SATrends Issue 47                                                                                                                  October 2004

  • Bioinformatics and Sequence analysis
  • Know thine enemy!
  • Peppered with salt!
  • Iron sharpens iron
  • 1. Bioinformatics and Sequence analysis

    Modern high throughput techniques employed in molecular biology laboratories have provided vast information on the genome in the form of nucleotide sequences in public databanks. Though these sequences are only strings of bases, locked up within them is a huge amount of information making them a highly valuable resource.

    A fundamental activity in Bioinformatics is the analysis of DNA and protein sequences. Proteins and genes in biology correspond to sequences and alignments in Bioinformatics. Alignment is the process of lining up two or more sequences to find the degree of similarity. There are several tools and techniques being used by bioinformaticians to derive meaning from sequences. This information can then be extended from one sequence to another through comparisons. This is how information from model crops can be extended to other food crops in silico. The challenge for Bioinformatics is three-fold: to rationalize the mass of sequence data, derive effective means of storage and design reliable analysis tools. More imperative is the need to convert sequence information into knowledge.

    There are two approaches to sequence analysis – (1) pattern recognition, used to detect similarity between sequences (this demands that some characteristic is already known and is housed in a database. Comparing a new sequence against those in the existing database using a search tool, such as BLAST, does this), and (2) ab initio prediction, used to deduce structure and to infer function directly from the sequence. This approach is still nascent. Given the variety of databases to search and the large number of freely available search tools, building a sensible analysis protocol for new sequences is important. Bioinformatics brings together a molecular biologist, biochemist, physiologist, biophysicist, statistician and computer scientist for interdisciplinary collaboration, and has enabled researchers from varied backgrounds to use databases and tools to make sense of all the available information.

    A multiple sequence alignment indicating conservation of amino acid residues across monocot and dicot plants (legumes in this case). The protein sequence over-expressed during stress is functionally annotated to be a lipid transfer protein.

    The Bioinformatics facility at ICRISAT has effectively used a combination of open source sequence analysis tools and parsing tools written in-house to analyze expressed sequence tags (ESTs) from biologically and agronomically relevant tissues in chickpea sequenced at ICRISAT, along with genomic sequences from groundnut and pigeonpea. The analysis protocol has allowed putative functional identification for most of the sequences, which have then been deposited into the public nucleic acid database - Genbank.

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    2. Know thine enemy!

    Knowledge of pathogen biology, epidemiology, host-pathogen interaction and genetics of resistance is essential to develop a disease management strategy. Man-made operations often result in ecological imbalance and lead to evolution of new virulent populations of pathogens. Eventually this breaks down host resistance and renders fungicide ineffective.

    Pearl millet (Pennisetum glaucum), a widely cultivated staple cereal, is the host of four major diseases: downy mildew (Sclerospora graminicola), ergot (Claviceps fusiformis), smut (Moesziomyces penicillariae), and rust (Puccinia substriata  var. indica). Downy mildew is the most widespread and economically significant. Research at ICRISAT and in partnership with other organizations has led to major advances in effective management of downy mildew.

    The downy mildew pathogen reproduces both sexually and asexually, and is thus genetically variable. The fungus is both soil- and seed-borne, and aerial dispersal occurs up to few hundred meters. With widespread cultivation of genetically uniform single-cross hybrids in India for the past 30 years, a few major epidemics occurred during the 1970s and the1980s resulting in boom-and-bust cycles of production. Several popular hybrids succumbed to the disease and were subsequently withdrawn from cultivation.

    A millet head rendered useless by downy mildew

    Techniques were developed and used to identify stable sources of genetic resistance through international collaborative nurseries. Several of these sources were used to breed disease resistant hybrid seed parents at ICRISAT, 60% of which are used by private seed companies and the All India Coordinated Pearl Millet Improvement Project (AICPMIP) to breed resistant hybrids. Both conventional and molecular breeding approaches are followed to transfer resistance genes in the parental lines.

    Monitoring virulence change in the pathogen populations provides proper guidance for the resistance breeding strategy. DNA markers have been identified for 60 different putative downy mildew resistance Quantitative Trait Locci (QTL) against various S. graminicola pathotypes in pearl millet. Using Restriction Fragment Length Polymorphism (RFLP)-based marker-assisted backcrossing, several QTLs have been transferred to the parental lines of a single-cross hybrid HHB 67. New hybrids based on improved parents have shown increased downy mildew resistance over the original HHB 67 in greenhouse and field evaluations. Two such hybrids that have agronomic traits superior to the original HHB 67 and improved downy mildew resistance, will soon be released by AICPMIP. No downy mildew epidemics in pearl millet have occurred during the past 10 years.

    Significant advances have been made, but the struggle continues. In view of the growing diversity in hybrids from private seed companies and the recent trend in climate change, we have to be more vigilant. Rapid and effective transfer and deployment of resistance should receive research priority to manage this disease.

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    3. Peppered with salt!

    Salinity-affected soils occupy about 10% of the world’s arable land. Soils are either naturally affected by salinity (costal and inland), or by mismanagement of irrigation water that builds up salt levels (secondary salinity). About 50% of irrigated land is affected by secondary salinity, and the salinity of arable lands is steadily increasing. Therefore, we urgently need to improve and develop salt-tolerant crops.

    An initiative to develop salinity-tolerant sorghum and pearl millet, two ICRISAT mandate crops, is supported by the OPEC Fund for International Development in collaboration with the International Center for Biosaline Agriculture (ICBA, Dubai). The work, initiated in mid 2002, has identified 17 sorghum and 11 pearl millet entries showing high tolerance to salinity, and 15 sorghum and 26 pearl millet entries that were very sensitive. The methodology involved growing plants in potted soils with 250 mM sodium chloride solution added to field capacity before sowing (resulting soil ECe 18.1 dS m-1). Harvests were made before anthesis and the selection was based on biomass production. The behavior of genotypes in glasshouse conditions at ICBA and at ICRISAT were largely similar.

    The salinity screening facility at ICRISAT-Patancheru.

    Several of the widely cultivated varieties and parental lines of widely cultivated hybrid sorghum were found to be tolerant to soil salinity. For example, variety ICSV 112 which is released and adopted in many countries in Asia, Africa and Latin America; drought-tolerant variety S 35 which is popular in western and central Africa; and a post rainy season variety, NTJ 2, which is also good for fodder.Similarly, several restorer lines (ICSR 90017, ICSR 56, ICSR 196, and ICSR 160) and A/B lines (ICS A/B 276, ICS A/B 300, ICS A/B 583 and ICS A/B 699) are parental lines of commercial hybrids.

    In pearl millet, several parental lines of commercial hybrids were found to be tolerant to highly tolerant of soil salinity. These include restorer lines such as ICMP 451 and HTP 94/54 (dual-purpose type); ICMR 356, and RIB 3135-15(drought tolerant); and maintainers of A-lines such as 841B and ICMB 91444.

    Simple screening procedures were developed, which allow the characterization of a large number of individuals – a requirement in molecular marker identification. The sodium and potassium contents and their ratios in shoots is a promising trait for salinity tolerance  (less sodium and more potassium = higher tolerance). Non-destructive methods of measuring salinity tolerance are being investigated to phenotype mapping populations. Identification of molecular markers for salinity tolerance, which has not yet been achieved for pearl millet and sorghum, is in progress.

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    4. Iron sharpens iron

    Iron sharpens iron: a farmer sharpens another farmer. Please take this message back home and apply it.” William Nhedzi, FFS member, Zvishavane, Zimbabwe

    New technologies diffuse slowly, especially in smallholder communities in semi-arid areas. Can new extension methods accelerate technology diffusion? The answer is yes, provided the extension approach involves genuine farmer participation.

    One participatory approach has proved successful in Zimbabwe: Farmer Field Schools (FFS), initially promoted by ICRISAT and FAO, and now being enthusiastically supported by government and NGO programs as well. An FFS is a group of farmers, led by a trained instructor. Each new management practice is applied jointly by the group on a ‘model’ field, and by individual farmers on their own plots. Performance is closely monitored throughout the season, the group discusses problems and solutions. Because they understand what they are doing, their profits are higher, and the risks (failure due to improper application of the technology) are lower.

    Farmers sharpening farmers!

    In the past 3 cropping seasons (2000/2001 to 2003/04), a total of 138 FFS on soil fertility and water management have been held in four drought-prone districts in southern Zimbabwe. These programs linked advice on soil water and nutrient technologies with development of agricultural input and product markets. The aim: provide farmers with information, and the means (market access) to utilize this information to increase their incomes. Over 3300 farmers have been trained and over 50,000 farmers exposed to new technologies through field days and farmer-to-farmer communication.

    ICRISAT worked with the national Department of Agricultural Research and Extension (AREX), to assess the impact of these efforts.

    • FFS “graduates” were more knowledgeable than non-FFS farmers about various resource conservation methods (see table)
    • Over 42% of FFS farmers, but only 27% of non-FFS farmers, had adopted improved soil fertility management practices
    • FFS farmers obtained higher yields during a drought year – 10% higher for sorghum, 40% higher for maize.

    Conclusion? FFS works. It’s a popular, practical way to communicate new ideas to farmers with little education and limited experience with new technologies. It’s particularly useful for knowledge-intensive technologies such as soil and water management. The national extension service has adopted FFS as a key part of their strategy to promote better management of natural resources

    .Knowledge scores (0-100) among FFS and non-FFS farmers
    Soil conservation
    Rainwater harvesting
    Soil fertility management
    Working together in groups

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