Introduction
Aromatic rice constitute a small but special group of rice, which are considered best for aroma and have occupied a prime position in society for aroma and cooking qualities (Ahuja et al., 1995). A large number of aromatic rice has already been disappeared and many are at the verge of extinction. Therefore, there is a utmost necessary to conserve the germplasm. 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. A 'barcode' gene that can be used to distinguish between the majorities of plant species on earth has been identified. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. In plant systematic, matK has recently emerged as an invaluable gene because of its high phylogenetic signal compared with other genes used so far (Muller et al., 2006).The 1500 bp matK gene is nested in the group II intron between the 50 and 30 exons of trnK in the large single copy region of the chloroplast genome of most of the green plants (Sugita et al., 1985; Steane, 2005; Daniell et al., 2006; Turmel et al., 2006). 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 an average per parsimony-informative site than the highly conserved chloroplast gene rbcL (Muller et al., 2006). Sequence information from matK alone has generated phylogenies as robust as those constructed from data sets comprised of 2-11 other genes combined (Hilu et al., 2003). Further, the molecular data generated from matK has been used to resolve phylogenetic relationships at taxonomic levels (Johnson and Soltis, 1994; Hayashi and Kawano, 2000; Hilu et al., 2003; Cameron, 2005). The matK gene stands out among plastid genes used in plant systematic for its distinct mode of evolution. The rate of substitution in matK is three times higher at the nucleotide level and six times higher at the amino acid level than that of rbcL which denoting it as a rapidly evolving gene (Johnson and Soltis, 1994; Olmstead and Palmer, 1994; Soltis and Soltis, 2004). Various scientist have been studied the plant evolution, and able to solve various anomalies in the taxonomic levels by using the chloroplast genes such as matK and rbcL. The matK gene has two unique features that emphasize its importance in molecular biology and evolution. It is characterized by its fast evolutionary rate and putative function as a group II intron maturase. It is a chloroplast encoded gene nested between the 5’ and 3’ exons of trnK, tRNA-lysine in the large single copy region of the chloroplast genome (Sugita et al., 1985).

Molecular sequence data has revolutionized evolutionary studies and enhanced the revolution of phylogenetic trees immensely. Genes used in plant systematic display different trends of evolution. Slow evolving genes such as rbcL and atpB, have high sequence conserve among plant groups. This high sequence conservation allows a good resolution that has been confined to the family level, but cannot solve the intricacies below this level (Goldman et al., 2001). The matK gene is considered to be fast evolving due to the fact that it has a high rate of substitution and more variable sites compared to other genes (Olmstead and Palmar, 1994). Some of the researchers reported that the matK has been considered as a pseudogene because they contain stop codons within the ORF, bear indels that create frame-shift mutations and display an equal level of substitution for all three codons position (Kores et al., 2000). Additionally, frame-shift mutations found in the 3’ region of matK of the  family Poaceae, which could also alter or destroy the reading frame; appear to be limited to the very 3’ region of this gene, not affecting the functionally of domain X (Hilu and Alice, 1999).

The genus Oryza, which includes rice and closely related wild relatives, has emerged as a powerful system to study the modes and mechanisms of genome evolution (Ammiraju et al., 2008). The genus Oryza comprises approximately 23 species that have been grouped into six diploid (AA, BB, CC, EE FF, GG) and four allotetraploid genome types (BBCC, CCDD, HHJJ and KKLL) (Lu et al., 2009). All Oryza polyploids are wild and contain important phenotypic traits that have the potential for use to improve the cultivated rice (Brar and Khush, 1997). In the present study implies the understanding of major evolutionary relationships of aromatic rice under the family, genus and species level by using the chloroplast derived matK gene.

Results and Discussion
Among the various approaches used in molecular systematic, DNA sequencing has become one of the most widely utilized, particularly above the genus level. Sequence variation in the coding and spacer regions of a number of chloroplast, mitochondrial, and nuclear genes has been utilized, with some genes more widely used, like the matK, Adh1, Adh2 and rbcL, and both coding and intergenic spacer region (ITS) of the ribosomal DNA (Clegg et al., 1994; Hsiao et al., 1994; Clark et al., 1995). The sequence data are cumulative, the potential sizes of informative data sets are immense, and the data are available in public computer databases. The matK gene is emerging gene as potential contributions to plant molecular systematic and evolution (Johnson and Soltis, 1994, 1995; Steele and Vilgalys, 1994; Liang and Hilu, 1996). This gene has been used effectively in addressing systematic questions in the number of families i.e. Polemoniaceae (Johnson and Soltis, 1995), Poaceae or Gramineae (Liang and Hilu, 1996), Orchidaceae tribe Vandeae (Jarrell and Clegg, 1995) and Myrtaceae.

In this investigation, sharp and bright bands of 1500 kbs of plastid matK gene fragment from template DNA of ten aromatic rice varieties were amplified by using matK specific primers (Figure 1). For the sequencing large scale amplification was done and amplified fragments were separated in 2.5% low agarose gel electrophoresis and subsequently used for elution though Gel Extraction Kit (Genei^TM). The ten eluted fragments of rice varieties were sequenced with an ABI 3730 XL genetic analyser with a BigDye terminator cycle sequencing kit (SciGenome, Kerala, India). For correctness of the sequences, DNAs isolated from independent varieties were analyzed for each genotype.

All nucleotide sequences from ten aromatic rice genotypes were subjected to multiple sequence alignment (MSA) in Muscle program. The conserved region was identified in all aromatic rice genotypes and found very little changes in the conserved region. With close observation of all ten genotypes sequences, it was found that the sequences of all genotype were muted during evolution and showed number of changes such as substitution, insertion and deletion in the cultivars (data not given). The number of substitution was higher between the 1110 bps to 1270 bps which might be the crucial during evolution. Similarly, Kron et al. (1999) was also found the insertion and deletion in matK of Epacrids and vaccinioids. Meanwhile MSA of ten aromatic rice genotypes along with wild relatives of rice and grasses revealed that the conserved nucleotide sequences as well as nucleotides changes during evolution of aromatic rice as compared to wild relatives and grasses under family Poaceae. The key mutational changes such as substitution ‘G’ to ‘C’ and ‘A’ was found in cultivars except ‘Banikunja’ and ‘Basaomati (Paikanapua)’ viz. ‘Basnasapuri’, ‘Basnaparijat, ‘Basumati dhan’,‘Chatiamaki - 1’, ‘Dhoiabankoi’, ‘Ganjam local - 2’, ‘Gatia’ and ‘Kalikati - 1’ at position 1352 bp respectively. Whereas, most variable nucleotide sequences was present in all aromatic rice genotypes at position 1389 to 1384 (Figure 2A) and complete deletion of nucleotide ‘T’ was observed at position 1761 and 1769 in all ten aromatic rice genotypes (Figure 2B). Relevant to these issues is also indicating that a gene is not a homogenous population of nucleotides since different parts of the gene assume different functional responsibilities and some sections might lack a function. Consequently, rates of nucleotide substitution can vary along the entire coding, or even the noncoding regions of a gene (Sastri et al., 1992; Clegg et al., 1994; Clark et al., 1995).

Figure 1 Amplification of 10 aromatic rice cultivars employing matk gene specific primer. Note: Ladder= Low range DNA ruler plus; Name of cultivars represents in the lower of the gel; Numbers on the right side of margin represents molecular weight marker DNA in base pairs (bps) and Number on left side represents size of amplified fragments in bps

Figure 2 Jalview of muscle multiple sequence alignment tool (www. ebi.ac.uk/ Tools/msa/ muscle/‎) based on nucleotide sequences of 10 aromatic rice cultivars and its wild relatives along with grasses progenitors. Note: A: Colored boxes in the MSA indicate the substitution nucleotide changes in aromatic rice cultivars; B: Red colored boxes indicates deletion of nucleotides with respect to its progenitors.

Phylogenetic tree for matK gene based on nucleotide sequences for both aromatic rice genotypes along with wild relatives and grasses under family Poaceae based on the alignment dataset was constructed by neighbour joining statistical method of bootstrap test with 1000 bootstrap replications in MEGA 6. Phylogenetic tree of ten aromatic rice genotypes showed one outgroup and two sub-clusters. Genotype ‘Banikunja’ was an outgroup, whereas genotypes ‘Basnasapuri’, ‘Ganjam local-2’, ‘Basumati dhan’, ‘Gatia’ and Basnaparijat’ were placed in cluster-IA and ‘Dhoiabankoi’, ‘Chatiamaki-1’, ‘Basaomati (Paikanapua)’ and ‘Kalikati-1’ was in cluster-IB (Figure 3). Divergence time scale from ‘Basnasapuri’ to ‘Banikunja’ was estimated which was found maximum for genotype ‘Banikunja’ i.e 0.015 and minimum for ‘Gatia’ and ‘Dhoiabanki’ i.e. 0.009. The maximum 47 and minimum 9 bootstrap values were observed. Evolutionary divergence between the sequences were estimated by using pair wise distance matrix for aromatic rice cultivars it was found that genotypes of ‘Banikunja’ and ‘Ganjam local-2’ was distantly related with maximum divergence value of 0.0296. whereas, Basumati dhan and Gatia was closely related with minimum divergence value of 0.0190 (Table 1).

Table 1 Pair wise evolutionary divergence of aromatic rice cultivars

Figure 3 Phylogenetic tree based on nucleotide sequences of 10 aromatic rice cultivars constructed by neighbor joining method Note: Out group indicates in red mark. Black bold fond on upper side of the tree node indicates bootstrap values in per cent. Values in black fond below the tree root indicates the divergence time

Phylogenetic analysis of aromatic rice along with related sequences was performed in order to find out the relationship between aromatic rice, wild relatives and grasses under family Poaceae in MEGA 6 program. Phylogenetic analysis of 41 other rice species and grasses along with ten aromatic rice genotypes were subjected to study the evolutionary pattern of aromatic rice. It observed that there are two main groups evolved from common ancestor. Phylogenetic tree was categorized into two main groups cluster-I and cluster-II. Cluster-I was further divided into two sub-clusters IA and IB and aromatic rice genotypes was placed in sub sub-cluster IA1 which indicates the aromatic rice was evolved after cultivated rice viz. Oryza sativa indica and japonica group. Similarly cluster-II was also divided into two sub clusters IIA and IIB. The 12 wild relatives of Oryza group was completely separated during evolution and placed differently in sub sub-cluster- IIB₁ (Figure 4). These 12 wild relatives of Oryza group might be evolved from closely related grasses such as Maltebrunia letestui, Prosphytocloa prehensilis and Leersia oryzoides. Whereas domesticated rice Oryza sativaindica group, Oryza sativa japonica group and its wild relatives were evolved from another group of grasses such as Leersiatis seranti, Chikusichloa aquatica, Potamophila parviflora, Rhynchoryza subulata and Zizania aquatica. Phylogenetic tree revealed that aromatic rice was evolved from Oryza sativa japonica group and Oryza sativa indica group along with the parallel evolution of Oryza glaberrima and Oryza rufipogal. Similarly, Khush (2000) was also suggested that the common rice and the African rice, Oryza glaberrima was an example of parallel evolution in crop plants. Oryza nivara was the possible ancestor of Oryza sativa indica and japonica group. The maximum bootstrap value 99 was calculated for Cluster-II including Oryza ridleyi and Oryza longiglumis which indicates the correctness of topology and minimum bootstrap value 1 was estimated for Oryza rufipogal which indicated the interior branch.

Figure 4 Phylogenetic tree based on the alignment of nucleotide sequences ofaromatic rice cultivars and its wild relatives along with progenitors grasses constructed by neighbor joining method. Note: Common rice indicates in red and green mark. Black bold

In the conclusion, this study indicates that the patterns and types of nucleotide substitutions, insertion and deletion in the matK gene are most comprehensive phylogenetic analysis using molecular sequence data. The phylogenetic study of the matK nucleotide sequences of ten aromatic rice genotypes including related wild species and other grasses family under Poaceae concludes that the genus Oryza was divided into two main clades and evolved from completely different group of grass families. Aromatic rice might be evolved from Oryza sativa (japonica and Indica group), Oryza glaberrima or Oryza rufipogal because of its low bootstrap value support. Oryza brachyantha has high affinity for grasses and should be treated as a progenitor for wild Oryza species. The study will also address the issues of optimal number of nucleotides essential for robust phylogenies and the consequences of utilizing different segments of the gene.

Materials and Methods
Genotypes
Ten promising upland as well as lowland cultivars of indigenous aromatic rice (Oryza sativa L.) viz. ‘Basnasapuri’, ‘Basnaparijat, ‘Basumati dhan’, ‘Banikunja’, ‘Basaomati (Paikanapura)’, ‘Chatianaki - 1’, ‘Dhoiabankoi’, ‘Ganjam local - 2’, ‘Kalikati - 1’, and ‘Gatia’ were collected from the Rice Research Station, Orissa university of Agriculture and Technology, Bhubaneswar, Odisha, India and used in the present study.

DNA extraction and PCR amplification
Total cellular DNAs were extracted from young leaftissues of ten aromatic rice genotypes using modified CTAB method (Edwards et al., 1991) and purified the DNA of each  genotype was subjected for PCR amplification using overlapping oligos viz. matKF1: 5’TAATTAAGAGGATTCACC AG 3’ and matKR1: 5‘ATGCAACACCCTGTTCTG AC3’ (Merck Bioscience, India) by examining the previous study on rice (Ge et al., 1999).PCR amplification was carried out with the template DNA (25~50 ng), 2.5 µl 10X PCR assay buffer (Merck Bioscience, India),1.5 µl each of 10 mM dNTPs (M/S Merck Bioscience, India), 1 µl of 5 µM forward primer, 1 µl of 5 µM primer and 1 µl of 1U Taq DNA polymerase (Merck Bioscience). M/s Peqlab, 96 universal gradient thermal cycler was used for the PCR amplification consisted ofa total of 35 cycles of initial denaturation (94°C for 4 min) followed by denaturation (94°C for 1 min), annealing (57°C for 2 min), elongation (72°C for 2 min) and final elongation (72°C for 15 min). The 1 X Tris-acetate ethylene diamine tetra acetic acid (TAE) buffer was used to electrophoreses PCR amplified products in 2.5% (w/v) agarose gel (Merck Bioscience, India) along with 3 kb ladder (Himedia Laboratories Pvt. Ltd., Mumbai). The gel image was documented using gel documentation system (UVITECH, Cambridge, UK).

PCR product purification and sequencing
Large-scale amplification was performed to elute bright PCR fragment. The single bright amplicon of 1500 kb was eluted using Gel Extraction Kit (Genei^TM).The PCR amplified DNA fragments were subjected for two way sequencing in order to get the full sequence and minimize the error in sequencing and was sequenced by using both 96 capillary high throughput sequencer; ABI 3730 XL system to generate sequences with accurate base calling which is an extension and refinement of Sanger’s dideoxy method (Sanger et al., 1977) at SciGenom, Cochin, Kerala, India.

Sequence Data Analysis
The sequenced nucleotide datasets were manually edited in BioEdit program (version 5.0.9 http://www. mbio.ncsu.edu/BioEdit/bioedit.html) and used for further analysis. Alignment of all ten sequences were toggled using Multiple Sequence Alignment (MSA) of muscle programme (www. ebi.ac.uk/Tools/MSA/ muscle/) and alignments were visualized in Jalview. Meanwhile BlastN program was used for preparation of dataset based on the alignment results of nucleotide for all related sequences were downloaded from the GeneBank database and analyzed in MSA along with ten aromatic rice cultivars, MEGA (Molecular Evolutionary genetic analysis) version 6 was used for phylogenetic analysis and calculations of pair wise distances [http://www.megasoftware.net] (Tamura et al., 2013). The phylogenetic tree was generated by bootstrap test Neighbour joining (NJ) with kimura two-parameter model (Kimura, 1980). The stability of internal nodes was assessed by bootstrap analysis with 1000 replicates.

Acknowledgement
The authors wish to acknowledge to Department of Biotechnology, Government of India for providing financial assistance under PG teaching HRD program.

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