Single base substitution causing the fragrant phenotype and development of a type-specific marker in aromatic coconut (Cocos nucifera).

The fragrance gene, betaine aldehyde dehydrogenase 2 (Badh2), has been well studied in many plant species. The objectives of this study were to clone Badh2 and compare the sequences between aromatic and non-aromatic coconuts. The complete coding region was cloned from cDNA of both aromatic and non-aromatic coconuts. The nucleotide sequences were highly homologous to Badh2 genes of other plants. Badh2 consisted of a 1512-bp open reading frame encoding 503 amino acids. A single nucleotide difference between aromatic and non-aromatic coconuts resulted in the conversion of alanine (non-aromatic) to proline (aromatic) at position 442, which was the substrate binding site of BADH2. The ring side chain of proline could destabilize the structure leading to a non-functional enzyme. Badh2 genomic DNA was cloned from exon 1 to 4, and from exon 5 to 15 from the two coconut types, except for intron 4 that was very long. The intron sequences of the two coconut groups were highly homologous. No differences in Badh2 expression were found among the tissues of aromatic coconut or between aromatic and non-aromatic coconuts. The amino acid sequences of BADH2 from coconut and other plants were compared and the genetic relationship was analyzed using MEGA 7.0. The phylogenetic tree reconstructed by the Bayesian information criterion consisted of two distinct groups of monocots and dicots. Among the monocots, coconut (Cocos nucifera) and oil palm (Elaeis guineensis) were the most closely related species. A marker for coconut differentiation was developed from one-base substitution site and could be successfully used.

Molecular barcoding of venomous snakes and species-specific multiplex PCR assay to identify snake groups for which antivenom is available in Thailand.

DNA barcodes of mitochondrial COI and Cytb genes were constructed from 54 specimens of 16 species for species identification. Intra- and interspecific sequence divergence of the COI gene (10 times) was greater than that of the Cytb gene (4 times), which suggests that the former gene may be a better marker than the latter for species delimitation in snakes. The COI barcode cut-off scores differed by more than 3% between most species, and the minimum interspecific divergence was greater than the maximum intraspecific divergence. Clustering analysis indicated that most species fell into monophyletic clades. These results suggest that these species could be reliably differentiated using COI DNA barcodes. Moreover, a novel species-specific multiplex PCR assay was developed to distinguish between Naja spp, Ophiophagus hannah, Trimeresurus spp, Hydrophiinae, Daboia siamensis, Bungarus fasciatus, and Calloselasma rhodostoma. Antivenom for these species is produced and kept by the Thai Red Cross for clinical use. Our novel PCR assay could easily be applied to venom and saliva samples and could be used effectively for the rapid and accurate identification of species during forensic work, conservation study, and medical research.

Isolation and characterization of novel microsatellite markers from Siamese fighting fish (Betta splendens, Osphronemidae, Anabantoidei) and their transferability to related species, B. smaragdina and B. imbellis.

Ten novel microsatellite markers were developed and characterized from Siamese fighting fish (Betta splendens). Nine of ten markers were polymorphic, exhibiting an allelic number (NA) from 2 to 6 alleles per locus. The effective number of alleles (NE) ranged from 1.60 to 3.08 (average of 2.30). The observed (HO) and expected (HE) heterozygosities ranged from 0.13 to 0.67 (average of 0.39) and 0.29 to 0.63 (average of 0.50), respectively. Linkage disequilibrium was not significantly detected for any pair of loci, and only two loci (BettaMS23 and BettaMS28) showed significant deviations from Hardy-Weinberg expectations. Of these, six loci could be amplified in genomic DNA of the closely related species B. imbellis and three loci in B. smaragdina. These microsatellite markers could be used as a tool to investigate genetic diversity and population structure, as well as breeding programs in hatcheries.

Development of microsatellite markers of vandaceous orchids for species and variety identification.

Vandaceous orchids are a group of orchid genera in the subfamily Vandoideae. Among this group, Mokara, Phalaenopsis, and Vanda are the most popular and commercially important orchids in Thailand. Novel microsatellite markers were developed from Mokara, the intergeneric hybrid from 3 genera Vanda, Ascocentrum, and Arachnis by using enriched method. Six primers from this study plus one primer previously developed from Vanda genome, a total of 7 markers, were selected to characterize 4 orchid genera (Mokara, Vanda, Rhynchostylis, and Ascocenda). The observed and expected heterozygosities varied in the 4 genera from 0.0000-1.0000 and 0.0000-0.8765, respectively. The transferability of these primers was also investigated in 76 vandaceous orchids from 12 genera. Three primer pairs, MOK26, MOK29, and MOK62, could successfully amplify the DNA of all samples, while MOK103 could be used with most of the samples. The total number of alleles from 76 samples ranged from 3 to 19 alleles per locus, with an average of 8.5714. Therefore, these markers could be used for variety/ species identification, certification and protection, genetic diversity, and evolutionary studies.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Squamata) by molecular cytogenetic approach.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L(4)(m) + L(2)(sm) + M(2)(m) + S(4)(m) + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.