Detection of genetic divergence among putative ethyl methane sulfonate mutants of super basmati using microsatellite markers

Seeds of super basmati were mutagenized with different ethyl methane sulphonate (EMS) doses for creating genetic variability. A total of 48 randomly selected putative EMS mutants of super basmati were analyzed to dissect the genetic diversity by using 25 SSR primers located on twelve chromosomes of rice. SSRs analysis revealed that wide-range of genetic diversity is present among mutants of super basmati. A sum of 91 alleles were identified, out of these, 82 alleles were polymorphic and the rest of nine alleles were monomorphic in nature. The range of allele number was 2–10 with mean of 3.64 alleles/locus. The value of polymorphic information content was range between 0.039 (RM5) and 0.878 (RM44) with mean of 0.439 for each locus. A number of polymorphic markers showed unique bands of various sizes ranges from 75 to 1000 bp, during genetic dissection of mutant population. Dendrogram divided whole mutant population into four major groups. Phylogenic analyses revealed that 40–96%genetic similarity is present among individuals of mutant population. It is concluded that EMS induced genetic variability and SSRs markers (RM44, RM154, RM1, RM252, RM334, RM487, RM110 and RM257) could be handy for the selection of rice mutants as parents for functional genomic and molecular breeding program.


Introduction
Rice (Oryza sativa) is a primary food for the whole world population.Basmati variety of rice is grown in different regions of Punjab, Pakistan [1] due to its enhanced grain quantity and quality.Overall, 40% of national rice production obtains by Basmati (fine) rice according to Food and Agriculture Organization of United Nations.It is estimated that food production (˃ 70%) and rice production (˃ 24%) must be increase upto 2050 to fulfill the needs of the growing population that would be almost nine billion expected at that time [2].Production of any crop can be improved by changing the inherited genetic makeup of the crop.Mutation breeding through physical and chemical mutagen incorporates the genetic variation in successive generations through recombinant hybridization events [3].Genetic variation in rice is prerequisite as the natural variation is limited due to domestic cultivation and selection of high yielding rice.Chemical mutagen (Ethyl methane sulphonate) is the most effective method than physical agents to induce genetic variability in crop plants [4] by 70-8% point mutation of GC to AT transition.Genetic markers have been widely used for the identification of genetic diversity and genetic relationships within and among various species including plants and animals [5,6] at molecular level.These markers lie inside the gene or nearby it that can be easily identified using newest sequencing techniques as genotyping by sequencing (GBS) [7].Microsatellites or Simple sequence repeats (SSR) are PCR based genetic markers that are widely used for evaluation of rice genetic diversity.SSR have been employed for identification of genetic diversity in numerous crops such as maize [8], cotton [9], wheat [10] and rice [1,11].Rice SSR markers have been developed for different purposes such as mutation study [12], genetic diversity [13], association and QTL mapping [14], marker assisted selection (MAS) [15] as well as for rice domestication [16].Rice SSR have been recognized as polymorphic between different [17], rice varieties [18,19] and among various rice subspecies [20].These markers are nominated as important tool for the analysis of genetic divergence and genetic relationships among various animal and plant species.Major goal of the current study was to find out that, EMS as a chemical mutagen could be used to induce desirable genetic variability in rice germplasm.Nature variation in rice is extremely limited that needed the EMS different doses have different level of variations as 0.1% and 0.3% was found to bring significant variations in cotton [21] studied through RAPD marker.The current study was conducted to determine the genetic relationship and genetic diversity among 48 EMS putative mutants of super basmati using simple sequence repeats (SSRs) markers and to categorize suitable SSR markers for genetic analysis of rice mutants.

Plant materials
Seeds of super basmati (Fine) were collected from RRI (Rice Research Institute; Kala Shah Kaku; Gujranwala; Punjab; Pakistan).

EMS mutagenesis
One hundred and fifty seeds were mutagenized with different EMS doses (0%, 0.25%, 0.5%, 1%, 1.25%, 1.5%, 1.75% and 2% v/v).EMS working solution (10 ml of 0%, 0.25%, 0.5%, 1%, 1.25%, 1.5%, 1.75% and 2% v/v) was added in each falcon and shifted to orbit shaker at 60 rpm for 24 h.Then, treated seeds were washed out three to four times by using distilled water as described by [22].Mutagenized seeds were grown under field conditions in a single seed progeny method to get the first mutagenic population (M 1 ).Each treatment was repeated five times with row-to-row distance (10 cm) and plant to plant distance (8 cm).

DNA isolation
DNA was extracted from 20 days old seedlings using Cetyl trimethyl ammonium bromide (CTAB) method [23].DNA quality was checked by running 0.8% agarose gel.The quantity of DNA was determined by using Nano Drop (ND-1000) spectrophotometer.

SSR markers analysis
PCR amplification of 48 rice mutant was carried out using 25 SSR primers covering all twelve chromosomes of the rice genome enlisted in Table 1.Chromosomes position, repeat motif and primer sequences of all SSR markers can be determined from rice genome annotation databases (http:// www.grame ne.org).SSR study was done following the method of [24] with minor modifications.PCR amplification was carried out in total volume of 25 µl reaction mixture, including template (25 ng), 10 × PCR buffer (2.5 µl), MgCl 2 (2.5 mM), dNTPs (200 µM of each set), 0.2 μM of each primer (10 pmol), Taq DNA polymerase (0.2 µl; Fermentas Life Sciences) and sterile distilled water.PCR profiling condition was optimized as 94 °C for 5 min as initial denaturation step.This was followed by 35 cycles of denaturation (94 °C for 1 min), primer annealing (52-67 °C for 1 min), extension (72 °C for 2 min) and final extension (72 °C for 5 min).Total of 4 µl of amplification product was loaded in 7% polyacrylamide gel for gel electrophoresis and stained with ethidium bromide to visualize the amplified bands under UV light.Amplified product was scored on the basis of presence (1) or absence (0) of band in binary coding format..The Polymorphic information content (PIC) value, genetic diversity, heterozygosity, major allele frequency was analyzed by using PowerMarker version 3.25 [32].POPGENE version 1.32 [33] was used to obtain the genetic relationship among mutants and wild type plants based upon Jaccard's similarity coefficient by Unweighted pair group of arithmetic means (UPGMA) [34].TreeView 32 software was used to construct the similarity based dendrogram of the mutant population [35].

Results
Twenty-five SSR markers (Table 1) distributed on all twelve rice chromosomes were utilized to assess genetic diversity among 48 putative EMS mutants of super basmati.A broad range of genetic variability was discovered among different mutants of super basmati for 23 SSR markers.The remaining two markers (RM5 and RM103) showed monomorphic results (Table 2).A sum of 91 alleles was identified out of these, just nine alleles (9.89%) were monomorphic and remaining 82 alleles (90.10%) were recognized as polymorphic with an average of 85.6 bands per primer.The level of polymorphism was calculated by the PIC value of each marker loci.The PIC value may differ from locus to locus and ranges from 0.04 (RM5) to 0.88 (RM44) with the average of 0.44 for each locus.The highest PIC value was observed in RM44 (0.88) followed by RM154 (0.87), RM1 (0.79), RM252 (0.71), RM334 (0.64), RM487 (0.63), RM110 (0.59) and RM257 (0.59) based upon SSRs data of mutants (Table 2).PIC value showed positive significant correlation with allele numbers in current study.The maximum numbers of alleles were ten in the RM44 marker with the highest PIC value as 0.88.Markers RM161, RM242, RM277, RM287, RM72, RM171, RM229, RM271, RM70, RM103 and RM235 showed minimum numbers of alleles as two (Table 2) and the mean allele number was 3.64 alleles/ locus.The overall amplified product range varied from 75 bp (RM1; RM252) to 1000 bp (RM154).Several bands amplified by these SSR markers were common among mutants and wild plant of super basmati.
Interestingly, several mutant loci exhibited presence or absence of discrete bands by different set of SSR markers (Table 3).These unique alleles were not produced by wild used plants.Every mutant is distinguished from each other either alone or by a combined set of SSR primers.Genotypic variations existed in putative mutants located on a particular chromosome (Table 3) because the selected primers sequence located on particular chromosomes.These genetic modifications occurred either in the form of discrete alleles or non-amplified PCR products due to mutation in the primer sequence.For instance, phenotypic putative mutants (xantha and albino; M56) (Fig. 1) showed genetic changes in various microsatellites with respect to particular base pair such  3).Correspondingly, microsatellites (RM110, RM154, RM152; RM44 and RM334) showed molecular variations on chromosome 2, 2, 8, 8 and 5 with range of 75-1000 bp in viridis and albino mutants (M71) of super basmati respectively (Fig. 1).The current outcomes pointed out that wild and mutants were genetically dissimilar to each other.
In the current study, putative mutants that decreased or increased in their agronomic features showed in the similar cluster.Dendrogram divided the whole mutant population into four main clusters with sub-clusters.Cluster one, two, three and four has 2, 38, 2, 6 mutants of super basmati respectively (Fig. 2).Phylogenetic study revealed that pair-wise similarity among wild and putative mutants ranging from 40% (M45 and M116) to 96% (M105 and W 1 ) at chemical dose of (0.25% and 0.5%) and (0.25% and 0%) respectively (Table 4).

Discussion
The genetic diversity of putative EMS mutant was studied using twenty-five SSR marker spread on all 12 chromosomes.Similar number of microsatellite markers previously used for genetic analysis of rice genome in various studies [17,25,28,36].PIC value is a mirror of allelic variability among and within varieties which was not always higher for every tested SSRs loci [17,28].Higher PIC value could be considered as an important criterion for the selection of   polymorphic markers for various phylogenetic and genetic diversity studies in natural as well as mutant populations.[19].
Low PIC value may be the product of repeated crossing among closely related germplasm having narrow genetic bases as well as the high PIC value might be the effect of diverse, outcrossing, among naturally occurring plant species belongs to different geographical regions [28,36].SSR markers are generally categorized into three different classes on the bases of PIC value i.e., moderately informative (PIC ≤ 0.25), informative (PIC > 0.25 ≤ 0.50), and highly informative (PIC ≥ 0.50 ≤ 1.0) respectively [37,38].The high PIC values suggested that microsatellites were polymorphic and suitable for the detection of genetic variation in rice cultivar.The range of PIC value in present finding was in line with PIC value of existing outcome [6,13,15].The PIC value for all SSRs loci ranges from 0.36 to 0.98 in rice putative mutant population.In current study the PIC value ranges from 0.04 to 0.88.Kumar et al. [39] reported that eight SSRs primers produced 100% polymorphic bands from total of 20 SSRs primers whereas Vikash and Bhagwat [17] studied SSRs primers that were distributed over all 12 chromosome of rice and found them polymorphic with PIC range varied from 0.125 (RM208) to 0.68 (RM1) across 20 dwarf, semi dwarf mutant and wild (WL112) type of rice variety.Higher PIC value of some SSR markers among wild type and EMS mutant might be the result of induced EMS mutation in the basmati variety.This sort of induced genetic variability could be an important tool for genetic improvement of crops for various agronomic and stress breeding programs.
The present PIC range is also comparable with the previously reported value of 0.36-0.78among colored and white rice genotypes [40].According to study of Sahu et al. [41] 83 loci (24.02%) revealed polymorphism out of 343 loci among rice genotypes and identified 28.98% (51) polymorphic ratio.The highest polymorphism was 41.67% and lowest polymorphic % was 6.67.The PIC value range of the current study lower from previous observation reports of rice by Jain et al. [42] and higher from the following findings [36,37].
Correlation of allele number and their PIC value might also depend upon repeat number and repetitive sequence of microsatellites [20].The present range and mean value of allele numbers were similar to those reported by [25] among basmati and non-basmatic rice varieties as the range of alleles was 1-8 with mean value of 3.51 alleles/ locus.RM252 marker showed the high PIC value (0.8) with the eight allele counts.Rahman et al. [37] concluded markers RM1 and RM334 as polymorphic among rice varieties of Bangladesh, with the PIC value of 0.862 and 0.863, respectively.
A major purpose of molecular analysis is to sort out a marker which can discriminate a desired genotype from control and rest of other genotypes used.Vikash and Bhagwat [17] described that distinct alleles were identified for 18 rice mutants either in a single or combination of two SSRs markers.A sum of 19 unique alleles was detected from 17 genotypes of weedy rice with the size range of 200 to1300 bp [43].The gene flow was caused by existing of unique bands in weedy rice.It was also reported that regular occurrence of gene flow from domesticated to weedy rice was happened due to presence of discrete alleles [44].Ethyl methane sulphonate (EMS) has been reported to be the most potent in producing chlorophyll mutation among chemical mutagens in rice [45] and other crops.Chlorophyll mutation were group into three classes including albino (white), xantha (yellow), viridis (light green) and the detectable mutation is valuable in mutant populations as clue of variability [46].
Similarity of mutants ranged from 40 to 96% whereas the cluster analysis divided the mutants into four main cluster.Previous finding of Kumar et al. [39] also supported the present study that similarity coefficient varied from 0.40 to 0.96 among genotypes of rice.Similarity index varied from 0.56 to 0.95 among rice genotypes for SSRs markers that described by Patel et al. [40].The similarity index was 35% as establishment of genetic diversity among dwarf mutant genotypes [17].It was also reported by that mean similarity was 0.48 among eighteen genotypes of all SSRs loci using microsatellite markers [47].A pair wise similarity ranges from 0.39 to 0.89 was observed by Naeem et al. [2] after mutation in rice varieties.The recent results are in synchronization with Arif et al. [48] who examined an increase or decrease in genetic diversity of mutated genotypes as compared to their control genotypes.Jaccard's genetic similarity ranged from 0.04 to 0.92 according to [49,50].The high degree of diversity was observed among rice cultivar with 21-86% similarity [6].The difference between the highest and lowest value of similarity coefficient indicated a high level of genetic diversity among rice genotypes depend upon different set of SSRs markers and diverse selection of rice genotypes [49][50][51].The high genetic diversity that was observed in dendrogram coefficient among mutant rice varieties predicted to be due to effect of induced mutation [51].
It was also suggested from current study that more diverse mutants exhibited using high EMS doses.Phenotypic and genotypic data correlated to each other indicating that EMS might be brought desired genetic variability in putative mutants of super basmati.These genetic variations might be useful in selection of desired mutant genotype of rice in future.Induced genetic variability was verified through phenotypic and genetic study of quantitative traits in rice [51,52].Moreover, the induced mutation could produce a significant quantity of genetic changes for development as well as diversification of crop.This genetic variability might be helpful in improvement of mutant lines with improved traits, which are deficient in wild [53].

Conclusion
The considerable amount of genotypic variability identified by genetic analysis through SSRs markers within mutant lines of super basmati.These genetic changes indicated that EMS might be helpful for the development of desired genetic changes in rice.It was also recommended that current SSRs markers could be suited for further analysis to estimate the genetic diversity of rice mutants.
chemical doses, C.N chromosome number, M.N markers name, B.P base pairs, A absence of unique bands, P presence of unique bands Table 3

Fig. 1 Fig. 2
Fig. 1 Effect of chemical treatment on mutant seedling (M56 and M71) of super basmati.V1 variety of super basmati, M mutant

Table 1
Detail description of simple sequence repeat (SSR) markers used for genetic diversity analysis of Super basmati mutants

Table 2
Genetic polymorphism revealed by SSRs markers among putative mutant population of Pakistani super basmati rice variety M* markers, C.N* chromosome number, P.R* product range, MAF* major allele frequency, A.N* allele number, TNAP* total number of alleles in population, TNL* total number of loci, TNPA* total number of polymorphic alleles, TNMA* total number of monomorphic allele, P* polymorphism %, G.D* genetic diversity, H* heterzygosity, PIC* polymorphism information content

Table 3
Identification of super basmati mutants based upon absence or presence of unique bands on chromosomes at particular base pair

Table 4
Similarity matrix based upon SSRs markers among of super basmati Nei's Unbiased Measures of Genetic Identity and Genetic distance Nei's identity (above diagonal) and genetic distance (below diagonal)