**2.3 Anatomical studies**

The free hand sections of the collected specimens were taken and observed under the light microscope. Measurements of anatomical characteristic features were made using standard methods.

## **2.4 Molecular studies**

Total genomic DNA was extracted from 7-day old seedlings of wild rice species; *O.rufipogon*, *O. nivara*, *O. eichingeri* and *O. rhizomatis* respectively using Promega Plant DNA extraction kit. A total of twelve SSR primer pairs were used (**Table 3**) for molecular study. SSR markers were obtained from Gramene (http://www.grame ne.org/). All SSR PCR amplification reactions were carried out in a total volume 30 μl of which consist 1 x PCR buffer, 1 mM dNTPs, 2 μM SSR primers, 2 mM


**Table 3.** *SSR markers used for the molecular studies.* MgCl2, 50 ng of genomic DNA and 0.5 Units of *Taq* DNA polymerase. SSR alleles were resolved on Poly Acrylamide Gel. The SSR banding patterns were identified using Poly Acrylamide Gel Electrophoresis (PAGE).

#### **2.5 Analysis of Data**

Gathered data were analyzed with univariate, bivariate and multivariate statistical procedures. Suitable statistical software was employed in the analysis of data. In addition, data mining analysis were also attempted for the data gathered from the study to reduce the noise in the data set.

Molecular data were analyzed using Genemapper 4.1 software and SSR profiles were analyzed using PowerMarker 3.25.

### **3. Results**

#### **3.1 Morphological studies**

The mean values of the parametric morphological measurements of wild rice species are given in **Table 4**. According to the table, the species *O. rufipogon* indicated highest mean for the plant height (153.23 cm) and the culm length (94.11 cm) and minimum plant height was observed in *O. eichingeri* (99.25 cm). Similarly, the highest leaf length and breadth were found in the samples of *O. nivara* and narrow leaves were occurred in samples of *O. rufipogon*. The variation of ligule length indicated that *O. nivara* possessed a higher ligule length with respect to other species included in the study. The summary of the ANOVA carried out on the parametric lamina morphological characters are shown in **Table 5**, except ligule length, panicle length, the rest of the characters are significantly varying across the wild rice species.

The association of the non-parametric characters with wild rice species included in the study is shown in **Table 6**. The characters such as leaf blade pubescent, awn after full heading and intermodal color after full heading are not significantly differ across the species (p > 0.05). However, the rest of the characters are significantly associated with the wild rice species and are of potential characters in separating wild rice species.


#### **Table 4.**

*Summary of the parametric morphological characters of the four wild rice species (Mean value and standard deviation below mean value).*

#### *Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*


#### **Table 5.**

*Summary of the ANOVA performed on the parametric morphological characters of the four wild rice species.*


#### **Table 6.**

*Result of χ<sup>2</sup> test performed on the non-parametric morphological characters of the wild rice species included in the study.*

A total of three clusters were resulted from the cluster analysis of morphological characters (**Figure 5**) and species were grouped under each cluster with respect to their similarities. The samples of *O. nivara* and *O. rufipogon* were intermingled and separated into two groups. Meanwhile the samples of *O. eichingeri* and *O. rhizomatis* well-separated from 80% similarity level and from rest of the clusters representing two populations. However, one sample of *O. eichingeri* was grouped with O*. rhizomatis.* The phylogenetic tree (**Figure 6**) constructed by morphological characters clearly showed a well separated cluster of *O. rhizamatis*. The samples of *O. nivara* and *O. rufipogon* were intermingled and separated into four groups. Findings of the study led to conclude that wild rice species in Sri Lanka are "ecological swarms" and represents allopatric or sympatric populations.

**Figure 5.**

*Dendrogram produced by cluster analysis of 22 morphological characters of wild rice species*, O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

#### **Figure 6.**

*Phylogenetic Tree constructed by 22 morphological characters of wild rice species,* O. nivara*,* O. rufipogan*,* O. rhizomatis*.*

#### **3.2 Anatomical studies**

The variation of anatomical characters, especially the laminar anatomical features across the wild rice species are given in **Table 7**. Comparatively, the magnitude of mean values of bundle sheath cell width indicated a considerable variation between the wild rice species *O. eichingeri* (11.77 μm) and *O. rufipogon* (10.74 μm). The summary of the ANOVA (**Table 8**), indicated that the all the anatomical

*Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*


#### **Table 7.**

*Summary of the parametric lamina anatomical characters of the wild rice species (Mean value and standard deviation below mean value).*


#### **Table 8.**

*Summary of the ANOVA performed on the laminar anatomical characters of the four wild rice species.*

characters except mesophyll height and bundle sheath height. The anatomy of the culm and leaf sheath of wild rice species indicated that the characteristic features of the structures reflect the habitat conditions (**Figures 7** and **8**).

The result of the cluster analysis of anatomical characters of wild rice species is shown in **Figure 9**. Comparatively, the Dendrogram resulted from the anatomical

**Figure 7.** *Laminar anatomical characters of 4 wild rice species collected during the study.*

**Figure 8.**

*(a) The transverse section of culm of* O. rhizomatis *(b) the section through a portion of* O. rufipogon *culm encircled by leaf sheath (c) the culm section of* O. nivara*.*

#### **Figure 9.**

*Dendrogram produced by cluster analysis of anatomical characters of wild rice species,* O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

features indicated that anatomical characters well-separate the samples of each wild rice species. The samples of *O. rhizomatis* formed a unique group at similarity level of 80%. The pattern of the sample grouping was similar to the results obtained from the cluster analysis of morphological characters. However, samples were homogenized representing each wild rice species by pure tree branch.

The dendrogram resulted from the morphological and anatomical characters are shown in **Figure 10**. The grouping pattern of wild rice samples obtained from the analysis of morphological characters and anatomical characters reflect the same pattern observed in previously (**Figures 5** and **9**).

#### **3.3 Molecular studies**

A total of three clusters were resulted from the cluster analysis of molecular data (**Figure 11**) and species were grouped under each cluster with respect to

### *Characterization of Wild Rice -* Oryza *Species Complexes in Sri Lanka DOI: http://dx.doi.org/10.5772/intechopen.97244*

**Figure 10.**

*Dendrogram produced by cluster analysis of morphological and anatomical characters of Wild rice cultivars,* O. nivara*,* O. rufipogan*,* O. rhizomatis *and* O. eichingeri*.*

#### **Figure 11.**

*Dendrogram produced by cluster analysis of molecular data of wild rice species,* O. nivara*,* O. rufipogan *and* O. rhizomatis*.*

their genetic similarities. The samples of *O. nivara*, *O. rufipogon* and *O. rhizomatis* were very well separated from 40% similarity level confirming their distant relationship with each other and of independent evolution within Sri Lanka.
