Assessment of genetic diversity among different sugarcane genotypes using internal transcribed spacer ( ITS ) region of the ribosomal DNA ( rDNA )

Internal transcribed spacer or ITS region of nuclear ribosomal DNA (rDNA) has been used to evaluate genetic assortment and phylogenetic relationship in nine sugarcane genotypes including Saccharum species and another related genus as Erianthus, Narenga and hybrid. DNA was extracted from selected genotypes and ITS (ITS-1 and ITS-2) regions were amplified using specific primers. The sequence lengths ITS-1 showed 205207 bp, while ITS2 was ranged from 211218 bp. However, G+C content (%) 65.2% 67% in ITS-1 and in ITS-2 68.4% 99.7%. The sequence lengths of fragment and GC content of ITS-1 and ITS-2 regions showed variable. To evaluate the phylogenetic association of both the region of ITS (ITS-1 and ITS-2) neighbor-joining (NJ) method was employed. The cluster A of ITS-1 and cluster B for ITS-2 and cluster C combined between ITS1+ ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II. The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species and hybrid. The ITS2 sequences in the group B showed better correlation amongst each other. The sequences ITS-1 & ITS-2 combined and compared with some selected sequences from NCBI database using NJ method. The results have confirmed that ITS region can be used for evaluating the genetic assortment in Saccharum and its closely related genes.


Introduction
Sugarcane (Saccharum spp.hybrids) is secure second rank an imperative crop and acting as significant role in agricultural and industrial economy [1], provides more than 70% sugar and more than 30% ethanol production in tropical and subtropical countries [2][3][4][5].Currently, sugarcane is cultivated in 20.42 million ha producing 1,333.2 million tonnes with an average cane productivity of 65.20 tonnes/ha [6].Sugarcane belongs to family Poaceae tribe Andropogoneae.
Universally in the entire organism gene coding is available for ribosomal DNA.The tremendous importance of the restriction fragment and sequence analysis of nuclear rDNA as a reliable approach for addressing the genomic relationships among cultivated species and its wild relatives has been well recognized [11][12].Nucleotide sequences of ITS (1, 2) of 18S to26S nr DNA has proved to be helpful in taxonomic studies [13][14].Then rDNA-ITS have been used in the evolutionary analysis of inter or intraspecific genetic variation and due to elevated rate of mutation it is also used in selection of parent in breeding program in different plants [15][16][17] and in sugarcane [10,18].The length of ITS regions and its sequences of rDNA repeats are considered to be quickly developing and hence may differ.Widespread PCR primers of ITS regions are developed from extremely conserved flanking regions, and comparatively, small size enables easy amplification of ITS region (600-700 bp) of rDNA repeats.To evaluate the ITS regions small quantity of DNA are necessary and it is easy to produce, quick and this whole thing makes the ITS region an exciting topic for phylogenetic and evolutionary investigations [13; 19] and additionally for biogeographic studies [20][21][22][23].The present investigation was to study the genetic diversity and phylogenetic relationship on the basis of ITS regions (ITS1 and ITS2) in sugarcane genotypes.

DNA isolation and PCR amplification of ITS regions
Collected leaves were frozen in liquid nitrogen and kept in -80°C for preservation.The total genomic DNA was isolated from each stored sample by using the CTAB method with little modification [24].The quantity and quality of extracted DNA were evaluated by UV spectrophotometric method and agarose gel electrophoresis.The final working concentration of DNA was diluted at 10 ng/µL was used in PCR.

Cloning and sequencing of ITS regions
The amplified PCR product was eluted and purified by using gene clean kit (Gene Clean II, Bio 101) by following manufacturer instructions.The isolated purified product was introduced in GMT vector (Promega) followed by transformed in E. coli (DH5α) and identified through blue/white screening as well as confirmed by PCR and sequencing of clones (minimum three) of every species were carried out in the sense and antisense direction.

Sequence and phylogenetic analysis
The sequences of ITS region obtained from the sugarcane varieties were subjected to sequencing and the raw sequences were edited and verified for vector sequence contamination followed by the sequence alignment and phylogeny analysis of final edited sequences.The sequence alignment was obtained using the ClustalW algorithm [26].The BioEdit software provided the graphic view of the sequence alignment generated.The sequence alignment assignment provided a brief description of our ITS sequences and the database sequences in terms of sequence identity and gap percentage and Evalue notes through the NCBI, BLAST analysis.The phylogeny software, MEGA6 [27][28][29] provided comparative analyses of our ITS sequences and that of the NCBI database ITS sequences.The phylogeny was generated using the MEGA6.0software using Neighbor-Joining method (1000 bootstraps) [30].

ITS-1 and ITS-2 sequences of nrDNA
All the amplified product from nine sugarcane are sequenced and have been submitted to nucleotide sequence database (EMBL) under the accession number LT220885 to LT220902.

Sequence length analysis of ITS-1 and ITS-2 and G+C content
All nine amplified ITS regions produce an inconsistent site, lengths, and percentage of GC content.The sequence lengths of ITS-1 ranged from 205 to 207 bp, while ITS-2 was ranged from 211 to 218 bp.However, the percentage of GC content is slightly lower in the ITS-1 region (65.2 to 67 %) compared to the ITS-2 region (68.4 to 99.7%) (Table 1).

Sequence alignment and phylogenetic analysis of ITS-1 and ITS-2
The sequence alignments of ITS-1 and ITS-2 generated by ClustalW showed the variable sites like T-A and C-G mismatch base insertion or base deletion and C-T and G-A transversion, as shown in Figure 2. The phylogenetic relationship of all nine sugarcane genotypes was carried out for ITS-1 and ITS-2 sequences using Neighbor-Joining method and combined nucleotide sequences data of ITS1 + ITS2 (Figure 3 A-C).Some of the sequences downloaded from NCBI database generated dendrogram for the comparison study with some sugarcane species and related genera as shown in (Figure 4 A-C).The phylogeny tree generated between the ITS-1 sequences revealed 3 groups; A, B and C. The A group comprised of the Erianthus species, the Saccharum series were clustered in group B and the C group was left only with the Erianthus cilliaris which showed a correlation with that of group A (Figure 3A).The phylogeny tree of ITS2 sequences showed better correlation amongst each other (Figure 3B).The phylogeny between combined ITS1 + ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II.The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species.The ITS2 sequences in the group B showed better correlation amongst each other (Figure 3C).

Discussion
ITS is the region of 18s-26s nrDNAis consisted of two spacer of ITS-1 and ITS-2 sequences have additional informative sites and it has been extensively utilized to investigate the intra-specific dissimilarity and inter-specific association of plants [13,[15][16][17][31][32][33][34].To measure the degree of relatedness in between species, variation in DNA sequences is utilized.Differences in nucleotide sequences can be used to definite the degree of relatedness between spp.[35].The extremely conserved character of 18s and 26s rRNA genes allows alleviate the primer construction and PCR amplification [36], and many investigations have revealed the ITS region to be adequately unpredictable, as a result, it is helpful in providing information to compare taxa at the molecular level.The nrDNA-ITS seem to be a very helpful source of data for considering phylogenetic association within the Saccharum.Internal transcribed spacer regions generally explain the advanced rate of variation that is necessary for extrapolating phylogenies of closely allied species and populations in a known species [36][37][38].Very recently [18] studied ITS-nrDNA to identify the genetic divergence and genetic variability of thirty sugarcane genotypes (Saccharum officinarum L.) for exploitation of the potential parent source in sugarcane improvement through breeding.Previously Internal transcribed spacer or nrDNA-ITS region were utilized to identify the intergeneric hybrids of Saccharum spp.× E. fulvus) [40] and evaluation of phylogenetic as well as the evolution of six Sorghum spp.[41].
In the current investigation, several variable sites were observed, with excellent reproducibility by repeated sequencing can be used as specific DNA fingerprinting sites.Similar kind of results was reported in sugarcane [18].The sequence of rnDNA-ITS has been used in the genetic relationship analysis of the spp.belong to genus Saccharum and another related genus as Erianthus, Narenga, and hybrid.The result is obtained in the present study have shown that GC percentage is lower in ITS1 compare to ITS2 and also sequence length is longer in ITS-2 region than in ITS-1.A similar result was reported by [18,42], but in the present investigation, the variation sites found higher in ITS-2 region than ITS-1 as is observed in other plants [16,43].The possible reason behind that the spp. of genus Saccharum are highly complex polyploidy in nature which leads to the divergent evolutionary process of Saccharum as compared to other crops.Therefore, ITS approach can be used for the assessment of the genetic diversity in sugarcane.
The phylogenetic analysis of ITS-1 sequences revealed the Saccharum series were clustered in one group and Erianthus cilliaris was left in the separate group which showed a correlation with Saccharum.The phylogenetic analysis of ITS-2 has shown better correlation amongst species compares to ITS-1.In ITS-2 Narenga porphyrocoma and Erianthus arundinaceus formed one group with 37% bootstrap values (Fig. 3B).Phylogenetic analysis between the sugarcane ITS-1, ITS-2 and combines sequences and their respective NCBI database showed the ITS-1 sequences revealed a fair distribution among the groups whereas ITS-2 sequences have shown to form a cluster among Narenga porphyrocoma and Erianthus forming separate groups.Our results are also correlated with phylogenetic tree generated in the investigation of the thirty genotypes of sugarcane [18] obtained that S. senseand S. barberi species formed a single cluster, and they were situated between S. spontaneum and S. officinarum.This study supported the hypothesis that S. sinense and S. barberi were coming from interspecific hybrid species of S. spontaneumand S. officinarum [44].According to classification by Irvine [45], derived that S. spontaneum and S. officinarum in two species similar observations in some extent observed in our study it supported by [18].This conclusion coincided with RAPD, ISSR and SSR markers [46][47].These results are supported by other marker systems as r-DNA spacers [48][49].

Conclusion
The present study shows that Saccharum and Narenga are closely related group whereas the Erianthus showed significant divergence.These outcomes are supported by other marker systems as r-DNA spacers.The information generated from sequence and its length from ITS region may be a helpful parameter for the evaluation of genetic diversity and phylogenetic studies in sugarcane species.

Figure 2
Figure 2 Sequence alignment among the ITS-1 (A) and ITS-2 (B) Code for the sugarcane genotypes as given inTable 1

The
Phylogeny of ITS (ITS-1, ITS-2, and ITS-1 + ITS-2) sequences both our sequences and other research group submitted sequences taken from NCBI database were used to study the phylogenetic relationship between sugarcane genotypes with some related species consider as out-group.The dendrogram clustered into 4 groups of (A, B, C, and D).The group A clustered the Erianthus species with the S. robustum and Saccharum arundinaceus.The group B consisted of Narenga, S. officinarum, S. spontaneum, S. officinarum hybrid and S. barberi which showed better clustering with Zea perennis, Saccharum fallax, S. robustum, S. barberi and S. spontaneum; whereas the group C formed a different group with Miscanthus and Zea mays.The group D showed the separate Saccharum fulvum which relates to the group A (Figure4A).The phylogeny ITS-2 sequences with their NCBI homology were clustered into 4 groups which clearly reflects that group An Erianthus showed association with the S. barberi and S. robustum ITS-2, group B shows major cluster of our ITS-2 sequences with that of, Saccharum fulvum, S. robustum and Saccharum arundinaceus.The group C showed only Narenga porphyrocoma (ITS-2) to show a relation with Zea mays, S. spontaneum and Narenga porphyrocoma (ITS-2, AF345233), whereas the group D was separated with Miscanthus and Narenga fallax from the other groups (Figure4B).The phylogeny generated between the ITS-1 + ITS-2 sequences and their NCBI homologies revealed that ITS1 sequences shown a fair distribution among the homologs whereas the ITS-2 sequences have shown to form a cluster among themselves with Narenga porphyrocoma and Erianthus forming separate groups (Figure4C).