Bananas in Thailand have been surveyed by our team to be at least 140 cultivars in the plantations, 10 wild species and, 4 introduced species. To characterize the genetic relationship of species and cultivars, a set of novel SSR markers was developed. Totaling 53 clones containing SSR motifs were isolated from SSR-enriched library of wild Musa balbisiana Colla 'Tani' (BB). Selected positive clones were used to design 28 primer pairs for amplification of 12 wild and 82 cultivar accessions with genome designations AA, AB, AAA, AAB, ABB, and BBB. These SSR markers loci were homology searched to the banana genomes to map their locations. The seven-sets multiplex PCR approach using four fluorescent-labeled universal primers were utilized for cost effectiveness. Capillary fragment analysis yielded the accurate size of amplicons for evaluation of particular patterns for each cultivar. Phylogram and Structure analysis presented the specific genotype of genome groups (A and B genotypes, polyploid hybrid genomes) and cultivar groups. By A:B specific alleles ratio, accurate genome designations of hybrids can be determined. Additionally, a marker, characterized to be partial plastid ycf2 gene, indicated the maternal identification of hybrid cultivars. One SSR marker was also preliminary tested with some wild species and advised to be the candidate fingerprinting marker for species identification. In conclusion, SSR marker sets developed here proved their exploitation in detailed identity and relationship of cultivated bananas, which would be useful for genetic conservation and ongoing breeding programs in Thailand and other areas.

Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. Methods: We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. Results: LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedlyhigher sensitivity than that of M-PCR (88% vs. 56%). Conclusions: LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.

Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica. The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.