Genetic Composition and Complexity of Virus Populations at Tungro-Endemic and Outbreak Rice Sites

We have recently demonstrated the geographic isolation of rice tungro bacilliform virus (RTBV) populations in the tungro-endemic provinces of Isabela and North Cotabato, Philippines. In this study, we examined the genetic structure of the virus populations at the tungro-outbreak sites of Lanao del Norte, a province adjacent to North Cotabato. We also analyzed the virus populations at the tungro-endemic sites of Subang, Indonesia, and Dien Khanh, Vietnam. Total DNA extracts from 274 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV. In the total population, 22 EcoRV-restricted genome profiles (genotypes) were identified. Although overlapping genotypes could be observed, the outbreak sites of Lanao del Norte had a genotype combination distinct from that of Subang or Dien Khanh but a genotype combination similar to that identified earlier from North Cotabato, the adjacent endemic province. Sequence analysis of the intergenic region and part of the ORF1 RTBV genome from randomly selected genotypes confirms the geographic clustering of RTBV genotypes and, combined with restriction analysis, the results suggest a fragmented spatial distribution of RTBV local populations in the three countries. Because RTBV depends on rice tungro spherical virus (RTSV) for transmission, the population dynamics of both tungro viruses were then examined at the endemic and outbreak sites within the Philippines. The RTBV genotypes and the coat protein RTSV genotypes were used as indicators for virus diversity. A shift in population structure of both viruses was observed at the outbreak sites with a reduced RTBV but increased RTSV gene diversity

Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites

• Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

Environmental and genetic control of arsenic accumulation and speciation in rice grain:comparing a range of common cultivars grown in contaminated sites across Bangladesh, China, and India

The concentration of arsenic (As) in rice grains has been identified as a risk to human health. The high proportion of inorganic species of As (As(i)) is of particular concern as it is a nonthreshold, class 1 human carcinogen. To be able to breed rice with low grain As, an understanding of genetic variation and the effect of different environments on genetic variation is needed. In this study, 13 cultivars grown at two field sites each in Bangladesh, India, and China are evaluated for grain As. There was a significant site, genotype, and site by genotype interaction for total grain As. Correlations were observed only between sites in Bangladesh and India, not between countries or within the Chinese sites. For seven cultivars the As was speciated which revealed significant effects of site, genotype, and site by genotype interaction for percentage As(i). Breeding low grain As cultivars that will have consistently low grain As and low As(i), over multiple environments using traditional breeding approaches may be difficult, although CT9993-5-10-1-M, Lemont, Azucena, and Te-qing in general had low grain As across the field sites.

Genome Wide Association Mapping of Grain Arsenic, Copper, Molybdenum and Zinc in Rice (Oryza sativa L.) Grown at Four International Field Sites

The mineral concentrations in cereals are important for human health, especially for individuals who consume a cereal subsistence diet. A number of elements, such as zinc, are required within the diet, while some elements are toxic to humans, for example arsenic. In this study we carry out genome-wide association (GWA) mapping of grain concentrations of arsenic, copper, molybdenum and zinc in brown rice using an established rice diversity panel of,300 accessions and 36.9 k single nucleotide polymorphisms (SNPs). The study was performed across five environments: one field site in Bangladesh, one in China and two in the US, with one of the US sites repeated over two years. GWA mapping on the whole dataset and on separate subpopulations of rice revealed a large number of loci significantly associated with variation in grain arsenic, copper, molybdenum and zinc. Seventeen of these loci were detected in data obtained from grain cultivated in more than one field location, and six co-localise with previously identified quantitative trait loci. Additionally, a number of candidate genes for the uptake or transport of these elements were located near significantly associated SNPs (within 200 kb, the estimated global linkage disequilibrium previously employed in this rice panel). This analysis highlights a number of genomic regions and candidate genes for further analysis as well as the challenges faced when mapping environmentally-variable traits in a highly genetically structured diversity panel.

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10−8 M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using 35S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [35S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent Kd values for [35S]PSK-α binding were found to be 1 × 10−9 M for the high-affinity type and 1 × 10−7 M for the low-affinity type, with maximal numbers of binding sites of 1 × 104 sites per cell and 1 × 105 sites per cell, respectively. Competition studies with [35S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.

Genetic Diversity of Rice Tungro Spherical Virus in Tungro-Endemic Provinces of the Philippines and Indonesia

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed

Inter-and Intra-Site Genetic Diversity of Natural Field Populations of Rice Tungro Bacilliform Virus in the Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host /environmental shifts.

Synchrony of planting and proportions of susceptible varieties affect rice tungro disease epidemics in the Philippines.

Surveys were conducted in the Philippines from 1995 to 1997 to examine relationships between production environment variables (agroecosystem, synchrony of planting, and varieties planted) and the occurrence of rice tungro disease epidemics using correspondence analyses. The sites covered were Isabela, Nueva Ecija, North Cotabato, and Bohol provinces as well as Bicol region. Tungro disease incidence in farmers’ fields was assessed visually based on typical symptoms. In addition, leaf samples were collected from each field and indexed serologically by enzyme-linked immunosorbent assay for the presence of Rice tungro bacilliform (RTBV) and Rice tungro spherical (RTSV) viruses. Thus, relationships between the production environment variables and four disease variables — visual incidence and double RTBV and RTSV, single RTSV, and single RTBV infections — were examined. A higher association was observed between site and varieties planted as well as site and synchrony of planting than between site and agroecosystem or site and disease variables (visual incidence, double RTBV and RTSV and single RTSV infections). Disease variables depended on both varieties planted and synchrony of planting and correspondence analysis revealed that the low disease incidence in Nueva Ecija was associated with synchronous planting while the high disease incidence in Isabela was associated with the planting of susceptible varieties and asynchronous planting. Such findings suggest that the relationship between the last two factors at a given site is critical to predicting tungro occurrence. Moreover, correspondence analysis of the relationship among disease variables revealed that tungro incidence is associated with not only double RTBV and RTSV infections but also single RTSV infections. Implications of these results on tungro epidemiology and management are discussed.

DNA Free Energy-Based Promoter Prediction and Comparative Analysis of Arabidopsis and Rice Genomes

The cis-regulatory regions on DNA serve as binding sites for proteins such as transcription factors and RNA polymerase. The combinatorial interaction of these proteins plays a crucial role in transcription initiation, which is an important point of control in the regulation of gene expression. We present here an analysis of the performance of an in silico method for predicting cis-regulatory regions in the plant genomes of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) on the basis of free energy of DNA melting. For protein-coding genes, we achieve recall and precision of 96% and 42% for Arabidopsis and 97% and 31% for rice, respectively. For noncoding RNA genes, the program gives recall and precision of 94% and 75% for Arabidopsis and 95% and 90% for rice, respectively. Moreover, 96% of the false-positive predictions were located in noncoding regions of primary transcripts, out of which 20% were found in the first intron alone, indicating possible regulatory roles. The predictions for orthologous genes from the two genomes showed a good correlation with respect to prediction scores and promoter organization. Comparison of our results with an existing program for promoter prediction in plant genomes indicates that our method shows improved prediction capability.

Characterization of rice small heat shock proteins targeted to different cellular organelles

Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure.

The ADH1B Arg47His polymorphism in East Asian populations and expansion of rice domestication in history

Background: The emergence of agriculture about 10,000 years ago marks a dramatic change in human evolutionary history. The diet shift in agriculture societies might have a great impact on the genetic makeup of Neolithic human populations. The regionally restricted enrichment of the class I alcohol dehydrogenase sequence polymorphism (ADH1BArg47His) in southern China and the adjacent areas suggests Darwinian positive selection on this genetic locus during Neolithic time though the driving force is yet to be disclosed. Results: We studied a total of 38 populations (2,275 individuals) including Han Chinese, Tibetan and other ethnic populations across China. The geographic distribution of the ADH1B*47His allele in these populations indicates a clear east-to-west cline, and it is dominant in south-eastern populations but rare in Tibetan populations. The molecular dating suggests that the emergence of the ADH1B*47His allele occurred about 10,000 similar to 7,000 years ago. Conclusion: We present genetic evidence of selection on the ADH1BArg47His polymorphism caused by the emergence and expansion of rice domestication in East Asia. The geographic distribution of the ADH1B*47His allele in East Asia is consistent with the unearthed culture relic sites of rice domestication in China. The estimated origin time of ADH1B*47His allele in those populations coincides with the time of origin and expansion of Neolithic agriculture in southern China.

Suitable sites for sustainable aquaculture development in Mymensingh, Bangladesh: A GIS methodological perspective

A GIS study was carried out to find out the sites suitable for aquaculture in Mymensingh region. A number of criteria were selected for GIS modeling followed by the approach of Kapetsky (1994). The criteria were developed from a range of existing data sources such as, surface and underground water level, availability of feed ingredients and animal wastes, sources of fish fry, market facilities, extension support and communication facilities for aquaculture development. The data were located, collected and compiled from different GOs and NGOs located in the region along with primary data from the field survey wherever necessary and then prepared for computer analyses. Using the database, a series of GIS models were developed in order to ascertain and prioritize the most suitable areas for aquaculture development in the region. Out of total 407,528 ha available lands in the region, the study identified 99,415 ha very suitable and 302,754 ha moderately suitable for aquaculture promotion. Result of the study is indicative to the modeling power of GIS for aquaculture application and could be used to refine the models in future, particularly if it is supported with further detail field data. To get a more concrete and complete model, detailed study should be made available on the availability of mustard oil cake, rice bran, wheat bran, and usable animal and urban wastes that could be used as low-cost feed for sustainable aquaculture.

Arsenic influence on genetic variation in grain trace-element nutrient content in Bengal delta grown rice

It has previously been shown that across different arsenic (As) soil environments, a decrease in grain selenium (Se), zinc (Zn), and nickel (Ni) concentrations is associated with an increase in grain As. In this study we aim to determine if there is a genetic element for this observation or if it is driven by the soil As environment. To determine the genetic and environmental effect on grain element composition, multielement analysis using ICP-MS was performed on rice grain from a range of rice cultivars grown in 4 different field sites (2 in Bangladesh and 2 in West Bengal). At all four sites a negative correlation was observed between grain As and grain Ni, while at three of the four sites a negative correlation was observed between grain As and grain Se and grain copper (Cu). For manganese, Ni, Cu, and Se there was also a significant genetic interaction with grain arsenic indicating some cultivars are more strongly affected by arsenic than others.

Arsenic limits Trace Mineral Nutrition (Selenium, Zinc, and Nickel) in Bangladesh Rice Grain

A reconnaissance of 23 paddy fields, from three Bangladesh districts, encompassing a total of 230 soil and rice plant samples was conducted to identify the extent to which trace element characteristics in soils and irrigation waters are reflected by the harvested rice crop. Field sites were located on two soil physiographic units with distinctly different As soil baseline and groundwater concentrations. For arsenic (As), both straw and grain trends closely fitted patterns observed for the soils and water. Grain concentration characteristics for selenium (Se), zinc (Zn), and nickel (Ni), however, were markedly different. Regressions of shoot and grain As against grain Se, Zn, and Ni were highly significant (P <0.001), exhibiting a pronounced decline in grain trace-nutrient quality with increasing As content. To validate this further, a pot experiment cultivar screening trial, involving commonly cultivated high yielding variety (HYV) rice grown alongside two U.S. rice varieties characterized as being As tolerant and susceptible, was conducted on an As-amended uniform soil. Findings from the trial confirmed that As perturbed grain metal(loid) balances, resulting in severe yield reductions in addition to constraining the levels of Se, Zn, and Ni in the grain.