Genet Resour Crop Evol (2011) 58:697711 DOI 10.

1007/s10722-010-9613-1

RESEARCH ARTICLE

Stratication and population structure of the genetic resources of ancient medicinal rice (Oryza sativa L.) landrace Njavara
Sreejayan U. Suresh Kumar George Varghese T. M. Jacob George Thomas

Received: 22 June 2010 / Accepted: 30 August 2010 / Published online: 1 October 2010 Springer Science+Business Media B.V. 2010

Abstract Njavara (shashtika in Sanskrit), a rice (Oryza sativa L.) landrace described in ancient Sanskrit treatises of Ayurveda for its nutritive and medicinal properties, is traditionally used in Kerala for Ayurveda treatments. We characterized the genetic resources of Njavara using 24 morphological traits and 664 amplied fragment length polymorphic (AFLP) markers. Different multivariate methods revealed four morphologically distinct types (morphotypes) in the Njavara germplasm, which were

Electronic supplementary material The online version of this article (doi:10.1007/s10722-010-9613-1) contains supplementary material, which is available to authorized users.
Sreejayan G. Varghese G. Thomas (&) Department of Plant Molecular Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India e-mail: gthomas@rgcb.res.in U. S. Kumar Regional Facility for DNA Fingerprinting, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India T. M. Jacob Department of Statistics, Nirmala College, Muvattupuzha 686 661, Kerala, India Present Address: Sreejayan Application Specialist, Labindia Instruments PVT.LTD, Bangalore 560055, Karnataka, India

highly differentiated (Qst [ 0.65) for several quantitative variables. Eight quantitative variables together with glume or bran color stably discriminated the four morphotypes. In accordance with ancient literature, most Njavara accessions exhibit a short maturity (\90 days). AFLP analysis supported the morphological grouping; however, it resolved morphotype I into three distinct clusters, revealing six genotypes under the four morphotypes identied. The genotypes were highly differentiated (Fst = 0.7276; Gst = 0.7202) and highly homozygous. QstFst comparison revealed a possible role for natural selection in structuring quantitative variables among Njavara populations. Data suggest that the Njavara germplasm represents a composite of highly homozygous genetically isolated units. The distinctness of Njavara accessions in the AFLP dendrogram in relation to other traditional rice strains further demonstrates that the genotypes are nevertheless genetically cohesive and perpetuated with minimum genetic admixing. Stabilizing selection traditionally performed by farmers for short maturity coupled with autogamous breeding may have retained the genetic purity and governed the genetic structure of Njavara. The morphotypes identied were named long yellow, short yellow, intermediate yellow and short black, after culm length and glume color, which differ strikingly among the Njavara accessions. The present comprehensive description, the rst of this kind in Njavara, yields vital insight into the genetic resources of this crop, which is relevant for designing methods

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for its effective utilization in different elds of research. Keywords AFLP Medicinal rice Morphological traits Njavara Oryza sativa Population structure Stratication

Introduction Although rice strains with medicinal properties occur in different parts of the world (Gutierrez 1980; Singh and Singh 2003), none is used for medicinal applications as extensively as Njavara (Oryza sativa landrace Njavara), being used in Ayurveda, the traditional health care system in India. Descriptions of the medicinal and nutritive qualities of Njavara rice can be found in various ancient treatises of Ayurveda such as Ashtanga Hrudayam (Vagbhatta, circa 400500 AD) (Murthy 2001). This shortduration landrace is currently cultivated and utilized only in Kerala state. The Dravidian name Njavara (Burrow and Emeneau 1961) and its Sanskrit name shashtika (Turner 1966) both mean rice variety that matures in 60 days. The traditional Ayurveda scholars of Kerala, called Vydyas, developed several health-conducive treatment systems in the past using Njavara grains. Njavarakizhi, a purported therapy for rheumatic pain and neural disorders, is the most important system among them (Manakkodan 1949; Singh 1992; Murthy 2001). Besides its curative and palliative effects, the Njavarakizhi is claimed to be rejuvenating also. Conned to Kerala until recently, Njavara-based healthcare practices and their health benets are now gaining wider attention. Despite its distinctness among traditional rice strains as a medicinal plant, only recently did Njavara become the focus of research interest. Deepa et al. (2008) reported higher amounts of protein, vitamins, minerals and dietary ber in Njavara grains compared to those of two commonly cultivated improved varieties, Jyothi and IR64. Similarly, Simi and Abraham (2008) found Njavara rice to be different from native Chamba rice with respect to the morphological, physiological and thermal properties of its starch. Because landraces are a unique and critical source of useful genetic variability for future rice improvement (Pusadee et al. 2009), the observed

differences between Njavara and other rice varieties in biochemical characteristics (Deepa et al. 2008; Simi and Abraham 2008) support efforts to locate novel genetic variations in Njavara for improvement of rice quality. Investigations in this direction may also provide leads to validate the traditional claims of the medicinal and nutritional superiorities of Njavara grains, the scientic basis of which is not yet understood. Characterized germplasm is a fundamental prerequisite for the systematic study of a crop plant. However, little is known about the germplasm diversity and genetic structure of Njavara (Sreejayan et al. 2003) and it is sparingly subjected to crop improvement methods (Elsy et al. 1992; Menon and Potty 1998, 1999). All previous reports relied on a single sample of Njavara seeds bought either from a market or a farmers eld (Elsy et al. 1992; Menon and Potty 1998, 1999; Deepa et al. 2008; Simi and Abraham 2008). In fact, development of a characterized germplasm is the beginning point for an in-depth study of various aspects of Njavara. Here we present the genetic diversity and population genetic characteristics of Njavara germplasm depicted by a combination of morphological traits and amplied fragment length polymorphisms (AFLPs). Such morphology AFLP combination studies linked with rigorous statistical analysis have been successfully employed for the genetic diversity analysis and population genetic studies of several other poorly characterized plant genetic resources (Adoukonou-Sagbadja et al. 2007; Hartings et al. 2008; Saccomani et al. 2009).

Materials and methods Plant materials and morphological evaluation Following intense expeditions from May 2001 to March 2003, 27 samples of Njavara seeds (hereafter refers to as accessions) were collected from 17 sites belonging to eight districts of Kerala (Table 1; Fig. 1). Distance between the collecting sites ranged from 329 km between Payyannur and Mithrakary to 3 km between Puthurvayal and Kalpetta (Supplementary Table 1). In order to ensure their genuineness, seed samples were collected only from those farmers and herbal medicine vendors who provide Njavara grains to reputed Ayurvedic practitioners for medicinal

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Genet Resour Crop Evol (2011) 58:697711 Table 1 List of Njavara accessions and outgroups used together with the number of electromorphs identied in each accession following RAPD analysis Accession No. & Name* N2, Pattambi B N4, Vengoor B N6, Kalpetta 1 B N8, Thuravoor B N17, Cheruthuruthy B N20, Calicut B N21, Puthurvayal B N22, Kalpetta 2 B N23, Kalpetta 2 Y N27, Thrutala B N1, Puthukkad Y N3, Pavaratty Y N9, Thuravoor Y N5 Vengoor Y N18, Payyannur 1 Y N19, Payyannur 2 Y N24, Kannur 5 Y N25A, Kannur 6 Y N25B, Kannur 6 Y N26, Thrutala Y N28, Kannur 7 Y N10, Mithrakary 2 Y N11, Kunnummel 2 Y N12, Kunnummel 1 Y N13, Mithrakary 1 Y N14, Vezhapra Y N15, Mulackamthuruthy Y N16, Padaharam Y Outgroups PTB-18 PTB-21 Pokkali Gandhakasala Jyothi Kochuvithu * B Black lemma and palea Y Yellow lemma and palea ** Electromorph/variety codes are in parenthesis (41) (42) (43) (44) (45) (46) No. of electromorphs** 1 (1) 2 (2,3) 3 (4,5,6) 1 (7) 1 (8) 1 (9) 2 (10,11) 2 (12,13) 1 (14) 1 (15) 1 (16) 2 (17,18) 3 (19,20,21) 0 1 (22) 2 (23,24) 3 (25,26,27) 1 (28) 1 (29) 1 (30) 1 (31) 1 (32) 2 (33,34) 1 (35) 1 (36) 1 (37) 2 (38,39) 1 (40)

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Fig. 1 Map of Kerala state showing districts and the 17 collecting sites of the Njavara accessions. 1Payyannur; 2Kannur; 3Puthurvayal; 4Kalpetta; 5Calicut; 6 Thrutala; 7Pattambi; 8Cheruthuruthy; 9Pavaratty; 10 Puthukkad; 11Thuravoor; 12Vengoor; 13Kunnummel; 14Vezhapra; 15Mulackamthuruthy; 16Padaharam; 17Mithrakary

and nutritional applications. Of the accessions, N2 was obtained from Regional Agricultural Research Station (RARS), Pattambi, Kerala. Two distinct glume colors, black and yellow, were evident among

the seeds collected. The accessions were named after the place of their cultivation and the color of their glume; B for black and Y for yellow. In our collection expeditions, we also enlisted the name and days to maturity of other traditional rice cultivars currently cultivated in Kerala in order to compare their days to maturity with that of Njavara. For morphological evaluation, variations for 20 quantitative and 11 qualitative characters were recorded from 15 individuals from each Njavara accessions raised in pots (pot experimentI). The quantitative traits included: leaf length (LL; cm), leaf width (LW; cm), ligule length (LiL; cm), culm length (CL; cm), culm number (CN), panicle length (PaL; cm), 100 grain weight (100 GW; g), grain length (GL; cm), grain width (GW; cm), panicle number (PaN), panicle weight/plant (PaW; g), grain number

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per panicle (GN), grain density (GD; grain number per panicle/panicle length), grain shape index (GS; grain length/grain width), internode length (InL; cm), internode number (InN), leaf sheath length (LsL; cm), ag leaf length (FlL; cm), ag leaf width (FlW; cm) and stem girth (SG; mm). The qualitative traits included leaf angle (LA), blade color (BC), ag leaf angle (FlA), culm angle (CA), internode color (InC), panicle type (PaT), panicle exsertion (PaE), panicle axis (PaA), lemma and palea (glume) color (LPC), bran color (BrC) and basal leaf sheath color (BlsC). The morphological data were scored according to descriptors for rice (IRRI/IBPGR 1980). Uniformity, stability and the discriminatory power of a set of traits, which discriminate different morphology classes identied within Njavara germplasm following various multivariate analyses of initial data sets, were validated following two more eld experiments: one again in pots (pot experimentII) and the other in plots (plot experimentI). For both validation experiments, variation for the selected traits was recorded from 15 individuals from each accession. Days to maturity were recorded during the plot experiment. Jyothy, an improved hybrid variety with a crop duration of 110125 days, released by Kerala Agricultural University (http://www.kau.edu/ varieties.htm), and Kochuvithu, a short-duration (*90 days) traditional cultivar in Kerala, were used as check varieties in the plot experiment. Genomic DNA isolation and molecular marker analysis Individuals used in the initial morphological evaluation were also used for DNA marker analysis. Genomic DNA was isolated from 100 mg of tender leaf tissues using a GenElute Plant Genomic DNA Miniprep Kit (Sigma) following the manufacturers instructions. The level of genetic polymorphism within each accession was rst examined by random amplied polymorphic DNA (RAPD) analysis of ve individuals from each accession using the method reported earlier (Thomas et al. 2001). Nine informative decamer oligonucleotide primers (OPC9, OPC13, OPC15, OPE2, OPE11, OPE16, OPF9, OPF10 and OPF14) selected following an initial screening of 36 primers randomly selected from C, E, F, J, AC and AO kits (Operon Technologies, Almeda, CA) were

used for this analysis. The nine RAPD proles generated in each accession were assessed visually and one representative individual from each of the accessions that yielded a monomorphic prole and one representative individual from each electromorph class in accessions that yielded polymorphisms were selected for AFLP analysis. AFLP analysis was carried out using an AFLP Analysis System II (Invitrogen) following the manufacturers instructions. Outgroups for AFLP analysis were chosen carefully to represent a cross-section of the rice gene pool in Kerala. These included ve traditional rice strains in Kerala: an aromatic rice, Gandhakasala; a highly salt-tolerant variety, Pokkali; a short-duration (90 days) landrace, Kochuvithu; selections from landraces PTB 18 and PTB 21; and one high-yielding hybrid variety, Jyothi. Accession N5, which yielded RAPD proles identical to those of accession N9 collected from a nearby location, was excluded from the AFLP analysis. Data analyses The level of homogeneity for the quantitative traits within an accession was tested using Levenes test of equality of variances. Principal component analysis (PCA) was performed using both quantitative and qualitative traits in order to reduce the number of variables examined to a common scale and to determine the importance of different traits in explaining the total variation. Hierarchical cluster analysis based on squared Euclidean distance using Wards method was done to identify natural groups in the Njavara germplasm. Multiple correspondence analysis (MCA) was performed on the qualitative traits to evaluate each ones contribution to the variation observed in the Njavara germplasm. Variation in quantitative traits among the groups identied by PCA was tested by multivariate analysis of variance (MANOVA). Discriminant function analysis (DFA) was carried out on quantitative traits to identify the characters that contribute most toward the discrimination of the morphology classes identied in the Njavara germplasm following multivariate analysis. The z-transformed values were used to perform PCA and cluster analysis. All computations of morphological data were carried out using the software package SPSS 13.0 for Windows. All of the analyses were tested at the 5% signicance level.

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The degree of differentiation of quantitative traits between morphology classes was assessed by using the Qst statistic (the Fst equivalent for quantitative traits) according to the method of Bonnin et al. (1996) for a completely selng species. Variance components were computed for each quantitative trait following a nested analysis of variance using type III SS using Proc GLM in the software SAS version 8.2. Each AFLP band was treated as a unit character and scored manually using independent binary codes (1 for presence and 0 for absence) and 1/0 matrices were generated. Two persons did the scoring independently, and bands which had conicting data between the two readings were eliminated from further analysis. Pairwise genetic distance between samples was computed from the 1/0 matrix based on the Dice coefcient using the software WinDist (a companion program of WinBoot, http://www.irri.org/winboot/html). The resulting genetic distance matrices were used to construct a UPGMA (unweighted pair-group method with arithmetic means) dendrogram using N-Join, SAHN and CONSEN modules in the software package NTSYSpc 2.1. The robustness of the clusters was estimated by performing a bootstrap analysis on 2,000 data replicates using WinBoot. Cophenetic correlation was estimated using the COPH and MXCOMP procedures of NTSYSpc to test the reliability of the topology of the AFLP-based dendrogram. POPGENE software version 1.32 (http://www. ualberta.ca/*fyeh/) was used to compute the population genetic parameters, percentage polymorphic loci (PPL), Shannons information index (I) (Lewontin 1972), Neis gene diversity (h) (Nei 1973) and gene ow (Nm), from the 1/0 matrices of the AFLP phenotype. The 1/0 matrices were also used to evaluate the degree of genetic differentiation among populations by using the Gst statistic and Fst statistic (signicance tested with 1,023 permutations) as implemented in software programs POPGENE and Arlequin version 3.11 (http:// cmpg.unibe.ch/software/arlequin3/), respectively. A Mantel test was performed between the pairwise Euclidian distance (morphological) matrices and Dice distance (AFLP) matrices using the MXCOMP procedure of NTSYSpc to test the correspondence between the groupings of Njavara germplasm revealed by the two marker systems. Correlation between Dice distance matrices and the pair-wise geographic distance (in kilometers) between the

locations of Njavara accessions was also computed by using the Mantel test.

Results Intra-accession homogeneity assessment and selection of traits and individuals Following Levenes test of equality of variance, 19 quantitative traits, which yielded a high degree of within-accession homogeneity, were selected for further analysis; one trait, internode number, which showed high within-accession variability was excluded. Of the 11 qualitative traits, ve traits ag leaf angle, panicle type, panicle exsertion, lemma and palea color and bran colorwere selected following thorough examination of the data; the remaining six traits (leaf angle, blade color, internode color, basal leaf sheath color, panicle axis and culm angle) were excluded either because accurate determination of the character states was difcult or the trait was monomorphic across the accessions. Within-accession genetic homogeneity was tested by RAPD analysis of ve individuals from each of 27 accessions using nine informative primers. Sixteen accessions yielded identical ngerprints for all ve individuals, whereas the remaining 11 accessions displayed electrophoretically distinct genotypes (electromorphs) for one or more primers (Table 1). In the case of accession N25, all nine primers yielded two distinct proles, indicating the existence of two distinct electromorphs within this accession (Supplementary Fig. 1). Morphological evaluation and RAPD analysis of additional individuals raised from N25 conrmed the existence of two distinct genotypes within N25. Based on this result, N25 individuals were separated into two groups, N25A and N25B, and were treated as separate accessions in all subsequent analysis; thus, the total number of accessions increased to 28, and the accessions yielding monomorphic proles to 18. Altogether, 40 electromorphs were selected for AFLP analysis (Table 1).

Multivariate analysis of morphological traits The 19 quantitative characters varied signicantly (P [ 0.05) among the 28 Njavara accessions

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examined (Supplementary Table 2). The ve qualitative characters also showed wide variations among the accessions. PCA of the 24 characters identied six principal components with Eigenvalues above 1.0, explaining altogether 85.48% of the total variation. The rst and second principal components (PC1 and PC2) showed sufciently non-random patterns to be interpreted and were able to separate the accessions into four groups: I, II, III and IV (Fig. 2). Hierarchical cluster analysis of the 28 accessions based on morphological variables similarly identied four clusters (sub-clusters A1, A2, B1 and B2) (Fig. 3), which are identical to PCA groups I, II, III and IV, respectively, except that accessions N4 and N8 that belong to PCA group I are clustered with sub-cluster A2. MCA of the ve qualitative traits detected three axes with Eigenvalues [ 1. Lemma and palea color and bran color, which dominated the rst axis, accounted for 52.17% of the total variation, and both traits recorded the highest loading (0.889 each) among the ve traits tested. A scatter plot of axis I and axis II values decisively separated the Njavara accessions into two distinct sets (Fig. 4). Set I comprises only group I accessions with black glume and light brown bran, and set II contains the accessions belonging to the remaining three groups, all of them with yellow glume and red bran.

Fig. 3 Dendrogram of Njavara accessions based on morphological data. The major clusters identied are indicated as A and B, and the sub-clusters as A1 and A2 and B1 and B2. Accession numbers are according to Table 1

Fig. 4 Multiple correspondence analysis of Njavara accessions based on six qualitative traits. Groups identied are numbered I and II. Accession numbers are according to Table 1

Characteristics of Njavara morphotypes

Fig. 2 Principal component analysis of 28 Njavara accessions based on morphological data. The groups identied are numbered IIV. Accession numbers are according to Table 1

Considering together the results of the PCA, cluster analysis and MCA, four morphologically distinct

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Table 2 Group-wise means of the quantitative variables in the four morphotypes (IIV) identied in Njavara germplasm and the Qst values of the quantitative traits Trait* LL (cm) LW (cm) CL (cm) CN PaL (cm) 100 GW (g) GL (cm) GW (cm) PaN GD GN InL (cm) LsL (cm) FlL (cm) FlW (cm) SG (mm) LPC BrC I, II 43.88 2.69 1.05 0.06 87.30 7.56 6.75 2.14 19.53 1.11 1.86 0.18 0.73 0.03 0.25 0.01 8.64 1.62 3.46 0.43 68.09 10.89 16.47 0.85 17.19 0.47 39.82 2.95 1.17 0.08 11.66 1.09 IBlack IIYellow ILight brown IIRed Morphotypes I and II are separated based on lemma and palea color and glume color * Trait code as given in Materials and Methods ** The 95% standard error estimates for Qst values obtained by bootstrapping Red Red III 51.67 4.59 1.14 0.09 115.66 10.98 4.58 0.80 22.81 1.56 1.83 0.09 0.70 0.01 0.25 0.01 5.94 0.93 4.44 0.43 102.39 14.61 17.99 0.46 19.24 0.44 41.34 1.33 1.21 0.05 12.90 0.97 Yellow IV 51.52 5.04 1.26 0.05 102.05 6.25 5.93 1.01 23.22 2.17 2.22 0.42 0.77 0.02 0.28 0.02 6.28 1.08 3.87 0.56 91.57 20.27 17.55 2.06 19.08 1.21 43.14 3.90 1.39 0.11 15.82 2.09 Yellow Qst 0.67 0.66 0.83 0.48 0.74 0.35 0.00 0.40 0.68 0.68 0.73 0.34 0.82 0.18 0.59 0.74 CI** 49.476.1 50.174.4 75.886.2 29.658.9 59.880.4 25.443.0 -28.064.3 31.3-46.9 47.174.4 43.880.5 57.880.1 13.947.8 65.788.3 -6.836.7 44.567.4 61.680.7

groups or morphotypes were discernible in Njavara germplasm and are hereafter referred to as morphotypes I-IV. The group-wise means of the 19 quantitative traits for the four Njavara morphotypes are given in Supplementary Table 3. Of the 19 quantitative traits, the group mean for 16 traits differed signicantly (P \ 0.05). However, none of the quantitative traits differed signicantly between morphotypes I and II (Supplementary Table 3). MANOVA using the quantitative traits detected signicant (P \ 0.001) population difference between a single group formed by merging morphotype I and morphotype II, morphotype III and morphotype IV according to several statistics: Wilks k (k = 0.004), Hotellings Trace (T2 = 33.723) and Roys Largest Root (klargest = 23.104). Thus, with respect to quantitative variations, there are only three groups in Njavara germplasm: a single group comprising of morphotypes I and II, morphotype III and morphotype IV, and these groups will be hereafter referred to as class IIII

respectively. Morphotype I and II accessions are differed each other only for their glume color and bran color; morphotype I accessions are distinguished by black glume and light brown bran and morphotype II by yellow glume and red bran (Fig. 4; Table 2). Otherwise, morphotypes I and II are characterized by short (87.30 7.56 cm) and thin culms (11.92 0.27 mm), and short panicles (19.53 1.11 cm) with lesser grains/panicle (68.09 10.89) but with the highest number of tillers (6.75 2.14) and the highest number of panicles/plant (8.64 1.62). These accessions are distributed in ve districts of Kerala, spanning from Ernakulam to Wayanad (Fig. 1; Table 1). According to the agroecological stratication of Kerala state (Anonymous 2002), the collecting sites of these accessions fall within the Central Midland and Malappuram agroecological zones. Accessions belonging to morphotypes III and IV all have yellow glumes and red bran. All of the accessions

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from the Alappuzha and Kottayam districts, which fall under the Kuttanad agroecological zone (Anonymous 2002), belong to morphotype III, whereas all of the accessions from Kannur district, which is categorized as a Northern Midland agroecological zone, belong to morphotype IV (Fig. 1; Table 1). Morphotype III accessions are the tallest accessions (115.66 4.15 cm), with the highest number of grains/panicle (102.39 5.52) and the fewest tillers (4.58 0.30), whereas morphotype IV accessions have an intermediate culm length (102.05 2.55 cm) with the longest (0.76 0.01 cm), thickest (0.28 0.01 cm) and heaviest (100 grain weight 2.22 0.17 g) grains and the thickest culms (15.82 0.85 mm). Days to maturity of morphotypes I, II and III were found to be lower than that of other traditional rice strains grown in Kerala including the short-duration traditional cultivar Kochuvithu, which matures within 90 days, whereas the days to maturity was relatively higher for all of the morphotype IV accessions (Supplementary Table 4). Discriminant function analysis of quantitative traits DFA was carried out to identify the key quantitative traits that separated the accessions into the three classes (class I, II, III) based on quantitative variations. The quantitative traits were initially subjected to a stepwise discriminant analysis, as implemented in the software SPSS, and two standardized canonical discriminant functions were extracted describing three traits, culm length, 100 grain weight, and stem girth. The three traits identied were able to classify 96.4% of the accessions into their original classes, with only one misclassication (Supplementary Table 5a). In the next step, other quantitative characters were analyzed one after the other by following the enter independents together method of discriminant analysis in combination with the three discriminatory traits already identied, in order to identify character combinations that show a superior prediction of classication. A combination of eight traits ligule length, culm length, 100 grain weight, grain length, grain width, grain number/panicle, grain shape index, and stem girthwere found to be able to classify the Njavara accessions with 100% accuracy to the original grouping (Supplementary Table 5b). The discriminatory powers of the traits

identied were very high, as indicated by the low values of Wilks k. The eight traits classied the Njavara accessions with 100% accuracy in pot experimentII (Supplementary Table 5c) and with 89.2% accuracy in plot experimentI (Supplementary Table 5d). Genetic differentiation of quantitative traits (Qst) Partitioning of the variation of the 16 quantitative traits that differed signicantly between the three classes is shown in Table 2. The Qst values for the individual traits ranged from 0.18 in ag leaf length to 0.83 in culm length, with an average of 0.55. The Qst values for nine traits were [ 0.65 (Table 2). Grain length was found not to be differentiated between the three classes. AFLPs and cluster analysis Eleven AFLP primer combinations generated a total of 664 bands with an average of 60.36 bands per primer combination, of which 343 (51.66%) were polymorphic (Table 3). A representative AFLP ngerprint is shown in Supplementary Fig. 2. The average pair-wise Dice distance estimates between Njavara accessions and out-groups (0.1366 0.0116) differed signicantly (P \ 0.05) from the average distance estimates within Njavara accessions (0.0633 0.0285) and within out-groups (0.0840 0.0405). The UPGMA dendrogram obtained following the cluster analysis of 40 Njavara electromorphs and the six out-groups using Dice distance estimates is shown in Fig. 5. High bootstrap values support the robustness of clustering. The high correlation (r = 0.97689) between the AFLP data matrix and the cophenetic matrix further suggests a good representation of the data matrix in the dendrogram produced. Two distinct nodes were evident in the UPGMA dendrogram: one major node consisting of 36 Njavara electromorphs and a minor one consisting of six outgroups and four Njavara electromorphs (three N24 electromorphs and one N25A electromorph) (Fig. 5). Under the major node, the 36 Njavara electromorphs were separated further into six clusters. Interestingly, more than one electromorphs used from the same accession (Table 1) invariably entered into the same cluster. AFLP data resolved morphotype I accessions into three clusters, cluster I, II and III, each cluster

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Genet Resour Crop Evol (2011) 58:697711 Table 3 Level of polymorphism revealed by AFLP analysis using 11 primer combinations in 40 Njavara electromorphs and six outgroups

705

AFLP primer combination

Total bands

Polymorphic bands 27 41 27 30 26 40 36 45 21 27 32 343

Percentage polymorphism 64.28 50.61 55.10 44.77 46.42 65.57 57.14 62.50 40.38 39.70 45.71 51.66

E-AA 9 M-CAC E-AA 9 M-CTA E-AC 9 M-CAG E-AC 9 M-CAT E-AG 9 M-CAG E-AT 9 M-CAC E-TA 9 M-CTT E-TC 9 M-CTA E-TG 9 M-CTA E-TG 9 M-CTG E-TG 9 M-CTT Total

42 68 49 67 56 61 60 72 52 67 70 664

II, III and IV, respectively (Fig. 2 vs Fig. 5). Morphotype II was relatively more reorganized in cluster IV. However, for the most part, cluster IV retained the two key characteristics of morphotype II, i.e. short plant form and yellow glume color. A Mantel test revealed a weak but signicant correlation (r = 0.357) between the morphological (Euclidian) and AFLP (Dice) distance matrices, whereas the correlation between Dice distance matrices and pair-wise geographic distance between Njavara accessions was not signicant (r = 0.2282) The six clusters, clusters IVI, identied in the AFLP-based dendrogram will be hereafter referred to as genotypes IVI, respectively. AFLP diversity and its partitioning Table 4 summarizes the population genetic statistics of the six Njavara genotypes revealed by AFLPs. The highest values for all the three diversity parameters (h, I, PPL) were recorded in genotype II and the lowest in genotypes IV and V. The pair-wise Fst values between the six Njavara genotypes varied from 0.5148 (between genotypes 1 and 2) to 0.8788 (between genotypes 3 and 5), and all the values were highly signicant (P \ 0.001). The average Gst and Fst values highlighted the partitioning at a very high level of 72% (Gst = 0.7202; Fst = 0.7276) of the total variation between the six Njavara genotypes, and this very high level of population partitioning was corroborated by a calculated very low gene ow (Nm = 0.1942) between the genotypes.

Fig. 5 UPGMA dendrogram of 40 Njavara electromorphs and six outgroups based on AFLP data. Bootstrap values are indicated at branch points. Roman numerals at the left side indicate the clusters of six Njavara genotypes (IV1) and the outgroup (VII). Arabic numerals at the end of each node correspond to the electromorphs/variety code in Table 1

consisting mostly of geographically closer accessions. To a greater extent, clusters IV, V and VI in the UPGMA dendrogram corresponded to morphotypes

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706 Table 4 Population genetic statistics of the six Njavara genotypes Genotypes I II III IV V VI Gst Fst Nm h 0.0336 0.0597 0.0201 0.0069 0.0069 0.0307 0.7202 0.7276 0.1942 I 0.0513 0.0891 0.0286 0.01 0.01 0.0458 PPL 10.69 17.02 4.52 1.66 1.81 8.58

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accession each from the six genotypes, were used for testing. The accessions chosen were N22, N9, N21, N3, N14 and N19, representing genotypes I, II, III, IV, V and VI, respectively. Of the 260 bands that were scored between the six parental individuals, 252 bands were reproduced uniformly across the two generations whereas altogether eight bands (3.08%) were not inherited uniformly (Supplementary Fig. 3; Supplementary Table 6).

Discussion Seed selection and cultivar permanence in Njavara Whereas rice landraces normally show high intraaccession heterogeneity (Fukuoka et al. 2006; Tu et al. 2007; Barry et al. 2007; Pusadee et al. 2009), Levenes test of equality of variances for quantitative traits and RAPD analysis revealed very high intra-collection homogeneity in Njavara. Electromorphs identied within some of the Njavara accessions following RAPD analysis were not substantially different from each other as they clustered together in the AFLP-based dendrogram. This result, in conjunction with the ancientness of Njavara cultivation, implies that Njavara has not been subjected to seed replacement or seed mixing, the two practices performed extensively in traditional agriculture to ensure better harvest and food security (Zeven 1999; Cleveland et al. 2000). While the ancient Ayurveda literature cite a 60 days maturity for Njavara (Murthy 2001), the 8088 days maturity recorded for Njavara accessions in this study may be due to the consequence of changes occurred in climatic and ecogeographic factors over centuries. However, it is to be noted that the Njavara has the shortest crop duration among the traditional rice strains cultivated in Kerala. Farmers retain a landrace if it is characterized by a distinct trait not seen elsewhere (Berg 2009). Nonetheless, how do farming communities precisely maintain purity and authenticity of a landrace over generations? Stabilizing selection performed by farmers has been highlighted as a powerful force in the retention of a landrace as well as the maintenance of its purity (Louette and Smale 2000; Perales et al. 2003). In Kerala, in compliance with the description for Njavara in Ayurveda texts, farmers may have

h Neis gene diversity (Nei 1973) I Shannons information index (Lewontin 1972) PPL Percent polymorphic loci

Molecular discrimination of morphotypes or genotypes The three genotypes (genotypes I, II, III) identied within morphotypeI were discriminated among each other and from others by seven fragments in total: the fragments E-AA x M-CAG166 and E-AA x M-CTA222 were unique to the genotype I and genotype II accessions, respectively, and E-AC 9 M-CAT125, E-AT 9 M-CAC264, E-TA 9 MCTT246, E-TC 9 M-CTA105 and E-TC 9 M-CTA188 were unique to genotype III. An AFLP that is able to discriminate between morphotype II and morphotype III was not identied. However, as a group, morphotypes II and III could be discriminated from other morphotypes with E-TC 9 M-CTA101 and E-TG 9 M-CTG162. Two AFLPs, E-AT 9 M-CAC104 and E-TA 9 M-CTT157, discriminated morphotype IV from the other morphotypes. E-AA 9 M-CAC154 and E-TG 9 M-CTA288 discriminated Njavara accessions from the out-groups, including the Njavara accessions N24 and N25A that grouped with the out-groups. Uniformity and stability of AFLP proles Stability and uniformity of the ngerprint proles generated by ve (E-AA 9 M-CTA, E-AC 9 M-CAG, E-TA 9 M-CTT, E-TG 9 M-CTA, E-TG 9 M-CTG) of the 11 primer combinations used in the study were tested. Three individuals each, sampled from two consecutive generations of progeny raised from one

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used short-crop duration as a tool for the management of Njavara. Although further studies are essential for drawing a denite conclusion, the discrete clustering of most Njavara accessions in the AFLP-based dendrogram in relation to the outgroup indicates the purity of Njavara, despite its sympatric cultivation with other traditional rice varieties over several centuries. Asynchronous owering between shortduration Njavara and other rice strains co-planted in a eld, which are mostly long duration, may also have contributed to the seed purity of Njavara by preventing genetic admixing between Njavara and other rice strains. However, as discussed later, the data also suggest introgression, at least in a few Njavara accessions. Ayurveda is inextricably linked with spirituality (Chattopadhyay 2007), and different codes are traditionally followed for the selection of herbals for Ayurveda treatments. In our collection expedition, it was evident that most Njavara farmers and Vydyas use only their own seeds, which are handed down over generations, and they maintain the seeds ritualistically. Together the results suggest that, like the Tewa Indians of New Mexico and many other communities in the world who maintain the purity of their most favored seed lots by adhering to longheld customs (Zeven 1999), the farmers of Kerala carefully cultivated Njavara rice over several centuries and continue to do so today despite the rapid advancement of modern improved varieties.

Genetic structure of Njavara Multivariate analysis of morphological traits revealed four distinct Njavara morphotypes. Morphotypes are commonly encountered in autogamous and vegetative species as minimum gene ow and lack of sexual recombination perpetuate population characteristics intact in such species (Hempel and Peakall 2003; Pissard et al. 2008). AFLP analysis resolved the strata identied by morphological data further, as reported in other species (Cheng et al. 2004), revealing three distinct genotypes within morphotype I and separating N24 and N25A accessions from morphotype IV and placing them with the out-groups. Thus, the data, revealed a highly structured germplasm in Njavara, comprising at least six genotypes that can be clumped into four morphotypes. AFLP data support the

genetic distinctness of morphotype I and morphotype II, although they differ only with respect to glume color and bran color. Population genetic parameters h, I and PPL highlight different levels of heterogeneity within the morphotypes or genotypes identied in Njavara, as was observed within the morphotypes of Oxalis tuberosa (Pissard et al. 2008). Morphotype II, morphotype III, and the third genotype of morphotype I are highly homogeneous, whereas the remainder are relatively heterogeneous. Between-population partitioning of neutral genetic diversity is expected to be high in predominantly autogamous species such as rice (Hamrick and Godt 1996). However, the level of differentiation recorded in this study between Njavara populations (Fst = 0.7276; Gst = 0.7202) is exceptional, because it occurs between different populations of the same landrace and not between different landraces (Tu et al. 2007; Barry et al. 2007) or between different populations within a species, as reported earlier (Kuittinen et al. 1997; Stenien et al. 2005; Zhang et al. 2006). There are few studies in the literature which have assessed population genetic structure of a single rice landrace, but the one study which examined population parameters of the Thai landrace Bue Chomee (Pusadee et al. 2009) also found very high partitioning (Fst = 68%) of total variability among individuals. In Njavara, not only the neutral diversity but also the variability of several quantitative traits is strongly partitioned (Qst [ 65) between morphology classes, and the Mantel test yielded a signicant and positive correlation between morphological and AFLP-based distances (r = 0.357). Higher genetic and phenotypic cohesion is achieved in populations with a long history of reproductive isolation, because in addition to facilitating the accumulation of genetic differences between populations, continuous reproductive isolation also permits the divergence of adaptive traits, pushing them close to their tness maxima (Rieseberg and Willis 2007). Inheritance studies of the AFLPs further revealed that the genotypes identied within the Njavara germplasm are highly homozygous, too. Thus, the data indicate that continuous self-fertilization coupled with perpetuation over centuries under human management have rendered Njavara populations as a set of genetically isolated homozygous units. This is in agreement with the general assumption that, in rice, a landrace represents a conglomeration of highly differentiated

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homozygous lines (Garris et al. 2005). Stabilizing selection performed by farmers, which is a powerful force for retaining the status quo of gene frequencies (Perales et al. 2003), may have helped the highly differentiated Njavara populations to be genetically cohesive, preventing them from evolving as independent varietal types. A strong geographic structuring is evident in Njavara germplasm, with a localized distribution of genotypes II and III of morphotype I, morphotype III and morphotype IV to Ernakulam, Wayanad, Alappuzha and Kannur districts, respectively, and a predominant distribution of morphotype II accessions in the adjacent Thrissur and Palakkad districts. Geographic structuring of genetic diversity has also been reported in rice landraces of Cote d Ivoire (Sanni et al. 2008). However, a Mantel test between the geographic and genetic distance estimates failed to show geographical structuring in Njavara germplasm. This is because in highly autogamous species, especially in cultivated crops, where the gene ow between adjacent populations is low, the genetic and geographic distance between populations may not correlate, and populations show divergence independent of the geographic distance between them (Steinger et al. 2002; Pusadee et al. 2009). Geographical structuring of Njavara germplasm demonstrates that Njavara seeds were not exchanged much in the past, in contrast to the practices with other rice landraces (Nuijten and van Treuren 2007; Pusadee et al. 2009), and substantiates our observation that farmers prefer to cultivate known local or their own strains rather than unknown ones from other farmers. Possible factors governing genetic differentiation in Njavara populations Our data provide a wide scope for understanding the roles of different factors in shaping the genetic structure of Njavara populations, because, unlike a previous study on the population genetic structure of a traditional rice (Pusadee et al. 2009), we included both genetic and quantitative characteristics of Njavara populations in our study. In principle, the comparison of FstQst estimates, the method frequently used to infer the level and heterogeneity of selection in natural populations (Leinonen et al. 2008), could be applied to study the role of selection in shaping the genetic structure of Njavara populations,

because farmers selection practices affect the status of only one trait (days to maturity) whereas other traits may evolve freely, as shown in maize landraces (Louette and Smale 2000). Considerable scatter in the Qst values among the different quantitative traits of Njavara populations suggests that the balance between selection and drift is specic to individual traits (McKay and Latta 2002). Hence, we preferred to consider the Qst of individual traits, as was done in earlier reports (Leinonen et al. 2008), instead of averaging over traits in order to avoid hiding among-trait variability in Qst values. The Qst values of nine quantitative traits are very high and comparable to the overall Fst values in Njavara populations. When Fst and Qst values are comparable, it implies that the relative contribution of drift and selection in achieving the observed differentiation is unknown (Leinonen et al. 2008). The accessions of morphotypes I and II do not differ signicantly with respect to quantitative traits (Qst = 0), but genetically composed of four highly differentiated, geographically localized genotypes. This structuring pattern may be an outcome of continuous selection of populations under homogenous conditions, because the Central Midland and Malappuram agroecological zones, where these accessions are distributed, share many agroecological features such as rainfall, topography and soil type (Anonymous 2002). Under homogenous selection, in populations with restricted gene ow, tness maxima of a quantitative trait are likely to converge, but the multi-locus genotypes are likely to diverge from one population to another by the accumulation of neutral variation, resulting in low Qst but high Fst values (Latta 1998; McKay and Latta 2002). Conversely, under heterogeneous conditions, quantitative traits undergo divergent selection and their trait maxima may move to different tness levels depending on their relative role in governing local adaptation (Latta 1998; McKay and Latta 2002; Leinonen et al. 2008). Morphological divergence of morphotype III accessions may be an outcome of diversifying selection under the selective forces occurring in the Kuttanad agroecological zone, whose agroclimatic conditions are strikingly different from those of the Midland and Malappuram agroecological zones in many parameters (Anonymous 2002). Thus, the data reveal a role for natural selection in accelerating genetic differentiation at least in some of the Njavara populations,

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similar to the case of the Thai traditional rice, Bue Chomee (Pusadee et al. 2009). However, the contribution of recurrent genetic drift exerted by farmers by sampling a small portion of the harvest every season as seed (Perales et al. 2003; Alvarez et al. 2005) and farming practices (Zhang et al. 2009) cannot be ignored in shaping the genetic structure of Njavara populations. Additional data are needed to address the role of such components in determining the dynamics of Njavara populations. However, the morphotype IV accessions showed high differentiation despite their distribution in the Northern Midland agroclimatic zone, which also has many agroclimatic factors in common with the Central Midland and Malappuram agroecological zones (Anonymous 2002). Unlike the morphotype III accessions, the morphologically similar morphotype IV accessions were not genetically cohesive. Of the six morphotype IV accessions, N24 and N25A clustered with the outgroup, whereas the others remained in the Njavara cluster. Morphological traits of all morphotype IV accessions yielded high values, relatively closer to those of other traditional rice strains (data not shown). These data point to a recent episode of gene introgression into some of the Njavara accessions from other traditional rices grown sympatrically and the local spread of the introgressants. Introgressants showing varying levels of morpho-molecular discordance between themselves and with their parental lines have been reported in many other plant species (Lihova et al. 2007; Greene et al. 2008). The observed longer crop duration in all morphotype IV accessions substantiates such introgression. Empirical records of inter-cultivar gene ow in rice are rare in the literature (Cleveland et al. 2000 and references therein). It is interesting to note that such gene ow, which usually accelerates population differ entiation (Alvarez and Wendel 2006) and on-farm evolution of new cultivars in cross-pollinating species (Cleveland et al. 2000; Perales et al. 2003), is likely to have a role in the genetic differentiation of Njavara populations. Stabilizing selection exerted by farmers may have prevented the evolution of introgressants into the level of a new cultivar. Morphotypes within Njavara and their discrimination Plant height and glume color are the two traits that differed strikingly between morphotypes, and they

are also the ones easily recognizable by farmers. Combining these two traits, we hereby name morphotypes I to IV as Short black (SB), Short yellow (SY), Long yellow (LY), and Intermediate yellow (IY). For easy application in the farming community we also propose alternate vernacular (Malayalam) names for the morphotypes based on their geographic afnity. We propose the names Wayanadan for Short black, because it is the predominant type in Wayanad district; Palakkadan for Short yellow, considering the predominance of this type in Palakkad district and nearby areas as well as the position of the Palakkad region as one of the rice bowls of Kerala; Kuttanadan for Long yellow, because this is the only type in Kuttanad paddy elds, another rice bowl in Kerala; and Vadakkan for Intermediate yellow, because this type is localized to northern Kerala. Genotypes I to III (clusters I to III in the UPGMA dendrogram; Fig. 5) will be Short black I to III (SB I to III). Until the phytoceutical factors contributing to the medicinal and nutritional attributes of Njavara are elucidated and the effect of introgression on such factors is determined, the morphotype IV accession should perhaps be avoided from the specialty applications of Njavara. A combination of eight quantitative traits can effectively discriminate the four morphotypes of Njavara in conjunction with one of the qualitative traits, either glume color or bran color. These quantitative traits hold great potential in the distinctness, uniformity and stability (DUS) test of Njavara germplasm, as they were stable over three generations and discriminated the morphotypes with almost the same effectiveness in every generation. Germplasm management in Njavara following the morphological approach alone could result in the loss of subtle genotypes of morphotype I and acceptance of introgressants of morphotype IV. Therefore, in practice, regular sampling could be performed using morphological parameters, and ner evaluation could be done using molecular markers, particularly for morphotype I and IV accessions. In conclusion, we have provided a framework for the genetic structure of Njavara. This framework forms a basis for future research initiatives in Njavara. Furthermore, we believe our studies will help this rice strain to be better known to the rice world and attract concerted efforts to dissect out its

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Genet Resour Crop Evol (2011) 58:697711 Elsy CR, Rosamma CA, Potty NN (1992) Njavara-a rice variety with special characters. Oryza 29:5556 Fukuoka S, Tran SD, Ebana K, Luu TN, Nagamine T, Okuno K (2006) Genetic organization of aromatic rice as revealed by RAPD markers: a case study in conserving crop genetic resources on farm. Euphytica 149:6171 Garris AJ, Tai TH, Coburn J, Kresovich S, McCouch S (2005) Genetic structure and diversity in Oryza sativa L. Genetics 169:16311638 Greene SL, Kisha TJ, Dzyubenko NI (2008) Conserving alfalfa wild relatives: is past introgression with Russian varieties evident today? Crop Sci 48:18531864 Gutierrez HG (1980) An illustrated manual of Philippine Materia Medica. 1:162164 Hamrick JL, Godt MJW (1996) Effects of life history traits on genetic diversity in plant species. Phil Trans R Soc Lond B 351:12911298 Hartings H, Berardo N, Mazzinelli GF, Valoti P, Verderio A, Motto M (2008) Assessment of genetic diversity and relationship among maize (Zea mays L.) Italian landraces by morphological and AFLP proling. Theor Appl Genet 117:831842 Hempel K, Peakall R (2003) Cross-species amplication from crop soybean Glycine max provides informative microsatellite markers for the study of inbreeding wild relatives. Genome 46:382393 IRRI/IBPGR (1980) Descriptors for rice (Oryza sativa L.), IRRI, Manila Kuittinen H, Mattila A, Savolainen O (1997) Genetic variation at marker loci and in quantitative traits in natural populations of Arabidopsis thaliana. Heredity 79:144152 Latta RG (1998) Differentiation of allelic frequencies at quantitative trait loci affecting locally adaptive traits. Am Nat 151:283292 Leinonen T, OHara RB, Cano JM, Merila J (2008) Comparative studies of quantitative trait and neutral marker divergence: a meta-analysis. J Evol Biol 21:117 Lewontin RC (1972) The apportionment of human diversity. Evol Biol 6:381398 ` Lihova J, Kucera J, Perny M, Marhold K (2007) Hybridization between two polyploidy Cardamine (Brassicaceae) species in north-western Spain: discordance between morphological and genetic variation pattern. Ann Bot 99: 10831096 Louette D, Smale M (2000) Farmers seed selection practices and traditional maize varieties in Cuzalapa, Mexico. Euphytica 113:2541 Manakkodan KKV (1949) Pancha Karma adhava shodhana cikitsa. Vidyarambham Press, Alappuzha McKay JK, Latta RG (2002) Adaptive population divergence: markers, QTL and traits. Trends Ecol Evol 17:285 291 Menon MV, Potty NN (1998) Variation in production pathway for quantitative and qualitative characteristics in medicinal rice, Njavara. Oryza 35:208210 Menon MV, Potty NN (1999) Nutritional specicity and quality properties of medicinal rice, Njavara. Oryza 36:315317 Murthy KRS (2001) Vagbattas Ashtanga Hrdayam (Text, English translation, notes, appendix, indices), 5th edn. Krishna Das Academy, Varanasi

various attributes. Such initiatives may eventually yield useful genetic variation for rice improvement.
Acknowledgments Funding in the form of a Senior Research Fellowship to Sreejayan from the Council for Scientic and Industrial Research (CSIR), Government of India is gratefully acknowledged. GT gratefully acknowledges the funding received from the International Foundation for Science (IFS), Sweden, to carry out the AFLP analysis. We thank Kreara Solutions, Thiruvananthapuram for the analysis using SAS software.

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