rugayah 1994),it is in agreement with Valeton classification (1918).Based on phenotyphic characters, Rostiana et al.(1990) distinguished ginger into three groups, i.e. bigginger, small ginger, and red ginger. Big ginger has bigrhizome size, less pungent and less fibrous, averageplant height 68.63 + 12.75 cm. Small ginger has smallerrhizome size, fibrous and pungent, plant height 49.16+ 7.29 cm, while red ginger has small rhizome size withred skin color, more pungent and more fibrous, plantheight 48.23 + 14.05 cm, and has darker green leavescompared with two others. According to the usage,small ginger and red ginger are generally used formedicinal purposes and cooking spices, while bigginger is used for food, beverage, and cooking spices.De Guzman and Siemonsma (1999) reported that thereare three types of ginger in Indonesia, namely (1) jahebadak = jahe gajah = jahe putih besar, (2) jahe merah= jahe sunti, and (3) jahe putih kecil = jahe emprit.Their rhizome differs in shape, color, aroma, andchemical composition, and all types can be consideredas cultivars.

Genetic diversity of ginger germplasm collected fromseveral locations in Indonesia was low (Bermawie etal. 2001). Although they consisted of big, small andred gingers, morphologically they showed considera-ble phenotypic variations for many traits such asrhizome size, color, flesh color, flavor/pungency, yield,fiber, and rhizome chemical content. However, it isdifficult to distinguish accessions within the groupcompared to accessions of different group. Therefore,the relationships between and within the groups ofginger have to be studied to pursue genetic improve-ment of ginger.

Polymerase chain reaction (PCR)-based methods forgenetic diversity analyses have been developed, suchas random amplified polymorphic DNA (RAPD),random fragment length polymorphism (RFLP),amplified fragment length polymorphism (AFLP), andinter simple sequence repeat (ISSR/SSR). Eachtechnique is not only differed in principal, but also inthe type and amount of polymorphism detected. AFLPtechnique is based on the selective PCR amplificationof restriction fragments from a total digest of genomicDNA. The technique involves three steps: (1) restric-

tion of the DNA and ligation of oligonucleotideadapters, (2) selective amplification of sets of re-striction fragments, and (3) gel analysis of theamplified fragments (Vos et al. 1995). Typically, 50-100restriction fragments are amplified and detected ondenaturing polyacrylamide gel. AFLP has beenrecognized as a reliable and efficient DNA markersystem (Vos et al. 1995). It has been proven the mostefficient for estimating diversity in barley (Russel etal. 1997), provides detailed estimates of the geneticvariation of papaya (Kim et al. 2002), and have beenused to analyze the genetic diversity of various plantssuch as tea (Lai et al. 2001), eggplant (Mace et al.1999), peach (Manubens et al. 1999), apple (Guolao etal. 2001), rapeseed (Lombard et al. 1999), wild radish(Man and Ohnishi 2002), and Musa sp. (Wong et al.2001; Ude et al. 2002). Another marker system whichcan be used for genetic diversity analysis is ISSR. Theuse of this technique for genetic diversity analysishas been reported on tea (Lai et al. 2001; Mondal2002), and Botrycum pumicola (Camacho and Liston2001)

The objective of this study was to understand thegenetic relationships between and within the types ofginger by using AFLP marker.

MATERIALS AND METHODS

Plant materials

A total of 22 accessions of cultivated ginger grown inIndonesia including small, red and big gingers wereused as a material for analysis. More over 3 accessionsof African ginger (Ivory Coast, Bouake) and 3 acces-sions of Japanese ginger were used as a test sample.The list of materials used were shown in Table 1.

DNA extraction

Total genomic DNA was extracted from rhizomes byusing cetyltrimethyl ammonium bromide (CTAB)methods (Doyle and Doyle 1990). The DNA was thenpurified with silica beads (MagExtractor Plant GenomeKit, Toyobo. Co.) to obtain a better quality of DNAwhich appropriate for AFLP. A few sample of DNAobtained were loaded in 0.8% agarose gel, andλÉDNA were loaded too as a standard for estimationof the quantity and quality of the DNA. The gel wasfinally stained with ethidium bromide and viewed onthe UV transiluminator light at gel documentationsystem.

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Amplified Fragment Length Polymorphism (AFLP)

AFLP was carried out according to the protocoldescribed in AFLPTM plant mapping (PE AppliedBiosystems). Genomic DNA was digested with 5 unitsEcoRI and 1 unit MseI at 37oC for 6 hours, then ligatedwith 5 pmol EcoRI and 50 pmol MseI adaptor in a totalvolume of 20 µl by one weiss unit of T4 DNA ligasein a PCR core mix (dNTPs, MgCl2, PCR buffer, rTagpolymerase; TOYOBO), then incubated for overnightat 16oC. Pre-amplification was performed in a totalvolume of 20 µl containing 3 µl DNA template, 0.125µM EcoRI +A and MseI + C primers, 0.2 mM dNTPs,0.4 unit rTag polymerase, 1.5 mM MgCl2 and 1x PCRbuffer, and amplified on the thermocycler. The reactioncondition was as follow: 25 cycles of 94oC for 20seconds, 56oC for 30 seconds and 72oC for 2 minutes;and one cycle of 60oC for 30 minutes. Pre-amplificationproduct was then diluted ten times as template forselective amplification. It was carried out in a totalvolume of 20 ml consisted of 3 µl diluted pre-selectiveamplification product, 1 µl 10 pmol MseI and 1 µl 2pmol EcoRI primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1x

PCR buffer and 0.4 unit rTaq polymerase thenperformed on the following program: one cycle ofdenaturation at 94oC for 20 seconds, annealing at 65oCfor 30 seconds and extension at 72oC for 2 minutes,followed by 8 cycles of a 1oC decreasing annealingtemperature per cycle, and 23 cycles of 94oC for 20seconds, 65oC for 30 seconds and 72oC for 2 minutesand finally at 60oC for 30 minutes. The final PCRproducts were separated on 5% denatured poly-acrylamide gel and electrophoresed on ABI 373sequencer (Perkins Elmer/Applied Biosystem, Foster,USA). Twenty one selective primer combinations wereused in this study.

Data scoring and analysis

A band was considered polymorphic if it was presentin at least one genotype and absent in the others. Eachaccession was scored for the present (1) or absent (0)of the polymorphic bands. Estimates of similarity werebased on simple matching (SM) coefficient (Sokal andMichener 1958): Sij = a+d/a+b+c+d, where Sij is thesimilarity between two individuals (i and j), a is the

Table 1. Description of ginger accessions used in this study.

Accession code Origin Rhizome size Rhizome color Inner part color

A Ina, West Java, Sukabumi Big Whi te Yellowish creamB Ina, West Java, Cianjur Big Whi te Yellowish creamC Ina, West Java, Garut Big Whi te Yellowish creamD Ina, West Java, Sumedang Big Whi te Grayish creamE Ina, Central Java, Salatiga Big Whi te Grayish creamF Ina, Central Java, Boyolali Big Whi te Grayish creamG Ina, West Java, Sukabumi Small Red Purple-redH Ina, West Java, Cianjur Small Red Light purpleI Ina, West Java, Cisewu Small Whi te Yellowish creamJ Ina, West Java, Sumedang Small Whi te Yellowish creamK Ina, West Java, Garut Small Whi te Yellowish creamL Ina, West Java, Maja Small Whi te Yellowish creamM Ina, West Java, Cianjur Small Whi te Yellowish creamN Ina, Central Java, Boyolali Small Whi te Yellowish creamO Ina, West Java, Wado Small Whi te Yellowish creamP Ina, West Java, Sukabumi Small Whi teQ Ina, Central Java, Boyolali Small Whi te Yellowish creamR Ina, Central Java, Salatiga Small Whi te Yellowish creamS Ina, West Java, Wado Big Whi te Yellowish creamT Ina, West Java, Bogor Big Whi te Grayish creamU Ina, Papua, Manokwari Small Red Light purpleV Ina, Papua, Manokwari Small Whi te Yellowish creamW Ivory Coast, Africa Big Whi te Yellowish creamX Ivory Coast, Africa Small Whi te Grayish yellowY Ivory Coast, Africa Big Whi te Grayish creamZ Japan Small Whi teAA Japan Small Whi teAB Japan Big Whi te Yellowish cream

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number of bands present both in i and j, b is thenumber of bands present in i and absent in j, c is thenumber of bands present in j and absent in i, and d isthe number of bands absent both in i and j. The matrixof similarity was then clustered using unweighted pairgroup methods of arithmetic average (UPGMA) usingNTSYS-pc version 2.1 (Exeter software). Diversityvalues were calculated for each locus as 1 - Σpi2,where pi is the phenotypic frequency for each assayunit of AFLP primer combinations (Russell et al. 1997).

RESULTS AND DISCUSSION

AFLP analysis

The bands produced by one primer combination withthree E+nnn and M+nnnn of selective bases wereranging from 47 to 137 with an average of 96 bands(Table 2). Primer combinations of E-ATC/M-CAGAand AGA/CGGA perform the clearest amplified andpolymorphic bands. Figure 1 shows an example of agel image for the 28 accessions studied with the primercombination E-ATC/M-CAGA.

A total of 221 polymorphic bands were obtained byAFLP analysis with the average of 10.5 polymorphicbands per primer combination or equal with 11.5%.

The amplified bands in ginger are generally not sharpand clear. It might be due to the large genome sizeof ginger (23,618 Mbp) (RBG Kew 2000). Theachievement of polymorphic bands was relatively low(11.45%) compared with another plants such as 72.8%on wild radish (Man and Ohnishi 2002), 57.2% onapple (Goulao 2001), 42% on papaya (Kim et al. 2002),and 46.8% on barley (Russel et al. 1997).

The genetic variation among 28 ginger accessionswas estimated using pair-wise comparison of geneticsimilarity. The average pair-wise genetic similarity was0.801, ranged from 0.527 to 0.992. About 37.3% of thepair-wise comparison among the ginger accessionsexhibited genetic similarity greater than 0.90, and lessthan 16% showed genetic similarity 0.70.

The most closely accessions sharing geneticsimilarity were big ginger D (accession collected fromWado, Sumedang, West Java) and E (accessioncollected from Salatiga, Central Java). Both of theplaces are the main production areas of ginger.Morphologically both accessions have similar per-formance. The lowest similarity was between smallginger K (accession collected from Garut, WestJava) and Z (commercial ginger from Japan). Thesimilarity matrix among the accessions is presented inTable 3.

Table 2. Number of polymorphic bands of ginger in each assay unit of AFLP.

AFLP primer Number of Number of Percentage ofcombination amplified bands polymorphic bands polymorphic bands

ACC/CAAG 96 12 12 .50ACC/CAAC 1 0 5 4 3.81ACC/CAAT 83 14 16 .86AAC/CACG 120 12 10.00AAC/CAAC 92 4 4.35AAC/CACA 105 23 21 .90AAC/CAGA 123 8 6.50AAC/CAAT 130 11 8.46ATA/CAGA 80 14 17.50ATC/CGGA 75 10 13.3ATC/CGGT 79 12 15.19ATC/CTGA 90 16 17.78AGA/CAGA 135 12 8.89AGA/CTGA 1 1 1 17 15 .32AGA/CGGA 92 7 7.61AGG/CATA 47 5 10.64AGG/CGGA 86 8 9.30AGG/CACG 62 8 12.90AAG/CAGA 1 0 6 9 8.33ACG/CAGA 60 9 15 .00ACT/CAGA 137 6 4.38

Average 96 10.523 11.446Range 60-137 4-23 3.81-21.90

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The dendrogram based on UPGMA produced threemajor clusters (Fig. 2). The first cluster consisted ofJapanese ginger, the second cluster consisted of allaccessions of red and three accessions of small ginger,while the third cluster consisted of small ginger andbig ginger. The correlation coefficient between thecophenetic matrix computed from the dendrogram andthe original similarity matrix was 0.960 (t = 7.496, p =1), suggesting very good fit of the three representa-tions to the rough data value.

Based on the cluster analysis, among red, small, andbig types of ginger are not clearly defined. It is not inagreement with the common classification that dis-tinguishes ginger into three types (big, small and red).In the second cluster, red ginger and some accessionsof small ginger clustered together, while in the third

cluster the small and big types of ginger are not wellseparated. Rhizome size and color were conspiciouscharacters in ginger, but AFLP result showed thatseveral accessions of small and big ginger were highlysimilar to each other genetically, and several acces-sions of small ginger genetically close to red ginger.The similar result was obtained in Musa sp. (Wong etal. 2001). Both Musa ssp. truncata and microcarpahave similar character which can be seen clearly inblack pseudostem, but AFLP result showed that thetwo subspecies were genetically less similar to eachother. Subspecies microcarpa (black stem with non-waxy leaves) was genetically more similar tosubspecies malaccensis (green stem with waxyleaves). These indicated that differences in mor-phological characters in ginger based on rhizome size

Fig. 1. Gel image of AFLP of 28 ginger accessions produced by primer combination E-ATC/M-CAGA.

Data point 2732Size 285 bp

Data point 2395Size 250 bp

l▼

l▼

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Table 3. Genetic similarity between 28 ginger accessions.

A B C D E F G H I J K LA 1.000B 0.954 1 .000C 0.946 0 .950 1 .000D 0.925 0 .963 0 .954 1 .000E 0 .934 0 .971 0 .963 0 .992 1 .000F 0 .946 0 .967 0 .950 0 .963 0 .971 1 .000G 0.701 0 .722 0 .722 0 .726 0 .726 0 .722 1 .000H 0.698 0 .736 0 .728 0 .732 0 .740 0 .745 0 .940 1 .000I 0 .929 0 .934 0 .909 0 .913 0 .921 0 .925 0 .705 0 .702 1 .000J 0 .693 0 .739 0 .730 0 .743 0 .751 0 .747 0 .925 0 .940 0 .722 1 .000K 0.693 0 .714 0 .722 0 .718 0 .726 0 .730 0 .917 0 .923 0 .705 0 .959 1 .000L 0 .905 0 .950 0 .917 0 .946 0 .946 0 .950 0 .722 0 .728 0 .909 0 .730 0 .714 1 .000M 0.917 0 .946 0 .938 0 .942 0 .942 0 .938 0 .718 0 .740 0 .929 0 .743 0 .734 0 .954N 0.909 0 .954 0 .946 0 .950 0 .959 0 .963 0 .726 0 .749 0 .946 0 .759 0 .751 0 .963O 0.781 0 .824 0 .820 0 .820 0 .828 0 .828 0 .601 0 .621 0 .811 0 .627 0 .601 0 .825P 0 .905 0 .934 0 .909 0 .929 0 .921 0 .934 0 .722 0 .715 0 .950 0 .730 0 .722 0 .934

A B C D E F G H I J K LQ 0.913 0 .917 0.884 0 .913 0 .913 0 .925 0 .705 0 .719 0 .942 0 .739 0 .739 0 .925R 0.871 0 .909 0 .884 0 .913 0 .913 0 .917 0 .714 0 .719 0 .892 0 .730 0 .730 0 .934S 0 .921 0 .943 0 .943 0 .947 0 .947 0 .961 0 .719 0 .730 0 .943 0 .732 0 .728 0 .965T 0 .913 0 .950 0 .917 0 .929 0 .929 0 .942 0 .714 0 .719 0 .934 0 .722 0 .714 0 .950U 0.672 0 .701 0 .710 0 .714 0 .714 0 .710 0 .913 0 .885 0 .693 0 .896 0 .905 0 .710V 0.697 0 .718 0 .710 0 .722 0 .722 0 .726 0 .913 0 .902 0 .693 0 .896 0 .896 0 .718W 0.876 0 .916 0 .903 0 .898 0 .898 0 .898 0 .673 0 .677 0 .898 0 .690 0 .673 0 .912X 0.867 0 .896 0 .896 0 .892 0 .892 0 .896 0 .701 0 .698 0 .896 0 .710 0 .701 0 .905Y 0.880 0 .884 0 .834 0 .855 0 .855 0 .867 0 .647 0 .643 0 .900 0 .656 0 .647 0 .876Z 0 .643 0 .680 0 .656 0 .676 0 .676 0 .680 0 .544 0 .562 0 .656 0 .535 0 .527 0 .689AA 0.676 0 .722 0 .689 0 .718 0 .718 0 .714 0 .552 0 .570 0 .705 0 .560 0 .535 0 .722AB 0.871 0 .909 0 .892 0 .913 0 .913 0 .917 0 .680 0 .694 0 .884 0 .705 0 .680 0 .925

M N O P Q R S T U V W XM 1.000N 0.975 1 .000O 0.820 0 .845 1 .000P 0 .946 0 .954 0 .820 1 .000Q 0.929 0 .938 0 .785 0 .934 1 .000R 0.929 0 .938 0 .833 0 .917 0 .942 1 .000S 0 .961 0 .978 0 .832 0 .952 0 .943 0 .939 1 .000T 0 .963 0 .963 0 .811 0 .959 0 .925 0 .917 0 .961 1 .000U 0.722 0 .730 0 .588 0 .710 0 .685 0 .685 0 .715 0 .718 1 .000V 0.730 0 .730 0 .579 0 .710 0 .710 0 .701 0 .711 0 .718 0 .934 1 .000W 0.929 0 .929 0 .826 0 .898 0 .876 0 .876 0 .934 0 .929 0 .690 0 .690 1 .000X 0.917 0 .925 0 .798 0 .905 0 .880 0 .888 0 .921 0 .921 0 .730 0 .705 0 .942 1 .000Y 0.880 0 .880 0 .725 0 .884 0 .900 0 .851 0 .890 0 .892 0 .643 0 .668 0 .881 0 .871Z 0 .685 0 .693 0 .584 0 .656 0 .656 0 .672 0 .693 0 .672 0 .556 0 .548 0 .681 0 .710AA 0.718 0 .734 0 .605 0 .705 0 .705 0 .697 0 .719 0 .714 0 .564 0 .581 0 .717 0 .734AB 0.921 0 .929 0 .790 0 .900 0 .892 0 .909 0 .939 0 .925 0 .701 0 .710 0 .934 0 .938

Y Z AA ABY 1.000Z 0.697 1 .000AA 0.730 0 .859 1 .000AB 0.892 0 .722 0 .763 1 .000

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and color are not always an indication of the similarityor difference in DNA marker (AFLP).

The level of genetic variation among gingeraccessions tested was low, revealed by diversity indexvalue of only 0.22 from the AFLP data. This wascomparable with clonally propagated species suchElymus repens, D = 0.33 (Szczepaniak et al. 2002) orPotentilla spp., D = 0.20 (Hansen et al. 2000). The lowgenetic variation of ginger was possibly caused by themode of ginger propagation which is mostly pro-pagated by rhizome. Ginger sometimes producedflower, but rarely beared fruit (Purseglove et al. 1981).By AFLP analysis, genetic variation within smallginger (Ht = 0.255) was broader compared with bigginger (Ht = 0.079).

Big ginger was collected from 7 locations, whilesmall ginger from 9 locations and mostly from WestJava. The Sundanese (West Java tribe) call jahe badak(means big) for their garden plantation to distinguishit from the common ginger which run wild (Burkill1935). Later big ginger is popular to the farmer andwidely cultivated for export purposes. On the otherhand small ginger is usually cultivated for domestic

uses such a cooking spice and medicinal purpose.Farmer provided their own seed for small ginger, andtrade over region sometimes for big ginger. It is morefare understandable why the diversity index of bigginger was lower than that of small ginger.

Marker specific to accession

There is no unique molecular marker band which canbe used to distinguish red, small or big ginger. Not allprimer pairs produced specific bands. The specificbands produced by certain primer combination wereonly detected on small ginger accessions collectedfrom Wado (O) (Fig. 3). A specific band related to redginger was also observed, but only one band, oneprimer combination and not a major band, so it was notreliably used for marker yet. Mostly unique bands wereon red ginger included some accessions of smallginger (Table 4). Both the red and small types of gingerwhich have the same specific marker bands, mor-phologically have similar shape and size of rhizomeand smaller compared to others.

Fig. 2. Dendrogram of 28 ginger accessions generated by AFLP analysis; S = small ginger, B = big ginger, R = red ginger.

B. SukabumiB. CianjurB. BoyolaliB. SumedangB. SalatigaB. GarutS. MajalengkaS. CianjurS. BoyolaliB. WadoB. BogorS. CisewuS. SukabumiS. BoyolaliS. SalatigaB. AfricaS. AfricaB. Japan

S. WadoR. SukabumiR. CianjurS. SumedangS. GarutS. PapuaR. PapuaS. JapanS. Japan

0.66 0.74 0.83 0.91 0.99

Coefficient

S. Africa

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Genetic relationships among ginger accessions based on AFLP marker 67

Table 4. Molecular marker bands specific to certain accession of ginger.

AccessionPrimer Number of

Size (kb)combinations specific bands

Red ginger and 3 accessions ATC/CGGA 2 154.2; 223.2of small ginger ATC/CGGT 1 81 .53

AGA/CTGA 4 80.4; 158.2; 183.8; 225AGA/CGGA 2 82; 292.4ATC/CTGA 2 68.9; 108.4.ATC/CAGA 1 108 .1

Small ginger collected AAC/CACA 2 248.8; 46.from Wado ACC/CAAT 4 279.4; 329.8; 331.9; 431.5.

ATC/CAGA 1 285 .5AGG/CGGA 2 113.4; 202.4ATC/CGGA 3 105; 108; 162.4

Red ginger AAC/CACA 1 > 500

CONCLUSION

Based on AFLP marker, the 28 ginger accessions couldbe classified into three major clusters. The red gingerwas genetically far from the big ginger, but close to

some accessions of small ginger. There was no cleargenetic differentiation between the small and bigtypes of ginger. The genetic diversity of small ginger(H = 0.25) is higher than that of the big ginger (Ht =0.08). Specific molecular marker bands of small gingeraccessions collected from Wado can be recognized.

Fig. 3. Specific molecular bands (arrow) of small ginger collected from Wado (15) with ATC/CAGA primer (lower) and ATC/CGGA (upper).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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ACKNOWLEDGEMENT

The authors gratefully acknowledge the financial andtechnical support by JIRCAS (Japan InternationalCentre for Agrobiological Sciences) for this research.

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