The ribulosebiphosphate carboxylase rbcL sequence method has been extensively used

The ribulosebiphosphate carboxylase (rbcL) sequence method has been extensively used in studies of evolution, phylogeny, biogeography, population genetics, and systematics because it can be readily copied and not strikingly different for related species (Sheng-Guo et al., 2008; Doyle et al., 1997). The sequence of rbcL has been recorded in many studies and it is clear that this marker has great potential and benefit in terms of studying the genetic variations of the natural populations (Hamdam et al., 2013). This gene is far more variable in sequence. Because of the relatively rapid rate at which new mutants are fixed, these regions may be distinguished closely with other related species that otherwise would show little genetic divergence (Hamdam et al., 2013).
Our study aimed to determine the molecular identification, genetic relationships, and development of DNA markers of S. ellipsospora, using microsatellite markers and rbcL sequencing.

Materials and methods

Results
The general morphology of Spirogyra is characterized by a coiled chloroplast and a light green color. The cell is cylindrical. Apical HMBA Linker cost are tapering, with rounded tips and thick cell walls. There are five different morphological triads of the Spirogyra specimens. The arrangement of chloroplast spirals and granules of patterns 1 and 5 was highly condensed and compacted, while patterns 2, 3 and 4 were relatively scattered, as indicated (Triads 1): condensed and slightly compacted chloroplast spiral, (Triads 2): short cell with scattered chloroplast spiral, (Triads 3): long cell with less chloroplast spiral, (Triads 4): short cell with less chloroplast spiral and (Triads 5): long cell with condensed and compacted chloroplast spiral (see Fig. 1).
In terms of the molecular investigation, ninety-two scorable markers were produced using ten ISSR primers. The cluster analysis of the ISSR markers separated S. ellipsospora, other Spirogyra species and Cladophora sp. as out-groups into two district clusters, which included (cluster 1): S. ellipsospora, Spirogyra sp.1, and Spirogyra sp.4 and (cluster 2): Spirogyra sp.3, Spirogyra sp.2, and Cladophora sp. (Fig. 2).
Nucleotide amplification of rbcL revealed about 570bp fragments in each Spirogyra specimen. Based on the rbcL sequencing data we obtained, they were trimmed to provide an equivalence sequence among each morphological triad. The specific DNA fragment of rbcL was analyzed using the BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. Sequence data of S. ellipsospora from our study revealed definitive identity matches for S. ellipsospora for consensus sequences with 2 accession numbers of S. ellipsospora that are available on the NCBI database.
Phylogenetic trees were analyzed for the rbcL sequences using UPGMA. The phylogram could be separated into two district clusters (cluster 1): S. ellipsospora, Spirogyra sp.2, and Spirogyramaxima and (cluster 2): Spirogyra sp.1, Spirogyra sp.3, Spirogyra sp.4, and Spirogyra sp. (Fig. 3).

Discussion
At present, classical morphologically based methods and molecularly based methods are used for the identification of Spirogyra specimens, which are wildly distributed throughout all parts of Thailand. However, the phenotypic traits may lead to misidentifications and they may be more sensitive than with the molecular identification approach. The Spirogyra specimens were collected and then classified into five patterns under a light microscope.
The species concept of Spirogyra is based on morphological characteristics, which are probably not accurately distinguishable in terms of classification, except by a specially trained individual (McCourt et al., 1986). Moreover, difficulties arise because they are small and soft and also have only a few stable morphological characteristics and are subject to phenotypic variations. Thus, an identification of the closely related species of Spirogyra has only been based on morphological characteristics and as a result they can be confused or misidentified.