The genus Oryza (Poaceae) consisting of only two cultivated species, (Oryza glaberrima Steud. and Oryza sativa L.) and 22 wild species. Cultivated species O. glaberrima is only available in Western Africa, but O. sativa is distributed globally (Sanchez et al. 2013) due to its wide adaptability to different habitats and growing conditions. The species O. sativa has two subspecies such as Oryza sativa ssp. japonica and Oryza sativa ssp. indica. The grouping was based on physiological and morphological features (Oka, 1998). Asian cultivated rice (Oryza sativa L.) is one of the most important crops in the world. Rice not only serves as a primary food source  (supplying 20% to 40% of the world’s total caloric intake) for more than half of the world’s population (Khush 1997), but also provides an excellent model system for studying a wide range of biological questions (Zhang et al. 2008). More than 1 20 000 distinct rice varieties recognized worldwide (Khush 1997; Sang and Ge 2007 and Vaughan et al. 2008). Although two subspecies indica and japonica have been widely accepted without dispute, the genetic subdivision or substructure of the crop has been inconsistent. The six varietal groups (I to VI) has been proposed by Glaszmann (1987) based on enzyme polymorphism. Garris et al (2005) detected five distinct groups based on SSR markers and referred to them as indica, aus, aromatic, temperate japonica and tropical japonica. Ebana et al (2010) identified seven rice groups based on SNP data. The 3,000 Rice Genomes project (2014) classified the rice varieties into five groups (indica, japonica, aus, basmati/sandri and intermediate group). Main target of this 3000 rice genome project is that rice scientists will soon be able to identify many key genes which govern traits (quality traits and stress resistance traits). Thus, the genetic subdivision and classification of rice germplasm remain unclear, with three to seven groups indentified by different studies. Despite its essential role in world agriculture and food security, the history of cultivated rice’s domestication from its progenitors (ancestors) and evolutionary pathways remains unclear. Phylogeographic studies indicate that India and Indochina may represent the center of diversity of one of the 22 wild rice species O. rufipogon Griff. This wild rice species, O. rufipogon, grows across this entire range. These two species, O. sativa L. and O. rufipogon Griff. share the similar vegetative growth and other eco-geographical parameters including the same AA genome and is widely recognized as a progenitor of cultivated rice (O. sativa L) (Khush 1997; Londo et al. 2006). O. rufipogon is a perennial species and cultivated rice is an annual species, it has been proposed that the annually occurring form of O. rufipogon, Oryza nivara (Sharma et Shastry 1965), may represent the most recent ancestor of O. sativa. During the course of domestication from wild rice to cultivated rice, many genes were lost through natural and human selection, leading to the lower genetic diversity of the cultivated rice (Zhu et al. 2007). These wild species are the reservoirs of many useful genes/QTLs particularly for resistance to major biotic and abiotic stresses (Xiao et al., 1996; Sanchez et al., 2013). Population of Oryza rufipogon Griff. (Poaceae) have been gradually reduced due to ecological stresses although it has proven to be a valuable reservoir of genes for rice genetic improvement as a progenitor of cultivated rice (O. sativa L.). It needs immediate attention with high priority so that we could not loss this agriculturally important Oryza genepool and identified not only based on morphological traits but based on DNA barcoding. Biological specimens were identified using morphological features and most cases an experienced professional taxonomist is needed. DNA barcoding can serve a dual purpose as a new tool in the taxonomists toolbox supplementing their knowledge as well as being an innovative device for non-experts who need to make a quick identification of biological species.

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. A 'barcode' gene that can be used to distinguish between the majorities of plant species on Earth has been identified. DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. DNA barcoding, a concept that has recently become popular, is characterized by using one or a few DNA fragments to identify different species (Kress et al. 2005). The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was selected to be a DNA barcode for animal species (Hebert et al. 2003). The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a “barcode” for land plants. Seven leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbl spacer, and trnH-psbAspacer) may be used in DNA barcoding (Hollingsworth et al. 2009; Lahaye et al. 2008; Jing et al. 2011). A region of the chloroplast gene rbcL- RuBisCo large subunit – is used for barcoding plants. The most abundant protein on earth, RuBisCo catalyzes the first step of carbon fixation. A region of the mitochondrial gene CO1 (cytochrom c oxidase subunit) is used for barcoding animals. Cytochrome c oxidase is involved in the electron transport phase of respiration. Thus, the genes used for barcoding are involved in the key reactions of life: storing energy in carbohydrates and releasing it to form ATP. CO1 in fungi is difficult to amplify, insufficiently variable, and some fungal groups lack mitochondria. Instead, the nuclear internal transcribed spacer (ITS), a variable region that surrounds the 5.8s ribosomal RNA gene, is targeted. Like organelle genes, there are many copies of ITS per genome, and the variability in fungi allows for their identification. In plant systematics, matK has recently emerged as an invaluable gene because of its high phylogenetic signal compared with other genes used in this field (Müller et al., 2006). The 1500 bp matK gene is nested in the group II intron between the 5′ and 3′ exons of trnK in the large single copy region of the chloroplast genome of most green plants. Phylogenetic analysis of a data set composed of matK, rbcL, and trnT-F sequences from basal angiosperms demonstrated that matK contributes more parsimony informative characters and significantly more phylogenetic structure on average per parsimony-informative site than the highly conserved chloroplast gene rbcL (Müller et al., 2006). Maturases are enzymes that catalyze nonautocatalytic intron removal from premature RNAs. Maturases generally contain three domains: a reverse-transcriptase (RT) domain, domain X (the proposed maturase functional domain), and a zinc-finger-like domain. By generating and comparing two nuclear gene (Adh1 and Adh2) trees and a chloroplast gene (matK) tree of all rice species, phylogenetic relationships among the rice genomes were inferred. Genome types of the maternal parents of allotetraploid species were determined based on the matK gene tree. An additional genome type, HHKK, was recognized for Oryza schlechteri and Porteresia coarctata, suggesting that P. coarctata is an Oryza species. The AA genome lineage, which contains cultivated rice, is a recently diverged and rapidly radiated lineage within the rice genus (Song et al. 1999).

The sequencing results are then used to search a DNA database. A close match quickly identifies a species that is already represented in the database. However, some barcodes will be entirely new, and identification may rely on placing the unknown species in a phylogenetic tree with near relatives. Novel DNA barcodes can be submitted to GenBank (www.ncbi.nlm.nih.gov). The dehydration-responsive element binding (DREB) factor was identified as a cis-acting promoter element in regulating gene expression in response to drought, high-salt, and cold stresses in plants including rice (Dubouzet et al. 2003) and encode transcription activators. The fragrance gene encodes non-function betain aldehyde dehydrogenase 2 (BAD2) responsible for aroma production in rice studied by Bradbury et al. (2005a) and reported that a stretch of mutations (a SNP haplotype) in the exon 7 of the fragrance gene is responsible for the aroma. During the transcription of the BAD2 gene, this haplotype would encode for a premature stop signal resulting in the production of a non-functional truncated BAD2 enzyme (Bradbury et al. 2005a; 2005b). This truncated BAD2 enzyme is deficient in three conserved protein motifs needed for its substrate binding activity and subsequently results in the accumulation of 2-acetyl-1-pyrroline (Bradbury et al. 2005a; 2005b). Thus, detection of the haplotype alleles in the BAD2 gene would enable discrimination between aromatic and non-aromatic rice and thus assist marker-assisted introgression of the aromatic trait into local rice varieties. Many rice scientists have been studied the genetic structure within and among the O. rufipogon based on morphology, ecology and DNA markers (Dong et al. 2010).

After exhaustive reviewing the literature and considering the importance of origin of problem of wild rice genetic resources, the present investigation was made first time to characterize the germplasm of Oryza rufipogon Griff. exists in the campus of North Bengal University (NBU) based on DNA barcoding including genetic variation based on BAD2 and DREB gene supported by morphological parameters to provide much needed data to maintain and implement in situ conservation strategies for this precious germplasm for crop improvement.

Material and Methods
Plant material
Asian common wild rice, Oryza rufipogon Griff. is naturally growing in the campus of the University of North Bengal (NBU) due to existence of desired ecological habitat and environment on the little river side of Magurmari, which is located at Latitude of 26º 84́ North and Longitude of 88º 44́ East, near Siliguri, Dist- Darjeeling, WB, India. The area is water logged and swampy covering small ponds and ditches.

Morphological traits as supportive data
Taxonomic tool was used to characterize the plant material (Oryza rufipogon Griff.) based on morphological features following standard protocol for wild rice characterization of IBV: ww.cgiar.org/cgiar-consortium/research-centers/bioversity-international/. Twenty-six morphological features were studied in this investigation.

DNA extraction and PCR amplification for BAD2 gene
Genomic DNA isolation for PCR amplification Genomic DNA was extracted from leaf material of 10 days old seedling of wild rice O. rufipogon of NBU campus using a Qiagen DNeasy® 96 Plant Kit (Qiagen GMbH, Germany). Purified genomic DNA was dissolved in 1xTE buffer at a concentration of 2.5 ng/μl and stored at -20º C until further use. Oligonucleotide primers were synthesised by Proligo Australia Ptv Ltd.

Primer for BAD2 gene: Two pairs of primers were used in fragrant gene amplification. One set was used for positive control as external primer to produce amplified product of 580 bp and second set of primer (internal primer) is then annealed to the first product (580 bp) to amplify the allele of desired size according to fragrant and non-fragrant genotypes (to identify mutated or not). External Sense Primer (ESP) 5́-TTGTTTGGAGCTTGCTGATG-3́; Internal Fragrant Antisense Primer (IFAP) 5́-CATAGGAGCAGCTGAAATATATACC-3́; Internal Non-fragrant Sense Primer (INSP) 5́-CTGGTAAAAAGATTATG
GCTTCA-3́; External Antisense Primer (EAP) 5́-AGTGCTTTACAAAGTCCCGC-3́.

PCR was performed using 0.2 μl Platinum Taq DNA Polymerase (Gibco BRLÒ), 1 μl of genomic DNA 10 ng μl-1, 2.5 μl of 10X buffer (Gibco BRLÒ), 1 μl of 50 mM MgCl₂ (Gibco BRLÒ), 1 μl of dNTPs [5 mM], 2.5 μl of each primer (ESP, IFAP, INSP and EAP)[2 μM], in a total volume of 25 μl. PCR was performed using a MJ Mini Gradient Thermal Cycler (BioRad, USA). Cycling conditions were an initial denaturation of 94 ºC for 2 min followed by 30 cycles of 40 s at 94 ºC, 30 s at 55 ºC, 40 s at 72 ºC; concluding with a final extension of 72 ºC for 5 min. PCR products were analysed by electrophoresis in ethidium bromide stained (0.5 ug ml-1) in 1.0% agarose gels.

Amplification of OsDREB gene: Primer sequences were as follows- OsDREB F 5́-CATCGTGGCGCAACATGAAAAAGA-3́ and R 5́-CCACAGTGCACTCAACACACAGTACAA-3́ and all the procedure were same as BAD2 amplification.The PCR products were sequenced bidirectionally in Applied Biosystem ABI 3700 sequencer using Sanger technology (SciGenom Pvt. Ltd, Cochin, Kerala, India).

 Amplification of matK gene for DNA Barcoding
Standard primer pairs for matK gene was used in the PCR amplification reaction to amplify the desired product approximately 1500 bp. The primer sequences were as follows- forward - matK Xf 5’- TAATTTACGATCAATTCATTC-3’ (Ford et al., 2009) and reverse matK-MALP 5’-ACAAGAAAGTCGAAGTAT-3’ (Dunning and Savolainen, 2010).  Thermal cycling conditions were as follows- 98 ºC for 45s followed by 30 cycles of 30 s at 98 ºC, 30 s at 54 ºC, 40 s at 72 ºC; concluding with a final extension of 72 ºC for 7 min. The PCR products were verified by electrophoresis in 1% agarose gels followed by staining with ethidium bromide. Amplification was carried out on MJ Mini Gradient Thermal Cycler (BioRad, USA). The PCR products were sequenced bidirectionally in Applied Biosystem ABI PRISM 3700 sequencer using Sanger technology (SciGenom Pvt. Ltd, Cochin, Kerala, India).

Database: There are two main barcode databases for DNA barcoding of plants (http://www.boldsystems.or
g/ and http://barcoding.si.edu/dnabarcoding.htm.). The international nucleotide sequence databases such as GenBank, EMBL, and DDBJ, have agreed to CBOL’s data standards (www.barcodeoflife.org) for barcode records. Second one i.e., Barcode of life database (BOLD), that was created and maintained by University of Guelph in Ontario to analyze DNA barcode data.

Data analysis: Specimens are identified by finding the closest matching reference record in the database. CBOL’s data analysis working group (COBOL 2009) has created the Barcode of life data portal which offers researchers new way to analyze the barcode data. Once the barcode sequence has been obtained, it is placed in the Barcode of Life Data System (BOLD system) database- a reference library of DNA barcode that can be used to assign identities to unknown specimens.

Results and Discussion
Morphological features of wild rice Oryza rufipogon
Pl  ant is annual cross pollinated, photosensitive and height varies from 64-145 cm (Figure 1). Eighteen quantitative and eight qualitative morphological traits were considered in the present study. Data were analysed using SPSS-15 software and summarized (Table 1). The wild rice taxon of NBU campus has been identified as Oryza rufipogon based on morpho-ecological parameters (Roy, 2014) although it is not matching all the characteristics of its previous reports (Table 2). Morphological variation were observed in the present study may be due to the introgression of genetic factors from Oryza sativa to O. rufipogon (Dong et al., 2010). The two taxa (O. nivara and O. rufipogon) are recognized as distinct species (Sharma et Shastry, 1965; Banaticla-Hilario et al., 2013) but others considered O. nivara to be the annual ecotype or subspecies of O. rufipogon (with O. rufipogon sensu stricto as the perennial ecotype) due to their continuous variation (morphological and genetic) including interfertility. Thus, the wild taxon of NBU campus showing deviated morphological features from O. rufipogon and may be considered as intermediate form (intergrade) (Table 2). Morphological differentiateon and variation within and among population has been studied to diagnosing species boundary (Elizabeth et al. 2008). Genetic diversity assessment within the species has been basis to utilize and manage germplasm resources (Wright and Gaut, 2005) and particularly crucial to its conservation. DNA barcoding based on matK gene sequence can provide some conclusive idea about the taxonomic position of this wild rice.

Figure 1 Morphological features of the wild rice Oryza rufipogon Griff. of NBU campus[A-Plant height and morphological traits, B-Node region with rootings, C- Seeds with long awn, D-Panicle morphology, E- Ligule shape and size, F- Seed and awn length measured, G- Panicles shape and colours in natural field, H- Seedling with root and shoot, and I- Deep pink coloration at the junction of awn with palea

Table 1 Morphological data of wild rice Oryza rufipogon of NBU campus were analyzed using SPSS-15 statistical software for mean, Sd, and SE.

 

Table 2 Comparison of NBU wild rice with reference data of other wild rice species.

MatK gene sequence of 1420 bp was used into the BOLD systems identification tool to search against plant sequence database. The BOLD identification system (IDS) is a tool for default identification of plant barcode. When sequence of Maturase K (matk) gene was uploaded in to the online BOLD system, it gives a species level identification of the taxon and further validated the identification with independent genetic markers (www.boldsystems.org). The BLAST algorithm search tool was available in BOLD identification search engine and matK gene sequencewas matched with the database. It was matched with species Oryza rufipogon Griff. As a result of BOLD identification systems search, the following reference information was obtained for the submitted query sequence matK (Fig.2) and Illustrative unique DNA Barcode of the plant taxon was shown in Figure 3. A phylogenetic tree was formed, when the matK gene sequence was run in MEGA6 software indicating the high degree of genetic relationship and closeness with other wild rice species particularly with O. rufipogon, O. nivara and also with cultivated rice O. sativa (Fig. 4)

Figure 2 PCR amplified products of DREB and BAD2 gene were fractionated on 1% agarose gel electrophoresis following staining in ethidium bromide. Lanes: 1 for genomic DNA, 2 for BAD2 357 bp, 3 for marker HindIII & EcoRI cut lambda DNA, 4 for DREB, 5 for BAD2 580 bp, 6 for same marker DNA and 7 for same BAD2 357 bp

Figure 3 Illustrative reference barcode (matK marker) obtained from matK gene sequence of Oryza rufipogon of NBU campus

Figure 4 The evolutionary phylogenetic history was inferred based on matk gene sequence information using the UPGMA method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree (run MEGA6)

DNA barcoding using BOLD system
DNA sequences of amplified matK gene compared with NCBI database through BLAST algorithm and matched with maturase (matK) gene of chloroplast of wild rice Oryza rufipogon Griff. Partial matK gene sequence (1420 bp) was annotated and submitted into the GenBank of NCBI and allotted GenBank accession no. KM516199. 
 

Assessment of genetic variation based on BAD2 and DREB gene
Phylogenetic analyses were done based on DREB(916 bp) (Table 3) and BAD2 gene sequences of NBU campus (Fig.5 and Fig.6). Phylogram showing that 916 bp DREBgene of O. rufipogon of NBU campus is closely related to other O. rufipogon taxon also with other varieties of O. sativa. The DNA sequence of 916 bp of DREB gene of O. rufipogon of NBU campus was BLAST run in www.gramene.org database, it was matched with DREB gene sequence of wild rice O. rufipogon in the database and aligned in chromosome 1, 4 and 9. The transcription factors DREBs or CBFs specially interact with the dehydration-responsive element/C repeat (DRE/CRT) cis-acting element and control the expression of many stressinducible genes in plants (Dubouzet et al. 2003). It has observed that the factor was present in O. rufipogon also and supporting this wild rice to survive in the adverse climatic habitat.

Table 3 Estimates of Evolutionary Divergence among five partial sequences of DREB gene

Figure 5 Maximum Parsimony analysis of five taxa based on partial sequence of DREB gene. The evolutionary history was inferred using the Maximum Parsimony method. The most parsimonious tree with length = 660 is shown. The consistency index is 0.950000 (0.914286), the retention index is 0.861345 (0.861345), and the composite index is 0.818277 (0.787515) for all sites and parsimony-informative sites (in parentheses). The tree is drawn to scale, with branch lengths calculated using the average pathway method and are in the units of the number of changes over the whole sequence. The analysis involved 5 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 468 positions in the final dataset. Evolutionary analyses were conducted in MEGA6

Figure 6 The evolutionary relationship of taxa was inferred using the UPGMA method [Sneath and Sokal 1973] based on BAD2 sequence of Oryza rufipogon of NBU campus. The optimal tree with the sum of branch length = 3.31974659 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [Tamura et al. 2004] and are in the units of the number of base substitutions per site. The analysis involved 6 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 58 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [Tamura et al. 2013]

In case of BAD2 amplification system it was a single tube allele specific PCR assay for the rice fragrant gene (Betain aldehyde dehydrogenase). Two primer pairs were used in this assay. One pair anneals to sequences common to both fragrant and non-fragrant varieties and external to the area where the mutation occurs and other pair that are specific to one of the two possible alleles were designed and synthesised. The two external primers were designed to act as an internal positive control amplifying a region of approximately 580 bp in both fragrant (577 bp) and non-fragrant (585 bp) genotypes. Individually, these external primers also pair with internal primers to give products of varying size, depending upon the genotype of the DNA sample. The internal primers, IFAP and INSP, will anneal only to their specified genotype producing DNA fragments with their corresponding external primer pair, ESP and EAP respectively. Using these four primers in a tube PCR, results in three possible outcomes. In all cases a positive control band of approximately 580 bp is produced. In the first case a band of 355 bp is produced indicating a variety or individual is homozygous non-fragrant. It is confirming that wild rice Oryza rufipogon of NBU campus is non-fragrant. For fragrant, it could produce a band of 257 bp. In case both bands of sizes 355 bp and 257 bp are produced indicating an individual is heterozygous non-fragrant. Non-fragrant rice varieties possess what appears to be a fully functional copy of the gene encoding BAD2 for the synthesis of γ-Aminobutyric acid (GABA) while fragrant varieties possess a copy of the gene encoding BAD2 which contains the deletion and SNPs, resulting in a frame shift that generates a premature stop codon that presumably disables the BAD2 enzyme that leads to the synthesis of 2-acetyl-1-pyrroline (2AP). This polymorphism provides an opportunity for the construction of a perfect marker for fragrance in rice (Bradbury et al. 2005a; 2005b).

Conclusion
The wild rice ecotype of NBU campus has been identifie
d as Oryza rufipogon Griff. based on morphology and taxonomic data was supported by DNA barcoding information. DNA barcoding has been carried outusing the matK gene of chloroplast. It was successfully identify the plant taxon when run in BOLD System as wild rice O. rufipogon. Phylogenetic analysis carried out using matK gene sequences of five different O. rufipogon accessions in MEGA6 software (Phylogenetic analysis software) and showed that NBU wild rice placed in separate cluster along with O. nivara.   DNA sequence of matK gene (1420 bp) of chloroplast was deposited into the NCBI GenBank and allotted accession number KM516199. Gene specific PCR amplification and sequencing was performed using DREB transcription factor and BAD2 fragrant gene information for study their relationship with other species considering the same genes.  MEGA6 analysis of DREB transcription factor gene (916 bp) showed that NBU wild rice placed in different cluster along with other O. rufipogon species. Same result was observed while BAD2 gene was analyzed in MEGA6 software for phylogenetic tree construction. The NBU wild rice Oryza rufipogon is non-fragrant based on BAD2 gene specific amplification, because it has given product of 357 bp when both the primer pairs were added in the PCR reaction instead of 257 bp (fragrant). DNA sequence of 916 bp of DREB transcription factor gene of O. rufipogon (NBU campus) was aligned in the chromosome 1 of O. rufipogon between 2.8 MB - 2.9 MB region when run BLAST algorithm in Gramene Genome Browser (www.gramene.org).

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