Comparative cytogenetic mapping of rRNA genes among naked catfishes: implications for genomic evolution in the Bagridae family.

In the present study, the karyotype and chromosomal characteristics of 9 species of the Bagridae fish family were investigated using conventional Giemsa staining as well as dual-color fluorescence in situ hybridization to detect the 18S and 5S rDNA sites. In addition to describing the karyotype of several Bagridae catfishes, we established molecular cytogenetic techniques to study this group. The 9 species contained a diploid chromosomal number, varying from 50 (Pseudomystus siamensis) to 62 (Hemibagrus wyckii), while none contained heteromorphic sex chromosomes. 18S rDNA sites were detected in only 1 chromosomal pair among all species evaluated. However, 3 different patterns were observed for the distribution of the 5S rDNA: 2 sites were found in the genus Mystus and in P. siamensis, multiple sites were observed in the genus Hemibagrus, and a syntenic condition for the 18S and 5S rDNA sites was identified in H. wyckii. The extensive variation in the number and chromosomal position of rDNA clusters observed among these Bagridae species may be related to the intense evolutionary dynamics of rDNA-repeated units, which generates divergent chromosomal distribution patterns even among closely related species. In summary, the distribution of repetitive DNA sequences provided novel, useful information regarding the evolutionary relationships between Bagridae fishes.

RAPD and barcode analyses of groupers of the genus Epinephelus.

The diversity of Epinephelus species was investigated throughout Thailand. Random amplified polymorphic DNA successfully produced 1300 bands that were phylogenetically informative and used to construct cladograms. Values of pairwise genetic similarity (S) within species ranged from 0.65 in E. erythrurus to 0.99 in E. malabaricus. The interspecific values of S ranged from 0.23 between E. malabaricus and E. bleekeri to 0.66 between E. coeruleopunctatus and E. erythrurus. The intraspecific nucleotide variation ranged from 0.037 to 0.159 in the mitochondrially encoded 16S RNA (MT-RNR2) region and from 0.003 to 0.157 for the mitochondrially encoded cytochrome c oxidase I (MT-CO1) region. All sequences were submitted individually to GenBank. The barcode sequences of Thai species of Epinephelus were aligned to the same species found in GenBank. For the MT-RNR2 gene region, intraspecific nucleotide variation ranged from 0.000 to 0.121, and interspecific nucleotide variation ranged from 0.003 to 0.146. For the MT-CO1 gene region, intraspecific nucleotide variation ranged from 0.000 to 0.140, and interspecific nucleotide variation ranged from 0.000 to 0.166. The MT-RNR2 data indicate that some species, including E. bleekeri from India and E. malabaricus from Thailand are not monophyletic. Additionally, the MT-CO1 data indicated that E. bleekeri, E. quoyanus and E. coeruleopunctatus are not monophyletic. The sequences of E. lanceolatus from each country are highly conserved, with genetic distances ranging from 0.000 to 0.003. Another important result from this study is that the barcode sequence from Thai E. erythrurus was previously not present in the GenBank.

A molecular marker for in situ genetic resource conservation of Capsicum annuum var. acuminatum (Solanaceae).

The Thailand cultivar pepper 'phrik man bangchang' (Capsicum annuum var. acuminatum, Solanaceae) was originally cultivated in the Bangchang Subdistrict, Amphawa District in Samut Songkhram Province. The cultivated areas are limited; we verified its distribution in Thailand for in situ 'phrik man bangchang' genetic resource conservation. Samples were collected from the original cultivation area of Bangchang Subdistrict (Or) and were randomly explored in Ratchaburi Province (RB), Khon Kaen Province (KK), and Sakon Nakhon Province (SN). A pure line from The Tropical Vegetable Research Center at Kasetsart University was used as the standard indicator. Two more Capsicum species, C. chinensis and C. frutescens, and a species from another genus in the family, Solanum melongena, were included. A dendrogram constructed from random amplified polymorphic DNA fingerprints indicated that the Or, RB, KK, and SN samples were C. annuum var. acuminatum with supportive similarity coefficients of 0.79 to 0.98. Finally, DNA barcodes, from psbA-trnH spacer region, were provided for the 3 wild species, C. annuum var. acuminatum, C. chinensis, and C. frutescens under GenBank accession Nos. JQ087869-JQ087871. The nucleotide variations between species were 0.23 to 0.26. In summary, 'phrik man bangchang' is still being planted in Bangchang Subdistrict, but only in small areas. The distribution of planting areas is expected to be throughout Thailand.

Molecular identification and barcodes for the genus Nymphaea.

Nymphaea species, the most popular decorative plants, were collected for specificity of inter-simple sequence repeat (ISSR) analyses in species identification and differentiation of cultivars and natural populations. Dendrogram constructed from ISSR analyses separated out wild species, namely Nymphaea cyanea, N. nouchali, N. capensis, N. lotus and an outgroup N. mexicana, and cultivars. The dendrogram indicates that the cultivars should be differentiated from N. capensis, as they are sister individuals of N. capensis. The ISSR banding data and the dendrogram are concordantly concluded that wild N. capensis would be an effective type species for producing different cultivars. After plant identification by ISSR markers, DNA barcodes of all sample materials were done to provide species specific markers which can be used for rapid and accurate further plant identification without morphological characters. DNA barcoding sequence analysis indicates genetic distance values. All sequences were recorded in GenBank database.

Molecular analysis for genetic diversity and distance of introduced Grus antigone sharpii L. to Thailand.

The genetic relationship was examined in a population of Grus antigone sharpii L. using DNA markers from the ISSR technique for applying towards breeding purposes for conservation of species. Since their extinction from Thailand, sixteen eastern sarus cranes: Grus antigone sharpii L. provided from Cambodia were fed and bred to sixty individuals at Nakhonratchasima Zoo, Northeastern Thailand to re-exist in Thai natural sites. Their genetic diversity and distance were examined to test their possibility to adapt to environmental variation. Blood samples from 27 individuals of Grus antigone sharpii L. were collected and DNA was extracted. These DNA samples were amplified using the successful fifteen from twenty four primers inter simple sequences repeat markers. A dendrogram was constructed and shows distance values of the species between 12.1 and 53.5. The samples produced 63.96% polymorphic banding profiles. The genetic diversity (H') in this population was estimated using Shannon's index. The high H' value of 0.501 reflected the somewhat wide range of distribution sites, which would adapt to environmental variations. Genetic evenness is 0.152. This value supports that all the studied samples have a small equal genetic abundance.

Development of SCAR markers for species identification of the genus Nepenthes (Nepenthaceae).

Nepenthes species in Thailand, namely N. mirabilis Druce, N. gracilis Korth., N. smilesii Hemsl., N. ampullaria Jack and N. kampotiana Lecomte, were collected for development of Sequence Characterized Amplified Region (SCAR) marker, a genotype identification tool. Forty Random Amplified Polymorphic DNA (RAPD) primers were screened and three successful primers produced different banding patterns including five candidate species-specific markers. The candidate markers were cloned and sequenced. The marker sequences are 602, 379, 420, 473 and 1017 bp for N. mirabilis, N. gracilis, N. smilesii, N. ampullaria and N. kampotiana, respectively. Then the sequences were used to design primers for development of a species-specific band being a SCAR marker, including Mir 1, Mir 2 and Mir 3 for N. mirabilis; Gra 1 and Gra 2 for N. gracilis; Smi 1, Smi 2 and Smi 3 for N. smilesii; Amp 1 and Amp 2 for N. ampullaria and Kam 1 and Kam 2 of N. kampotiana. The primers were evaluated with each other Nepenthes species. Finally, species-specific SCAR markers were successfully developed for N. gracilis, N. ampullaria and N. kampotiana. Application of these markers is feasible for identification of Nepenthes species in Thailand.