issr polymorphism in indian wild orange (citrus indica tanaka, rutaceae) and related wild species in...

Scientia Horticulturae 123 (2010) 350–359

ISSR polymorphism in Indian wild orange (Citrus indica Tanaka, Rutaceae) andrelated wild species in North-east India

Susheel Kumar, Satya Narayan Jena, Narayanan K. Nair *

Plant Biodiversity & Conservation Biology, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India

A R T I C L E I N F O

Article history:

Received 28 March 2009

Received in revised form 25 September 2009

Accepted 13 October 2009

Keywords:

Citrus indica

Genetic diversity

Polymorphism

Inter simple sequence repeats (ISSR)

Rutaceae

A B S T R A C T

Inter simple sequence repeats (ISSR) polymorphism in Citrus indica Tanaka (Rutaceae), an endemic and

threatened wild species, was examined along with three other closely related wild taxa (C. medica L., C.

latipes (Swingle) Tanaka, and C. sp. ‘Memang athur’) by analyzing 53 representative accessions sampled

from North-east India. Jaccard’s similarity values among 53 accessions of Citrus ranged from 0.46 to 0.97

(average = 0.75). Genetic similarity values among all the 34 accessions of C. indica were found in the

range of 0.82 to 0.97 with an average of 0.90. Heterozygosity (Ht = 0.123) and Shannon’s information

index (I = 0.188) values estimated for C. indica revealed significantly low level of genetic variation within

the species. UPGMA dendrogram grouped all 53 accessions of Citrus into four major clusters: Cluster I – C.

latipes; Cluster II – C. medica; Cluster III – ‘Memang athur’ and Cluster IV – C. indica. The dendrogram

placed all the 34 accessions of C. indica in five sub-clusters under Cluster IV. The placement of C. indica

accessions in various sub-clusters and groups in the dendrogram was based on molecular differentiation

of individual accessions rather than their geographical origin. Very low genetic diversity and destruction

of its natural habitat pose serious threat to C. indica even in the Citrus Gene Sanctuary in Nokrek

Biosphere Reserve (NBR) in Meghalaya. Low genetic variability, heterozygosity and Shannon’s

information index in C. medica, C. latipes and ‘Memang athur’ are also concerns that need to be

addressed for developing appropriate strategies to conserve the genetic diversity extant in these

valuable genetic resources.

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1. Introduction

The genus Citrus L. (family Rutaceae; subfamily Aurantioideae)includes some of the principal fruit crops of the world, such as thecitrons [C. medica L.], lemons [C. � limon (L.) Osbeck], limes [C. �aurantifolia (Christm.) Swingle], mandarins [C. reticulata Blanco],sour oranges [C. � aurantium L.], sweet oranges [C. � aurantium L.;C. sinensis (L.) Osbeck], grapefruits [C. � aurantium L.; C. paradisi

Macf.] and pummelos [C. maxima (Burm.) Merr.]. Citrus fruits arewell-known for their dietary, nutritional, medicinal and cosmeticproperties and are also good sources of citric acid, flavonoids,phenolics, pectins, limonoids, ascorbic acid, etc. (Dugo and DiGiacomo, 2002).

There are two widely used classification systems in Citrus:Swingle (Swingle, 1943; Swingle and Reece, 1967) and Tanaka(Tanaka, 1977). The Swingle system included 16 species undertwo subgenera – Citrus and Papeda, while the Tanaka system

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fax: +91 0522 2205836.

E-mail address: [email protected] (N.K. Nair).

0304-4238/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2009.10.008

recognized 162 species under the subgenera Archicitrus andMetacitrus. Advanced studies based on biochemical and mor-phological characterization, suggest that there are only threebasic species, i.e. citron (C. medica L.), mandarin (C. reticulata

Blanco), and pummelo [C. maxima (Burm.) Merr.] within thesubgenus Citrus and that the other edible citrus, e.g. lemon, lime,sour orange, sweet orange, grapefruit, etc. are apomicticallyperpetuated biotypes with probable hybrid origin (Scora, 1975;Barrett and Rhodes, 1976). Mabberley (1998, 2004) treated Citrus

in a broader sense by merging three of its closely allied genera –Fortunella Swingle, Eremocitrus Swingle and Microcitrus Swinglewithin it. Citrus is thus believed to have its primary center oforigin in North-east India, China, Malaysia and Australia (Swingleand Reece, 1967; Scora, 1975; Gmitter and Hu, 1990; Mabberley,2004). Citrus fruit trees are now commercially grown in morethan 100 countries in the tropical and sub tropical regions ofthe world, many of them located far away from their actualcenters of origin.

India has a distinct position in the ‘Citrus belt’ of the world dueto remarkable diversity in citrus genetic resources, both incultivation and wild. Apart from the most commonly cultivatedspecies/cultivars/hybrids of citrons, lemons, limes, mandarins,sour oranges, sweet oranges, pummelos and grapefruits, three wild

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S. Kumar et al. / Scientia Horticulturae 123 (2010) 350–359 351

species and one variety, viz. Citrus indica Tanaka (Indian wildorange), C. latipes (Swingle) Tanaka (Khasi Papeda), C. ichangensis

Swingle (Ichang Papeda) (a synonym of C. cavaleriei H. Leveille exCavalerie; cf. Zhang and Mabberley, 2008) and C. hystrix DC. (C.

macroptera Montrouz. var. annamensis Tanaka – MelanesianPapeda) were reported to occur in the subtropical forests ofNorth-east India and the foot hills of the East Himalayas (Tanaka,1928, 1937; Bhattacharya and Dutta, 1956; Nair and Nayar, 1997;Sharma et al., 2004; Malik et al., 2006). C. medica, one of thecommonly known citrus fruits of the world, is considered a nativeof India (Scora, 1975; Mabberley, 2004) and is found to grow in awild and semi-wild state in primary as well as secondary forestsalong the foothills of the East Himalayas and North-east India(Hooker, 1875; Tanaka, 1977; Bhattacharya and Dutta, 1956; Nairand Nayar, 1997). The genetic resource spectrum of Indian Citrus

also includes several interesting indigenous varieties or land races,such as jambhiri (C. � taitensis Risso; C. jambhiri Lush.), karna orkhatta orange (C. karna Raf.), sour pummelo (C. megaloxycarpa

Lush.), hill lemon (C. pseudolimon Tanaka), ada-jamir or gingercitrus of Assam (C. assamensis Dutta and Bhattacharya), etc., whichare found mostly in home gardens in North-east India and theNorth Himalayas.

The indigenous genetic resources of Citrus have great utilityin citriculture and citrus industry. Unfortunately, like in manyother crop plants, the genetic base of indigenous and wildspecies of Indian Citrus is being eroded due to habitatdestruction, introduction of new exotic cultivars/varieties, andlack of appropriate conservation and management strategies.The Indian wild orange (C. indica) is a good example todemonstrate the above. Citrus indica, one of the primitive wildspecies of Citrus, is endemic to the Tura ranges in Garo hills ofMeghalaya in North-east India. A Citrus Gene Sanctuary forpreserving the indigenous germplasms of Citrus, particularly of C.

indica has been established in the Nokrek Biosphere Reserve(NBR) in the Garo Hills along the Tura ridge in Meghalaya (Singh,1981). The Indian wild orange is popularly known among theGaro tribes as ‘Memang-narang’ (i.e. fruit for ghosts or departedhuman spirits; in Garo language ‘memang’ means ghost and‘narang’ means fruit). The plant is thus revered with religioussentiments and grown in the backyards of several Garo hamletsfor medicinal uses (Malik et al., 2006). The Garo settlements arelocated along the forest fringes and they do not practice anorganized farming or cultivation system for C. indica. The Garosusually nurture the wild plants of C. indica that are foundgrowing naturally in and around their dwellings.

Despite its medicinal and genetic resource values, no detailedstudy has ever been carried out to evaluate the extent and patternof genetic diversity found within C. indica in its native distribu-tional range. The only available information on this importantplant genetic resource pertains to a few occasional inventories andmorphological characterization (Nair and Nayar, 1997; Sharmaet al., 2004; Malik et al., 2006). Detailed characterization andevaluation, including molecular analysis of genetic diversity is,therefore, needed to assess the conservation requirements andutilitarian prospects of C. indica. Assessment of genetic diversityusing morphological markers alone has serious limitations,especially in species of a complex genus like Citrus, whosetaxonomy is otherwise in a chaotic state due to frequent incidencesof hybridization, apomixis, polyploidy and bud mutations. Geneticdiversity assessment in plants has now become far more simple,cost effective, reliable and reproducible, thanks to the advent ofPCR-based DNA marker techniques such as random amplifiedpolymorphic DNA (RAPD), amplified fragment length polymorph-ism (AFLP), inter simple sequence repeats (ISSR), simple sequencerepeats (SSR), directed amplification of minisatellite DNA (DAMD),etc. (Weising et al., 2005). ISSR is a widely used technique for

molecular analysis of genetic and species diversity in various plantgroups (Zietkiewicz et al., 1994; Gupta et al., 1994). This markertechnique often reveals a much larger number of polymorphicfragments per primer (Qian et al., 2001) and enables higherstringency amplifications, leading to more reproducible bandingpatterns (Wolfe and Liston, 1998). Recent ISSR studies on naturalpopulations of several plant species, including rare and endan-gered species have demonstrated the hyper variable nature ofthese markers and their potential use in species diversity andpopulation level studies (Deshpande et al., 2001; Qiu et al., 2004;Apte et al., 2006; Cao et al., 2006; Syamkumar and Sasikumar,2007; Han et al., 2007; Xia et al., 2007).

Several earlier molecular studies in Citrus have utilized ISSRmarkers for assessing genetic diversity in clementine (Breto etal., 2001) and in lemon (Gulsen and Roose, 2001), cultivaridentification in Citrus and Poncirus (Fang and Roose, 1997;Capparelli et al., 2004), constructing genetic linkage map inCitrus grandis and Poncirus trifoliata (Shankar and Moore, 2001),in management of citrus germplasm resources (Krueger andRoose, 2003), discrimination of clones of sour oranges (Pasqualeet al., 2006), and molecular differentiation in Citrus germplasmresources of Iran (Shahsavar et al., 2007). However, there arefew previous studies, wherein C. indica formed a part ofmolecular diversity analysis using RFLP & RAPD (Federici etal., 1998) RAPD, SCAR & cpDNA markers (Nicolosi et al., 2000)and chloroplast DNA (Bayer et al., 2009; Jena et al., 2009). Allthese studies were based only on a few accessions of C. indica.The present study, based on an extensive sampling from itsnatural habitat, is the first attempt to assess the ISSRpolymorphism and genetic variability in C. indica along withother closely related species, viz. C. medica, C. latipes and a newvariant (C. sp. ‘Memang athur’) occurring in NBR and other partsof North-east India.

2. Materials and methods

2.1. Plant materials

Thirty-four accessions of C. indica were collected from wild aswell as from Garo home gardens/backyards from five localities(Daribokgre, Nokrek peak, Chandgre, Dura Kalakgre, and Oragi-tok) in NBR. Four of these collection sites (Daribokgre, Nokrekpeak, Chandgre and Dura Kalakgre) were located at an averagedistance of 7–10 km, while the distance between the two farthestlocales, i.e. Oragitok and Nokrek peak was 15 km. Of the 34accessions, 21 were from a single locality, Daribokgre, whereC. indica has its maximum concentration, both wild and planted.In other localities, the species is extremely rare and has a verysparse and scattered occurrence with only a few individual plants.The sampling of genotypes was representative of the distributionof C. indica in the study site, and special attention was paid toinclude morphologically diverse materials and wild and culti-vated specimens in the samples. The age of cultivated materialscollected from Garo home gardens/backyards in Daribokgre,Chandgre and Oragitok was in the range of 15–20 years old. Theseplants in cultivation were raised from seeds or bud sticks collectedfrom the wild.

For a comparative molecular differentiation, three other wildtaxa [C. medica (seven accessions), C. latipes (seven accessions) and‘Memang athur’ (five accessions)] occurring in NBR and other partsof North-east India were also included in the present analysis(Table 1). Atalantia monophylla (L.) DC., a close relative of Citrus,was included as an outgroup taxon. All the materials were criticallystudied to determine their correct taxonomic identity andrepresentative voucher specimens were deposited in the herbar-ium of NBRI (LWG). Young leaf tissues from all the samples were

Table 1Details of the accessions of Citrus used for ISSR analysis.

Lane no. Accession code Taxon identity Origin/source Altitude Latitude Longitude Voucher no. Status

1 CIND-D01 Citrus indica Tan. Daribokgre, NBR 1216 m N 25828.8660 E 090819.0280 228023 Wild

2 CIND-D02 C. indica Tan. Daribokgre, NBR 1226 m N 25828.8480 E 090819.0100 228024 Wild







9 CIND-D09 C. indica Tan. Daribokgre, NBR 1103 m – – 228389 Wild

10 CIND-D10 C. indica Tan. Daribokgre, NBR 1216 m N 25828.8660 E 090819.0280 228011 Cult.











21 CIND-D21 C. indica Tan. Daribokgre, NBR 1118 m – – 228388 Cult.

22 CIND-N22 C. indica Tan. Nokrek peak, NBR 1182 m N 25028.3550 E 090018.8550 228366 Wild




26 CIND-K26 C. indica Tan. Dura Kalakgre 1200 m – – 228377 Wild



29 CIND-C29 C. indica Tan. Chandgre, NBR 798 m N 25832.1490 E 090819.6640 228044 Cult.

30 CIND-C30 C. indica Tan. Chandgre, NBR 797 m N 25002.1600 E 090019.5420 228392 Cult.

31 CIND-C31 C. indica Tan. Chandgre, NBR 757.5 m N 25002.1600 E 090019.5420 228393 Cult.

32 CIND-C32 C. indica Tan. Chandgre, NBR 754.5 m N 25002.1600 E 090019.5420 228394 Cult.

33 CIND-O33 C. indica Tan. Oragitok, NBR 849 m N 25033.1090 E 090019.6560 228398 wild

34 CIND-O34 C. indica Tan. Oragitok, NBR 845.5 m N 25033.1090 E 090019.6560 228399 wild

35 CSMA-D01 C. sp. (Memang athur) Daribokgre, NBR 1082 m – – 228363 Wild

36 CSMA-D02 C. sp. (Memang athur) Daribokgre, NBR 1082 m – – 228364 Wild

37 CSMA-D03 C. sp. (Memang athur) Daribokgre, NBR 1132 m N 25029.4710 E 090019.3690 228354 Cult.



40 CMED-D01 C. medica L. Daribokgre, NBR 1112 m N 25029.5170 E 090019.4390 228348 Wild

41 CMED-D02 C. medica L. Daribokgre, NBR 1091 m – – 228360 Wild

42 CMED-D03 C. medica L. Daribokgre, NBR 1085 m – – 228362 Wild

43 CMED-C04 C. medica L. Chandgre, NBR 798 m N 25832.1490 E 090819.6640 228045 Wild

44 CMED-T05 C. medica L. Tipi, Arunachal Pradesh N 25002.0500 E 092086.4070 228320 Wild

45 CMED-A06 C. medica L. Along valley, Arunachal Pradesh – – – – Wild

46 CMED-P07 C. medica L. Pathali Pahar, Assam – – – – Wild

47 CLAT-S01 C. latipes (Swingle) Tan. Thinmal, Shillong, Meghalaya 1613 m N 25833.7970 E 091851.2560 228001 Cult.

48 CLAT-S02 C. latipes (Swingle) Tan. Mawkdok, Shillong, Meghalaya 1835 m N 25825.7160 E 091847.5090 228012 Cult.

49 CLAT-C03 C. latipes (Swingle) Tan. Cherrapunji, Meghalaya 1666 m N 25819.7170 E 091843.9180 228013 Cult.

50 CLAT-B04 C. latipes (Swingle) Tan. ICAR, Basar, Meghalaya 791 m N 27059.6110 E 094042.3780 228344 Cult.

51 CLAT-N05 C. latipes (Swingle) Tan. Nokrek peak, NBR 1410 m N 25027.7000 E 090019.0410 228372 Wild

52 CLAT-N06 C. latipes (Swingle) Tan. Nokrek peak, NBR 1410 m N 25027.7000 E 090019.0410 228373 Wild

53 CLAT-U07 C. latipes (Swingle) Tan. ICAR, Umiam, Meghalaya 669 m N 27059.6110 E 094042.3780 228305 Cult.

54 AMON-K1 Atalantia monophylla (L.) DC. Quilon, Kerala – – – 225639 Wild

S. Kumar et al. / Scientia Horticulturae 123 (2010) 350–359352

stored in silica gel (20–60 mesh size) and used subsequently forDNA isolation.

2.2. Morphological characterization

Seventy-six discrete morphological characters (33 quantitativeand 43 qualitative characters) were selected from taxonomicliterature (Barrett and Rhodes, 1976; IPGRI, 1999; Nair and Nayar,1997) and by examination of living plants and herbariumcollections of the four target taxa of Citrus (Appendix A). Thecharacters were converted in to bi-states and multi-states(interval) code. Standardization of morphological data was donebased on YBAR option with the software NTSYS ver. 2.10e (Rohlf,2000). A pairwise similarity matrix was generated using SimpleMatching coefficient and an NJ tree was constructed based onEuclidean distance method with the same software.

2.3. DNA extraction

Total genomic DNA was isolated from all the 54 accessionsthrough hexadecyl trimethyl ammonium bromide (CTAB) methodwith some modifications (Rogstad, 1993). Quantitation of isolatedDNA was done spectrophotometrically and its quality checked byelectrophoresis on 0.8% agarose gel.

2.4. ISSR analysis

A total of 105 primers were used for ISSR-PCR optimizationtrials, of which 100 were procured from University of BritishColumbia (UBC # 9) and five, based on sequence data from twoearlier published works on Citrus (Fang and Roose, 1997; Shankarand Moore, 2001), were custom synthesized from Bangalore Genie,India. Fifteen primers, which gave the best amplification results

Fig. 1. NJ tree of four taxa of Citrus generated from morpho-metric data based on

Euclidean distance.

Table 2Pairwise similarity matrix of four target taxa of Citrus generated from morpho-

metric analysis.

C. indica Memang athur C. medica C. latipes

C. indica 1.000

Memang athur 0.452 1.000

C. medica 0.193 0.435 1.000

C. latipes 0.177 0.274 0.339 1.000


with the entire sample DNA, were selected for the final ISSR-PCRanalysis. PCR-amplification was carried out in 25 ml reactionvolume containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.0–2.5 mM MgCl2, 0.2 mM dNTP each, 1.0 U Taq DNA polymerase(Bangalore Genie, India), 0.2 mM primer and 25–30 ng genomicDNA. The amplification was performed in a PTC-200 Thermocycler(MJ Research, Massachusetts, USA), with reaction conditionsprogrammed as initial pre-denaturation at 94 8C for 4 minfollowed by 35 cycles of denaturation at 94 8C for 1 min, annealingat optimized temperature for 1 min, and extension at 72 8C for2 min. A final 7 min extension at 72 8C followed the completion of35 cycles. Aliquots of the amplification products were separated byelectrophoresis on 1.5% agarose gels stained with ethidiumbromide at 100 V for 3 h, and bands were visualized anddocumented in UVITec Gel Documentation System (UVITecLimited, Cambridge, UK). PCR amplification of the samples witheach primer was carried out in triplicate so as to ensure theconsistency and reproducibility of the results.

2.5. Data analysis

Amplified fragments were scored for each accession as presence(1) or absence (0) of homologous bands on the basis of sizecomparison with external standards (DNA Ruler: low range DNALadder-provided by Bangalore Genie, India). Molecular weight ofthe marker bands was calculated using the software programUVIgelstart (UVITec Limited Cambridge, UK). A pairwise geneticsimilarity matrix between accessions was estimated usingJaccard’s coefficient and a dendrogram was constructed basedon UPGMA (Unweighted Pair Group Method by Arithmeticaverages) with the Software FreeTree ver. 0.9.1.50 (Pavlicek etal., 1999), and the dendrogram was viewed in TreeView ver. 1.6.6(Page, 2001). Two-way Mantel test for goodness of fit for UPGMAcluster to the binary data and principal co-ordinate analysis(PCOA) were also performed using the NTSYS ver. 2.01e software(Rohlf, 2000). The binary data were used to calculate theheterozygosity (Ht) and Shannon’s information index (I) usingthe software POPGENE 1.31 (Yeh et al., 1999). Considering theuneven distribution of the target taxa in the study sites, and theasymmetry in the number of sampled genotypes from differentlocalities, the genetic diversity parameters were calculated usingthe single population approach under the assumption of HardyWeinberg equilibrium.

3. Results

3.1. Morphological characterization

All accessions of C. indica collected from five localities weresimilar in their morphology, although some showed minorvariations in the following characters: habit-small bushy shrubsor stragglers or scandent shrubs; leaves-lamina lanceolate oroblong-lanceolate in shape, long acuminate or short-abruptlyacuminate at apex; petals-generally pure white or occasionallywith purplish tinge. Plants growing inside the canopy of denseforests were relatively robust with profusely branched stem andscandent or straggling branches, whereas those found growing inthe open areas or along forest fringes were rather slow growingwith stunted bushy habit. No significant morphological variationswere seen among sample genotypes collected from wild andcultivation.

C. indica prefers black humus and iron-rich slightly acidic (pH5.0) loamy soil. It is a very slow growing plant and thrives well incool, shady places under the canopy of Dillenia indica, D. trigyna,Macranga peltata, etc. The flowering in C. indica is usually duringMarch–April and fruiting begins around May–June and the fruits

mature during October–February. It is an insect-pollinated plantwith honey bees as the most common pollinators (personalobservation). Although its breeding system is yet to be studied,preliminary observations on honey bee-pollination and structureof its bisexual flower, especially the positioning of anthers in closeproximity to stigma suggest both out-crossing and selfing in thisspecies. Fruit and seeds dispersal mechanism is by gravity.

Citrus indica shows similarity with C. medica in having bushyhabit with older branches arising from the base, and in the long andstout spines with brownish tip. However, both the species differ inthe floral and fruit characters.

C. indica, however, has close affinity with ‘Memang athur’ inhaving bushy habit, oblong to elliptic leaves with abruptlyacuminate apex, subpyriform (ca. 4.5 cm long, 5.5 cm broad)and scarlet red fruits and large to medium sized plump seeds (ca.10 mm � 5.0 mm). ‘Memang athur’ shares the following characterswith C. medica: short petioles (ca. 5 mm long), serrations on leafmargin; flowers in clusters, thick, fleshy petals with purplish tinge;mammiform fruit apex, longitudinal furrows and ridges on thefruit surface, white pulp colour; and reddish chalazal cap. C. latipes

is quiet distinct from all other target taxa in having a large treehabit, leaves with a broad petiole, which is as long and broad as thelamina; larger flower with stamens in groups of 2 or 3, sometimes 1or 2 free; larger globose to oblate fruits with pitted-glandularsurface, white and spongy mesocarp, endocarp with 9–15segments, containing acrid oil droplets in their pulp-vesicles.

Comparative analysis of 76 morphological characters examinedin the four target taxa of Citrus revealed moderate to significantvariation among them. Maximum similarity was observedbetween C. indica and ‘Memang athur’ (0.452), while minimumbetween C. indica and C. latipes (0.177) (Table 2). C. indica alsoshowed low morphological similarity (0.193) with C. medica.Similarity value was found 0.435 (between Memang athur and C.

medica) and 0.274 (Memang athur and C. latipes). The NJ tree(Fig. 1), based on Euclidean distance, resolved two main clusters:

Table 3List of total amplified bands and polymorphic bands generated by 15 ISSR primers.

S. no. Primer Sequence

(50 to 30)a

AT (8C) TB PB PPB No. of bands in

C. indica (PB)

No. of bands in

Memang athur (PB)

No. of bands

in C. medica (PB)

No. of bands

in C. latipes (PB)

Range of

fragment

size (bp)

1 PD01 HVH(CA)7T 51 15 13 87 10(4) 9(4) 8(6) 8(4) 397–2134

2 PD03 HVH(TCC)5 52 13 11 85 10(8) 6(2) 6(2) 6(3) 355–2459

3 PD05 HVH(TG)7T 53 18 14 78 15(6) 10(2) 16(6) 13(8) 295–2000

4 801 (AT)8T 52 13 11 85 9(5) 8(5) 8(3) 10(5) 359–1792

5 807 (AG)8T 52 12 9 75 6(2) 4(1) 8(3) 9(5) 351–2500

6 808 (AG)8C 54 17 16 94 8(4) 8(3) 12(9) 13(9) 250–2500

7 810 (GA)8T 54 15 14 93 13(5) 11(2) 9(6) 7(3) 244–2213

8 811 (GA)8C 55 12 10 83 12(8) 9(4) 9(5) 8(3) 305–2500

9 812 (GA)8A 52 14 13 92 10(7) 9(4) 10(7) 10(4) 254–2336

10 834 (AG)8YT 48 10 8 80 6(2) 8(3) 8(3) 8(4) 320–3000

11 836 (AG)8YA 52 17 16 94 13(8) 13(8) 8(5) 12(7) 329–2800

12 842 (GA)8YG 52 13 12 92 7(6) 5(4) 8(5) 10(6) 310–1850

13 868 (GAA)6 46 14 13 92 11(4) 11(1) 7(5) 9(7) 250–2994

14 880 (GGGTG)3 54 11 10 90 9(6) 6(2) 5(2) 6(2) 650–3000

15 881 VBV(AT)7 54 10 9 90 4(2) 5(1) 7(5) 8(5) 445–2500

Total 204 179 87% 143(77) 122 (46) 129(72) 137(75)

PPB 54 38 56 55

a V: non-T; B: non-A; H: non-G; D: non-C; Y: C or T; AT: annealing temperature; TB: total number of bands; PB: polymorphic bands; PPB: percentage of polymorphic bands.


Cluster I with C. indica, ‘Memang athur’ and C. medica; Cluster IIwith C. latipes.

3.2. ISSR polymorphism and genetic diversity

A total of 204 bands were generated from 15 ISSR primers, ofwhich 179 bands were polymorphic (87%) with an average of 12polymorphic bands per primer. The fragment size ranged from250 bp to 3000 bp (Table 3). Each primer generated 10–18 bandswith an average of 13.6 bands per primer. PD05 primer generatedthe maximum number of 18 bands, whereas UBC834 and UBC881generated the minimum of 10 bands with the lowest number ofpolymorphic bands. 30-anchored primers with AG-repeats, i.e.UBC808 and UBC836 generated the highest number of 16polymorphic bands. In Citrus indica, 15 ISSR primers generated atotal of 143 bands, of which 77 bands were polymorphic (54%) with

Fig. 2. ISSR profiles of 53 accessions of Citrus and the outgroup Atalantia monophylla gener

indicate specific band found only in Memang athur & C. indica (470 bp) and Memang a

an average of 5 polymorphic bands per primer. The percentage ofpolymorphism in related species, ‘Memang athur’, C. medica and C.

latipes was 38, 56 and 55, respectively. Fig. 2a and b showsrepresentative ISSR profiles generated by primers UBC868 andUBC881, respectively.

Pattern of distribution of bands across all candidate taxarevealed that the primer PD01 generated a unique taxon-specificmarker band (430 bp) for C. indica, whereas primers PD05, 812 and836 generated three specific bands in C. medica with fragment sizeof 719 bp, 668 bp, and 329 bp, respectively (Table 4). In C. latipes,five specific marker bands were amplified by the followingprimers: PD05 (580 bp), UBC808 (300 bp), UBC834 (320 bp),UBC880 (650 bp) and UBC812 (254 bp). UBC868 (430 bp) andUBC881 (1000 bp) marker bands were shared between C. indica &‘Memang athur’, and C. medica & ‘Memang athur’, respectively(Fig. 2a and b). Some primers were able to generate unique marker

ated by ISSR primers, (a) 868, (b) 881. Lane M stands for low range DNA ruler. Arrows

thur & C. medica (1000 bp).

Table 4Species-specific bands in Citrus taxa generated by ISSR primers.

Taxa Primer Fragment size (bp)

C. indica PD01 430

C. medica PD05 719

812 668

836 329

C. latipes PD05 580

808 300

812 254

834 320

880 650


bands in certain accessions of C. indica. A maximum of six suchspecific bands of 518 bp, 560 bp, 620 bp, 675 bp, 680 bp, and1500 bp size were amplified by the primers PD05, UBC812,UBC836, UBC807, UBC801, and UBC811, respectively, in anaccession CIND-D08 sampled from the Garo backyard at Dar-ibokgre. Similarly, two unique bands (305 bp, 680 bp) weregenerated in a wild sample accession CIND-D06 (Daribokgre) byprimers UBC811 and UBC880, respectively, while primers UBC842,UBC811 and UBC812 were able to amplify a single specific band of395 bp, 600 bp, and 1150 bp size, respectively, in three othercultivated samples-CIND-D20 (Daribokgre), CIND-C32 (Chandgre)and CIND-O33 (Oragitok).

Pairwise Jaccard’s similarity values among all the 53 accessionsof Citrus ranged from 0.46 to 0.97 (average = 0.75) (Table 5).Similarity values among all the 34 accessions of C. indica rangedfrom 0.82 (between CIND-D20 and CIND-N25; CIND-D08 andCIND-C29) to 0.97 (between CIND-D11 and CIND-D12; CIND-D21and CIND-N22) with an average of 0.90.

The mean values of heterozygosity (Ht) and Shannon’sinformation index (I) within C. indica was found 0.123 and0.188, respectively. Ht and I were also calculated for ‘Memangathur’ (Ht = 0.105, I = 0.154), C. medica (Ht = 0.160, I = 0.239) and C.

latipes (Ht = 0.153, I = 0.225) (Table 6).A dendrogram (Fig. 3) was generated based on UPGMA method,

which grouped all 53 accessions of Citrus into four major clusters:Cluster I – C. latipes; Cluster II – C. medica; Cluster III – ‘Memangathur’ and Cluster IV – C. indica. All the five wild as well ascultivated accessions of ‘Memang athur’ grouped to form aseparate Cluster III, showing high genetic similarity (0.81) withC. indica.

All the 34 accessions of C. indica in Cluster IV formed five sub-clusters (IVa, IVb, IVc, IVd and IVe): IVa comprising two accessionsfrom Chandgre (CIND-C29 and CIND-C30); IVb with 15 accessionsfrom two localities – Nokrek peak (CIND-N24, CIND-N25) andDaribokgre (CIND-D01, CIND-D02, CIND-D03, CIND-D04, CIND-D05, CIND-D06, CIND-D07, CIND-D10, CIND-D11, CIND-D12,CIND-D13, CIND-D14 and CIND-D15); IVc containing 15 accessionsfrom five localities – Daribogkre (CIND-D21, CIND-D18, CIND-D17,CIND-D19, CIND-D16, CIND-D20 and CIND-D09), Nokrek peak(CIND-N22 and CIND-N23), Dura Kalakgre (CIND-K26, CIND-K27and CIND-K28), Chandgre (CIND-C31 and CIND-C32) and Oragitok(CIND-O33); IVd with a single accession from Oragitok (CIND-O34); and the last sub-cluster IVe comprising a single accessionfrom Daribokgre (CIND-D08). C. indica accessions from Daribokgreand Nokrek Peak populations were grouped together and did notshow much genetic isolation. However, accessions of C. indica fromDura Kalakgre and Chandgre formed separate subgroups showingboth genetic and geographical isolation.

Based on Mantel Z-statistics (Mantel, 1967), the correlationcoefficient (r) was estimated as 0.98. A value of r > 0.90 isconsidered a very good fit of the UPGMA cluster pattern to thedata. Two-dimensional plot (Fig. 4) generated from principal

co-ordinate analysis (PCOA) of ISSR data also supported theclustering pattern of UPGMA dendrogram. 1st and 2nd principalco-ordinate axes accounted for 28.93% and 15.88%, respectively, ofthe total variation (Table 7).

4. Discussion

ISSR data generated from representative accessions of four taxa(C. indica, C. medica, C. latipes and ‘Memang athur’) with 15 primerswere sufficient to provide inferences on genetic differentiation andrelationships among and within each taxon. ISSR analysis resultedin clear cut segregation of C. indica and all other target taxa inseparate clusters with high bootstrap support (60–100). ISSRmarkers were also able to differentiate the individual accessionswithin each taxon with moderate polymorphism (38–56%).

Jaccard’s genetic similarity value was found in the range of0.82–0.97 with an average value of 0.90 among all the 34accessions of C. indica, suggesting low level of genetic diversity inthis species. This inference was correspondingly congruent withthe very low value of heterozygosity (Ht = 0.123) and Shannon’sinformation index (I = 0.188) of C. indica. ISSR analysis alsorevealed one C. indica specific marker band (PD01–430 bp). Theplacement of C. indica in various sub-clusters and groups wasapparently based on molecular differentiation of individualaccessions rather than their geographical origin. However,formation of accessions from Dura Kalakgre and Chandgre inseparate subgroups in sub-clusters IVb showed both genetic andgeographical isolation. Morphological variation in plant habit andfloral petal colour in a few accessions were not reflected in themolecular differentiation pattern of the individual accessionsanalyzed.

According to Hogbin and Peakall (1999), breeding system,genetic drift or genetic isolation of populations can cause high levelof genetic differentiation among plant species populations.Similarly, scattered distribution of a species and topographicalbarriers can lead to difficulties in pollen and seed dispersal,consequently to limited gene flow among populations (Qiu and Fu,2001). This holds true for C. indica, whose natural habitat in NBRand vicinity is facing severe fragmentation due to jhuming(Talukdar et al., 2005) and clearance of forests in and aroundthe Garo hamlets for introduction of agri-horticrops other thanCitrus for better economic income. The physical barriers betweenseveral separate hill blocks within the entire Tura ridge coupledwith poor fruit and seed dispersal mechanisms in C. indica are otherapparent reasons for the current scattered distribution and highhabitat differentiation. The present study has shown no significantcorrelation between accessions sampled from Garo home gardensand truly wild stock of C. indica with regard to any of the geneticdiversity data drawn from an analysis of ISSR. This would meanthat the cultivation of C. indica by Garos has not been able to bringin any substantial genetic improvement in this plant.

‘Memang athur’ is a hitherto unidentified citrus fruit, which wecould locate in one of the Garo tribal settlements in Daribokgre inNBR and its vicinity. ISSR markers revealed low genetic diversity(P = 38%, GD = 11%) within ‘Memang athur’, which was supportedwith low heterozygosity (Ht = 0.105), and Shannon’s informationindex (I = 0.154). Morphologically, ‘Memang athur’ looks moresimilar to C. indica in the leaf shape, small and scarlet red fruits, andmedium to large sized plumpy seeds. Some characters, like petiolesize, serrations on leaf margin, flowers with thick fleshy, 4 or 5purplish tinged petals, mammiform fruit apex, longitudinal furrowand ridge on the surface, and reddish chalazal cap bring ‘Memangathur’ much closer to C. medica. Based on the morphologicalcharacters and partial seed sterility, ‘Memang athur’ appears to be aprobable hybrid. Our ISSR analysis revealed close genetic relation-ship of ‘Memang athur’ with C. indica with high genetic similarity

Table 5Pairwise similarity matrix of 53 accessions of Citrus and the outgroup, Atalantia monophylla, derived from ISSR data.

S.K

um

ar

eta

l./Scientia

Ho

rticultu

rae

12

3(2

01

0)

35

0–

35

93

56

Table 6Heterozygosity (Ht) and Shannon’s information index (I) derived from ISSR data of

four Citrus taxa.

Taxa Ht I

C. indica 0.123 0.188

Memang athur 0.105 0.154

C. medica 0.160 0.239

C. latipes 0.153 0.225

Ht: Total heterozygosity, estimated as Ht = 1�SPi2, where Pi is the frequency of ith

allele (Nei, 1973). I: Shannon’s information index as a measure of gene diversity,

I = Hpop/Hsp where Hpop and Hsp are population and species diversity indices,

respectively (Shannon and Weaver, 1949).


(0.81). The presence of one unique fragment of 430 bp generated by868 primer only in C. indica and ‘Memang athur’, and anotherfragment (1000 bp), generated by 881 primer only in C. medica and‘Memang athur’ indicates the possible role of C. indica and C. medica

as the putative parents in the origin of ‘Memang athur’. This

Fig. 3. UPGMA dendrogram generated from ISSR data of 54 accessions of Citrus including

on 500 resampling.

assumption, however, needs confirmation through further mole-cular studies using microsatellites or sequence analysis of specificchloroplast or nuclear genes.

C. medica (citron), one of the basic species of Citrus, isconsidered a native of India. ISSR polymorphism and averagegenetic similarity within C. medica were 56% and 0.84, respectively,indicating low genetic diversity within this species, which was alsosupported by low heterozygosity (Ht = 0.160), and Shannon’sinformation index (I = 0.239). However, genetic variability withinC. medica was comparatively higher than that of C. indica and‘Memang athur’. Three species-specific ISSR fragments were alsogenerated in C. medica (PD05–719 bp; 812–668 bp; 836–329 bp).Low genetic diversity and high number of species-specificfragments support the treatment of C. medica as a true basicspecies of Citrus (Federici et al., 1998).

C. latipes showed high genetic similarity (0.84) and lowpolymorphism (55%), indicating low genetic diversity within this

the outgroup Atalantia monophylla. Numbers at the node are bootstrap values based

Fig. 4. 2D plot of the first (28.93%) and second (15.88%) principal co-ordinate axes,

derived from principal co-ordinate analysis of ISSR data of 53 accessions of Citrus

and the outgroup, Atalantia monophylla (Note: Numbers are equivalent to those

listed in Table 1).

Table 7Eigenvalues, differences, percentage of proportions and cumulative for 10 principal

co-ordinate axes, derived from ISSR data of 53 accessions of Citrus and the outgroup,

Atalantia monophylla.

Axis Eigen value Difference Percentage Cumulative

1 15.3348 6.9146 28.9336 28.9336

2 8.4202 2.6480 15.8872 44.8208

3 5.7722 3.4313 10.891 55.7118

4 2.3409 0.3832 4.4169 60.1287

5 1.9577 0.2401 3.6940 63.8227

6 1.7176 0.3454 3.2408 67.0635

7 1.3722 0.1403 2.5892 69.6527

8 1.2319 0.1513 2.3244 71.9771

9 1.0806 0.1597 2.0390 74.0161

10 0.9209 0.0404 1.7377 75.7538


species. This result was also supported by low heterozygosity(Ht = 0.153), and Shannon’s information index (I = 0.225) within C.

latipes.The polymorphism (P) and Heterozygosity (Ht) values obtained

for C. indica, Memang athur, C. medica and C. latipes were foundsimilar with those of ISSR derived data in some rare and threatenedplant species, like Cycas guizhouensis (Cycadaceae) (Ht: 0.1082,P: 35.90%) (Xiao et al., 2004), Ammopiptanthus (Leguminosae)(Ht: 0.1832, P: 39.39% in A. mongolicus; Ht: 0.1026, P: 25.89% inA. nanus) (Ge et al., 2005), Changium synrmioides (Ht: 0.240) (Qiuet al., 2004), Monimopetalum chinense (Ht: 0.183) (Xie et al., 2005).Low genetic diversity within Citrus taxa warrants urgent needfor conservation of the natural populations in North-east India.

The species-specific markers generated by some of the ISSRprimers can be developed in to SCAR markers for rapididentification of these taxa.

5. Conclusions

Very low genetic diversity and destruction of its natural habitatpose serious threat to C. indica even in the Citrus Gene Sanctuary inNBR. Narrow distributional range, high habitat specificity, lownatural regeneration and extraction of fruits from wild by Garos forlocal use and sale in local markets are the other constraintsassociated with in situ germplasm conservation and managementof C. indica. Complementary ex situ conservation, propagation andcultivation methods need to be urgently undertaken for protectionand maintenance of genetic diversity in this rapidly declininggenetic resource. NBPGR, New Delhi has taken initiatives for long-term preservation of pollen and seeds of C. indica in Cryogene Bank

(Malik et al., 2006). C. indica can be propagated from seeds, but itsgrowth, survival and establishment outside its natural habitat hasbeen difficult to achieve (personal observation). Establishment ofex situ germplasm conservatories in the form of a field gene bankand associated propagation centers in the vicinity of NBR will be analternate strategy for augmentation of germplasm materials of C.

indica, which can serve as important resources for recovery andrehabilitation of this threatened plant species. Low geneticvariability, heterozygosity and Shannon’s information index in C.

medica and C. latipes are also concerns that need to be addressed fordeveloping appropriate strategies to conserve the genetic diversityextant in these genetic resources.

Acknowledgements

The authors thank Dr. Rakesh Tuli, Director, National BotanicalResearch Institute (NBRI) for providing facilities to carry out thiswork, and to the officers and staff of Nokrek Biosphere Reserve(NBR), Tura, Meghalaya for their help, support and facilitiesextended to us during our field study in NBR. The financial supportreceived from the Department of Biotechnology, New Delhi tocarry out this work is gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.scienta.2009.10.008.

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