Molecular Identification, Genetic Relationships and Development of DNA Markers of Spirogyra spp.

Abstract

This study was aimed to investigate the molecular identification, genetic relationships and development of DNA markers of Spirogyra spp. The 36 Spirogyra specimens were collected randomly in all part of Thailand during February 2009 to May 2011 including Chiang Rai, Chiang Mai, Phayao, Lampang, Lamphun, Phrae, Nan, Mae Hong Son, Tak, Uttaradit, Phitsanulok, Lop Buri, Ang Thong, Prachin Buri, Nakhon Sawan, Suphan Buri, Ratchaburi, Chainat, Kanchanaburi, Saraburi, Phetchaburi, Nakhon Phanom, Udon Thani, Loei, Maha Sarakham, Kalasin, Mukdahan, Chanthaburi, Rayong, Chumphon, Surat Thani, and Prachuap Khiri Khan provinces. The water quality from each collecting sites were recorded including pH, dissolve oxygen (DO), conductivity, biochemical oxygen demand (BOD), salinity and total dissolve solid (TDS), which was ranged from 4.09 – 9.04, 113 - 752 μs, 63 – 671 ppm, 0.1-0.8, and 5.5 - 11.2 mg/l, respectively. After that, all specimens of Spirogyra were investigated morphological characteristics under both of light microscope and scanning electron microscope. The results shown that, the Spirogyra specimens were classified into 5 patterns as fallows; Pattern 1: condensed and slightly compacted chloroplast spiral, Pattern 2: short cell with scattered chloroplast spiral, Pattern 3: long cell with less chloroplast spiral, Pattern 4: short cell with less chloroplast spiral and Pattern 5: long cell with condensed and compacted chloroplast spiral. In addition, DNA markers of 36 Spirogyra specimens were investigated using 10 ISSR primers (UBC 809, UBC 826, UBC 835, UBC 808, UBC 825, UBC 827, UBC 864, UBC 857, UBC 880 and UBC 807) and 5 primers of HAT-RAPD PCR (O 15, O 16, V 14, V 15 and B 01). The results shown that, both of PCR techniques were amplified 111 and 69 fragments, respectively. The analysis of the DNA fragments can be grouped 36 Spirogyra specimens by ISRR-PCR and HAT-RAPD PCR which are separated into five groups. Based on the 5 morphological patterns of DNA sequences of rbcL gene was sequenced and analyzed data by BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. The sequences data of Pattern 1 revealed definitive identity matches in the range of 99% for consensus sequences of S. ellipsospora Kütz, while Pattern 3 indicated definitive identity matches in the range only 93% with S. neglecta Kütz, which DNA sequences of rbcL gene could use as DNA markers of Spirogyra spp. The DNA sequences of the ITS 2 region of 5 patterns of Spirogyra are close to the Chlorella with 89-96%. Because of the sequences data of this ITS 2 was not available on NCBI database. The analysis of sequences data of both DNA sequences using UPGMA can be divided into five groups according to morphological characteristics.