Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

QTL analysis of morphological and developmental traits in the Ler × Cvi population of Arabidopsis thaliana

Drought frequently constrains production of wheat (Triticum aestivum L.), but development of tolerant cultivars is hampered by low heritability for drought tolerance and a lack of effective selection strategies. Our objective was to identify an optimum selection regime for wheat in drought-prone environments. Six-hundred entries derived from 10 crosses were developed by selection under continuous high moisture, alternating high with low moisture, alternating low with high moisture, and continuous low moisture conditions for five generations. The selections were evaluated in two low-yield, a medium-yield, and a high-yield environment in the Yaqui Valley, Sonora, Mexico. The mean performance of entries derived from a particular selection regime was dependant on the stress level of the evaluation environment. Lines developed and selected under continuous high moisture and continuous low-moisture regimes produced the highest mean yields in the low moisture evaluation environment. There was no relationship between continuous selection under either high yielding conditions or low yielding conditions and the mean performance of the resultant lines in their respective high and low yielding evaluation environments. The mean yield of lines selected using the alternating high/low moisture regime as well as the five highest yielding lines were superior in the HY environment, and had similar performance with other regimes under the low yielding evaluation environment. Our results indicate that alternating selection between high and low yielding environments is the most effective way to develop wheat germplasm adapted to environments where intermittent drought occurs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of a CSR10 (indica) × Taraori Basmati F3 population segregating for salt tolerance using ISSR markers

Segregation for salinity tolerance and ISSR markers based molecular polymorphism were investigated in a F₃ plant population raised via single-seed descent method from a cross between salt-tolerant indica rice variety CSR10 and salt-susceptible premium traditional Basmati rice variety Taraori Basmati HBC19. A total of 130 F₃plants were evaluated individually for salinity tolerance on 1–9 scale on the basis of seedling growth parameters; the average score ranged between 1.7 to 8.3. Frequency distribution curve obtained using the salinity tolerance data of F₃ population and a chi-square analysis, showed a good fit to a normal distribution. Eleven plants each in the category of salt-tolerant and salt-susceptible were selected from the segregating F₃ population for ISSR marker analysis. A total of 149 bands (4–11 bands per primer) ranging from 200 to 3530 bp were scored for the two rice varieties and the selected CSR10 × HBC19 segregating F₃ plants using 26 ISSR primers. Of these, 89 were monomorphic and 60 were polymorphic. Of the 60 polymorphic bands,36 and 20 bands were specific to CSR10 andHBC19 respectively. The remaining four bands were amplified using UBC primers 810,848, 853 and 886 and present in only some of the CSR10 × HBC19 F₃ plants. Notably, ISSR primers with dinucleotide repeat motif and 5'-anchored end amplified more number of bands (7.0 bands/primer) compared to3'-anchored dinucleotide primers (5.4bands/primer), but 3'-anchored dinucleotide primers revealed higher level of polymorphism (2.6 polymorphic bands/primer) compared to 5'-anchoreddinucleotide primers (1.43 polymorphic bands/ primer). While distribution of majority of the polymorphic bands were more or less in the expected ratios in salt-tolerant and/or salt-sensitive F₃segregating plants, but some of the bands amplified using UBC ISSR primers 823, 825,826, 849, 853, 864, 866 and 884 showed highly skewed distribution. Such polymorphic bands stand greater chances of having a linkage with the genes/ QTLs for salinity tolerance and shall be the target for further studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Water Stress on Physiological Attributes and their Relationship with Growth and Yield of Wheat Cultivars at Different Stages

The effects of water stress on physiological attributes of drought‐sensitive (Kalyansona) and drought‐tolerant (C‐306) wheat cultivars were studied in a pot experiment. Water stress was imposed by withholding irrigation at boot and anthesis stages. Leaf water potential, leaf osmotic potential and leaf turgor potential (measured with pressure chamber and osmometer), as well as leaf diffusive resistance, leaf transpiration rate and leaf‐to‐air transpiration gradient (measured with a steady‐state porometer) were measured diurnally. Growth and yield parameters were recorded after harvesting of the crop. Triplicate data were analysed using a completely randomized design and correlations amongst these parameters were computed. Water stress was found to reduce diurnal leaf water potential and leaf osmotic potential in both the genotypes but leaf osmotic potential was significantly higher in the drought‐tolerant cultivar C‐306 than in the drought‐sensitive cultivar Kalyansona. Positive turgor was recorded in both the genotypes under water stress and non‐stress conditions. Water‐stressed plants showed significantly lower turgor potential than control plants. In diurnal observations, water‐stressed plants exhibited significantly higher leaf diffusive resistance in both genotypes at both stages. The diffusive resistance of C‐306 was predominantly higher than that of Kalyansona. Water stress decreased leaf transpiration rate at both stages but the reduction was higher at the anthesis stage. The leaf‐to‐air temperature gradient was much higher in C‐306 than in Kalyansona at the boot stage but at the anthesis stage genotypic variation was non‐significant. The capacity to maintain cooler foliage was lower at the anthesis stage than at the boot stage in both the cultivars. Shoot dry weight, number of grains, test weight, grain yield, biological yield and harvest index decreased to a greater extent when water stress was imposed at the anthesis stage, while imposition of water stress at the boot stage caused a greater reduction in plant height and number of tillers. Similarly, water stress caused a smaller reduction in growth, yield and yield attributes in C‐306 than in Kalyansona. In general, the correlation coefficient of grain and biological yield with water potential and its components was positive and highly significant. Similarly, turgor potential was also correlated positively and significantly with grain yield at both the stages, but with biological yield it was significant only at the anthesis stage. A negative and significant correlation was obtained for diffusive resistance and leaf‐to‐air temperature gradient with grain yield at the boot and anthesis stages. The rate of transpiration was also positively and significantly correlated to grain and biological yields at both the stages. Amongst the yield attributes, number of leaves and number of tillers were positively correlated at the anthesis stage, whereas leaf area and shoot dry weight were significantly correlated with grain and biological yields at both the stages.     

Pre-Treatment of Currency Printing Ink Wastewater through Coagulation-Flocculation Process

Attempt has been made to study the treatability of printing ink wastewatergenerated from an Indian currency printing press using coagulation-flocculation process. Coagulant agents, viz. ferrous sulphate, ferric chloride, aluminium sulphate and polyaluminium chloride were studied to select the most suitablecoagulant for effective treatment, and attain the optimum coagulant concentration. Cationic polyeletrolyte in conjunction with the most effective coagulant was also studied to assess its effect on floc settleability. Polyaluminium chloride (PAC) was found to be the most efficient coagulant, achieving removals of colour, suspended solids (SS), biochemical oxygen demand (BOD) and chemical oxygen demand (COD) of 95.9–96.5%, 96.5–97.0%, 61.3–65.8%and 54.8–61.8%, respectively at an optimum concentration of 1500 mg L^-1. Other coagulants, viz. ferrous sulphate, ferric chloride and aluminium sulphate did not show substantial removals of colour, BOD and COD, except suspended solids in comparison to the performance of PAC. Cationic ploylectrolyte in combination with PAC at an optimum concentration of 1500 mg L^-1 of PAC and 1.0 mg L^-1 of polyelectrolyte further improved the removal efficiency of various parameters studied, in addition to improving the floc settling rate, and reduction in quantity of sludge generation.Effect of rapid mixing intensity expressed as mean temporal velocity gradient (G), and mixing time (t) on flocculation was also investigated. Rapid mixing at an impeller rotational speed (n) of 300 rpm and mixing time (t) of 60 sec indicated good floc formation, which resulted in achieving velocity gradient of 821 s^-1, and was found to be an optimum combination. This was also indicated by relatively high settling velocity of sludge formed and low residual colour in the supernatant. However, slow mixing beyond 5 min, adopted after initial optimum rapid mixing, indicated negligible effect on flocculation.     

Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.     

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular‐based phylogenetic inference and sequence analysis. Twenty‐two valid species and several species complexes were identified among nematodes included in the analysis. PCR‐RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR‐RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.     

Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells

A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.     

Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]

Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non‐availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty‐six clones were sequenced to generate 41.82‐kb high‐quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR‐containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F₂ mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross‐species amplification (~67%) in 10 millets and non‐millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.