Cryptococcus albidus and C. laurentii were the predominant non-neoformans cryptococci isolated during an environmental sampling study for C. gattii at Klang Valley, Malaysia. Cryptococcus gattii was not isolated from any of the environmental samples. Cryptococcus albidus and C. laurentii were isolated mainly from vegetative samples of Eucalyptus trees and bird droppings. Upon testing on canavanine-glycine-bromothymol blue (CGB) agar, all the C. albidus isolates remained unchanged. Interestingly, a total of 29 (76.3%) C. laurentii isolates formed blue colours on the CGB agar. Sequence analysis of ITS1-5.8rDNA-ITS2 gene sequences (468 bp) of four CGB-blue C. laurentii isolates demonstrated the closest match (99%) with that of C. laurentii CBS 7140. This study demonstrated the diverse environmental niche of C. albidus and C. laurentii in Malaysia.
The molecular types and genetic heterogeneity of Cryptococcus neoformans and C. gattii clinical isolates in Malaysia were determined in this study. Of 44 C. neoformans collected between 1980 and 2003, 42 (95.5%) were molecular type VNI, 2 (4.5%) were molecular type VNII. Of 17 C.gattii isolates, 13 (76.5%) were molecular type VGI, and 4 (23.5%) were molecular type VGII. A difference was noted when comparing the molecular types of cryptococcal isolates in the earlier and recent cases of cryptococcosis. While both molecular types VNI and VGI were equally predominant in the earlier cases of cryptococcosis, VNI was the most predominant molecular type isolated from the recent cases. VNII was a new molecular type, isolated from 5.1% of the recent cases. All the bird dropping isolates were molecular type VNI. The genetic heterogeneity of the two predominant molecular types, i.e., VNI, VGI clinical isolates and bird dropping isolates of C. neoformans were further determined by polymerase chain reaction (PCR) fingerprinting method, using (GTG)5 as single primer. Two clusters of cryptococcal isolates were distinguished at 68.5% of similarity, with cluster I consisting of VNI isolates and cluster II consisting of VGI isolates. Each cluster was further subdivided into three subtypes at >/=80% of similarity. Fourteen bird dropping isolates were grouped into a subtype within VN1, sharing 82.7% of similarity with the clinical isolates. A higher degree of similarities, ranging from 93.4-97.6% was noted between 3 bird dropping isolates with the clinical isolates in another subtype. This study demonstrated the existence of various molecular types of C. neoformans isolates in Malaysia and the genetic heterogeneity within the predominant molecular types. The study also provides evidence for genetic relatedness of clinical isolates with bird dropping isolates in the environment.
Sago plant (Metroxylon sagu Rottb.) is one of the most carbohydrate-producing plants in the world. Microsatellites or simple sequence repeats (SSRs) play an important role in the genome and are used extensively compared to other molecular markers. For the first time, we are exploiting data expressed sequence tags (EST) of sago plants to identify and characterise markers in this species. EST data about sago plants are obtained through the EST database on the National Center for Biotechnology Information (NCBI) website. We obtained data of 458 Kb (412 contig) with a maximum and minimum length of 1,138 and 124 nucleotides, respectively. We successfully identified 820 perfectly patterned SSR using Phobos 3.3.12 software. The type characterisation of EST-SSR was dominated by tri-nucleotides 36% (294), followed by hexa-nucleotides 24% (202), tetra-nucleotides 15% (120), penta-nucleotides 13% (108) and di-nucleotides 12% (96). The most frequency of SSR motifs in each type is AG, AAG and AAAG. Analysis of synteny on the EST sequence with the online application Phytozome found that sequences were distributed on 12 Oryza sativa chromosomes with a likeness percentage between 63% to 100% and e-value between 0 to 0.094. We developed the primer and generated 19 primers. Furthermore, we validated 7 primers that all generated polymorphic alleles. To our knowledge, this report is the first identification and characterisation of EST-SSR for sago species and these markers can be used for genetic diversity analysis, marker assisted selection (MAS), cultivar identification, kinship analysis and genetic mapping analysis.