Rice and Arabidopsis BBX proteins: toward genetic engineering of abiotic stress resistant crops.

Productivity of crop plants are enormously affected by biotic and abiotic stresses. The co-occurrence of several abiotic stresses may lead to death of crop plants. Hence, it is the responsibility of plant scientists to develop crop plants equipped with multistress tolerance pathways. A subgroup of zinc finger transcription factor family, known as B-box (BBX) proteins, play a key role in light and hormonal regulation pathways. In addition, BBX proteins act as key regulatory proteins in many abiotic stress regulatory pathways, including Ultraviolet-B (UV-B), salinity, drought, heat and cold, and heavy metal stresses. Most of the BBX proteins identified in Arabidopsis and rice respond to more than one abiotic stress. Considering the requirement of improving rice for multistress tolerance, this review discusses functionally characterized Arabidopsis and rice BBX proteins in the development of abiotic stress responses. Furthermore, it highlights the participation of BBX proteins in multistress regulation and crop improvement through genetic engineering.

Association of Kiss1 and GPR54 Gene Polymorphisms with Polycystic Ovary Syndrome among Sri Lankan Women.

Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder affecting women of reproductive age. Its aetiology, though yet unclear, is presumed to have an oligogenic basis interacting with environmental factors. Kisspeptins are peptide products of Kiss1 gene that control the hypothalamic pituitary (HPG) axis by acting via G protein-coupled receptor known as GPR54. There is paucity of data on the role of Kiss1 and GPR54 gene in PCOS. We aimed to identify the polymorphisms in Kiss1 and GPR54 genes and explore their association with serum kisspeptin levels among Sri Lankan women with well-characterized PCOS. Consecutive women with PCOS manifesting from adolescence (n=55) and adult controls (n=110) were recruited. Serum kisspeptin and testosterone levels were determined by ELISA method. Whole gene sequencing was performed to identify the polymorphisms in Kiss1 and GPR54 genes. Serum kisspeptin and testosterone concentrations were significantly higher in women with PCOS than controls: kisspeptin 4.873nmol/L versus 4.127nmol/L; testosterone 4.713nmol/L versus 3.415 nmol/L, p<0.05. Sequencing the GPR54 gene revealed 5 single nucleotide polymorphisms (SNPs), rs10407968, rs1250729403, rs350131, chr19:918686, and chr19:918735, with two novel SNPs (chr19:918686 and chr19:918735), while sequencing the Kiss1 gene revealed 2 SNPs, rs5780218 and rs4889. All identified SNPs showed no significant difference in frequency between patients and controls. GPR54 gene rs350131 polymorphism (G/T) was detected more frequently in our study population. The heterozygous allele (AG) of GPR54 gene novel polymorphism chr19:918686 showed a marginal association with serum kisspeptin levels (p=0.053). Genetic variations in GPR54 and Kiss1 genes are unlikely to be associated with PCOS among Sri Lankan women manifesting from adolescence. Meanwhile the heterozygous allele of chr19:918686 is probably associated with serum kisspeptin concentrations, which suggests a potential role in the aetiology of PCOS.

Unveiling Members of Colletotrichum acutatum Species Complex Causing Colletotrichum Leaf Disease of Hevea brasiliensis in Sri Lanka.

Colletotrichum is an important fungal genus with great diversity, which causes anthracnose of a variety of crop plants including rubber trees. Colletotrichum acutatum and Colletotrichum gloeosporioides have been identified as the major causative agents of Colletotrichum leaf disease of rubber trees in Sri Lanka based on morphology, pathogenicity, and the analysis of internally transcribed spacer sequences of the nuclear ribosomal DNA. This study has been conducted to investigate the members of the C. acutatum species complex causing rubber leaf disease using a morphological and multi gene approach. For the first time in Sri Lanka, Colletotrichum simmondsii, Colletotrichum laticiphilum, Colletotrichum nymphaeae, and Colletotrichum citri have been identified as causative agents of Colletotrichum leaf disease in addition to C. acutatum s. str. Among them, C. simmondsii has been recognized as the major causative agent.

Cascade Screening for Fragile X Syndrome/CGG Repeat Expansions in Children Attending Special Education in Sri Lanka.

Fragile X syndrome (FXS) is the commonest cause of inherited mental retardation and clinically presents with learning, emotional and behaviour problems. FXS is caused by expansion of cytosine-guanine-guanine (CGG) repeats present in the 5' untranslated region of the FMR1 gene. The aim of this study was to screen children attending special education institutions in Sri Lanka to estimate the prevalence of CGG repeat expansions. The study population comprised a representative national sample of 850 children (540 males, 310 females) with 5 to 18 years of age from moderate to severe mental retardation of wide ranging aetiology. Screening for CGG repeat expansion was carried out on DNA extracted from buccal cells using 3' direct triplet primed PCR followed by melting curve analysis. To identify the expanded status of screened positive samples, capillary electrophoresis, methylation specific PCR and Southern hybridization were carried out using venous blood samples. Prevalence of CGG repeat expansions was 2.2%. Further classification of the positive samples into FXS full mutation, pre-mutation and grey zone gave prevalence of 1.3%, 0.8% and 0.1% respectively. All positive cases were male. No females with FXS were detected in our study may have been due to the small sample size.

Differentiation of Setaria digitata and Setaria labiatopapillosa using molecular markers.

5S rRNA intergenic regions of Setaria digitata and Setaria labiatopapillosa were PCR amplified with primers designed from the 5S rRNA gene of Brugia malayi. The ladder-like banding patterns obtained for the amplifications were distinctly different for the two species. Four amplified products were cloned into the pBS vector and completely sequenced. DNA clones from two individual samples of S. digitata, Sd4 and Sd6, showed 97% sequence homology to each other. All sequenced clones showed the presence of the spliced leader (SL) RNA gene with a 22 nucleotide spliced leader sequence. The phylogenetic tree constructed using these data and the 5S rRNA intergenic regions of several other filarial nematodes showed the Setaria species sharing a branch with Dirofilaria. RAPD-PCR analyses identified 107 bands of which 86 were polymorphic (80%). A dendrogram constructed for S. digitata and S. labiatopapillosa separated the two species into two distinct clusters. The polymorphic loci identified by the RAPD-PCR analyses can be studied further to develop species-specific probes/PCR primers for the identification of each species.

A sensitive polymerase chain reaction based assay for the detection of Setaria digitata: the causative organism of cerebrospinal nematodiasis in goats, sheep and horses.

A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previously reported oligonucleotide based chemiluminescent detection of microfilariae in infected host blood samples and L3 larvae in mosquitoes.

A simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.

Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.

Development of a rapid, nonradioactive, oligonucleotide-based assay for the detection of Setaria digitata.

Setaria species are filarial parasites which inhabit the peritoneal cavity of cattle and other ungulates. The parasite is generally considered to be nonpathogenic in its natural hosts, but the transmission of the infective larvae through mosquito vectors to its abnormal hosts (goats, sheep, or horses) can result in a serious and often fatal neuropathological disorder commonly referred to as cerebrospinal nematodiasis. We have previously described the cloning and characterization of a repetitive DNA sequence of Setaria digitata that could be used as a diagnostic probe to detect the parasite in host and vector populations. Here we report the development of a rapid nonradioactive hybridization assay using an oligonucleotide probe based on the above cloned repetitive sequence.

Development of a diagnostic DNA probe to detect Setaria digitata: the causative parasite of cerebrospinal nematodiasis in goats, sheep and horses.

Two repetitive sequences (IpSdM and IpSdS) have been cloned and sequenced from the genome of Setaria digitata. When IpSdM (214 bp) and IpSdS (201 bp) were aligned, a high degree of homology (85%) was observed, indicating that they belong to the same family of repeats. IpSdM represents a complete repeating element while IpSdS consists of two partial repeating elements arranged in tandem. The elements are present in about 10 000 copies comprising 2.8% of the S. digitata genome. As a diagnostic probe IpSdM detects as little as 100 pg DNA of both S. digitata and S. labiato-papillosa. It can also detect a single microfilaria and a L3 larva making it a valuable tool to monitor cattle and mosquito vector populations in the prevention of cerebrospinal nematodiasis.