A UPLC-MS/MS method for quantification of ß-N-methylamino-L-alanine (BMAA) in Cycas sphaerica roxb. and its use in validating efficacy of a traditional BMAA removal method.

The presence of neurotoxin ß-N-Methylamino-L-alanine (BMAA) in the seeds of Cycas sphaerica is reported for first time. We developed a UPLC-MS/MS method for BMAA quantification by derivatizing with dansyl chloride. The method successfully differentiated L-BMAA from its structural isomer 2,4-diaminobutyric acid (DAB). The extracting mixture 0.1M TCA: ACN 4:1 v/v had a recovery level of >95%. The method is a high throughput sensitive chromatographic technique with 16.42 ng g-1 Limit of Quantification. BMAA was present in the endosperm of C. sphaerica, and was not detected in the leaves and pith. Washing of seeds in running cold water for 48 h reduced BMAA content by 86%. The local communities also treat the seeds under running cold water, but only for 24 h. The results of the study thus validated the traditional BMAA removal process through cold water treatment, but recommend for increase in the treatment period to 48 h or more.

Molecular phylogenetic relationships among four species of the mangrove tree genus Bruguiera (Rhizophoraceae), as revealed by chromosome and RAPD markers.

Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (approximately 17.0 pg) in the second group than in the first (approximately 8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (approximately 1,100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of approximately 600 bp and approximately 2,200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits ofBruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy.

Molecular phylogenetic relationships among four species of the mangrove tree genus Bruguiera (Rhizophoraceae), as revealed by chromosome and RAPD markers

Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (~17.0 pg) in the second group than in the first (~8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (~ 1 100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of ~600 bp and ~2 200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits of Bruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy. Rev. Biol. Trop. 55 (2): 437-448. Epub 2007 June, 29.

Estudiamos cuatro especies del mangle Bruguiera (Rhizophoraceae) en Orissa, India. Los cromosomas indican queB. cylindrica y B. parviflora son un grupo taxonómico, y que B. gymnorrhiza y B. sexangula son otro. Genéticamente, el par B. sexangula y B. gymnorrhiza está cercanamente emparentado con B. parviflora and B. cylindrica. Nuestros datos indican que el uso combinado de marcadores genéticos y evidencia citológica permiten discernir el polimorfismo genético interespecífico en los mangles, tanto para resolver problemas taxonómicos como para diseña estrategias eficaces de conservación.