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scattered between the traditional Basmati and indica rice groups with CSR30, Super, Kasturi and Pusa Basmati 1 being closer to the TB, (vi) a number of SSR ...
Theme area: 1. Application of biotechnological and molecular tools in genetic and cytogenetical research, in evaluation, conservation and use of plant biodiversity and in plant breeding. Running title: Genetic Status of Basmati in Genus Oryza

Phylogenetic relationships among various rice groups in genus Oryza sativa L. as revealed by microsatellite and transposable element based marker analysis A. Kaushik1, S. Jain2, S. R. McCouch3 and R.K. Jain1 1 Department of Biotechnology and Molecular Biology, 2Bioinformatics Section, CCS Haryana Agricultural University, Hisar-125004, INDIA; 3Department of Plant Breeding, Cornell University, 240 Emerson Hall, Ithaca, NY 14853-1901, USA. Abstract Molecular markers provide novel tools for varietal identification, diversity analysis and assessing phylogenetic relationships among various rice groups in genus Oryza. A DNA fingerprint database has been developed for 50 rice genotypes representative of the traditional Basmati (TB), cross-bred Basmati, indica, japonica and wild rice groups using fifty SSR and thirty transposable element (TE) based markers. The salient features of marker data analyzed using various clustering algorithms, principal component analysis and Mantel test are as given below: (i) SSR generated higher levels of polymorphism (mean PIC value = 0.698) than TE based markers (PIC = 0.258), (ii) a total of 341 alleles were generated with an average of 6.8 allele per locus using SSR markers of which 40 were rare/unique alleles being present in only one of 50 genotypes with 17 unique alleles in the nine wild rice genotypes, (iii) analysis of SSR database clearly exhibited the formation of four distinct groups of Basmati, indica, japonica and wild rice genotypes, (iv) traditional Basmati rice varieties except Basmati 217 were genetically distinct from indica, japonica and wild rice types and invariably formed a separate cluster, (v) twelve Basmati rice varieties developed from indica x Basmati crosses/backcrosses were scattered between the traditional Basmati and indica rice groups with CSR30, Super, Kasturi and Pusa Basmati 1 being closer to the TB, (vi) a number of SSR markers were identified which can be used to differentiate within/among the various rice groups, (vii) genetic relationships assessed using TE based markers (mPing and Dasheng) were essentially the same as obtained using SSRs except that it also differentiated between the temperate and tropical japonica rice genotypes into separate clusters, and (vii) SSR and TE based marker data-set showed high levels of positive correlation (Mantel test, r = 0.655). The study demonstrate that SSRs are best for varietal identification especially for differentiating between the closely related Basmati, indica or japonica rice varieties, while TE based markers may provide vital clues about evolution/speciation in rice. Keywords: Basmati, microsatellite, Dasheng, mPing, Oryza sativa L.

*corresponding author; E-mail: [email protected]

Introduction Among the cultivated rice (Oryza sativa L.) types, Basmati rice has long been preferred in Indian subcontinent, Middle-east, Europe, United States and Australia for its aroma, and excellent cooking qualities [1]. Historically, genetic diversity and intraspecific classification in Asian rice has been studied using morpho-geographical, serological and/or hybrid fertility parameters [2-5]. The common rice O. sativa and the African rice O. glaberrima are thought to be an example of parallel evolution in crop plants [5]. O. rufipogon is considered to be the wild progenitor of Asian rice O. sativa, which shows a range of variation from perennial to annual types [6]. Genus O. sativa has two major varietal groups, indica (Hsien) and japonica (Keng) [3]. The genetic status of Basmati rice varieties vis-à-vis indica and japonica rices in O. sativa L. is still not clear. Phylogenetic analysis using microsatellite DNA markers led to the clustering of most of the Basmati rice cultivars into a separate group distinct from those of indica and japonica rice varieties [7-10]. The results in fact showed that Indian Basmati germplasm may have a long, independent and complex pattern of evolution that distinguishes it from other groups within O. sativa [8]. In surveys of diverse scented rice accessions belonging to Group V (i.e. Basmati), indica (e.g. Jasmine) and tropical japonica (e.g. Azucena), nearly all of them have been shown to possess the same aroma (betaine aldehyde dehydrogenase 2.1, BADH2.1) allele, suggesting that this allele is common by descent in scented rice varieties [10-13]. Despite its small genome, rice has also been a model organism for the study of transposable elements [TE, 14]. The transposon content of rice is at least 35% and is populated by representatives from all known transposon superfamilies [15]. TE based markers such as (Dasheng and mPing elements), provides a ubiquitous source of polymorphism and genome size variation in eukaryotes [16]. Dasheng is one of the recently amplified high-copy-number family of 800-1300 nonautonomous elements mostly concentrated in the gene-poor pericentromeric regions of the chromosomes, which have been reported to generate high levels of polymorphism within indica and japonica subspecies [17]. “Master or limited amplification model” given by Deininger et al. [18] concluded that most TE copies (of retro types) in the genome arouse from a few active master copies and that different sub-families were active at different evolutionary periods. Therefore, TE sub-families that are active after the radiation of two sub-species (or two phylogenetic groups) should generate new copies at specific loci not shared between the species. Such transposable elements may also provide useful markers for evaluating the evolutionary status of different rice types. In this paper, we report the phylogenetic status of Basmati rice with regard to indica and japonica rice types as revealed by polymorphism generated by SSR and TE (Dasheng and mPing) based markers. The SSR and TE-marker database sets so developed may broaden the list of markers being used to differentiate premium traditional Basmati from cheaper cross-bred Basmati or nonBasmati rice supplies at commercial level.

Materials and Methods A total of 50 rice genotypes/accessions representing the traditional Basmati, cross-bred Basmati, indica, japonica, Aus and wild rice (O. glaberrima, O. meridionalis, O. barthii, O. rufipogon, O. nivara and O. glumalpatula) groups, were selected for molecular marker analysis: the characteristics and origin of these genotypes are given in Table 1. Genomic DNA of all rice genotypes was isolated from bulked leaf samples (20 mg each) from five one-month-old plants grown in a net house using CTAB method [19]. DNA was dissolved in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) and checked for its quality and quantity by 1% (w/v) agarose gel electrophoresis using a standard containing 100 ng per µl genomic λ DNA. Eighty primer pairs, fifty microsatellite and thirty TE based (16 Dasheng elements specific, 9 Nipponbare specific mPing elements and 5 indica rice cv. 9311 specific mPing elements) covering all the twelve chromosomes were used. The rice microsatellite primer pairs were obtained from Research Genetics, Inc. (Huntsville, AL, USA) (Table 2). The original source, repeat motifs, primer sequences and chromosomal position of SSR markers can be found in Temnykh et al. [20] and in the RiceGenes database (http://www.gramene.org/micrsat/RMprimers.html). The sequence information for TE based

primers was provided by Dr. Susan McCouch, Department of Plant Breeding, 252 Emerson Hall, Cornell University, Ithaca, NY, USA (Table 3). PCR amplification, denaturing polyacrylamide gel electrophoresis and silver staining and band scoring was essentially carried out as described earlier by Jain et al. [9]. TE amplifications were performed essentially as described above for SSR analysis. The PCR reaction mix contained 1 X PCR buffer, 200 µM dNTPs, 0.4 µM of primer, 2 mM MgCl2, 1 unit Taq DNA polymerase and 50 ng template DNA. PCR amplification was performed with initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 2 min and final extension at 72°C for 15 min before cooling at 4°C. The PCR products were resolved by electrophoresis on 1.5% (w/v) agarose gels stained with ethidium bromide, photographed under UV light. The size (in nucleotide base pairs) of the amplified band was determined based on its migration relative to molecular weight size markers (wide range DNA ladder 50 bp to 1000 bp from Sigma chemicals Co. USA and Lambda DNA/EcoRI + HindIII double digested from Bangalore, Genei Pvt. Ltd.). The frequency of molecular marker polymorphism was calculated based on presence or absence of common bands [21]. The polymorphism information content (PIC) value was calculated according to Anderson et al. [22] based on the allelic patterns of all the genotypes analysed. The two different sets of data gathered (based on SSR and TE markers) were subjected to cluster analysis. Data analysis was done using NTSYS-PC version 2.02 [23]. Allele molecular weight data were also used to determine the genetic distance for phylogeny reconstruction based on the neighbor-joining method [24] implemented in PowerMarker ([email protected]). The Mantel test of significance [25] was also used to compare each pair of similarity matrices.

Results Microsatellite marker analysis A total of 343 alleles were detected at 50 SSR loci in 50 rice varieties (Table 2). The number of alleles ranged from two (RM321 and RM323) to sixteen (RM252), with an average of 6.86 alleles per locus. SSRs with complex mixed repeat motifs generated highest number of alleles (7.75 alleles per locus) followed by those with di-nucleotide (7.17), tri-nucleotide (6.3) and tetra-nucleotide (5.0) repeat motifs (data not shown). The overall size of amplified products ranged from 74 bp (RM248) to 389 bp (RM422). The size difference between the smallest and largest allele at a given SSR locus varied from 3 (RM323) to 150 (RM582). As many as 42 unique alleles (12.2%; 4 in TB, 7 in crossbred Basmati, 5 in indica, 8 in japonica, one in Aus, and 17 in wild rice genotypes) were observed at 26/50 SSR loci. Of the four unique alleles in TB group, two were noticed in Basmati 217. A total of 69 null alleles were observed at 30/50 SSR loci in 33/50 genotypes. O. meridionalis and O. rufipogon-81885 showed the largest proportion of null alleles of 7 and 5, respectively. Multiple alleles were observed in 1-10 of the 40/50 genotypes at 29/50 SSR loci. Relatively higher number of multiple alleles were observed in cross-bred Basmati (3.46 multiple alleles per genotype) in comparison to indica (2.5), japonica (1.0), TB (2.0) and wild rice (1.0) genotypes. Pusa Sugandha 4 and CSR30 had multiple alleles at eight and five loci, respectively. The frequency of most common allele ranged from 10% (RM252) to 66% (RM323). On average 38.5% of the rice genotypes shared a common allele at any given locus. PIC values, which are indicative of genetic diversity, ranged from 0.348 to 0.922, with an overall average of 0.698 per locus. PIC values showed a positive correlation of 0.54 with number of alleles at a SSR locus (Significant at 0.01). As many as 55 alleles (at 39 loci) were present in both TB and indica rice accessions but were absent in japonica rice varieties. Similarly, at 11 loci, 12 alleles were common in TB and japonica rice accessions; but these were missing in indica rice varieties. Notably, Basmati 217 had five alleles and Karnal Local had one allele which were present in two or more indica rice accessions but were absent in other TB and japonica rice varieties. Such alleles were not noticed in Ranbir Basmati. Azucena (tropical scented japonica rice variety) and TB rice varieties shared 7 alleles at seven loci; these alleles were absent in other japonica rice varieties. At RM547 (chromosome 8), two alleles were noticed both in TB and japonica rice accessions; but were absent in indica rices.

Jaccard similarity coefficient values Among the TB rice varieties, Basmati 370/HBC19 and Basmati 370/Type III were quite close with similarity coefficient values of 0.54 and 0.50, respectively. However, Basmati 370, was relatively diverse from the rest of the three TB rice varieties, Basmati 217 (0.177), Karnal local (0.272) and Ranbir Basmati (0.423). Cross-bred Basmati rice variety, CSR30 and TB rice cultivar HBC19, showed a high similarity coefficient of 0.60. Group wise mean similarity coefficient values were also calculated for the various rice groups including TB, indica, japonica, O. glaberrima, O. rufipogon, O. nivara, O. glumalpatula, O. barthii and O. meridionalis (Table 3). TB group had greater similarity with indica (0.171) compared to japonica (0.112) rice groups. Among the japonica rice accessions, Azucena was relatively closer (0.136) to TB group. Japonica and indica groups had a mean similarity of 0.086. Among the six wild rice groups, TB had maximum similarity with O. glumalpatula (0.127) followed by O. glaberrima (0.121), O. barthii (0.116), O. rufipogon (0.092), O. nivara (0.084) and O. meridionalis (0.060). These similarity values were higher compared to corresponding values involving indica or japonica rice group instead of TB. Genetic relationships within and among various rice groups Genetic relationships as determined by two-dimensional PCA scaling and PowerMarker analysis of various rice genotypes are shown in Fig. 1. Two-dimensional PCA scaling clearly shows high-level of differentiation between Basmati, japonica, indica and wild rice varieties and placed them in four distinct clusters (Fig. 1a). TB rice varieties except Basmati 217 and Karnal Local and some of the cross-bred Basmati rice varieties (CSR30 and Super) were clustered together in one group. Japonica rice varieties except Azucena formed another distinct group. Indica rice varieties except 9311? with one TB (Basmati 217) and some of the cross-bred Basmati varieties (Pusa Sugandha 2, Pusa Sugandha 3, Sabarmati, Improved Sabarmati and Mahi Sugandha) formed a separate cluster. The other cross-bred Basmati rice varieties were interspersed between indica and TB groups with Basmati 385, Kasturi and Pusa Basmati 1 being closer to TB group. All the wild rice genotypes formed a separate group placed between indica and japonica rice groups. Aromatic japonica rice variety, Azucena, was placed closer to the wild rice group. The PCA analysis also showed a close relationship between two salt-tolerant „Aus‟ genotypes, KR1-24 and BR4-10, which were closer to the wild rice group. The radial phylogenetic tree based on PowerMarker analysis depicted a similar picture of genetic relationships as observed by PCA analysis but with some differences (Fig. 1b). Most of the Basmati rice varieties including TB (except Basmati 217) and cross-bred Basmati rice varieties (CSR30, Super, Kasturi, Basmati 385, Pusa Sugandha 2, Pusa Sugandha 3, HKR228, Pusa Basmati 1 and Pusa Sugandha 4) formed one major group. Basmati rice varieties, Basmati 217, Sabarmati, Improved Sabarmati and Mahi Sugandha, clustered in indica rice group. The wild rice genotypes showed variable clustering with O. nivara 1, O. nivara 2 and O. rufipogon 1 in one group and O. glaberrima, O. rufipogon 2 and O. nivara 3 in another group. All of the japonica rice genotypes formed a separate cluster which merged with the two clusters of wild rice genotypes to form a big cluster. TE based marker analysis Dasheng elements were present in high frequencies in japonica rice group (54.2%) followed by crossbred Basmati (24%) and traditional Basmati (21%) rice groups. In wild rice genotypes, frequencies of Dasheng elements were minimum (13.9%). In these genotypes null alleles were detected at higher frequencies (44.4%). In traditional Basmati and indica rice groups, Nipponbare mPing elements were present at very low frequencies ranging from 1.9% to 3.0%. The frequencies of 9311 mPing elements were maximum in indica rice (44%) followed by cross-bred Basmati (21.6%) and traditional Basmati (12.7%). PIC values, which is indicative of genetic diversity varied, from one TE locus to another and ranged from 0.110 to 0.374, with an overall average of 0.258 per locus (data not shown).

Jaccard similarity coefficient values Highest similarity coefficient of 0.9655 was observed between traditional Basmati rice genotypes (Basmati 385 and Basmati 370). Azucena rice variety showed a similarity coefficient of 0.8125 and 0.7143 with Basmati 370 and Super Basmati, respectively. Cross-bred Basmati rice genotypes Kasturi/Pusa Basmati 1 and HKR 93-401/Mahisugandha showed a similarity coefficient of 0.7857. Lowest similarity coefficient of 0.1026 was observed between TNG67/Improved Sabarmati, followed by Nipponbare/Karnal Local (0.1346). Group wise mean similarity values were calculated for the various rice groups (Traditional Basmati, indica, japonica, O. glaberrima, O. rufipogon, O. nivara, O glumalpatula, O. barthii, and O. meridionalis) (Table 3) using the TE similarity indices. Similarity values of 0.669, 0.420 and 0.545 were observed for traditional Basmati/traditional Basmati, japonica/japonica and indica/indica, respectively. Traditional Basmati had grater mean similarity with indica (0.514) compared to japonica (0.400) rice groups. Among the wild rice types, traditional Basmati had grater similarity with O. glumalpatula (0.348) followed by O. meridionalis (0.327) and O. nivara (0.038); these values were higher compared to corresponding mean similarity values involving indica or japonica rice groups in place of traditional Basmati group. Genetic relationships within and among various rice groups Genetic relationship as determined by NTSYS-PC two-dimensional PCA scaling of various rice genotypes is shown in Fig. 2a. The three temperate japonica rice and 9311? varieties formed a distinct cluster at left upper corner of the PCA. All the wild rice genotypes formed a distinct cluster at the left bottom corner and all the other rice genotypes were scattered in the right half. Traditional Basmati except Basmati 217 and Karnal Local and cross-bred Basmati were clustered in the right upper region. The two Aus rice genotypes BR 4-10 and KR 1-24 are not that close as identified by SSR analysis. The TE fingerprint database was used to generate radial and phylogenetic tree based on PowerMarker analysis (Fig. 2b). The analysis showed a clear distinct group comprising three temperate japonica rice varieties i.e. Nipponbare, TNG67, Taipei 309 and 9311?. Most of the wild rice genotypes including KR 1-24 formed a separate cluster. The traditional Basmati rice varieties (except Basmati 217 and Karnal Local) and some of the cross-bred Basmati rice varieties formed a small cluster. Most of the indica rice varieties, some of the cross-bred Basmati and the remaining two traditional Basmati (Basmati 217 and Karnal Local) formed a separate cluster. The tropical japonica rice varieties, NPTII and NPTIII, were quite divergent from the temperate japonica group. The third tropical aromatic japonica rice variety, Azucena, grouped with traditional Basmati group. Correlation between the similarity values measured using two marker system Mantel test of significance [25] was used to compare each pair of similarity matrices produced using the two marker systems and level of correlation between the two. The values of the Mantel test correlation showed a positive correlation between the two marker types. The correlation coefficient (r) was 0.655 between SSR and TE based markers.

Discussion Microsatellite marker analysis In the present set of fifty rice genotypes, SSR marker analysis revealed a high level of polymorphism with an average of 6.86 alleles per locus. These results were comparable to 7.4 and 7.9 alleles per locus reported by Olufowote et al. [26] and Jain et al. [8], respectively but are higher compared to 2.0-5.5 alleles per locus for various classes of microsatellites reported by Cho et al. [27]. High number of alleles may be attributed to the constitution of genotypes targeted in this study, which

comprises of several genetically diverse rice groups (indica, japonica, Aus and Basmati) and wild species of rice. Numerous unique (rare) and null alleles observed in this collection of TB and some of the crossbred Basmati rice genotypes that were absent in indica, japonica and wild rice genotypes which indicates that Basmati rices represent a unique source of genetic diversity [7, 8]. Occurrence of such rare or unique alleles in rice varieties may have resulted from unequal crossing-over, translocation or other types of mutations [8]. While the length variation at microsatellite loci generally results from a change in the number of repeats, null alleles are the consequence of polymorphism in the primer binding site(s) [27]. The prevalence of high number of null alleles in O. meridionalis and O. rufipogon strongly suggests that sequence divergence in flanking regions may have played a significant role in interspecific SSR variation [20]. Allelic mixtures were detected in 58% (29/50) of the rice genotypes at one or more loci with relatively high frequency in cross-bred Basmati rice varieties. The detection of multiple alleles at high frequency is probably due to heterogeneity rather than the genetic heterozygosity. Rice is predominantly self-fertilized and inbred crop, but genetic heterogeneity has been widely reported in rice accessions and could be due to out-crossing or residual heterozygosity in case of cross-bred varieties, remnant heterozygosity in some varieties, inadvertent seed mixing or when a variety consists of a mixture of pure lines [8, 28]. SSR dataset also showed that two of the TB varieties (Basmati 217 and Karnal Local) were quite divergent from the rest of TB and closer to indica group. Interestingly, Basmati 217 had two unique alleles. Nagaraju et al. [7] reported low level of genetic similarity between Basmati 217 and other TB rice varieties using both SSR and ISSR markers. Basmati 217 and Karnal Local may have resulted from Basmati/indica rice crosses (natural out-crossing or manual) and probably represents separate lineages. This point is further supported by the fact that these varieties shares relatively large number of alleles with indica varieties. Among the thirteen cross-bred (indica × Basmati) Basmati rice varieties, there were two groups: the first group comprising of CSR30, Super, Kasturi, Pusa Basmati 1 and Basmati 385 which invariably clustered with TB group and second group with Improved Sabarmati, Mahi Sugandha, Pusa Sugandha 2, Pusa Sugandha 3, Pusa Sugandha 4, Sabarmati, HKR228 and 93-401 was closer to indica rice group. The variable degree of diversity between TB and cross-bred Basmati rice varieties may be due to the complex parentage of cross-bred varieties involving several recombination events and is the indicative of different levels of genomic fractions from their respective indica rice parent(s) [7, 29, 30]. The present study shows that TB rice landraces/varieties (Basmati 370, HBC19, Type III, Karnal Local and Ranbir Basmati) are genetically distinct from indica (HKR120, CSR10, IR24, IR36 and IR64) and japonica (Nipponbare, Azucena, NPTII, NPTIII, TNG67 and Taipei 309) rice varieties. Higher levels of genetic diversity between Basmati and non-Basmati (indica, japonica and wild genotypes) support the hypothesis that Basmati rice may have a long history of independent evolution and diverged from non-Basmati rices a long time ago through conscious selection and patronage for this cluster of germplasm [8, 10]. The results also demonstrate that Basmati rice varieties are relatively closer to geographically associated indicas but genetic exchange with japonica rice accessions may have also contributed towards the evolution of Basmati rice. It is noteworthy that TB varieties shared 55/343 alleles at 39 loci with indica rice gene pool, which were absent in japonica rice varieties. In comparison, only 12 such alleles (missing in indica gene pool) were shared between TB and japonica rice genotypes. This observation is contrary to earlier isozymatic [31] and microsatellite marker [8, 9, 32] analyses reports depicting a close evolutionary relationship between Basmati and japonica rice sub-populations compared to that between Basmati and indica rices. These could be due to the differences in the constitution of rice genotypes and microsatellite loci targeted. Kovach et al. [10] reported the presence of same mutant alleles at BADH2 locus in scented rice accessions of Basmati, indica and tropical japonica rice groups. In this study also, the tropical scented japonica rice variety, Azucena, was relatively closer to TB and shared seven alleles with TB rice varieties, which were missing in other japonica rice varieties. More elaborate research involving large number of geographically associated indica, japonica and Basmati rice accessions/landraces will be required to solve the paradox of evolution of Basmati rices in Indian sub-continent.

TE based marker analysis The rice genome is populated by representatives from all known transposon super-families, including elements that cannot be easily classified into either class I or II [33]. Availability of several wellcharacterized wild relatives provides the material necessary to analyze the impact of TEs on genome evolution and speciation. The results obtained in this study were in agreement with Jiang et al. [14] as only temperate japonicas (Nipponbare, TNG67 and Taipei 309) and cv. 9311? rice genotypes showed the presence of most of the mPing elements. These elements were largely missing in indica and Basmati rice genotypes but were present in wild rice genotypes (O. glaberrima, O. nivara, O. barthii, O. rufipogon, O. glumalpatula and O. meridionalis) at variable frequencies. Kikuchi et al. [34] studied the mobilization of mPing and found that japonica has pattern with many bands, indica and O. rufipogon have a pattern consistent with fewer mPing elements. These results indicate that mPing was amplified in the japonica lineage after divergence from O. rufipogon. None of the indica and O. rufipogon cultivars had a ping sequence. This might account for the lower number of mPing elements in these species than in japonica rice. Presence of mPing elements in some of wild rice genotypes though at low frequency indicates that these elements may have evolved long back but had greater amplification during speciation of japonica lineages, temperate and tropical. Dasheng elements have been shown to widely spread in Oryza genus which supports the hypothesis that this family originated earlier than or around the time of speciation, approximately 5 to 10 million years ago [14]. However, their abundance in japonica rice genotypes is indicative of its recent activity and amplification after speciation of sub-species japonica. The values of Mantel test correlation showed a positive correlation (r = 0.655) between the two marker types which are known to target different sequences in the genomic fractions. SSR markers more or less equally distributed on the entire genome amplify short repeat sequences whereas TE based markers amplifies the sequences inserted in the genome during the course of evolution. Marker-based differences in the genetic relationships between rice genotypes do emphasize the need of using a combination of different marker systems for a comprehensive genetic analysis.

Acknowledgements This research was supported by University Grants Commission, New Delhi (F.32-542/2006) and Council of Scientific and Industrial Research, New Delhi (JRF to A. Kaushik). Thanks are due to Dr. Kuldeep Singh (Punjab Agri. University, India) for providing seed of wild rice genotypes and Dr. R.K. Behl (CCS Haryana Agri. University, India) for useful discussion.

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Table 1. A brief description of rice varieties/accessions used for molecular marker analysis _____________________________________________________________________________________________________ Rice types Varieties Abbreviation Origin/ description ___________________________________________________________________________________________________________________ Traditional Basmati 217 Bas217 Traditional Basmati rice variety Basmati Basmati 370 Bas370 Traditional Basmati rice variety Karnal Local KLocal Traditional Basmati rice variety HBC19 HBC19 Pure line selection from local Taraori Basmati Ranbir Basmati RanBas Pure line selection from Basmati-370-90-95 Dehradun Basmati Type III TypeIII A selection from Dehradun Basmati rice Cross-bred Basmati 385 Bas385 Obtained from crosses and backcrosses of T(N)1/Bas370 Basmati Traditional Basmati rice variety CSR30 CSR30 Salt tolerant, obtained by crosses and backcrosses of BR410/Pakistan Basmati Improved Sabarmati ImpSabar Released in 1970 by IARI, N. Delhi, obtained from crosses and backcrosses of T(N)-161/Basmati 370 HKR228 HKR228 Released in 1991, obtained from crosses and backcrosses of Sona/Basmati 370 Kasturi Kasturi Released in 1989 by DRR, obtained from crosses and backcrosses of Basmati 370/CR-88-17-1-5 Mahisugandha Mahi Obtained from crosses and backcrosses of BK-79 x Basmati 370 Pusa Basmati 1 PB1 Released in 1989 by IARI, N. Delhi, obtained from crosses and backcrosses of Pusa 150/Karnal local Pusa Sugandha 2 PS2 Cross-bred Basmati Pusa Sugandha 3 PS3 Cross-bred Basmati Pusa Sugandha 4 PS4 Cross-bred Basmati (Pusa Basmati 1121) Sabarmati Sabar Released in 1970 by IARI, N. Delhi, obtained from crosses and backcrosses of T(N)1/Basmati 370 Super Basmati Super Released in 1996 by IRRI, obtained from crosses and backcrosses of IR-661/Basmati 370 HKR93-401 93-401 Obtained from crosses and backcrosses of Basmati/indica varieties Indica Punjab Rice 118 PR118 Indica rice variety Punjab Rice 106 PR106 Obtained from (IR-8 x Peta 5) x Bella Patna IR36 IR36 Obtained from IR-8 x Tadukan x TKM-62 x T(N)1 x IR-243 x O. nivara-4 x IR-8 x PTB-21 and PTB-18 CSR1 CSR1 Indica rice variety CSR10 CSR10 Salt tolerant cross-bred indica , developed from crosses and backcrosses of M-40-431-24-114 x Jaya CSR21 CSR21 Indica rice variety HKR120 HKR120 Obtained from PTB-33/4 x IR-3403-267-1 IR24 IR24 Indica rice variety IR72 IR72 Indica rice variety

Japonica

Aus Wild rice

MI48 Pokkali IR64 9311? Sharbati Azucena

MI48 Pokkali IR64 9311? Sharbati Azu

Indica rice variety Salt tolerant indica from South-India (Kerela) Obtained from IR-5857-33-2-1 x IR-2061-465-1-5-5 Indica, genome sequencing accomplished, authenticity not clear A local selection from Uttar Pradesh

Nipponbare New Plant Type II IR68552-100-1-2-2 New Plant Type III Taipei 309 TNG67 BR4-10 Kala Ratta 1-24 O. glaberrima -102206 O. barthii- 100117 O. rufipogon- 81885 O. rufipogon -103404 O. nivara -81847 O. nivara- 81859 O. nivara -104688 O. glumalpatula -104387 O. meridionalis -101146

Nippon NPTII

Tropical japonica rice variety Temperate japonica rice variety Tropical japonica rice accession developed at IRRI

NPTIII T309 TNG67 BR4-10 KR1-24 Oglab Obart Orufi1 Orufi2 Oniv1 Oniv2 Oniv3 Oglumal Omerdio

Tropical japonica rice accession developed at IRRI Temperate japonica rice variety Temperate japonica rice variety Salt-tolerant landrace Salt-tolerant landrace Liberia, Africa Guinea India Bangladesh India India India Brazil, South America Australia

________________________________________________________________________________________________________

Table 2. Data on number of total and unique alleles, genotypes with multiple and null alleles, allele size, highest frequency allele and polymorphic information content (PIC) obtained using 50 SSR markers in 50 rice genotypes ___________________________________________________________________________________________ Marker

Chr. location

Total no. of Alleles

No. of unique Alleles

Genotypes with null alleles

Genotypes Allele Size Highest PIC with _______________ frequency value multiple Range Difference allele (%) alleles (bp) (bp) _____________________________________________________________________________________________________ RM213 2 7 2 0 0 127-163 36 58 0.598 RM 215 9 9 1 0 1 136-158 22 28 0.788 RM 220 1 7 0 1 2 104-124 20 28 0.769 RM 222 10 9 1 0 1 199-221 22 22 0.829 RM 224 11 8 0 1 5 124-172 48 30 0.825 RM 229 11 9 3 2 1 106-138 32 40 0.715 RM 234 7 8 2 2 3 126-164 38 38 0.681 RM 242 9 5 1 3 0 193-225 32 38 0.649 RM 248 7 10 1 0 8 74-150 76 32 0.818 RM 252 4 16 1 8 3 194-246 52 10 0.922 RM 255 4 8 4 0 0 127-151 24 60 0.544 RM 259 1 10 1 3 0 148-176 28 24 0.841 RM 282 3 3 0 3 0 127-135 8 38 0.559 RM 286 11 8 1 0 0 104-130 26 44 0.715 RM 316 9 6 2 2 0 192-212 20 44 0.639 RM 321 9 2 0 0 0 200-206 6 50 0.375 RM 323 1 2 0 0 0 241-244 3 66 0.348 RM 400 6 9 2 2 7 180-321 141 36 0.765 RM 410 9 3 0 0 1 169-185 16 44 0.562 RM 422 3 5 0 2 1 363-389 26 40 0.665 RM 423 2 5 0 3 2 250-297 47 28 0.720 RM 424 2 5 0 4 0 239-281 42 40 0.671 RM 426 3 11 1 2 5 148-184 36 32 0.814 RM 432 7 4 0 0 0 167-187 20 42 0.590 RM 439 6 6 0 3 0 230-278 48 52 0.584 RM 440 5 8 0 2 0 149-220 71 26 0.812 RM 444 9 6 0 0 5 162-226 64 56 0.634 RM 450 2 7 1 0 1 127-143 16 36 0.749 RM 472 1 5 1 1 0 206-270 64 32 0.698 RM 475 2 8 2 0 2 178-235 57 34 0.746 RM 488 1 8 0 3 10 165-195 30 32 0.828 RM 490 1 5 0 0 7 93-107 14 30 0.744 RM 500 7 3 0 1 2 253-259 6 50 0.558 RM 511 12 4 0 1 0 118-133 15 38 0.657 RM 518 4 8 1 0 0 151-173 22 40 0.744 RM 525 2 7 1 0 5 107-140 33 42 0.706 RM 526 2 5 0 1 3 220-264 44 46 0.660 RM 527 6 4 0 5 0 214-239 25 44 0.568 RM 528 6 5 0 2 5 232-278 46 46 0.660 RM 536 11 6 1 3 1 215-243 28 42 0.652 RM 541 6 10 1 1 3 146-210 64 30 0.821 RM 547 8 13 4 2 4 199-277 78 28 0.842 RM 551 4 5 1 1 0 186-220 34 38 0.677 RM 562 1 7 0 0 9 219-267 48 30 0.800 RM 567 4 4 0 1 7 239-261 22 52 0.639 RM 575 1 7 1 0 2 187-207 20 46 0.709 RM 580 1 11 3 2 0 179-230 51 40 0.761 RM 582 1 9 2 0 0 181-331 150 48 0.686 RM 585 6 7 0 0 0 170-270 100 28 0.797 RM 594 1 6 0 2 3 288-312 24 24 0.780 Total 343 42 69 109 74-389 3-150 Mean 6.86 0.84 1.38 2.18 38.4 ______________________________________________________________________________________________________

Table 3. A brief description of Transposon element based markers used in this study Sr. No.

Primer number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

DashengD9 DashengD7 DashengD11 Dasheng E3 Dasheng C11 Dasheng A3 Dasheng G5 Dasheng A7 Dasheng C7 Dasheng C9 Dasheng E11 Dasheng G7 Dasheng F1 Dasheng C5 Dasheng G11 Dasheng F3 mPing8-2 mPing7-1 mPing6-1 mPing12-1 mPing3-3 mPing3-4 mPing3-2 mPing3-1 mPingU1 mPing9311-1 mPing9311-4 mPing9311-3 mPing9311-6 mPing9311-5

Forward Sequence ACgTACTCTCgCTTggATgg ggTggTgTCgTgTgTTACTgA ggggTCCTCACTCgTCAATA ATTgCAATTCgCACACAAAA ACACACACCCCCAAATTgTT ATATTTgAggggCgCTTTTT AAgTTggACCggCAAAgAAT AAATTgCCTgTgCgTTACAA TggACgCTgTCAAgTTCAgA TgCgggATgTgTACAgTgAC ACACAAATgCCACACACAgAg gTTggCTTTggTCAgAgCTT TCgAgCATTgAgTgTTgAgC ggTggTgTCgTgTgTTACTgA AACAAAAggAgAgCCCACAA ACCTTACCCCTCCACAAAgg AAAAAgTgTCggAAgCTCTg CCTCgATACTgTTgCCTgTT ACAATCTATggCggAAACAg ggCAAgTggAAgATCATACg CCACgAgTcAAgCTAAAggT gCTgAAgTTTggACACAACC AAAgTCAgAAgCgAATgTgg CTTCAgCAgCCTATgTTAgTCg CAggAgTgTTgggTTATTgg TATACCCACTTTATCCCATTgC gCCTTgAAACATgTCCACAC CgAgAAgTCCgACACAAATg AgTACTTCATCTCCAgggACAg ATTCTCgCCTCTTggTTCTC

Reverse Sequence CgATCCATgCTCATCATCAC ggCTATggTCCACATCgACT TAgCCCTTTCATgCAACCAT CgAggAACATCCCgTCTAAT TgTgAgTATgAgAAggggTgA TgCTACgATCACCAgTCCAA TgAAAgAtgATgggCATgTg TCAACATAggCTggACCAAA AgATTgTAgCAgCgCCCTAA AggCTTCCgTggTgACTTC ACTCCgTCCCAAAAAgAATC gTCCggCCTTgTgTTgTAgT gTgTgAgCATTgTggCAgAg ggCTATggTCCACATCgACT gTACgTACgTCgCATgTggT AAgCACCTAgAgggTgACCA ATCgCTgTACTCCACTCTgC TAgAgCTggTAgTgggCTgT gCCCTCCCATAggATTAgAA CCTCAAgAACAgTgCCAACT CgTCTCTCggTgACACAgTA CCTTCgCTCTTTggACATAA CATTTTgCCTTTCTgCTgAT gCAggCAgATgTTgATggTA gAATTATTTggggACgACCT ACgATTTCAgTgACCTCATCA AgAggCAAgAgCTACTCCAAAC ggTATCCCTgTAgCTAgATgTgC gCgTCAgCAAgAAggTTAAg TTACTTCAgCTgTACCCgTAgC

Table 4. Average similarity (Jaccard Similarity Coefficient) between various rice types based on SSR and TE based marker analysis _________________________________________________________________________ Rice types SSR-J TE-J Rice types SSR-J TE-J _________________________________________________________________________ TB/TB 0.317 0.669 O. glaberrima/Japonica 0.079 0.175 Japonica/Japonica 0.301 0.420 O. barthii/Japonica 0.065 0.230 Indica/Indica 0.229 0.545 O. rufipogon/Japonica 0.082 0.221 Japonica/TB 0.112 0.400 O. nivara/Japonica 0.100 0.261 Japonica/Indica 0.086 0.269 O. glumalpatula/Japonica 0.094 0.269 Indica/TB 0.171 0.514 O. meridionalis/Japonica 0.084 0.173 O. glaberrima/TB 0.121 0.215 O. glaberrima/Indica 0.086 0.227 O. barthii/TB 0.116 0.214 O. barthii/Indica 0.091 0.274 O. rufipogon/TB 0.092 0.284 O. rufipogon/Indica 0.084 0.235 O. nivara/TB 0.084 0.308 O. nivara/Indica 0.086 0.264 O. glumalpatula/TB 0.127 0.348 O. glumalpatula/Indica 0.081 0.304 O. meridionalis/TB 0.060 0.327 O. meridionalis/Indica 0.088 0.277 _________________________________________________________________________

0.60

HBC19

CSR30 Bas370

Basmati

TypeIII

a

Super 0.39

PB1

Kasturi Bas385

RanBas

KLocal Dim-2 0.17 PS4

Indica -0.04

PR118 PS2 PS3

HKR228

Wild species Oglab

CSR1 ImpSabar Bas217 Mahi PR106 CSR21 IR36 IR72 CSR10 Sabar HKR120 MI48 IR64 Pokkali Sharbati

Obart

IR24

-0.25 -0.33

Japonica

Azu

93-401

-0.09

KR1-24 BR4-10

Oglumal

Oniv2 Orufi2 Orufi1 Oniv3 Oniv1 Omerdio

0.15

NPTII

T309

9311? NPTIII

0.39

TNG67

Nippon

0.63

Dim-1

b

Fig. 1. Two-dimensional scaling by Principal Component Analysis (a) and PowerMarker phylogeny reconstruction tree analysis (b) of 50 rice genotypes using allelic diversity data for 50 SSR markers

Fig. 2. Two-dimensional scaling by Principal Component Analysis (a) and PowerMarker phylogeny reconstruction tree analysis (b) of 50 rice genotypes based on genetic distance matrix data obtained using 30 TE primers.